[0001]This application claims priority to U.S. Provisional Application Nos. 60/782,428 filed Mar. 14, 2006 and 60/821,639 filed Aug. 7, 2006, the entire contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002]The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able to confer the trait of altered flowering time in plants. The present invention further relates to using the nucleic acid molecules and polypeptides to make transgenic plants or plant cells, plant materials or seeds of a plant capable of producing plants having flowering times that are altered with respect to wild-type plants grown under similar conditions. Altered flowering time includes both plants having an early flowering time and a late flowering time with respect to wild-type plants grown under similar conditions. Flowering time in plants may be altered either due to change of the timing of the developmental transition from vegetative to reproductive growth or because of acceleration or prolonging of the entire life cycle of the plant with respect to wild-type plants using the nucleic acid molecules and polypeptides of the present invention.
BACKGROUND OF THE INVENTION
[0003]The life cycle of plants is highly regulated at the genetic level and very sensitive to environmental variables, such as day length and temperature (Koomneef et al. (1998) Annu. Rev. Plant. Physiol. Mol. Biol., 49: 345-370). Plant life cycles are complex and must be regulated to maximize chances of survival. There must be a careful balance of investment of time in various stages of the life cycle of a plant, such as dormancy, germination, reproduction, vegetative growth and maturation, to insure survival. To implement this balance, each of these stages requires the expression or repression of various genes or groups of genes.
[0004]The life cycle of flowering plants in general can be divided into three growth phases: vegetative, reproductive, and seed development. In the vegetative phase, the shoot apical meristem (SAM) generates leaves that later will ensure the resources necessary to produce fertile offspring. Upon receiving the appropriate environmental and developmental signals the plant switches to floral, or reproductive, growth and the SAM enters the inflorescence phase and gives rise to an inflorescence with flower primordia. During this phase the fate of the SAM and the secondary shoots that arise in the axils of the leaves is determined by a set of meristem identity genes, some of which prevent and some of which promote the development of floral meristems. Once the floral organs are produced and fruits are formed, the plant enters seed development phase (Xu et al. (1995) Plant Mol. Biol. 27.237). If the appropriate environmental and developmental signals that induce the plant to switch to floral, or reproductive, growth are disrupted, the plant will not be able to enter reproductive growth, and will maintain vegetative growth.
[0005]Temporal coordination of life cycle stages depends on factors such as energy requirements, environmental variables and reproductive strategy. Maximization of any one life cycle period or stage is usually at the cost of one of the other periods or stages in the life cycle. For instance, trade-offs exist between the initiation of the reproductive growth stage and the length of the vegetative growth stage, the early flowering and the later growth and reproduction stages. The transition of a plant from a vegetative growth stage into a reproductive, or flowering, stage is pivotal because the fitness of a plant, and therefore its chances of survival, can be highly sensitive to this transition.
[0006]Thus, early flowering in plants can provide discernable advantages over other later flowering plants. For instance, early flowering usually results in early maturity and eventually shortens growth duration from sowing to harvest in plants. This allows farmers to avoid environmental adversity, such as freezing temperatures, in the early or later growing seasons at high latitude or altitude. This competitive advantage can also help crop rotation schedules. Alternatively, if cold weather or crop rotation is not a problem, later flowering has other advantages, such as a robust vegetative state, yielding higher amounts of plant material.
[0007]These advantages to the plant also translate into economic and production advantages providing more efficient human use of plants and especially plant crops critical to human survival. Generally, early flowering plants have smaller plant stature due to shortened vegetative growth compared to wild-type plants. Small plant stature is desirable in some cases. However, technologies enabling alteration of the moment within the life cycle at which a plant flowers are more advantageous if this alteration is accomplished without other disadvantageous trade-offs, such as a reduction in plant mass or other changes in phenotypically discernable traits.
[0008]Availability and maintenance of a reproducible stream of food to feed people has been a high priority throughout the history of human civilization. Specialists and researchers in the fields of crop science, horticulture and forest science are even today constantly striving to find and produce plants with an increased growth potential to feed an increasing world population and to guarantee a supply of reproducible raw materials. The robust level of research in these fields of science indicates the level of importance leaders in every geographic environment and climate around the world place on providing sustainable sources of food for their people.
[0009]Manipulation of crop performance has been accomplished conventionally for centuries through selection and plant breeding. The breeding process is, however, both time-consuming and labor-intensive. Furthermore, appropriate breeding programs must be specially designed for each relevant plant species.
[0010]On the other hand, great progress has been made in using molecular genetics approaches to manipulate plants to provide better crops. Through introduction and expression of recombinant nucleic acid molecules in plants, researchers are now poised to provide people with plant species tailored to grow more efficiently and produce more product despite unique geographic and/or climatic environments. These new approaches have the additional advantage of not being limited to one plant species, but instead being applicable to multiple different plant species (Zhang et al. (2004) Plant Physiol. 135:615).
[0011]Despite this progress, today there continues to be a great need for generally applicable processes that improve forest or agricultural plant growth to suit particular needs depending on specific environmental conditions. To this end, the present invention is directed to advantageously manipulating plant life cycles to maximize the benefits of various crops depending on the benefit sought and the particular environment in which the crop must grow, characterized by expression of recombinant DNA molecules in plants. These molecules may be from the plant itself, and simply expressed at a higher or lower level, or the molecules may be from different plant species, providing advantages from one species to another allowing the new plant species to better adapt to a challenging geographic or climatic environment.
SUMMARY OF THE INVENTION
[0012]The present invention, therefore, relates to isolated nucleic acid molecules and polypeptides and their use in making transgenic plants or plant cells, plant materials or seeds of plants capable of generating plants having life cycles, particularly flowering times, that are altered with respect to wild-type plants grown under similar or identical conditions.
[0013]Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
BRIEF DESCRIPTION OF THE FIGURES
[0014]FIG. 1. Amino acid sequence alignment of homologues of Clone 241491 transformed in ME08427 (SEQ ID NO: 95). Conserved regions are enclosed in a box.
[0015]FIG. 2. Amino acid sequence alignment of homologues of Clone 246416 transformed in ME03153 (SEQ ID NO: 139). Conserved regions are enclosed in a box.
[0016]FIG. 3. Amino acid sequence alignment of homologues of Clone 1010174 transformed in ME04928 (SEQ ID NO: 195). Conserved regions are enclosed in a box.
[0017]FIG. 4. Amino acid sequence alignment of homologues of Clone 22339 transformed in ME04522 (SEQ ID NO: 237). Conserved regions are enclosed in a box.
[0018]FIG. 5. Amino acid sequence alignment of homologues of Clone 13660 transformed in ME07393 (SEQ ID NO: 284). Conserved regions are enclosed in a box.
[0019]FIG. 6. Amino acid sequence alignment of homologues of Clone 708396 (SEQ ID NO: 298) transformed in ME03198, Clone 231890 (SEQ ID NO: 319) transformed in ME11261 and Clone 101876 (SEQ ID NO:564) transformed in ME04402. Conserved regions are enclosed in a box.
[0020]FIG. 7. Amino acid sequence alignment of homologues of At1g21340 transformed in ME11030 (Lead 107; SEQ ID NO: 326). Conserved regions are enclosed in a box.
[0021]FIG. 8. Amino acid sequence alignment of homologues of At2g44680 transformed in ME09573 (SEQ ID NO: 341). Conserved regions are enclosed in a box.
[0022]FIG. 9. Amino acid sequence alignment of homologues of Clone 32842 transformed in ME05718 (SEQ ID NO: 383). Conserved regions are enclosed in a box.
[0023]FIG. 10. Amino acid sequence alignment of homologues of Clone 251923 transformed in ME02194 (SEQ ID NO: 400). Conserved regions are enclosed in a box.
[0024]FIG. 11. Amino acid sequence alignment of homologues of Clone 149496 transformed in ME03847 (SEQ ID NO: 435). Conserved regions are enclosed in a box.
[0025]FIG. 12. Amino acid sequence alignment of homologues of Clone 21240 transformed in ME06492 (SEQ ID NO: 449). Conserved regions are enclosed in a box.
[0026]FIG. 13. Amino acid sequence alignment of homologues of Clone 19080 transformed in ME06561 (SEQ ID NO: 463). Conserved regions are enclosed in a box.
[0027]FIG. 14. Amino acid sequence alignment of homologues of Clone 33231 transformed in ME03027 (SEQ ID NO: 472). Conserved regions are enclosed in a box.
[0028]FIG. 15. Amino acid sequence alignment of homologues of Clone 33877 transformed in ME05742 (SEQ ID NO: 555). Conserved regions are enclosed in a box.
[0029]FIG. 16 Amino acid sequence alignment of homologues of clone 100516 (SEQ ID NO: 321). Conserved regions are enclosed in a box.
[0030]FIG. 17 Amino acid sequence alignment of homologues of clone 156373 (SEQ ID NO: 574). Conserved regions are enclosed in a box.
DETAILED DESCRIPTION OF THE INVENTION
1. The Invention
[0031]The invention of the present application may be described by, but not necessarily limited to, the following exemplary embodiments.
[0032]The present invention discloses novel isolated nucleic acid molecules, nucleic acid molecules that interfere with these isolated nucleic acid molecules, nucleic acid molecules that hybridize to these nucleic acid molecules, and isolated nucleic acid molecules that encode the same protein due to the degeneracy of the DNA code. Additional embodiments of the present application further include the polypeptides encoded by the isolated nucleic acid molecules of the present invention.
[0033]More particularly, the nucleic acid molecules of the present invention comprise: (a) a nucleotide sequence that encodes an amino acid sequence that is at least 85% identical to any one of the amino acid sequences in the sequence listing (SEQ ID NOS: 96, 140, 196, 238, 285, 299, 327, 342, 384, 401, 436, 320, 450, 464, 473, 556, 384, 399, 322, 565 and 575), (b) a nucleotide sequence that is complementary to any one of the nucleotide sequences according to (a), (c) a nucleotide sequence according to any one of SEQ ID NOS. 95, 139, 195, 237, 284, 298, 326, 341, 383, 400, 435, 319, 449, 463, 472, 555, 383, 398, 321, 564 and 574, (d) a nucleotide sequence able to interfere with any one of the nucleotide sequences according to (a), (e) a nucleotide sequence able to form a hybridized nucleic acid duplex with the nucleic acid according to any one of paragraphs (a)-(d) at a temperature from about 5° C. to about 10° C. below a melting temperature of the hybridized nucleic acid duplex, (f) a nucleotide sequence encoding any one of amino acid sequences corresponding to SEQ ID NOS. 96, 140, 196, 238, 285, 299, 327, 342, 384, 401, 436, 320, 450, 464, 473, 556, 384, 399, 322, 565 and 575, and (g) a nucleotide sequence encoding any one of the amino acid sequences that fits an HMM based on the sequences aligned in any one of FIGS. 1-17 with an HMM bit score greater than 20.
[0034]Additional embodiments of the present invention include those polypeptide and nucleic acid molecule sequences disclosed in SEQ ID NOS: 95-601.
[0035]Another embodiment of the present invention may additionally comprise amino acid sequences that are at least 85% identical to any one of SEQ ID Nos. 96, 140, 196, 238, 285, 299, 327, 342, 384, 401, 436, 320, 450, 464, 473, 556, 384, 399, 322, 565 and 575.
[0036]The present invention further embodies a vector comprising a first nucleic acid having a nucleotide sequence encoding a plant transcription and/or translation signal, and a second nucleic acid having a nucleotide sequence according to the isolated nucleic acid molecules of the present invention. More particularly, the first and second nucleic acids may be operably linked. Even more particularly, the second nucleic acid may be endogenous to a first organism, and any other nucleic acid in the vector may be endogenous to a second organism. Most particularly, the first and second organisms may be different species.
[0037]In a further embodiment of the present invention, a host cell may comprise an isolated nucleic acid molecule according to the present invention. More particularly, the isolated nucleic acid molecule of the present invention found in the host cell of the present invention may be endogenous to a first organism and may be flanked by nucleotide sequences endogenous to a second organism. Further, the first and second organisms may be different species. Even more particularly, the host cell of the present invention may comprise a vector according to the present invention, which itself comprises nucleic acid molecules according to those of the present invention.
[0038]Other embodiments of the present invention include methods of introducing an isolated nucleic acid of the present invention into a host cell. More particularly, an isolated nucleic acid molecule of the present invention may be contacted to a host cell under conditions allowing transport of the isolated nucleic acid into the host cell. Even more particularly, a vector as described in a previous embodiment of the present invention, may be introduced into a host cell by the same method.
[0039]Methods of detection are also available as embodiments of the present invention. Particularly, methods for detecting a nucleic acid molecule according to the present invention in a sample. More particularly, the isolated nucleic acid molecule according to the present invention may be contacted with a sample under conditions that permit a comparison of the nucleotide sequence of the isolated nucleic acid molecule with a nucleotide sequence of nucleic acid in the sample. The results of such an analysis may then be considered to determine whether the isolated nucleic acid molecule of the present invention is detectable and therefore present within the sample.
[0040]A further embodiment of the present invention comprises a plant or a plant cell, plant material or seeds of plants comprising an isolated nucleic acid molecule and/or vector of the present invention. More particularly, the isolated nucleic acid molecule of the present invention may be exogenous to the plant, plant cell, plant material or seed of a plant.
[0041]A further embodiment of the present invention includes a plant regenerated from a plant cell or seed according to the present invention. More particularly, the plant, or plants derived from the plant, plant cell, plant material or seeds of a plant of the present invention flowers early as compared to a wild-type plant cultivated under identical conditions. Furthermore, the transgenic plant may comprise a first isolated nucleic acid molecule of the present invention, which encodes an early flowering component, and a second isolated nucleic acid molecule which functions as a plant promoter, wherein the early flowering component and the plant promoter are operably linked. Alternatively, the early flowering component may be mis-expressed in the transgenic plant of the present invention, and the transgenic plant exhibits an earlier flowering time than a progenitor plant devoid of the gene, when the transgenic plant and the progenitor plant are cultivated under identical environmental conditions. In another embodiment of the present invention the modulated growth and phenotype characteristics may be due to the inactivation of a particular sequence, using such techniques as, for example, an interfering RNA.
[0042]A further embodiment consists of a plant, plant cell, plant material or seed of a plant according to the present invention which comprises an isolated nucleic acid molecule of the present invention, wherein the plant, or plants derived from the plant, plant cell, plant material or seed of a plant, flowers earlier than a wild-type plant cultivated under identical conditions.
[0043]Another embodiment of the present invention includes methods of inducing altered flowering time in plants. These methods may comprise transforming a plant with an isolated nucleic acid molecule according to the present invention.
[0044]Polypeptides of the present invention include sequences belonging to the consensus sequence families shown in FIGS. 1 to 17 as delineated by Hidden Markov Models (HMMs).
2. Definitions
[0045]The following terms are utilized throughout this application:
[0046]Altered flowering time: Altered flowering time may mean either that: (a) flowering time of a plant is earlier than a wild-type plant (i.e., early flowering) or (b) the flowering time is later than a wild-type plant (i.e., late flowering).
[0047]Early Flowering: Plant species vary in the temporal lengths of their life cycles. Plants can have life cycles that may be completed within one year or span across several years. Plants generally flower late in their life cycle, after embryogenesis, seedling development and a period of vegetative growth (Walbot (1985) Trends Genet. 1:165-169.). Flowering time in plants is influenced by many endogenous and environmental factors, including gibberellin biosynthesis and signaling, autonomous controls, light quality and intensity, photoperiod, temperature, and availability of nutrients (Gamer and Allard (1920) J. Agric. Res. 18:553-606; Bernier (1988) Annu. Rev. Plant Physiol. 39:175-219; Millar (1999) New Phytol. 14:175-197.; Battey (2000) J. Exp. Bot. 51:1769-1780; Samach and Coupland (2000) Bioessay, 22:38-47.). Early flowering may mean: (a) that the plant has begun to flower at a time statistically significantly earlier than a wild-type plant, grown under the same conditions because the transition from the vegetative, pre-flowering phase to the reproduction phase occurs earlier; or that the plant has begun to flower at a time statistically significantly earlier than a wild-type plant grown under the same conditions because the growth rate of the plant prior to flowering and/or in the entire life cycle has been enhanced.
[0048]Early flowering may also be described as a plant flowering at a moment in its life cycle that is at least 1% and 10% earlier in the plant's life cycle compared to a corresponding wild-type plant grown under identical conditions. Alternatively, the plant may begin to flower at a moment in the plant's life cycle that is at least 10% to 25% earlier or at least 25% and 50% earlier or at least 50% and 99% earlier.
[0049]Early Maturity: Seeds or fruits are formed after a plant flowers. Seeds or fruits undergo the processes of formation, development, in some cases desiccation, and maturation. Maturation means that the growth and development of the seed or fruit has been fulfilled such that it is ready for harvesting for consumption and/or reproducing the next generation of plants.
[0050]Early maturity may also be described as the seed or fruit of a plant maturing at a moment in its life cycle that is at least 1% to 10% earlier in the plant's life cycle compared to a corresponding wild-type plant grown under identical conditions. Alternatively, the seed or fruit may begin to mature at a moment in the plant's life cycle that is at least 2% to 5% earlier, 10% to 25% earlier or at least 25% to 50% earlier or at least 50% to 99% earlier.
[0051]Functionally Comparable Proteins: Functionally Comparable Proteins or Functional Homologs This phrase describes a set of proteins that perform similar functions within an organism. By definition, perturbation of an individual protein within that set (through misexpression or mutation, for example) is expected to confer a similar phenotype as compared to perturbation of any other individual protein. Such proteins typically share sequence similarity resulting in similar biochemical activity. Within this definition, homologs, orthologs and paralogs are considered to be functionally comparable.
[0052]Functionally comparable proteins will give rise to the same characteristic to a similar, but not necessarily the same, degree. Typically, comparable proteins give the same characteristics where the quantitative measurement due to one of the comparables is at least 20% of the other; more typically, between 30 to 40%; even more typically, between 50-60%; even more typically between 70 to 80%; even more typically between 90 to 100% of the other.
[0053]Heterologous sequences: “Heterologous sequences” are those that are not operatively linked or are not contiguous to each other in nature. For example, a promoter from corn is considered heterologous to an Arabidopsis coding region sequence. Also, a promoter from a gene encoding a growth factor from corn is considered heterologous to a sequence encoding the corn receptor for the growth factor. Regulatory element sequences, such as UTRs or 3′ end termination sequences that do not originate in nature from the same gene as the coding sequence, are considered heterologous to said coding sequence. Elements operatively linked in nature and contiguous to each other are not heterologous to each other. On the other hand, these same elements remain operatively linked but become heterologous if other filler sequence is placed between them. Thus, the promoter and coding sequences of a corn gene expressing an amino acid transporter are not heterologous to each other, but the promoter and coding sequence of a corn gene operatively linked in a novel manner are heterologous.
[0054]Hidden Markov Model (HMM): HMM is a statistical description of a sequence family's consensus. The model is indicative of similarity of a polypeptide sequence to a group of structurally and functionally related polypeptides (Durbin, R., Eddy, S. R., Krogh, A. & Mitchison, G. J. Biological Sequence Analysis. Probabilistic Models of Proteins and Nucleic Acids Cambridge University Press, Cambridge UK, 1998).
[0055]HMM based on specified sequences: An HMM profile based on specified sequences is the output model generated by the program HMMER 2.3.2 (released Oct. 3, 2003 under a GNU general public license, and available from various sources on the internet, such as the “HMMER” website) configured with default parameters, the model being built by the program using the specified sequences as input. The program outputs the model as a text file.
[0056]HMM bit score: An HMM bit score is a probabilistic indication of confidence that a sequence fits the model. The bit score reflects whether the sequence is a better fit to an HMM of interest than to a null model of nonhomologous sequences. A significant HMM bit score is greater than zero, but is typically greater than 20. The HMM bit score of a polypeptide sequence fitted to an HMM profile can be determined by fitting the polypeptide to the HMM with program HMMER 2.3.2 configured for glocal alignments.
[0057]Late flowering: In contrast to early flowering, late flowering may mean: (a) that the plant has begun to flower at a time statistically significantly later than a wild-type plant, grown under the same conditions because the transition from the vegetative, pre-flowering phase to the reproduction phase occurs later; or that the plant has begun to flower at a time statistically significantly later than a wild-type plant, grown under the same conditions because the growth rate of the plant prior to flowering and/or in the entire life cycle has been decreased.
[0058]Late flowering can also be described as a plant flowering at a moment in its life cycle that is at least 1% and 10% later in the plant's life cycle compared to a corresponding wild-type plant grown under identical conditions. Alternatively, the plant may begin to flower at a moment in the plant's life cycle that is at least 10% to 25% later or at least 25% and 50% later or at least 50% and 99% later.
[0059]Misexpression: The term “misexpression” refers to an increase or a decrease in the transcription of a coding region into a complementary RNA sequence as compared to transcription in the wild-type plant. This term also encompasses expression and/or translation of a gene or coding region or inhibition of such transcription and/or translation for a different time period as compared to that in the wild-type plant and/or from a non-natural location within the plant genome, including a gene coding region from a different plant species or from a non-plant organism.
[0060]Percentage of sequence identity: As used herein, the term “percent sequence identity” refers to the degree of identity between any given query sequence and a subject sequence. A subject sequence typically has a length that is from about 80 percent to 250 percent of the length of the query sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 percent of the length of the query sequence. A query nucleic acid or amino acid sequence is aligned to one or more subject nucleic acid or amino acid sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or protein sequences to be carried out across their entire length (global alignment). Chema et al. (2003) Nucleic Acids Res. 31(13):3497-500.
[0061]ClustalW calculates the best match between a query and one or more subject sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a query sequence, a subject sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: percentage; number of top diagonals: 4; and gap penalty: 5. For an alignment of multiple nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1; window size: 5; scoring method: percentage; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, and Lys; residue-specific gap penalties: on. The output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher website and at the European Bioinformatics Institute website on the World Wide Web.
[0062]To determine a percent identity for polypeptide or nucleic acid sequences between a query and a subject sequence, the sequences are aligned using Clustal W and the number of identical matches in the alignment is divided by the query length, and the result is multiplied by 100. The output is the percent identity of the subject sequence with respect to the query sequence. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
[0063]Plant Stature Generally, the whole plant body is comprised of the above-ground portion known as the shoot and the underground portion known as the root. The shoot may consist of stems, leaves, flowers, fruits, and other organs. The size and number of these plant organs determine the stature of the plant. Usually, plants with smaller stature than the wild-type plant produces less biomass than the wild-type plant.
[0064]Regulatory Regions The term “regulatory region” refers to nucleotide sequences that, when operably linked to a sequence, influence transcription initiation or translation initiation or transcription termination of said sequence and the rate of said processes, and/or stability and/or mobility of a transcription or translation product. As used herein, the term “operably linked” refers to positioning of a regulatory region and said sequence to enable said influence. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5′ and 3′ untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns. Regulatory regions can be classified in two categories, promoters and other regulatory regions.
[0065]Stringency: “Stringency,” as used herein is a function of nucleic acid molecule probe length, nucleic acid molecule probe composition (G+C content), salt concentration, organic solvent concentration and temperature of hybridization and/or wash conditions. Stringency is typically measured by the parameter Tm, which is the temperature at which 50% of the complementary nucleic acid molecules in the hybridization assay are hybridized, in terms of a temperature differential from Tm. High stringency conditions are those providing a condition of Tm-5° C. to Tm-10° C. Medium or moderate stringency conditions are those providing Tm-20° C. to Tm-29° C. Low stringency conditions are those providing a condition of Tm-40° C. to Tm-48° C. The relationship between hybridization conditions and Tm (in ° C.) is expressed in the mathematical equation:
[0000]
Tm=81.5−16.6(log10[Na+])+0.41(% G+C)−(600/N) (I)
[0000]where N is the number of nucleotides of the nucleic acid molecule probe. This equation works well for probes 14 to 70 nucleotides in length that are identical to the target sequence. The equation below, for Tm of DNA-DNA hybrids, is useful for probes having lengths in the range of 50 to greater than 500 nucleotides, and for conditions that include an organic solvent (formamide):
[0000]
Tm=81.5+16.6 log {[Na+]/(1+0.7[Na+])}+0.41(% G+C)−500/L 0.63(% formamide) (II)
[0000]where L represents the number of nucleotides in the probe in the hybrid (Bonner et al. (1973) J. Mol. Biol. 81:123). The Tm of Equation II is affected by the nature of the hybrid: for DNA-RNA hybrids, Tm is 10-15° C. higher than calculated; for RNA-RNA hybrids, Tm is 20-25° C. higher. Because the Tm decreases about 1° C. for each 1% decrease in homology when a long probe is used (Frischauf et al. (1983) J. Mol Biol, 170: 827-842), stringency conditions can be adjusted to favor detection of identical genes or related family members.
[0066]Equation II is derived assuming the reaction is at equilibrium. Therefore, hybridizations according to the present invention are most preferably performed under conditions of probe excess and allowing sufficient time to achieve equilibrium. The time required to reach equilibrium can be shortened by using a hybridization buffer that includes a hybridization accelerator such as dextran sulfate or another high volume polymer.
[0067]Stringency can be controlled during the hybridization reaction, or after hybridization has occurred, by altering the salt and temperature conditions of the wash solutions. The formulas shown above are equally valid when used to compute the stringency of a wash solution. Preferred wash solution stringencies lie within the ranges stated above; high stringency is 5-8° C. below Tm, medium or moderate stringency is 26-29° C. below Tm and low stringency is 45-48° C. below Tm.
[0068]For example, the hybridization step may be performed in aqueous hybridization solution at a temperature between 63° C. and 70° C., more preferably at a temperature between 65° C. and 68° C. and most preferably at a temperature of 65° C. Alternatively, the high stringency hybridization step may be performed in formamide hybridization solution at a temperature between 40° C. and 46° C., at a temperature between 41° C. and 44° C. and most preferably at a temperature of 42° C.
[0069]A wash step follows hybridization, and an initial wash is performed with wash solution 1 at 25° C. or 37° C. Following the initial wash, additional washes are performed with wash solution 1 at a temperature between 63° C. and 70° C., more preferably at a temperature between 65° C. and 68° C. and most preferably at a temperature of 65° C. The number of additional wash steps can be 1, 2, 3, 4, 5 or more. The time of both the initial and additional wash steps may be 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours, 2 hours or more.
[0070]Set forth below are the composition of the hybridization and wash solutions and their components. A person of ordinary skill in the art will recognize that these solutions are typical and exemplary of high stringency hybridization solutions.
- Aqueous Hybridization Solution: 6×SSC or 6×SSPE
- 0.05% Blotto or 5×Denhardt's Reagent
- 100 μg/ml denatured salmon sperm DNA
- 0.05% SDS
- Formamide Hybridization Solution: 50% Formamide
- 6×SSC or 6×SSPE
- 0.05% Blotto or 5×Denhardt's Reagent
- 100 μg/ml denatured salmon sperm DNA
- 0.05% SDS
- Wash Solution 1: 2×SSC or SSPE
- Wash Solution 2: 0.1×SSC or SSPE
- 20×SSC: 175.3 g NaCl
- 88.2 g Sodium Citrate
- Bring to 800 ml with H2O
- Adjust to pH 7 with 10 n NaOH
- Bring to 1 L with H2O
- 20×SSPE: 175.3 g NaCl
- 27.6 g NaH2PO4
- Bring to 800 ml with H2O.H2O
- 7.4 g EDTA
- Adjust to pH 7.4 with 10 n NaOH
- Bring to 1 L with H2O
- 1×BLOTTO: 5% Non-fat dry milk
- 50×Denhardts's Reagent: 5 g Ficoll
- 5 g Polyvinylpyrrolidone
- 5 g BSA
- Adjust to 500 ml with H2O
[0101]Superpool: As used in the context of the current invention, a “superpool” contains an equal amount of seed from 500 different events, representing 100 distinct exogenous nucleotide sequences. An event is a plant carrying a unique insertion of a distinct exogenous sequence which misexpresses that sequence. Transformation of a single polynucleotide sequence can result in multiple events because the sequence can insert in a different part of the genome with each transformation.
[0102]T0: The term “T0” refers to the whole plant, explant or callus tissue, inoculated with the transformation medium.
[0103]T1: The term T1 refers to either the progeny of the T0 plant, in the case of whole-plant transformation, or the regenerated seedling in the case of explant or callous tissue transformation.
[0104]T2: The term T2 refers to the progeny of the T1 plant. T2 progeny are the result of self-fertilization or cross-pollination of a T1 plant.
[0105]T3: The term T3 refers to second generation progeny of the plant that is the direct result of a transformation experiment. T3 progeny are the result of self-fertilization or cross-pollination of a T2 plant.
[0106]T4: As used in the current application, the term T4 refers to third generation progeny of the plant that is the direct result of a transformation experiment. T4 progeny are the result of self-fertilization or cross pollination of a T3 plant.
[0107]Transformation: Examples of means by which this can be accomplished are described below and include Agrobacterium-mediated transformation (of dicots (Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. (USA) 85:2444), of monocots (Yamauchi et al. (1996) Plant Mol. Biol. 30:321-9; Xu et al. (1995) Plant Mol. Biol. 27:237; Yamamoto et al. (1991) Plant Cell 3:371), and biolistic methods (P. Tijessen, “Hybridization with Nucleic Acid Probes” In Laboratory Techniques in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam), electroporation, in planta techniques and the like. Such a plant containing the exogenous nucleic acid is referred to here as a T0 for the primary transgenic plant and T1 for the first generation.
3. Important Characteristics of the Polynuceotides and Polypeptides of the Invention
[0108]The nucleic acid molecules and polypeptides of the present invention are of interest because when the nucleic acid molecules are misexpressed (i.e., when expressed at a non-natural location or in an increased or decreased amount relative to wild-type) they produce plants that exhibit an altered flowering time with respect to wild-type plants, as evidenced by the results of various experiments disclosed below. This trait can be used to exploit or maximize plant products. For example, the nucleic acid molecules and polypeptides of the present invention are used to increase the expression of genes that cause the plant to flower earlier or decrease the expression of genes that may cause the plant to flower later than the wild-type plant. As a consequence, these transgenic plants produce seed more quickly and/or exhibit altered biomass production characteristics that may be useful in certain climates and geographic regions.
[0109]As more and more transgenic plants are developed and introduced into the environment, it can be important to control the undesired spread of the transgenic trait(s) from transgenic plants to other traditional and transgenic cultivars, plant species and breeding lines, thereby preventing cross-contamination. The use of a conditionally lethal gene, i.e. one which results in plant cell death under certain conditions, has been suggested as a means to selectively kill plant cells containing a recombinant DNA (see e.g., WO 94/03619 and US patent publication 20050044596A1). Polypeptides comprised of a sequence belonging to the consensus sequence families shown in FIGS. 1 to 17 as delineated by HMMs can be utilized for the purposes of the invention, namely to make transgenic plants with altered flowering times.
[0110]Male or female sterile genes can also be used to control the spread of certain germplasm, such as by selective destruction of tissue, such as of the tapetum by fusing such a gene to a tapetum-specific promoter such as, TA29. Further examples of such promoters are described below.
4. The Polynucleotides/Polypeptides of the Invention
[0111]The polynucleotides of the present invention and the proteins expressed via translation of these polynucleotides are set forth in the Sequence Listing, specifically, SEQ ID NOS: 95-601. The Sequence Listing consists of functionally comparable proteins. Polypeptides comprised of a sequence within and defined by one of the consensus sequences in FIGS. 1 to 17 can be utilized for the purposes of the invention, namely to make transgenic plants with altered flowering times.
5. Use of the Polynucleotides to Make Transgenic Plants
[0112]The nucleic acid molecules of the present invention encode appropriate proteins from any organism, but may be found, for instance, in plants, fungi, bacteria or animals. The nucleic acid molecules of the present invention may be used to confer the trait of an altered flowering time.
[0113]To use the sequences of the present invention or a combination of them or parts and/or mutants and/or fusions and/or variants of them, recombinant DNA constructs are prepared that comprise the polynucleotide sequences of the invention inserted into a vector and that are suitable for transformation of plant cells. The construct can be made using standard recombinant DNA techniques (Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, New York.) and can be introduced into the plant species of interest by, for example, Agrobacterium-mediated transformation, or by other means of transformation, for example, as disclosed below.
[0114]The vector backbone may be any of those typically used in the field such as plasmids, viruses, artificial chromosomes, BACs, YACs, PACs and vectors such as, for instance, bacteria-yeast shuttle vectors, lamda phage vectors, T-DNA fusion vectors and plasmid vectors (Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA, 89:8794-8797; Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA, 93:9975-9979; Burke et al. (1987) Science 236:806-812); Sternberg N. et al. (1990) Proc Natl. Acad. Sci. USA, 87:103-7; Bradshaw et al., (1995) Nucl. Acids Res. 23, 4850-4856; Frischauf et al. (1983) J. Mol. Biol, 170:827-842; Huynh et al., Glover N. Mex. (ed) DNA Cloning: A practical Approach, Vol. 1, Oxford: IRL Press (1985); Walden et al. (1990) Mol. Cell. Biol. 1: 175-194).
[0115]Typically, the construct comprises a vector containing a nucleic acid molecule of the present invention with any desired transcriptional and/or translational regulatory sequences such as, for example, promoters, UTRs, and 3′ end termination sequences. Vectors may also include, for example, origins of replication, scaffold attachment regions (SARs), markers, homologous sequences, and introns. The vector may also comprise a marker gene that confers a selectable phenotype on plant cells. The marker may encode a biocide resistance trait, particularly antibiotic resistance, such as resistance to, for example, kanamycin, bleomycin, or hygromycin, or herbicide resistance, such as resistance to, for example, glyphosate, chlorosulfuron or phosphinotricin.
[0116]It will be understood that more than one regulatory region may be present in a recombinant polynucleotide, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements. Thus, more than one regulatory region can be operably linked to said sequence.
[0117]To “operably link” a promoter sequence to a sequence, the translation initiation site of the translational reading frame of said sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). For example, a suitable enhancer is a cis-regulatory element (−212 to −154) from the upstream region of the octopine synthase (ocs) gene (Fromm et al., The Plant Cell, 1:977-984 (1989).
[0118]Some suitable promoters initiate transcription only, or predominantly, in certain cell types. For example, a promoter that is active predominantly in a reproductive tissue (e.g., fruit, ovule, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo, zygote, endosperm, integument, or seed coat) can be used. Thus, as used herein a cell type- or tissue-preferential promoter is one that drives expression preferentially in the target tissue, but may also lead to some expression in other cell types or tissues as well. Methods for identifying and characterizing promoter regions in plant genomic DNA include, for example, those described in the following references: Jordano, et al. (1989) Plant Cell 1:855-866; Bustos et al. (1989) Plant Cell 1:839-854; Green et al. (1988) EMBO J. 7: 4035-4044; Meier et al. (1991) Plant Cell 3: 309-316; and Zhang et al. (1996) Plant Physiology 110: 1069-1079.
[0119]Examples of various classes of promoters are described below. Some of the promoters indicated below are described in more detail in U.S. patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140; 10/950,321; 10/957,569; 11/058,689; 11/172,703; 11/208,308; and PCT/US05/23639. It will be appreciated that a promoter may meet criteria for one classification based on its activity in one plant species, and yet meet criteria for a different classification based on its activity in another plant species.
[0120]Other Regulatory Regions: A 5′ untranslated region (UTR) can be included in nucleic acid constructs described herein. A 5′ UTR is transcribed, but is not translated, and lies between the start site of the transcript and the translation initiation codon and may include the +1 nucleotide. A 3′ UTR can be positioned between the translation termination codon and the end of the transcript. UTRs can have particular functions such as increasing mRNA stability or attenuating translation. Examples of 3′ UTRs include, but are not limited to, polyadenylation signals and transcription termination sequences, e.g., a nopaline synthase termination sequence.
[0121]Various promoters can be used to drive expression of the polynucleotides of the present invention. Nucleotide sequences of such promoters are set forth in SEQ ID NOS: 1-94. Some of them can be broadly expressing promoters; others may be more tissue preferential.
[0122]A promoter can be said to be “broadly expressing” when it promotes transcription in many, but not necessarily all, plant tissues. For example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems. As another example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds. Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326 (SEQ ID NO:76), YP0144 (SEQ ID NO:55), YP0190 (SEQ ID NO:59), p13879 (SEQ ID NO:75), YP0050 (SEQ ID NO:35), p32449 (SEQ ID NO:77), 21876 (SEQ ID NO:1), YP0158 (SEQ ID NO:57), YP0214 (SEQ ID NO:61), YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO:26), and PT0633 (SEQ ID NO:7) promoters. Additional examples include the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34S promoter, actin promoters such as the rice actin promoter, and ubiquitin promoters such as the maize ubiquitin-1 promoter. In some cases, the CaMV 35S promoter is excluded from the category of broadly expressing promoters.
[0123]Root-active promoters confer transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues. In some embodiments, root-active promoters are root-preferential promoters, i.e., confer transcription only or predominantly in root tissue. Root-preferential promoters include the YP0128 (SEQ ID NO:52), YP0275 (SEQ ID NO:63), PT0625 (SEQ ID NO:6), PT0660 (SEQ ID NO:9), PT0683 (SEQ ID NO: 14), and PT0758 (SEQ ID NO:22) promoters. Other root-preferential promoters include the PT0613 (SEQ ID NO:5), PT0672 (SEQ ID NO:11), PT0688 (SEQ ID NO:15), and PT0837 (SEQ ID NO:24) promoters, which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds. Other examples of root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et al., Proc. Natl. Acad. Sci. USA, 86:7890-7894 (1989)), root cell specific promoters reported by Conkling et al., Plant Physiol., 93:1203-1211 (1990), and the tobacco RD2 promoter.
[0124]In some embodiments, promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used. Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin promoter (Bustos et al., Plant Cell, 1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs et al., Plant Cell, 1(6):609-621 (1989)), the ACP promoter (Baerson et al., Plant Mol. Biol., 22(2):255-267 (1993)), the stearoyl-ACP desaturase promoter (Slocombe et al., Plant Physiol., 104(4):167-176 (1994)), the soybean α′ subunit of β-conglycinin promoter (Chen et al., Proc. Natl. Acad. Sci. USA, 83:8560-8564 (1986)), the oleosin promoter (Hong et al., Plant Mol. Biol., 34(3):549-555 (1997)), and zein promoters, such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al., Mol. Cell. Biol., 13:5829-5842 (1993)), the beta-amylase promoter, and the barley hordein promoter. Other maturing endosperm promoters include the YP0092 (SEQ ID NO:38), PT0676 (SEQ ID NO:12), and PT0708 (SEQ ID NO:17) promoters.
[0125]Promoters that are active in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, the melon actin promoter, YP0396 (SEQ ID NO:74), and PT0623 (SEQ ID NO:94). Examples of promoters that are active primarily in ovules include YP0007 (SEQ ID NO:30), YP0111 (SEQ ID NO:46), YP0092 (SEQ ID NO:38), YP1003 (SEQ ID NO:43), YP0028 (SEQ ID NO:33), YP0121 (SEQ ID NO:51), YP0008 (SEQ ID NO:31), YP0039 (SEQ ID NO:34), YP0115 (SEQ ID NO:47), YP0119 (SEQ ID NO:49), YP0120 (SEQ ID NO:50), and YP0374 (SEQ ID NO:68).
[0126]To achieve expression in embryo sac/early endosperm, regulatory regions can be used that are active in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell. A pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
[0127]Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see, Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No. 6,906,244). Other promoters that may be suitable include those derived from the following genes: maize MAC1 (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant Mol. Biol., 22:10131-1038). Other promoters include the following Arabidopsis promoters: YP0039 (SEQ ID NO:34), YP0011 (SEQ ID NO:41), YP0102 (SEQ ID NO:42), YP0110 (SEQ ID NO:45), YP0117 (SEQ ID NO:48), YP0119 (SEQ ID NO:49), YP0137 (SEQ ID NO:53), DME, YP0285 (SEQ ID NO:64), and YP0212 (SEQ ID NO:60). Other promoters that may be useful include the following rice promoters: p530c10 (SEQ ID NO:100), pOsFIE2-2 (SEQ ID NO:101), pOsMEA (SEQ ID NO:102, pOsYp102 (SEQ ID NO:103) and pOsYp285 (SEQ ID NO:104).
[0128]Regulatory regions that preferentially drive transcription in zygotic cells following fertilization can provide embryo-preferential expression. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable. Embryo-preferential promoters include the barley lipid transfer protein (Ltp1) promoter (Plant Cell Rep (2001) 20:647-654), YP0097 (SEQ ID NO:40), YP0107 (SEQ ID NO:44), YP0088 (SEQ ID NO:37), YP0143 (SEQ ID NO:54), YP0156 (SEQ ID NO:56), PT0650 (SEQ ID NO:8), PT0695 (SEQ ID NO:16), PT0723 (SEQ ID NO:19), PT0838 (SEQ ID NO:25), PT0879 (SEQ ID NO:28), and PT0740 (SEQ ID NO:20).
[0129]Promoters active in photosynthetic tissue confer transcription in green tissues such as leaves and stems. Most suitable are promoters that drive expression only or predominantly in such tissues. Examples of such promoters include the ribulose-1,5-bisphosphate carboxylase (PbcS) promoters such as the PbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al., Plant Cell Physiol., 35:773-778 (1994)), the Cab-1 promoter from wheat (Fejes et al., Plant Mol. Biol., 15:921-932 (1990)), the CAB-1 promoter from spinach (Lubberstedt et al., Plant Physiol., 104:997-1006 (1994)), the cab1R promoter from rice (Luan et al., Plant Cell, 4:971-981 (1992)), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al., Proc. Natl. Acad. Sci. USA, 90:9586-9590 (1993)), the tobacco Lhcb1*2 promoter (Cerdan et al., Plant Mol. Biol., 33:245-255 (1997)), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al., Planta, 196:564-570 (1995)), and thylakoid membrane protein promoters from spinach (psaD, psaF, psae, PC, FNR, atpC, atpD, cab, rbcS). Other photosynthetic tissue promoters include PT0535 (SEQ ID NO:3), PT0668 (SEQ ID NO:2), PT0886 (SEQ ID NO:29), YP0144 (SEQ ID NO:55), YP0380 (SEQ ID NO:70) and PT0585 (SEQ ID NO:4).
[0130]Examples of promoters that have high or preferential activity in vascular bundles include YP0087 (SEQ ID NO:86), YP0093 (SEQ ID NO:87), YP0108 (SEQ ID NO:88), YP0022 (SEQ ID NO:89), and YP0080 (SEQ ID NO:90). Other vascular tissue-preferential promoters include the glycine-rich cell wall protein GRP 1.8 promoter (Keller and Baumgartner, Plant Cell, 3(10):1051-1061 (1991)), the Commelina yellow mottle virus (CoYMV) promoter (Medberry et al., Plant Cell, 4(2):185-192 (1992)), and the Rice Tungro Bacilliform Virus (RTBV) promoter (Dai et al., Proc. Natl. Acad. Sci. USA, 101(2):687-692 (2004)).
[0131]Inducible promoters confer transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought. Examples of drought-inducible promoters include YP0380 (SEQ ID NO:70), PT0848 (SEQ ID NO:26), YP0381 (SEQ ID NO:71), YP0337 (SEQ ID NO:66), PT0633 (SEQ ID NO:7), YP0374 (SEQ ID NO:68), PT0710 (SEQ ID NO:18), YP0356 (SEQ ID NO:67), YP0385 (SEQ ID NO:73), YP0396 (SEQ ID NO:74), YP0388 (SEQ ID NO:92), YP0384 (SEQ ID NO:72), PT0688 (SEQ ID NO:15), YP0286 (SEQ ID NO:65), YP0377 (SEQ ID NO:69), PD1367 (SEQ ID NO:78), and PD0901 (SEQ ID NO:93). Examples of nitrogen-inducible promoters include PT0863 (SEQ ID NO:27), PT0829 (SEQ ID NO:23), PT0665 (SEQ ID NO:10), and PT0886 (SEQ ID NO:29). Examples of shade-inducible promoters include PR0924 (SEQ ID NO:91) and PT0678 (SEQ ID NO:13).
[0132]A basal promoter is the minimal sequence necessary for assembly of a transcription complex required for transcription initiation. Basal promoters frequently include a “TATA box” element that may be located between about 15 and about 35 nucleotides upstream from the site of transcription initiation. Basal promoters also may include a “CCAAT box” element (typically the sequence CCAAT) and/or a GGGCG sequence, which can be located between about 40 and about 200 nucleotides, typically about 60 to about 120 nucleotides, upstream from the transcription start site.
[0133]Other classes of promoters include, but are not limited to, shoot-preferential, callus-preferential, trichome cell-preferential, guard cell-preferential such as PT0678 (SEQ ID NO: 13), tuber-preferential, parenchyma cell-preferential, and senescence-preferential promoters. Promoters designated YP0086 (SEQ ID NO:36), YP0188 (SEQ ID NO:58), YP0263 (SEQ ID NO:62), PT0758 (SEQ ID NO:22), PT0743 (SEQ ID NO:21), PT0829 (SEQ ID NO:23), YP0119 (SEQ ID NO:49), and YP0096 (SEQ ID NO:39), as described in the above-referenced patent applications, may also be useful.
[0134]A promoter can be said to be “broadly expressing” when it promotes transcription in many, but not necessarily all, plant tissues or plant cells. For example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the shoot, shoot tip (apex), and leaves, but weakly or not at all in tissues such as roots or stems. As another example, a broadly expressing promoter can promote transcription of an operably linked sequence in one or more of the stem, shoot, shoot tip (apex), and leaves, but can promote transcription weakly or not at all in tissues such as reproductive tissues of flowers and developing seeds. Non-limiting examples of broadly expressing promoters that can be included in the nucleic acid constructs provided herein include the p326 (SEQ ID NO: 76), YP0144 (SEQ ID NO: 55), YP0190 (SEQ ID NO: 59), p13879 (SEQ ID NO: 75), YP0050 (SEQ ID NO: 35), p32449 (SEQ ID NO: 77), 21876 (SEQ ID NO: 1), YP0158 (SEQ ID NO: 57), YP0214 (SEQ ID NO: 61), YP0380 (SEQ ID NO: 70), PT0848 (SEQ ID NO: 26), and PT0633 (SEQ ID NO: 7). Additional examples include the cauliflower mosaic virus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1′ or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, the figwort mosaic virus 34S promoter, actin promoters such as the rice actin promoter, and ubiquitin promoters such as the maize ubiquitin-1 promoter. In some cases, the CaMV 35S promoter is excluded from the category of broadly expressing promoters.
[0135]Root-active promoters drive transcription in root tissue, e.g., root endodermis, root epidermis, or root vascular tissues. In some embodiments, root-active promoters are root-preferential promoters, i.e., drive transcription only or predominantly in root tissue. Root-preferential promoters include the YP0128 (SEQ ID NO: 52), YP0275 (SEQ ID NO: 63), PT0625 (SEQ ID NO: 6), PT0660 (SEQ ID NO: 9), PT0683 (SEQ ID NO: 14), and PT0758 (SEQ ID NO: 22). Other root-preferential promoters include the PT0613 (SEQ ID NO: 5), PT0672 (SEQ ID NO: 11), PT0688 (SEQ ID NO: 15), and PT0837 (SEQ ID NO: 24), which drive transcription primarily in root tissue and to a lesser extent in ovules and/or seeds. Other examples of root-preferential promoters include the root-specific subdomains of the CaMV 35S promoter (Lam et al. (1989) Proc. Natl. Acad. Sci. USA 86:7890-7894), root cell specific promoters reported by Conkling et al. (1990) Plant Physiol. 93:1203-1211 and the tobacco RD2 gene promoter.
[0136]In some embodiments, promoters that drive transcription in maturing endosperm can be useful. Transcription from a maturing endosperm promoter typically begins after fertilization and occurs primarily in endosperm tissue during seed development and is typically highest during the cellularization phase. Most suitable are promoters that are active predominantly in maturing endosperm, although promoters that are also active in other tissues can sometimes be used. Non-limiting examples of maturing endosperm promoters that can be included in the nucleic acid constructs provided herein include the napin promoter, the Arcelin-5 promoter, the phaseolin gene promoter (Bustos et al. (1989) Plant Cell 1(9):839-853), the soybean trypsin inhibitor promoter (Riggs et al. (1989) Plant Cell 1(6):609-621), the ACP promoter (Baerson et al. (1993) Plant Mol Biol, 22(2):255-267), the stearoyl-ACP desaturase gene (Slocombe et al. (1994) Plant Physiol 104(4):167-176), the soybean α′ subunit of β-conglycinin promoter (Chen et al. (1986) Proc Natl Acad Sci USA 83:8560-8564), the oleosin promoter (Hong et al. (1997) Plant Mol Biol 34(3):549-555), and zein promoters, such as the 15 kD zein promoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zein promoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoter from the rice glutelin-1 gene (Zheng et al. (1993) Mol. Cell. Biol. 13:5829-5842), the beta-amylase gene promoter, and the barley hordein gene promoter. Other maturing endosperm promoters include the YP0092 (SEQ ID NO: 38), PT0676 (SEQ ID NO: 12), and PT0708 (SEQ ID NO: 17).
[0137]Promoters that drive transcription in ovary tissues such as the ovule wall and mesocarp can also be useful, e.g., a polygalacturonidase promoter, the banana TRX promoter, and the melon actin promoter. Other such promoters that drive gene expression preferentially in ovules are YP0007 (SEQ ID NO: 30), YP0111 (SEQ ID NO: 46), YP0092 (SEQ ID NO: 38), YP0103 (SEQ ID NO: 43), YP0028 (SEQ ID NO: 33), YP0121 (SEQ ID NO: 51), YP0008 (SEQ ID NO: 31), YP0039 (SEQ ID NO: 34), YP0115 (SEQ ID NO: 47), YP0119 (SEQ ID NO: 49), YP0120 (SEQ ID NO: 50) and YP0374 (SEQ ID NO: 68).
[0138]In some other embodiments of the present invention, embryo sac/early endosperm promoters can be used in order drive transcription of the sequence of interest in polar nuclei and/or the central cell, or in precursors to polar nuclei, but not in egg cells or precursors to egg cells. Most suitable are promoters that drive expression only or predominantly in polar nuclei or precursors thereto and/or the central cell. A pattern of transcription that extends from polar nuclei into early endosperm development can also be found with embryo sac/early endosperm-preferential promoters, although transcription typically decreases significantly in later endosperm development during and after the cellularization phase. Expression in the zygote or developing embryo typically is not present with embryo sac/early endosperm promoters.
[0139]Promoters that may be suitable include those derived from the following genes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsis atmycl (see Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); Arabidopsis MEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No. 6,906,244). Other promoters that may be suitable include those derived from the following genes: maize MAC1 (see, Sheridan (1996) Genetics, 142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) Plant Mol. Biol., 22:10131-1038). Other promoters include the following Arabidopsis promoters: YP0039 (SEQ ID NO: 34), YP0101 (SEQ ID NO: 41), YP0102 (SEQ ID NO: 42), YP0110 (SEQ ID NO: 45), YP0117 (SEQ ID NO: 48), YP0119 (SEQ ID NO: 49), YP0137 (SEQ ID NO: 53), DME, YP0285 (SEQ ID NO: 64), and YP0212 (SEQ ID NO: 60). Other promoters that may be useful include the following rice promoters: p530c10, pOsFIE2-2, pOsMEA, pOsYp102, and pOsYp285.
[0140]Promoters that preferentially drive transcription in zygotic cells following fertilization can provide embryo-preferential expression and may be useful for the present invention. Most suitable are promoters that preferentially drive transcription in early stage embryos prior to the heart stage, but expression in late stage and maturing embryos is also suitable. Embryo-preferential promoters include the barley lipid transfer protein (Ltp1) promoter (Plant Cell Rep (2001) 20:647-654, YP0097 (SEQ ID NO: 40), YP0107 (SEQ ID NO: 44), YP0088 (SEQ ID NO: 37), YP0143 (SEQ ID NO: 54), YP0156 (SEQ ID NO: 56), PT0650 (SEQ ID NO: 8), PT0695 (SEQ ID NO: 16), PT0723 (SEQ ID NO: 19), PT0838 (SEQ ID NO: 25), PT0879 (SEQ ID NO: 28) and PT0740 (SEQ ID NO: 20).
[0141]Promoters active in photosynthetic tissue in order to drive transcription in green tissues such as leaves and stems are of particular interest for the present invention. Most suitable are promoters that drive expression only or predominantly such tissues. Examples of such promoters include the ribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcS promoter from eastern larch (Larix laricina), the pine cab6 promoter (Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778), the Cab-1 gene promoter from wheat (Fejes et al. (1990) Plant Mol. Biol. 15:921-932), the CAB-1 promoter from spinach (Lubberstedt et al. (1994) Plant Physiol. 104:997-1006), the cabIR promoter from rice (Luan et al. (1992) Plant Cell 4:971-981), the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuoka et al. (1993) Proc Natl Acad. Sci. USA 90:9586-9590), the tobacco Lhcb1*2 promoter (Cerdan et al. (1997) Plant Mol. Biol. 33:245-255), the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit et al. (1995) Planta 196:564-570), and thylakoid membrane protein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Other promoters that drive transcription in stems, leafs and green tissue are PT0535 (SEQ ID NO: 3), PT0668 (SEQ ID NO: 2), PT0886 (SEQ ID NO: 29), PR0924 (SEQ ID NO: 78), YP0144 (SEQ ID NO: 55), YP0380 (SEQ ID NO: 70) and PT0585 (SEQ ID NO: 4).
[0142]In some other embodiments of the present invention, inducible promoters may be desired. Inducible promoters drive transcription in response to external stimuli such as chemical agents or environmental stimuli. For example, inducible promoters can confer transcription in response to hormones such as giberellic acid or ethylene, or in response to light or drought. Examples of drought inducible promoters are YP0380 (SEQ ID NO: 70), PT0848 (SEQ ID NO: 26), YP0381 (SEQ ID NO: 71), YP0337 (SEQ ID NO: 66), PT0633 (SEQ ID NO: 7), YP0374 (SEQ ID NO: 68), PT0710 (SEQ ID NO: 18), YP0356 (SEQ ID NO: 67), YP0385 (SEQ ID NO: 73), YP0396 (SEQ ID NO: 74), YP0384 (SEQ ID NO: 72), PT0688 (SEQ ID NO: 15), YP0286 (SEQ ID NO: 65), YP0377 (SEQ ID NO: 69), and PD1367 (SEQ ID NO: 78). Examples of promoters induced by nitrogen are PT0863 (SEQ ID NO: 27), PT0829 (SEQ ID NO: 23), PT0665 (SEQ ID NO: 10) and PT0886 (SEQ ID NO: 29). An example of a promoter induced by salt is rd29A (Kasuga et al. (1999) Nature Biotech 17: 287-291).
[0143]Other Promoters: Other classes of promoters include, but are not limited to, leaf-preferential, stem/shoot-preferential, callus-preferential, guard cell-preferential, such as PT0678 (SEQ ID NO: 13), and senescence-preferential promoters. Promoters designated YP0086 (SEQ ID NO: 36), YP0188 (SEQ ID NO: 58), YP0263 (SEQ ID NO: 62), PT0758 (SEQ ID NO: 22), PT0743 (SEQ ID NO: 21), PT0829 (SEQ ID NO: 23), YP0119 (SEQ ID NO: 49), and YP0096 (SEQ ID NO: 39), as described in the above-referenced patent applications, may also be useful.
[0144]Alternatively, misexpression can be accomplished using a two component system, whereby the first component consists of a transgenic plant comprising a transcriptional activator operatively linked to a promoter and the second component consists of a transgenic plant that comprise a nucleic acid molecule of the invention operatively linked to the target-binding sequence/region of the transcriptional activator. The two transgenic plants are crossed and the nucleic acid molecule of the invention is expressed in the progeny of the plant. In another alternative embodiment of the present invention, the misexpression can be accomplished by having the sequences of the two component system transformed in one transgenic plant line.
[0145]Another alternative consists in inhibiting expression of a growth or phenotype-modulating polypeptide in a plant species of interest. The term “expression” refers to the process of converting genetic information encoded in a polynucleotide into RNA through transcription of the polynucleotide (i.e., via the enzymatic action of an RNA polymerase), and into protein, through translation of mRNA. “Up-regulation” or “activation” refers to regulation that increases the production of expression products relative to basal or native states, while “down-regulation” or “repression” refers to regulation that decreases production relative to basal or native states.
[0146]Another alternative consists in inhibiting expression of a biomass or vigor-modulating polypeptide in a plant species of interest under saline conditions. The term “expression” refers to the process of converting genetic information encoded in a polynucleotide into RNA through transcription of the polynucleotide (i.e., via the enzymatic action of an RNA polymerase), and into protein through translation of mRNA. “Up-regulation” or “activation” refers to regulation that increases the production of expression products relative to basal or native states, while “down-regulation” or “repression” refers to regulation that decreases production relative to basal or native states.
[0147]A number of nucleic-acid based methods, including anti-sense RNA, ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be used to inhibit protein expression in plants. Antisense technology is one well-known method. In this method, a nucleic acid segment from the endogenous gene is cloned and operably linked to a promoter so that the antisense strand of RNA is transcribed. The recombinant vector is then transformed into plants, as described above, and the antisense strand of RNA is produced. The nucleic acid segment need not be the entire sequence of the endogenous gene to be repressed, but typically will be substantially identical to at least a portion of the endogenous gene to be repressed. Generally, higher homology can be used to compensate for the use of a shorter sequence. Typically, a sequence of at least 30 nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500 nucleotides or more).
[0148]Thus, for example, an isolated nucleic acid provided herein can be an antisense nucleic acid to one of the aforementioned nucleic acids encoding a flowering-modulating polypeptide. A nucleic acid that decreases the level of a transcription or translation product of a gene encoding a growth or phenotype-modulating polypeptide is transcribed into an antisense nucleic acid similar or identical to the sense coding sequence of the growth or phenotype-modulating polypeptide. Alternatively, the transcription product of an isolated nucleic acid can be similar or identical to the sense coding sequence of a growth or phenotype-modulating polypeptide, but is an RNA that is unpolyadenylated, lacks a 5′ cap structure, or contains an unsplicable intron.
[0149]In another method, a nucleic acid can be transcribed into a ribozyme, or catalytic RNA, that affects expression of an mRNA. (See, U.S. Pat. No. 6,423,885). Ribozymes can be designed to specifically pair with virtually any target RNA and cleave the phosphodiester backbone at a specific location, thereby functionally inactivating the target RNA. Heterologous nucleic acids can encode ribozymes designed to cleave particular mRNA transcripts, thus preventing expression of a polypeptide. Hammerhead ribozymes are useful for destroying particular mRNAs, although various ribozymes that cleave mRNA at site-specific recognition sequences can be used. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target RNA contain a 5′-UG-3′ nucleotide sequence. The construction and production of hammerhead ribozymes is known in the art. See, for example, U.S. Pat. No. 5,254,678 and WO 02/46449 and references cited therein. Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA (tRNA) to increase cleavage efficiency in vivo (53-54). RNA endoribonucleases such as the one that occurs naturally in Tetrahymena thermophila, and which have been described extensively by Cech and collaborators can be useful. See, for example, U.S. Pat. No. 4,987,071.
[0150]Methods based on RNA interference (RNAi) can be used. RNA interference is a cellular mechanism to regulate the expression of genes and the replication of viruses. This mechanism is thought to be mediated by double-stranded small interfering RNA molecules. A cell responds to such a double-stranded RNA by destroying endogenous mRNA having the same sequence as the double-stranded RNA. Methods for designing and preparing interfering RNAs are known to those of skill in the art; see, e.g., WO 99/32619 and WO 01/75164. For example, a construct can be prepared that includes a sequence that is transcribed into an interfering RNA. Such an RNA can be one that can anneal to itself, e.g., a double stranded RNA having a stem-loop structure. One strand of the stem portion of a double stranded RNA comprises a sequence that is similar or identical to the sense coding sequence of the polypeptide of interest, and that is from about 10 nucleotides to about 2,500 nucleotides in length. The length of the sequence that is similar or identical to the sense coding sequence can be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to 300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25 nucleotides to 100 nucleotides. The other strand of the stem portion of a double stranded RNA comprises an antisense sequence of the biomass-modulating polypeptide of interest, and can have a length that is shorter, the same as, or longer than the corresponding length of the sense sequence. The loop portion of a double stranded RNA can be from 10 nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000 nucleotides, from 20 nucleotides to 500 nucleotides, or from 25 nucleotides to 200 nucleotides. The loop portion of the RNA can include an intron. See, e.g., WO 99/53050.
[0151]Transcriptional silencing of the target gene can also be achieved via the promoter through expression of an RNAi construct. This results in the synthesis of double stranded RNA molecules of which the nucleotides sequence is identical to a part of the promoter region of the target gene.
[0152]Another alternative method for suppression of the target gene may be achieved through a methodology generally referred to as Virus Induced Gene Silencing or VIGS (Ratcliff et al (2001) Plant J. 25, 237-245). Here, effective and specific gene silencing is achieved by infection of a plant with a plant virus carrying an insert which is homologous to the target gene. The advantage of the VIGS system is that there is no need to develop a plant transformation protocol for the plant species in which the target gene resides.
[0153]In all of these silencing methods, the silencing construct (antisense RNA, co-suppression, RNAi or hairpin construct or VIGs vector) preferably contains a DNA fragment that is identical to the target sequence (gene or promoter) that needs to be silenced. The percentage of identity may, however, range between 50-100%, preferably between 60-100%, more preferably between 70-100%, even more preferably between 80-100% and most preferably between 90-100%.
[0154]In some nucleic-acid based methods for inhibition of gene expression in plants, a suitable nucleic acid can be a nucleic acid analog. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugar moiety include modification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six-membered morpholino ring, or peptide nucleic acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained (for example, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195; Hyrup et al., 1996, Bioorgan. Med. Chem., 4:5-23.). In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite, or an alkyl phosphotriester backbone.
Transformation
[0155]Nucleic acid molecules of the present invention may be introduced into the genome or the cell of the appropriate host plant by a variety of techniques. These techniques, able to transform a wide variety of higher plant species, are well known and described in the technical and scientific literature (Vissenberg et al. (2005) Plant Cell Physiol 46:192; Husebye et al. (2002) Plant Physiol 128:1180.).
[0156]A variety of techniques known in the art are available for the introduction of DNA into a plant host cell. These techniques include transformation of plant cells by injection (Plesch et al. (2001) Plant J 28:455), microinjection (Griesbach (1987) Plant Sci. 50:69-77), electroporation of DNA (Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824), PEG (Paszkowski et al. (1984) EMBO J. 3:2717), use of biolistics (Klein et al. (1987) Nature 327:773), fusion of cells or protoplasts (Willmitzer, L. (1993) Transgenic Plants. In: iotechnology, A Multi-Volume Comprehensive treatise (H. J. Rehm, G. Reed, A. Püler, P. Stadler, eds., Vol. 2, 627-659, VCH Weinheim-New York-Base1-Cambridge)), and via T-DNA using Agrobacterium tumefaciens (Crit. Rev. Plant. Sci. 4:1-46; Fromm et al. (1990) Biotechnology 8:833-844) or Agrobacterium rhizogenes (Cho et al. (2000) Planta 210:195-204) or other bacterial hosts (Brootghaerts et al. (2005) Nature 433:629-633), for example.
[0157]In addition, a number of non-stable transformation methods that are well known to those skilled in the art may be desirable for the present invention. Such methods include, but are not limited to, transient expression (Lincoln et al. (1998) Plant Mol. Biol. Rep. 16:1-4) and viral transfection (Lacomme et al. (2001), “Genetically Engineered Viruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOS Scientific Publishers, Ltd. Oxford, UK).
[0158]Seeds are obtained from the transformed plants and used for testing stability and inheritance. Generally, two or more generations are cultivated to ensure that the phenotypic feature is stably maintained and transmitted.
[0159]A person of ordinary skill in the art recognizes that after the expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
[0160]In aspects related to making transgenic plants, a typical step involves selection or screening of transformed plants, e.g., for the presence of a functional vector as evidenced by expression of a selectable marker. Selection or screening can be carried out among a population of recipient cells to identify transformants using selectable marker genes such as herbicide resistance genes. Physical and biochemical methods can be used to identify transformants. These include Southern analysis or PCR amplification for detection of a polynucleotide; Northern blots, SI RNase protection, primer-extension, or RT-PCR amplification for detecting RNA transcripts; enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides; and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and/or polynucleotides. Methods for performing all of the referenced techniques are known.
[0161]A population of transgenic plants can be screened and/or selected for those members of the population that have a desired trait or phenotype conferred by expression of the transgene. For example, a population of progeny of a single transformation event can be screened for those plants having a desired level of expression of a heterologous salt tolerance polypeptide or nucleic acid. As an alternative, a population of plants comprising independent transformation events can be screened for those plants having a desired trait, such as salt tolerance. Selection and/or screening can be carried out over one or more generations, which can be useful to identify those plants that have a statistically significant difference in a protein level as compared to a corresponding level in a control plant. Selection and/or screening can also be carried out in more than one geographic location. In some cases, transgenic plants can be grown and selected under conditions which induce a desired phenotype or are otherwise necessary to produce a desired phenotype in a transgenic plant. In addition, selection and/or screening can be carried out during a particular developmental stage in which the phenotype is expected to be exhibited by the plant. Selection and/or screening can be carried out to choose those transgenic plants having a statistically significant difference in salt tolerance relative to a control plant that lacks the transgene. Selected or screened transgenic plants have an altered phenotype as compared to a corresponding control plant, as described elsewhere in this specification.
Transgenic Plant Phenotypes
[0162]Information that the polypeptides disclosed herein can modulate flowering are useful in breeding of crop plants. Based on the effect of the disclosed polypeptides on the timing of flowering, one can search for and identify polymorphisms linked to genetic loci for such polypeptides. Polymorphisms that can be identified include simple sequence repeats (SSRs), amplified fragment length polymorphisms (AFLPs) and restriction fragment length polymorphisms (RFLPs).
[0163]If a polymorphism is identified, its presence and frequency in populations is analyzed to determine if it is statistically significantly correlated to an alteration in timing of flowering. Those polymorphisms that are correlated with an alteration in timing of flowering can be incorporated into a marker assisted breeding program to facilitate the development of lines that have a desired alteration in the timing of flowering. Typically, a polymorphism identified in such a manner is used with polymorphisms at other loci that are also correlated with a desired alteration in timing of flowering or other desired trait.
[0164]The methods according to the present invention can be applied to any plant, such as, for example, higher plants, pertaining to the classes of Angiospermae and Gymnospermae. Plants of the subclasses of the Dicotylodenae and the Monocotyledonae are also suitable. Dicotyledonous plants belonging to the orders of the Magniolales, Illiciales, Laurales, Piperales Aristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales, and Asterales, for example, are also suitable. Monocotyledonous plants belonging to the orders of the Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales, Lilliales, and Orchidales also may be useful in embodiments of the present invention. Further examples include, but are not limited to, plants belonging to the class of the Gymnospermae are Pinales, Ginkgoales, Cycadales and Gnetales.
[0165]The methods of the present invention are preferably used in plants that are important or interesting for agriculture, horticulture, biomass for bioconversion and/or forestry. Non-limiting examples include, for instance, tobacco, oilseed rape, sugar beet, potatoes, tomatoes, cucumbers, peppers, beans, peas, citrus fruits, avocados, peaches, apples, pears, berries, plumbs, melons, eggplants, cotton, soybean, sunflowers, roses, poinsettia, petunia, guayule, cabbages, spinach, alfalfa, artichokes, sugarcane, mimosa, Servicea lespedera, corn, wheat, rice, rye, barley, sorghum and grasses such as switch grass, giant reed, Bermuda grass, Johnson grasses or turf grass, millet, hemp, bananas, poplars, eucalyptus trees and conifers. Of interest are plants grown for energy production, so called energy crops, such as broadleaf plants like alfalfa, hemp, Jerusalem artichoke and grasses such as sorghum, switchgrass, Johnson grass and the likes. Thus, the described materials and methods are useful for modifying biomass characteristics, such as characteristics of biomass renewable energy source plants. A biomass renewable energy source plant is a plant having or producing material (either raw or processed) that comprises stored solar energy that can be converted to fuel. In general terms, such plants comprise dedicated energy crops as well as agricultural and woody plants. Examples of biomass renewable energy source plants include: switchgrass, elephant grass, giant chinese silver grass, energycane, giant reed (also known as wild cane), tall fescue, bermuda grass, sorghum, napier grass, also known as uganda grass, triticale, rye, winter wheat, shrub poplar, shrub willow, big bluestem, reed canary grass and corn.
Homologues Encompassed by the Invention
[0166]It is known in the art that one or more amino acids in a sequence can be substituted with other amino acid(s), the charge and polarity of which are similar to that of the substituted amino acid, i.e. a conservative amino acid substitution, resulting in a biologically/functionally silent change. Conservative substitutes for an amino acid within the polypeptide sequence can be selected from other members of the class to which the amino acid belongs. Amino acids can be divided into the following four groups: (1) acidic (negatively charged) amino acids, such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids, such as arginine, histidine, and lysine; (3) neutral polar amino acids, such as serine, threonine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as glycine, alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, cysteine, and methionine.
[0167]Nucleic acid molecules of the present invention can comprise sequences that differ from those encoding a protein or fragment thereof selected from the group consisting of the nucleotide sequences in the sequence listing due to the fact that the different nucleic acid sequence encodes a protein having one or more conservative amino acid changes.
[0168]Biologically functional equivalents of the polypeptides, or fragments thereof, of the present invention can have about 10 or fewer conservative amino acid changes, alternatively about 7 or fewer conservative amino acid changes, and alternatively about 5 or fewer conservative amino acid changes. In another embodiment of the present invention, the polypeptide has between about 5 and about 500 conservative changes, alternatively between about 10 and about 300 conservative changes, and alternatively between about 25 and about 150 conservative changes, and alternatively between about 5 and about 25 conservative changes or between 1 and about 5 conservative changes.
Identification of Useful Nucleic Acid Molecules and Their Corresponding Nucleotide Sequences
[0169]The nucleic acid molecules, and nucleotide sequences thereof, of the present invention were identified by use of a variety of screens that are predictive of nucleotide sequences that provide plants with altered flowering times. One or more of the following screens were, therefore, utilized to identify the nucleotide (and amino acid) sequences of the present invention.
[0170]The present invention is further exemplified by the following examples. The examples are not intended to in any way limit the scope of the present application and its uses.
6. Experiments Confirming the Usefulness of the Polynucleotides and Polypeptides of the Invention
General Protocols
Agrobacterium-Mediated Transformation of Arabidopsis
[0171]Host Plants and Transgenes: Wild-type Arahidopsis thaliana Wassilewskija (WS) plants are transformed with Ti plasmids containing nucleic acid sequences to be expressed, as noted in the respective examples, in the sense orientation relative to the 35S promoter in a Ti plasmid. A Ti plasmid vector useful for these constructs, CRS 338, contains the Ceres-constructed, plant selectable marker gene phosphinothricin acetyltransferase (PAT), which confers herbicide resistance to transformed plants.
[0172]Ten independently transformed events are typically selected and evaluated for their qualitative phenotype in the T1 generation.
[0173]Preparation of Soil Mixture: 24 L Sunshine Mix #5 soil (Sun Gro Horticulture, Ltd., Bellevue, Wash.) is mixed with 16 L Therm-O-Rock vermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixer to make a 60:40 soil mixture. To the soil mixture is added 2 Tbsp Marathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE® 14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer 20-20-20 (J. R. Peters, Inc., Allentown, Pa.), which are first added to 3 gallons of water and then added to the soil and mixed thoroughly. Generally, 4-inch diameter pots are filled with soil mixture. Pots are then covered with 8-inch squares of nylon netting.
[0174]Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated. 25 drops are added to each pot. Clear propagation domes are placed on top of the pots that are then placed under 55% shade cloth and subirrigated by adding 1 inch of water.
[0175]Plant Maintenance: 3 to 4 days after planting, lids and shade cloth are removed. Plants are watered as needed. After 7-10 days, pots are thinned to 20 plants per pot using forceps. After 2 weeks, all plants are subirrigated with Peters fertilizer at a rate of 1 Tsp per gallon of water. When bolts are about 5-10 cm long, they are clipped between the first node and the base of stem to induce secondary bolts. Dipping infiltration is performed 6 to 7 days after clipping.
[0176]Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL each of carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stock concentration). Agrobacterium starter blocks are obtained (96-well block with Agrobacterium cultures grown to an OD600 of approximately 1.0) and inoculated one culture vessel per construct by transferring 1 mL from appropriate well in the starter block. Cultures are then incubated with shaking at 27° C. Cultures are spun down after attaining an OD600 of approximately 1.0 (about 24 hours). 200 mL infiltration media is added to resuspend Agrobacterium pellets. Infiltration media is prepared by adding 2.2 g MS salts, 50 g sucrose, and 5 μL 2 mg/mil benzylaminopurine to 900 ml water.
[0177]Dipping Infiltration: The pots are inverted and submerged for 5 minutes so that the aerial portion of the plant is in the Agrobacterium suspension. Plants are allowed to grow normally and seed is collected.
[0178]High-throughput Phenotypic Screening of Misexpression Mutants: Seed is evenly dispersed into water-saturated soil in pots and placed into a dark 4° C. cooler for two nights to promote uniform germination. Pots are then removed from the cooler and covered with 55% shade cloth for 4-5 days. Cotyledons are fully expanded at this stage. FINALE® (Sanofi Aventis, Paris, France) is sprayed on plants (3 ml FINALE® diluted into 48 oz. water) and repeated every 3-4 days until only transformants remain.
[0179]Phenotyping: Phenotyping is routinely performed at four stages: seedling, rosette, flowering and senescence.
- Seedling: the time after the cotyledons have emerged, but before the 3rd true leaf begins to form.
- Rosette—the time from the emergence of the 3rd true leaf through just before the primary bolt begins to elongate.
- Flowering—the time from the emergence of the primary bolt to the onset of senescence (with the exception of noting the flowering time itself, most observations should be made at the stage where approximately 50% of the flowers have opened).
- Senescence—the time following the onset of senescence (with the exception of “delayed senescence”, most observations should be made after the plant has completely dried). Seeds are then collected.
[0184]Validation: The early flowering validation was conducted in all experiments. The validation assays are implemented as follows.
[0185]Flowering time analyses are performed using at least two T2 or T3 events. Subsequently, the next generation (T3 or T4) seeds for the two events are evaluated under the same conditions. Briefly, soil is prepared as in the early flowering screen protocol. Soil in pots is subirrigated with water prior to adding seed. Five seeds are sown per pot. 24 pots are placed in a flat and covered with a clear plastic humidity dome. Flats are transferred to the dark at 4° C. for 3 days, then transferred to a growth chamber set at a 16:8 hour light:dark cycle or a 12:12 hour light:dark cycle, 150 μEinsteins, 70% relative humidity and 22° C.
[0186]After 5 days or when cotyledons are fully expanded, humidity domes are removed. On the 7th day seedlings are weeded such that only one seedling remains in each pot. In this assay the time of bolting is used to measure the flowering time. The “days to bolting” is the number of days post-sowing that are required for the inflorescence of the plant to reach 1 cm in height. The number of rosette leaves is also counted at this stage.
[0187]Five days post-bolting Finale™ resistant:sensitive analysis is conducted by harvesting a portion of a cauline leaf from each plant including the control. The leaf sections are placed on petrie plates containing the following media: 2.165 g/L MS salts, 0.5 g/L MES Hydrate, 7 g/L PhytoTech Agar and 200 μl/L filter sterilized Finale™. Plates are incubated for two days in a growth chamber set at a 16:8 hour light:dark cycle or a 12:12 hour light:dark cycle, 150 teinsteins, 70% relative humidity and 22° C. Plates are analyzed using a CF Fluorescent Imager. Transgenic plants (Finale™ resistant) appear red while non-transgenic plants (Finale™ sensitive) appear blue.
[0188]The days to bolting of the transgenic plants within an event in one generation was compared with that of non-transgenic segregants pooled across all events in the same experiment. A t-test is used to determine whether the events flower significantly earlier than wild-type Ws at p=0.05 assuming one-tailed distribution and unequal variance.
[0189]Determining Transgene Copy Number: T2 generation transformed plants are tested on BASTA™ plates in order to determine the transgene copy number of each transformed line. A BASTA™ resistant:BASTA™ sensitive segregation ratio of 3:1 generally indicates one copy of the transgene.
7. Results
MADS-box Proteins
[0190]MADS-box genes encode a family of transcriptional regulators that have been found in fungi, yeast, plants and animals. Members in this protein family contain a highly conserved ˜60 amino acid DNA-binding MADS domain. MADS-box proteins play pivotal roles in diverse biological processes in various organisms. In plants, MADS-box proteins are demonstrated to be involved in a number of developmental processes, such as flowering time, floral meristem identity, floral organogenesis and fruit formation (Parenicova et al. (2003) Plant Cell 15:1538-1551)
AP2 Domain Proteins
[0191]The AP2 domain is a DNA-binding motif of about 60 amino acid residues that is present in numerous proteins in plants. AP2 domain-containing proteins are involved in a wide range of biological functions such as flower and seed development (Jofuku et al. 1994), ethylene response (Ohme-Takagi and Shinshi 1995), temperature and dehydration response (Stockinger et al 1997) and cell expansion and differentiation (Wilson et al 1996).
Dof Domain Proteins
[0192]Dof (DNA-binding with one finger) domain polyupeptides are plant-specific transcription factor polypeptides with a highly conserved DNA-binding domain. The D of domain consists of 52 amino acid residues that shows sequence resemblance to the Cys2/Cys2 zinc finger domain in GATA1-type transcription factors, although it has a longer putative loop where an extra Cys residue is conserved (Umemura et al. (2004) Plant J. 37: 741-749).
Casein Kinase II Beta Chain Proteins
[0193]The casein kinase II (CK2) beta chain is a highly conserved subunit of casein kinase II, a ubiquitous, well-conserved serine/threonine protein kinase involved in cell metabolism and differentiation. Casein kinase II forms a heterotetrameric structure in vivo consisting of two catalytic alpha subunits and two regulatory beta subunits. The CK2 beta chain possesses an N-terminal auto-phosphorylation site, an internal acidic domain and a potential metal-binding motif. The beta subunit is a highly conserved protein of about 25 kD that contains, in its central section, a cysteine-rich motif that could be involved in binding a metal such as zinc. It has been shown that CK2 can modulate transcription factors that regulate light-responsive genes in plants (Sugano et al. (1998) Proc Natl Acad Sci USA 95: 11020-11025).
WD-40 Repeat Family Protein
[0194]WD-40 repeats are short, ˜40 amino acid motifs, often terminating in a Trp-Asp (W-D) dipeptide. WD-40-containing proteins usually have 4 to 16 repeating units, all of which are thought to form a circularized beta-propeller structure. WD-repeat proteins form a large family found in all eukaryotes and are implicated in a variety of functions ranging from signal transduction and transcription regulation to cell cycle control and apoptosis. An example is the pleiotropic developmental regulator LEUNING (LUG). LUG contains seven carboxyl-terminal WD motif repeats, internal polyglutamine tracts and an extended motif termed the single-stranded DNA-binding-protein. It has been shown that LUG functions in floral development together with SEUSS (SEU) (see Neer et al. (1994) Nature 371: 297-300).
60S Ribosomal Protein L21
[0195]Ribosomes are the particles in cells where mRNA-directed protein synthesis occurs in all organisms. L21 is one of the many subunits in ribosomes. It has been known that many of the proteins in the ribosome have other functions in addition to their roles in the ribosome (Crouzet and Begueret (1980) Mol Gen Genet. 180: 177-83).
Cyclin Family Proteins
[0196]Cyclins are key activators of cyclin-dependent Ser/Thr protein kinases, which play an essential role in controlling cell cycle progression in eukaryotes. In Arabidopsis, the cyclin family is comprised of over 50 genes. Overexpression of different cyclins has been demonstrated to enhance growth and development in plants. Cyclin may also play a role in the promoteion of cell division by the hormone cytokinin (Wnag et al (2004) Plant Physiol 135: 1084-1099).
PHD Finger Proteins
[0197]PHD finger proteins contain a PHD finger domain. This motif is similar to a C4HC3-type zinc finger motif and is often found in nuclear proteins which are thought to be involved in regulating gene transcription via chromatin remodeling. The PHD finger may act as a protein interaction domain although it is unclear whether PHD finger proteins have a common molecular function. In Arabidopsis, VIN3, a PHD-containing protein, functions as a suppressor of FLC (a negative regulator of flowering) in the vernalization pathway (Sung and Amasino, 2004).
Example 1
ME08427 (SEQ ID NO: 95)
[0198]The maize clone 241419 transformed in ME08427 encodes a MADS-box protein. An alignment of sequences showing similarity to the protein encoded by clone 241491 is presented in FIG. 1. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in both the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 1-1 and 1-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by a reduced number of rosette leaves, and a slight curly-leaf phenotype.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME08427 | ME08427-03 | T2 | 15.70 | 0.21 | 18 | 16.90 | 0.23 | 16 | 3.33E−04 |
| | T3 | 15.80 | 0.31 | 15 | 17.60 | 0.22 | 20 | 2.23E−05 |
| ME08427-05 | T2 | 15.90 | 0.24 | 14 | 16.90 | 0.23 | 16 | 3.86E−03 |
| | T3 | 15.10 | 0.25 | 13 | 17.60 | 0.22 | 20 | 5.07E−09 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME08427 | ME08427-03 | T2 | 5.72 | 0.13 | 18 | 6.63 | 0.13 | 16 | 1.33E−05 |
| | T3 | 4.93 | 0.27 | 15 | 5.90 | 0.10 | 20 | 9.03E−04 |
| ME08427-05 | T2 | 5.79 | 0.19 | 14 | 6.63 | 0.13 | 16 | 4.28E−04 |
| | T3 | 4.46 | 0.24 | 13 | 5.90 | 0.10 | 20 | 2.80E−06 |
|
Example 2
ME03153 (SEQ ID NO:139)
[0199]The maize clone 246416 transformed in ME03153 encodes a MADS-box protein. An alignment of sequences showing similarity to the protein encoded by clone 246416 is presented in FIG. 2. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in both the T2 and T3 generations. Data showing early flowering are presented in Tables 2-1 and 2-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves, and a slight curly-leaf phenotype.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME03153 | ME03153-01 | T2 | 15.20 | 0.32 | 14 | 16.80 | 0.26 | 21 | 3.59E−04 |
| | T3 | 14.40 | 0.16 | 18 | 15.30 | 0.41 | 6 | 2.42E−02 |
| ME03153-02 | T2 | 15.00 | 0.22 | 13 | 16.80 | 0.26 | 21 | 8.85E−06 |
| | T3 | 14.30 | 0.18 | 24 | 15.30 | 0.41 | 6 | 1.33E−02 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME03153 | ME03153-01 | T2 | 5.57 | 0.17 | 14 | 6.43 | 0.20 | 21 | 1.42E−03 |
| | T3 | 4.33 | 0.18 | 18 | 4.67 | 0.42 | 6 | 2.38E−01 |
| ME03153-02 | T2 | 5.15 | 0.19 | 13 | 6.43 | 0.20 | 21 | 3.31E−05 |
| | T3 | 4.08 | 0.08 | 24 | 4.67 | 0.42 | 6 | 9.28E−02 |
|
Example 3
ME04928 (SEQ ID NO: 195)
[0200]The Arabidopsis clone 1010174 transformed in ME04928 encodes a MADS-F box protein (AGL11). An alignment of sequences showing similarity to the protein encoded by clone 1010174 is presented in FIG. 3. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in both the T3 and the T4 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 3-1 and 3-2. The plants displayed a slight curly-leaf phenotype.
[0201]The Arabidopsis plants transformed with the maize clone 228787 and the Arabidopsis clone 32791 that have sequence homology to the clone 1010174 also showed early flowering. The transgenic plants are significantly smaller than the ME04928 transgenic plants.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME04928 | ME04928-02 | T3 | 16.10 | 0.15 | 21 | 17.90 | 0.14 | 49 | 4.43E−13 |
| | T4 | 15.68 | 0.11 | 46 | 17.33 | 0.31 | 6 | 3.08E−06 |
| ME04928-03 | T3 | 17.00 | 0.26 | 16 | 17.90 | 0.14 | 49 | 1.58E−03 |
| | T4 | 16.15 | 0.09 | 41 | 17.33 | 0.31 | 6 | 2.80E−04 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME04928 | ME04928-02 | T3 | 5.86 | 0.34 | 7 | 6.14 | 0.13 | 28 | 2.20E−01 |
| | T4 | 5.70 | 0.07 | 46 | 6 | 0.00 | 6 | 2.51E−05 |
| ME04928-03 | T3 | 5.62 | 0.18 | 13 | 6.14 | 0.13 | 28 | 1.19E−02 |
| | T4 | 5.93 | 0.05 | 41 | 6 | 0.00 | 6 | 9.10E−02 |
|
Example 4
ME04522 (SEQ ID NO:237)
[0202]The Arabidopsis clone 22339 transformed in ME04522 encodes a MADS-box K protein (AGL1, SHATTERPROOF1). An alignment of sequences showing similarity to the protein encoded by clone 22339 is presented in FIG. 4. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in both the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 4-1 and 4-2. Event -05 was noted having slightly shorter siliques than the control.
[0203]The Arabidopsis plants transformed with the maize clone 243227 that has sequence homology to the clone 22339 also showed early flowering . The transgenic plants are significantly smaller than the ME04522 transgenic plants
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME04522 | ME04522-02 | T2 | 18.13 | 0.15 | 20 | 18.94 | 0.26 | 18 | 5.22E−03 |
| | T3 | 16.24 | 0.19 | 21 | 17.92 | 0.23 | 26 | 7.70E−07 |
| ME04522-05 | T2 | 17.79 | 0.20 | 21 | 18.94 | 0.26 | 18 | 6.15E−04 |
| | T3 | 17.05 | 0.29 | 19 | 17.92 | 0.23 | 26 | 1.23E−02 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME04522 | ME04522-02 | T2 | 5.80 | 0.16 | 20 | 6.67 | 0.14 | 18 | 9.99E−05 |
| | T3 | 5.62 | 0.13 | 21 | 6.31 | 0.14 | 26 | 4.42E−04 |
| ME04522-05 | T2 | 6.19 | 0.15 | 21 | 6.67 | 0.14 | 18 | 1.25E−02 |
| | T3 | 5.84 | 0.19 | 19 | 6.31 | 0.14 | 26 | 2.93E−02 |
|
Example 5
ME07393 (SEQ ID NO:284)
[0204]The Arabidopsis clone 13660 transformed in ME07393 encodes a protein with unknown function. An alignment of sequences showing similarity to the protein encoded by clone 13660 is presented in FIG. 5. A 12:12 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in both the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 5-1 and 5-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME07393 | ME07393-03 | T2 | 26.29 | 0.20 | 21 | 27.71 | 0.47 | 7 | 4.94E−03 |
| | T3 | 24.38 | 0.31 | 16 | 27.71 | 0.47 | 7 | 3.96E−06 |
| ME07393-04 | T2 | 21.93 | 0.38 | 15 | 23.17 | 0.47 | 12 | 2.70E−02 |
| | T3 | 20.59 | 0.21 | 17 | 23.17 | 0.47 | 12 | 1.66E−05 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME07393 | ME07393-03 | T2 | 10.10 | 0.18 | 21 | 11.43 | 0.48 | 7 | 7.69E−03 |
| | T3 | 8.81 | 0.25 | 16 | 11.43 | 0.48 | 7 | 4.32E−05 |
| ME07393-04 | T2 | 7.47 | 0.27 | 15 | 8.33 | 0.45 | 12 | 5.60E−02 |
| | T3 | 6.88 | 0.15 | 17 | 8.33 | 0.45 | 12 | 2.41E−03 |
|
Example 6
ME03198 (SEQ ID NO: 298)
[0205]The soybean clone 708396 transformed in ME03198 encodes an AP2 domain-containing protein. An alignment of sequences showing similarity to the protein encoded by clone 708396 is presented in FIG. 6. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Three events were chosen to be analyzed in both the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 6-1 and 6-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Events | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME03198 | ME03198-01 | T2 | 16.84 | 0.14 | 19 | 17.82 | 0.15 | 17 | 1.86E−05 |
| | T3 | 16.67 | 0.15 | 21 | 17.99 | 0.13 | 41 | 5.79E−09 |
| ME03198-03 | T2 | 17.90 | 0.21 | 21 | 19.17 | 0.32 | 12 | 1.20E−03 |
| | T3 | 17.23 | 0.12 | 20 | 17.99 | 0.13 | 41 | 4.82E−05 |
| ME03198-04 | T2 | 17.27 | 0.19 | 11 | 17.82 | 0.15 | 17 | 1.78E−02 |
| | T3 | 16.10 | 0.16 | 21 | 17.99 | 0.13 | 41 | 4.30E−13 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME03198 | ME03198-01 | T2 | 5.21 | 0.12 | 19 | 6.35 | 0.15 | 17 | 4.81E−07 |
| | T3 | 6.05 | 0.05 | 21 | 6.93 | 0.12 | 41 | 1.73E−09 |
| ME03198-03 | T2 | 6.33 | 0.13 | 21 | 7.33 | 0.28 | 12 | 1.52E−03 |
| | T3 | 6.15 | 0.08 | 20 | 6.93 | 0.12 | 41 | 5.96E−07 |
| ME03198-04 | T2 | 5.45 | 0.16 | 11 | 6.35 | 0.15 | 17 | 1.50E−04 |
| | T3 | 5.10 | 0.17 | 21 | 6.93 | 0.12 | 41 | 6.32E−13 |
|
Example 7
ME11030 (SEQ ID NO:326)
[0206]The Arabidopsis genomic clone At1g21340 transformed in ME11030 encodes a Dof-type zinc finger domain-containing protein. An alignment of sequences showing similarity to the protein encoded by clone At1g21340 is presented in FIG. 7. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Three events were chosen to be analyzed in both the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 7-1 and 7-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Events | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME11030 | ME11030-01 | T2 | 17.71 | 0.23 | 17 | 18.83 | 0.26 | 18 | 1.32E−03 |
| | T3 | 17.10 | 0.12 | 20 | 18.30 | 0.11 | 53 | 6.79E−11 |
| ME11030-02 | T2 | 17.32 | 0.20 | 22 | 18.83 | 0.26 | 18 | 2.59E−05 |
| | T3 | 17.89 | 0.11 | 19 | 18.30 | 0.11 | 53 | 5.44E−03 |
| ME11030-03 | T2 | 16.92 | 0.14 | 13 | 18.24 | 0.23 | 21 | 1.16E−05 |
| | T3 | 17.66 | 0.13 | 16 | 18.30 | 0.11 | 53 | 1.22E−04 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME11030 | ME11030-01 | T2 | 6.18 | 0.18 | 17 | 6.94 | 0.10 | 18 | 2.93E−04 |
| | T3 | 6.15 | 0.13 | 20 | 7.28 | 0.09 | 53 | 2.53E−10 |
| ME11030-02 | T2 | 6.00 | 0.13 | 22 | 6.94 | 0.10 | 18 | 6.19E−07 |
| | T3 | 6.63 | 0.14 | 19 | 7.28 | 0.09 | 53 | 7.33E−05 |
| ME11030-03 | T2 | 4.93 | 0.13 | 14 | 5.76 | 0.19 | 21 | 5.22E−04 |
| | T3 | 6.75 | 0.17 | 16 | 7.28 | 0.09 | 53 | 3.50E−03 |
|
Example 8
ME09573(SEQ ID NO:341)
[0207]The Arabidopsis genomic clone At2g44680 transformed in ME11030 encodes a casein kinase II beta chain. An alignment of sequences showing similarity to the protein encoded by clone At2g44680 is presented in FIG. 8. A 12:12 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 or T4 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 8-1 and 8-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME09573 | ME09573-01 | T2 | 25.05 | 0.26 | 21 | 27.76 | 0.34 | 21 | 8.16E−08 |
| | T3 | 22.22 | 0.21 | 23 | 24.50 | 0.19 | 12 | 1.52E−09 |
| ME09573-02 | T3 | 23.00 | 0.38 | 18 | 27.76 | 0.34 | 21 | 1.90E−11 |
| | T4 | 21.35 | 0.15 | 31 | 24.50 | 0.19 | 12 | 3.45E−16 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME09573 | ME09573-01 | T2 | 8.24 | 0.28 | 21 | 11.10 | 0.32 | 21 | 2.92E−08 |
| | T3 | 8.22 | 0.22 | 23 | 11.33 | 0.26 | 12 | 5.18E−11 |
| ME09573-02 | T3 | 6.67 | 0.21 | 18 | 11.10 | 0.32 | 21 | 5.28E−14 |
| | T4 | 7.42 | 0.17 | 31 | 11.33 | 0.26 | 12 | 3.07E−16 |
|
Example 9
ME05718 (SEQ ID NO:383)
[0208]The Arabidopsis genomic clone 32842 transformed in ME05718 encodes a WD-40 repeat family protein. An alignment of sequences showing similarity to the protein encoded by clone 32842 is presented in FIG. 9. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 9-1 and 9-2. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Events | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME05718 | ME05718-02 | T2 | 15.17 | 0.17 | 23 | 18.54 | 0.17 | 35 | 4.33E−20 |
| | T3 | 16.00 | 0.00 | 11 | 19.54 | 0.10 | 39 | 9.00E−36 |
| ME05718-03 | T2 | 18.09 | 0.17 | 23 | 18.54 | 0.17 | 35 | 3.05E−02 |
| | T3 | 18.58 | 0.16 | 24 | 19.54 | 0.10 | 39 | 2.07E−06 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME05718 | ME05718-02 | T2 | 4.09 | 0.09 | 23 | 7.29 | 0.11 | 35 | 6.12E−31 |
| | T3 | 4.00 | 0.13 | 11 | 7.18 | 0.10 | 39 | 1.86E−24 |
| ME05718-03 | T2 | 7.13 | 0.10 | 23 | 7.29 | 0.11 | 35 | 1.39E−01 |
| | T3 | 6.67 | 0.10 | 24 | 7.18 | 0.10 | 39 | 2.13E−04 |
|
Example 10
ME02194 (SEQ ID NO:400)
[0209]The Arabidopsis genomic clone 251923 transformed in ME02194 encodes a putative 60S ribosomal protein L21. An alignment of sequences showing similarity to the protein encoded by clone 251923 is presented in FIG. 10. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. The same events were also analyzed in the early maturity validation assay. Data showing early flowering, early maturity and number of rosette leaves at bolting are presented in Tables 10-1, 10-2, and 10-3, respectively.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME02194 | ME02194-01 | T2 | 18.74 | 0.14 | 27 | 19.96 | 0.31 | 13 | 4.98E−04 |
| | T3 | 18.16 | 0.11 | 25 | 18.75 | 0.25 | 12 | 1.89E−02 |
| ME02194-06 | T2 | 18.03 | 0.19 | 20 | 19.96 | 0.31 | 13 | 4.97E−06 |
| | T3 | 17.38 | 0.11 | 26 | 18.75 | 0.25 | 12 | 7.88E−06 |
|
[0000] |
| T-test comparison of the days to silique maturing time between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Maturing | SE | n | Days to Maturing | SE | n | p-value |
|
| ME02194 | ME02194-01 | T2 | 37.42 | 0.11 | 26 | 38.69 | 0.46 | 13 | 5.35E−03 |
| | T3 | 38.08 | 0.08 | 25 | 38.58 | 0.19 | 12 | 1.07E−02 |
| ME02194-06 | T2 | 36.65 | 0.20 | 20 | 38.69 | 0.46 | 13 | 1.39E−04 |
| | T3 | 37.27 | 0.09 | 26 | 38.58 | 0.19 | 12 | 1.96E−07 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME02194 | ME02194-01 | T2 | 7.30 | 0.15 | 27 | 8.23 | 0.20 | 13 | 3.10E−04 |
| | T3 | 5.96 | 0.04 | 25 | 6.25 | 0.13 | 12 | 2.04E−02 |
| ME02194-06 | T2 | 7.70 | 0.18 | 20 | 8.23 | 0.20 | 13 | 2.89E−02 |
| | T3 | 5.96 | 0.12 | 26 | 6.25 | 0.13 | 12 | 5.46E−02 |
|
Example 11
ME03847 (SEQ ID NO:435)
[0210]The Arabidopsis genomic clone 149496 transformed in ME03847 encodes a putative zinc finger protein. An alignment of sequences showing similarity to the protein encoded by clone 149496 is presented in FIG. 11. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. The same events were also analyzed in the early maturity validation assay. Data showing early flowering, early maturity and number of rosette leaves at bolting are presented in Tables 11-1, 11-2 and 11-3, respectively.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME03847 | ME03847-02 | T2 | 17.64 | 0.16 | 29 | 18.78 | 0.19 | 27 | 1.50E−05 |
| | T3 | 17.03 | 0.11 | 60 | 18.31 | 0.17 | 13 | 1.74E−08 |
| ME03847-03 | T2 | 18.24 | 0.20 | 33 | 18.78 | 0.19 | 27 | 3.03E−02 |
| | T3 | 18.17 | 0.10 | 58 | 18.31 | 0.17 | 13 | 2.50E−01 |
|
[0000] |
| T-test comparison of the days to silique maturing time between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Maturing | SE | n | Days to Maturing | SE | n | p-value |
|
| ME03847 | ME03847-02 | T2 | 36.44 | 0.15 | 27 | 37.80 | 0.20 | 5 | 4.11E−06 |
| | T3 | 37.87 | 0.09 | 60 | 38.92 | 0.18 | 13 | 5.36E−07 |
| ME03847-03 | T2 | 36.91 | 0.18 | 33 | 37.80 | 0.20 | 5 | 1.09E−03 |
| | T3 | 38.53 | 0.11 | 58 | 38.92 | 0.18 | 13 | 3.38E−02 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME03847 | ME03847-02 | T2 | 6.76 | 0.16 | 29 | 7.63 | 0.19 | 27 | 5.46E−04 |
| | T3 | 5.77 | 0.06 | 60 | 6.31 | 0.13 | 13 | 2.09E−04 |
| ME03847-03 | T2 | 7.48 | 0.13 | 33 | 7.63 | 0.19 | 27 | 2.69E−01 |
| | T3 | 6.33 | 0.06 | 58 | 6.31 | 0.13 | 13 | 4.46E−01 |
|
Example 12
ME11261 (SEQ ID NO:319)
[0211]The Arabidopsis genomic clone 231890 transformed in ME11261 encodes a putative AP2-domain-containing protein. An alignment of sequences showing similarity to the protein encoded by clone 231890 is presented in FIG. 6. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. The same events were also analyzed in the early maturity validation assay. Data showing early flowering, early maturity and number of rosette leaves at bolting are presented in Tables 12-1, 12-2 and 12-3, respectively.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME11261 | ME11261-01 | T2 | 17.93 | 0.15 | 27 | 19.28 | 0.51 | 16 | 7.73E−03 |
| | T3 | 17.50 | 0.11 | 24 | 18.00 | 0.19 | 19 | 1.30E−02 |
| ME11261-04 | T2 | 18.26 | 0.22 | 23 | 19.28 | 0.51 | 16 | 3.77E−02 |
| | T3 | 17.17 | 0.20 | 23 | 18.00 | 0.19 | 19 | 2.01E−03 |
| ME11261-05 | T2 | 17.05 | 0.13 | 32 | 19.28 | 0.51 | 16 | 5.70E−05 |
| | T3 | 16.66 | 0.15 | 28 | 18.00 | 0.19 | 19 | 5.24E−07 |
|
[0000] |
| T-test comparison of the days to silique maturing time between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Maturing | SE | n | Days to Maturing | SE | n | p-value |
|
| ME11261 | ME11261-01 | T2 | 36.56 | 0.15 | 27 | 38.31 | 0.58 | 16 | 2.62E−03 |
| | T3 | 37.25 | 0.28 | 24 | 38.00 | 0.28 | 19 | 3.26E−02 |
| ME11261-04 | T2 | 37.52 | 0.20 | 23 | 38.31 | 0.58 | 16 | 1.01E−01 |
| | T3 | 37.30 | 0.28 | 23 | 38.00 | 0.28 | 19 | 4.14E−02 |
| ME11261-05 | T2 | 36.81 | 0.18 | 32 | 38.31 | 0.58 | 16 | 8.17E−03 |
| | T3 | 36.46 | 0.12 | 28 | 38.00 | 0.28 | 19 | 3.25E−06 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME11261 | ME11261-01 | T2 | 6.52 | 0.16 | 27 | 7.06 | 0.32 | 16 | 7.00E−02 |
| | T3 | 5.67 | 0.10 | 24 | 5.79 | 0.14 | 19 | 2.43E−01 |
| ME11261-04 | T2 | 6.48 | 0.12 | 23 | 7.06 | 0.32 | 16 | 4.95E−02 |
| | T3 | 5.35 | 0.10 | 23 | 5.79 | 0.14 | 19 | 8.34E−03 |
| ME11261-05 | T2 | 6.00 | 0.12 | 32 | 7.06 | 0.32 | 16 | 1.68E−03 |
| | T3 | 5.21 | 0.12 | 28 | 5.79 | 0.14 | 19 | 1.81E−03 |
|
Example 13
ME06492 (SEQ ID NO:449)
[0212]The Arabidopsis genomic clone 21240 transformed in ME06492 encodes a knotted-1 like homeobox protein (KNAT3). KNOX (knotted1-like homeobox) proteins have been implicated in roles in plant development including meristem maintenance and leaf primordial initiation (Serikawa (1997) Plant J 11: 863-869). An alignment of sequences showing similarity to the protein encoded by clone 21240 is presented in FIG. 12. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 13-1 and 13-2, respectively. The transgenic plants showed a slight decrease in average rosette diameter compared to the wild-type control, which is possibly caused by reduced number of rosette leaves
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Events | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME06492 | ME06492-04 | T2 | 16.05 | 0.11 | 22 | 16.97 | 0.24 | 16 | 6.53E−04 |
| | T3 | 15.74 | 0.13 | 29 | 17.66 | 0.19 | 16 | 1.06E−10 |
| ME06492-07 | T2 | 16.24 | 0.17 | 27 | 16.97 | 0.24 | 16 | 8.40E−03 |
| | T3 | 16.29 | 0.14 | 21 | 17.66 | 0.19 | 16 | 8.03E−07 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME06492 | ME06492-04 | T2 | 5.55 | 0.11 | 22 | 6.25 | 0.17 | 16 | 6.64E−04 |
| | T3 | 4.90 | 0.10 | 29 | 5.81 | 0.10 | 16 | 5.81E−08 |
| ME06492-07 | T2 | 5.85 | 0.14 | 27 | 6.25 | 0.17 | 16 | 3.86E−02 |
| | T3 | 5.14 | 0.08 | 21 | 5.81 | 0.10 | 16 | 3.78E−06 |
|
Example 14
ME06561 (SEQ ID NO:463)
[0213]The Arabidopsis genomic clone 19080 transformed in ME06561 encodes a putative Dof-type zinc finger protein. An alignment of sequences showing similarity to the protein encoded by clone 19080 is presented in FIG. 13. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 14-1 and 14-2, respectively.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | N | Days to Bolting | SE | N | p-value |
|
| ME06561 | ME06561-02 | T3 | 18.00 | 0.13 | 19 | 19.92 | 0.16 | 52 | 9.48E−14 |
| | T4 | 16.57 | 0.13 | 15 | 18.21 | 0.15 | 43 | 1.69E−11 |
| ME06561-03 | T2 | 18.68 | 0.23 | 19 | 19.92 | 0.16 | 52 | 2.06E−05 |
| | T3 | 16.86 | 0.13 | 18 | 18.21 | 0.15 | 43 | 3.27E−09 |
| ME06561-05 | T2 | 18.82 | 0.38 | 11 | 19.92 | 0.16 | 52 | 4.65E−03 |
| | T3 | 17.00 | 0.17 | 18 | 18.21 | 0.15 | 43 | 1.08E−06 |
|
[0000] |
| T-test comparison of the number of rosette leaves between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Leaves | SE | N | Leaves | SE | N | p-value |
|
| ME06561 | ME06561-02 | T3 | 6.11 | 0.13 | 19 | 7.02 | 0.11 | 52 | 8.79E−07 |
| | T4 | 5.40 | 0.13 | 15 | 6.02 | 0.07 | 43 | 9.95E−05 |
| ME06561-03 | T2 | 6.00 | 0.08 | 19 | 7.02 | 0.11 | 52 | 7.13E−11 |
| | T3 | 5.11 | 0.11 | 18 | 6.02 | 0.07 | 43 | 3.53E−09 |
| ME06561-05 | T2 | 6.27 | 0.24 | 11 | 7.02 | 0.11 | 52 | 5.71E−03 |
| | T3 | 5.11 | 0.11 | 18 | 6.02 | 0.07 | 43 | 3.53E−09 |
|
Example 15
ME03027(SEQ ID NO:472)
[0214]The Arabidopsis genomic clone 33231 transformed in ME03027 encodes a putative aquaporin protein. An alignment of sequences showing similarity to the protein encoded by clone 19080 is presented in FIG. 14. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 15-1 and 15-2, respectively.
[0000] |
| T-test comparison of the days to silique maturing time between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Maturing | SE | n | Days to Maturing | SE | n | p-value |
|
| ME03027 | ME03027-02 | T2 | 16.83 | 0.18 | 30 | 18.07 | 0.13 | 7 | 1.16E−06 |
| | T3 | 17.16 | 0.15 | 32 | 16.75 | 0.41 | 8 | −1.80E−01 |
| ME03027-05 | T2 | 17.09 | 0.20 | 27 | 18.07 | 0.13 | 7 | 1.31E−04 |
| | T3 | 17.71 | 0.24 | 24 | 16.75 | 0.41 | 8 | −2.73E−02 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME03027 | ME03027-02 | T2 | 6.03 | 0.03 | 30 | 7.43 | 0.20 | 7 | 3.32E−08 |
| | T3 | 5.44 | 0.10 | 32 | 5.88 | 0.23 | 8 | 4.26E−02 |
| ME03027-05 | T2 | 6.59 | 0.11 | 27 | 7.43 | 0.20 | 7 | 4.85E−04 |
| | T3 | 5.88 | 0.11 | 24 | 5.88 | 0.23 | 8 | 5.00E−01 |
|
Example 16
ME05742 (SEQ ID NO:555)
[0215]The Arabidopsis genomic clone 33877 transformed in ME05742 encodes a cyclin family protein. An alignment of sequences showing similarity to the protein encoded by clone 19080 is presented in FIG. 15. A 16:8 hour light:dark regime was employed in the early flowering validation assay. Two events were chosen to be analyzed in the T2 and the T3 generations. Data showing early flowering and number of rosette leaves at bolting are presented in Tables 16-1 and 16-2, respectively.
[0000] |
| T-test comparison of the days to silique maturing time between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Maturing | SE | n | Days to Maturing | SE | n | p-value |
|
| ME05742 | ME05742-03 | T2 | 22.50 | 0.20 | 18 | 23.20 | 0.33 | 10 | 3.99E−02 |
| | T3 | 26.24 | 0.27 | 21 | 26.00 | 0.17 | 22 | −2.29E−01 |
| ME05742-04 | T2 | 22.25 | 0.25 | 16 | 23.20 | 0.33 | 10 | 1.49E−02 |
| | T3 | 25.83 | 0.24 | 23 | 26.00 | 0.17 | 22 | 2.81E−01 |
|
[0000] |
| T-test comparison of the number of rosette leaves at bolting between |
| transgenic plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| | | Number of | | | Number of | | | |
| Line | Event | Generation | Rosette Leaves | SE | n | Rosette Leaves | SE | n | p-value |
|
| ME05742 | ME05742-03 | T2 | 10.33 | 0.24 | 18 | 10.47 | 0.22 | 15 | 3.42E−01 |
| | T3 | 11.05 | 0.18 | 21 | 11.27 | 0.25 | 22 | 2.31E−01 |
| ME05742-04 | T2 | 9.81 | 0.26 | 16 | 10.47 | 0.22 | 15 | 3.17E−02 |
| | T3 | 11.09 | 0.23 | 23 | 11.27 | 0.25 | 22 | 2.94E−01 |
|
Example 17
SALK 060638 (SEQ ID NO: 383)
[0216]The DNA sequence (SEQ ID NO: 383) that was used to produce plant line ME05718 (see Example 9) has been deleted or disrupted in the plant line SALK 060638. SALK 108846, where SEQ ID NO: 398 is disrupted or deleted, exhibits earlier flowering than wild type or SALK 060638. The double mutant (SALK 060638×SALK 108846) exhibits later flowering that the wild type or SALK 060638. SALK 060638 and the wild type exhibit larger number of rosette leaves at bolting than the double mutant, which in turn exhibits a higher number of rosette leaves at bolting than SALK 108846. These results suggest that the genes are involved in plant growth and development.
[0000] |
| Bolting time, leaf number and rosette size of the single |
| and double mutant in comparison to wild-type Col plants |
| Days | Rosette | Cauline |
| Lines | to bolting | leaf number | leaf number |
|
| Wild-type Col | 53.83 ± 1.96 | 48.00 ± 2.67 | 8.49 ± 0.95 |
| 22.94 ± 1.17 | 13.39 ± 1.40 | 3.28 ± 0.66 |
| At5g52250 KO | 52.67 ± 1.15 | 47.25 ± 1.96 | 8.75 ± 0.75 |
| (SALK 060638) | 22.83 ± 0.94 | 13.33 ± 0.98 | 3.33 ± 0.65 |
| At5g23730 KO | 41.42 ± 1.21 | 22.00 ± 1.87 | 5.38 ± 0.58 |
| (SALK 108846) | 23.79 ± 1.18 | 12.25 ± 1.15 | 3.08 ± 0.28 |
| Double mutant | 59.09 ± 3.41 | 37.45 ± 4.18 | 7.89 ± 1.15 |
| 26.69 ± 1.19 | 14.08 ± 1.13 | 3.74 ± 0.61 |
|
| Values in short-day (top) and long-day (bottom) conditions shown are mean ± standard deviation; At least 12 plants were scored for each line. |
Example 18
ME04402 (Clone 100156; SEQ ID NO:321)
[0217]ME04402 contains Clone 100516 and encode an AP2 domain protien. The product of Clone 100156 may interact with transcriptional activators or repressors to regulate expression of the genes controlling flowering time.
[0218]Two events, ME04402-02 and -04, were evaluated in the T2 and T3 generations and a t-test used to determine whether the events flower significantly earlier than wild-type Ws at p=0.05 assuming one-tailed distribution and unequal variance.
[0219]Events ME04402-02 and -04 showed statistically significant early flowering when compared to non-transgenic segregants in the same event in the same generation (Table 18-1). For ME04402-02 and -04, the days to bolting were decreased by 1.28 and 1.07 days, respectively, as compared to the pooled non-transgenic plants in the T2 generation. In the T3 generation, the values were 0.96 and 0.78 days, respectively. The differences between transgenic and non-transgenic plants were all statistically significant at p≦0.05 level.
[0000] |
| T-test comparison of the days to bolting between transgenic |
| plants and pooled non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | n | Days to Bolting | SE | n | p-value |
|
| ME04402 | ME04402-02 | T2 | 17.77 | 0.15 | 24 | 19.05 | 0.19 | 31 | 1.02E−06 |
| | T3 | 18.04 | 0.12 | 27 | 19.00 | 0.17 | 29 | 1.00E−05 |
| ME04402-04 | T2 | 19.23 | 0.30 | 11 | 20.30 | 0.25 | 10 | 6.13E−03 |
| | T3 | 18.17 | 0.29 | 18 | 18.95 | 0.15 | 46 | 9.66E−03 |
|
Example 19
ME07345 (Clone 101876, SEQ ID NO:564)
[0220]ME07345 contains a transgene from Arabidopsis that encodes a 156 amino acid protein with unknown function.
[0221]Five T3 events were evaluated and a t-test used to determine whether the events flower significantly earlier than wild-type Ws at p=0.05 assuming one-tailed distribution and unequal variance.
[0222]Of the five events, two (-02 and -05) showed early flowering while three did not flower earlier than wild-type and were not included in subsequent assays. The T4 generation seeds from the two events were evaluated under the same Early Flowering Assay conditions.
[0223]Statistical analysis showed that the T3 and T4 generations of both-02 and -05 had early flowering compared to non-transgenic plants (Table 19-1). For ME07345-02 and -05, the days to bolting were decreased by 0.47 and 0.98 days, respectively, as compared to the pooled non-transgenic plants in the T3 generation. In the T4 generation, the values were 0.91 and 0.9 days, respectively. The differences between transgenic and non-transgenic plants were all statistically significant at p≦0.05 level.
[0000] |
| T-test comparison of the days to bolting between transgenic plants and non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | N | Days to Bolting | SE | N | p-value |
|
| ME07345 | ME07345-02 | T3 | 18.39 | 0.14 | 19 | 18.86 | 0.16 | 22 | 1.65E−02 |
| | T4 | 17.95 | 0.12 | 31 | 18.86 | 0.25 | 11 | 1.17E−03 |
| ME07345-05 | T3 | 17.88 | 0.13 | 24 | 18.86 | 0.16 | 22 | 9.48E−06 |
| | T4 | 17.96 | 0.18 | 24 | 18.86 | 0.25 | 11 | 3.19E−03 |
|
Example 20
ME04195 (Clone 156373, SEQ ID NO:574)
[0224]ME04195 contains a transgene from Arabidopsis that encodes a 246 amino acid protein containing a homeodomain PHD finger motif at the C-terminus.
[0225]Two events in the T2 and T3 generations were evaluated and a t-test used to determine whether the events flower significantly earlier than wild-type Ws at p=0.05 assuming one-tailed distribution and unequal variance.
[0226]Events ME04195-01 and -02 showed statistically significant early flowering when compared to non-transgenic segregants in the same event in the same generation (Table 20-1). For ME04195-01 and -02, the days to bolting were decreased by 1.48 and 1.17 days, respectively, as compared to the pooled non-transgenic plants in the T2 generation. In the T3 generation, the values were 1.24 and 1.0 days, respectively. The differences between transgenic and non-transgenic plants were all statistically significant at p≦0.05 level.
[0000] |
| T-test comparison of the days to bolting between transgenic plants and non-transgenic segregants. |
| Transgenic | Non-transgenic | |
| Line | Event | Generation | Days to Bolting | SE | N | Days to Bolting | SE | N | p-value |
|
| ME04195 | ME04195-01 | T2 | 18.52 | 0.20 | 27 | 20.00 | 0.52 | 6 | 6.01E−03 |
| | T3 | 16.97 | 0.13 | 24 | 18.21 | 0.26 | 7 | 1.02E−04 |
| ME04195-02 | T2 | 18.50 | 0.35 | 18 | 19.67 | 0.49 | 6 | 3.30E−02 |
| | T3 | 16.83 | 0.41 | 12 | 17.83 | 0.16 | 20 | 1.47E−02 |
|
Example 21
Determination of Functional Homolog Sequences
[0227]The sequences described in the above Examples are utilized as query sequences to identify functional homologs of the query sequences and, together with those sequences, are utilized to define consensus sequences for a given group of query and functional homolog sequences. Query sequences and their corresponding functional homolog sequences are aligned to illustrate conserved amino acids consensus sequences that contain frequently occurring amino acid residues at particular positions in the aligned sequences, as shown in FIGS. 1-17.
[0228]A subject sequence is considered a functional homolog of a query sequence if the subject and query sequences encode proteins having a similar function and/or activity. A process known as Reciprocal BLAST (Rivera et al, (1998) Proc. Natl. Acad. Sci. USA 95:6239-6244) is used to identify potential functional homolog sequences from databases consisting of all available public and proprietary peptide sequences, including NR from NCBI and peptide translations from Ceres clones.
[0229]Before starting a Reciprocal BLAST process, a specific query polypeptide is searched against all peptides from its source species using BLAST in order to identify polypeptides having sequence identity of 80% or greater to the query polypeptide and an alignment length of 85% or greater along the shorter sequence in the alignment. The query polypeptide and any of the aforementioned identified polypeptides are designated as a cluster.
[0230]The main Reciprocal BLAST process consists of two rounds of BLAST searches; forward search and reverse search. In the forward search step, a query polypeptide sequence, “polypeptide A,” from source species SA is BLASTed against all protein sequences from a species of interest. Top hits are determined using an E-value cutoff of 10−5 and an identity cutoff of 35%. Among the top hits, the sequence having the lowest E-value is designated as the best hit, and considered a potential functional homolog. Any other top hit that had a sequence identity of 80% or greater to the best hit or to the original query polypeptide is considered a potential functional homolog as well. This process is repeated for all species of interest.
[0231]In the reverse search round, the top hits identified in the forward search from all species are used to perform a BLAST search against all protein or polypeptide sequences from the source species SA. A top hit from the forward search that returned a polypeptide from the aforementioned cluster as its best hit is also considered as a potential functional homolog.
[0232]Functional homologs are identified by manual inspection of potential functional homolog sequences. Representative functional homologs are shown in FIGS. 1-17. The Figures represents a grouping of a query sequence aligned with the corresponding identified functional homolog subject sequences. Query sequences and their corresponding functional homolog sequences are aligned to identify conserved amino acids and to determine a consensus sequence that contains a frequently occurring amino acid residue at particular positions in the aligned sequences, as shown in FIGS. 1-17.
[0233]An HMM was made based on SEQ ID NO:96, 100, 105, 106, 107 and 108, aligned in FIG. 1. When fit to the HMM profile, SEQ ID NO:96, 98, 100, 102, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 603, and 605 gave HMM bit scores of 627.2, 482.7, 590.6, 457.2, 558.2, 633.1, 633.6, 632, 625.7, 638.1, 632.2, 438.9, 434.8, 389.3, 457.2, 544.8, 365.7, 368.7, 365.9, 510.1, 623.6, 572.8, 429.6, 445.7, 469.5, 620.8, 516.6, 540.1, 627.5, 495.7, 518.5, 516.1, 581.6, 376.9, 449.8, 518.2, 482.7, 522.8, 625.1, 597, and 66.2 respectively.
[0234]An HMM was made based on SEQ ID NO:140, 144, 148, 150, 159, 160 and 161, aligned in FIG. 2. When fit to the HMM profile, SEQ ID NO:140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, and 194 gave HMM bit scores of 653, 652.9, 625.2, 561.2, 665, 665.9, 614.4, 604.7, 622.1, 606.9, 647.8, 652.9, 618, 661.8, 573.6, 635.8, 651.6, 511.9, 512, 637.3, 641.2, 633.2, 639.6, 638.1, 641, 628.3, 665, 532, 600.2, 594.4, 664.7, 614.1, 565.6, 536, 625.2, 587.4, 660, 594.5, 593.5, 557.2, 519.2, 513.6, 552.5, 545.8, 539.3, and 535 respectively.
[0235]An HMM was made based on SEQ ID NO:196, 198, 204, 207, 208, and 209 aligned in FIG. 3. When fit to the HMM profile, SEQ ID NO:196, 198, 200, 202, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, and 236 gave HMM bit scores of 497.8, 498.2, 497.8, 478.5, 508.1, 502.6, 510.9, 522.9, 521.4, 450.6, 477, 442.2, 486.2, 439.9, 439.4, 437, 484.1, 451.4, 502.4, 441, 481.3, 461.5, 489.9, 461.9, 480.4, 480.4, 452.7, 443.8, 465.8, 481.4, 507.4, 483.8, 462.6, 505.2, 435 and 440.2 respectively.
[0236]An HMM was made based on SEQ ID NO:238, 240, 244, 250, 251, 252, and 254 aligned in FIG. 4. When fit to the HMM profile, SEQ ID NO:238, 240, 242, 244, 246, 248, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, and 283 gave HMM bit scores of 572.1, 565.6, 558.5, 557.8, 524.5, 524.5, 579.8, 540.6, 589.4, 562.2, 516.4, 519.6, 524.5, 506.8, 558.5, 565.6, 528.7, 524.7, 523.3, 527.2, 544.5, 537.5, 553.8, 535.4, 537.5, 521.1, 536.7, 562, 557.8, 534.3, 506.6, 556.7, 558.8, 563.6, 563.6, 563.4, 548.6, 561.6, 519.8 and 511.8 respectively.
[0237]An HMM was made based on SEQ ID NO:285, 287, 291, 293, and 296 aligned in FIG. 5. When fit to the HMM profile, SEQ ID NO:285, 287, 289, 291, 293, 295, 296, and 297 gave HMM bit scores of 386.6, 429.1, 269.7, 636.2, 338.6, 542.2, 441.8 and 429.2 respectively.
[0238]An HMM was made based on SEQ ID NO:299, 301, 305, 307, 309, 311, 315, 316, 320, and 322, aligned in FIG. 6. When fit to the HMM profile, SEQ ID NO:299, 301, 303, 305, 307, 309, 311, 313, 315, 316, 317, 318, 320, 322, 324, and 325 gave HMM bit scores of 520, 519.5, 510.5, 510.5, 519.9, 1086.7, 529.7, 367.9, 531.7, 542.3, 520.5, 519.9, 587, 520.5, 511.9, and 542.3 respectively.
[0239]An HMM was made based on SEQ ID NO:327, 331, 333, 336, 338, 340, and 607, aligned in FIG. 7. When fit to the HMM profile, SEQ ID NO:327, 329, 331, 333, 334, 336, 338, 340, and 607 gave HMM bit scores of 555.4, 700.4, 680.3, 648.9, 687.7, 673.3, 624.9, 624.9, and 687.7 respectively.
[0240]An HMM was made based on SEQ ID NO:342, 366, 377, 379, 381, and 382, aligned in FIG. 8. When fit to the HMM profile, SEQ ID NO:342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 371, 372, 373, 374, 375, 377, 379, 381, and 382 gave HMM bit scores of 758, 758.2, 619.5, 764.3, 715.9, 698, 693.2, 698, 629.5, 698, 605.7, 634.4, 602.9, 610.3, 682.4, 731.1, 602.1, 723, 675.6, 648.3, 698, 710.4, 731.1, and 727.9 respectively.
[0241]An HMM was made based on SEQ ID NO:384, 394, 396, 397, and 399 aligned in FIG. 9. When fit to the HMM profile, SEQ ID NO:384, 386, 388, 390, 392, 394, 396, 397, 399 gave HMM bit scores of 904.9, 908, 916.6, 696.7, 744.9, 969.9, 955.3, 916.6, and 908 respectively.
[0242]An HMM was made based on SEQ ID NOs 575, 577, 585, 589, 591, 593, 596, 597, 598, 599 and 600, aligned in FIG. 17. When fit to the HMM, SEQ ID NOs 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 596, 597, 598, 599, 600 and 601 gave HMM bit scores of 587.2, 556.7, 547.6, 571.2, 543.1, 574.6, 572.1, 581.8, 549, 554.1, 545.4, 541.4, 553.7, 565, 562, 561.7 and 550 respectively.
[0243]An HMM was made based on SEQ ID NOs 565, 567, 571, and 573, aligned in FIG. 16. When fit to the HMM, SEQ ID NOs 565, 567, 569, 571, and 573 gave HMM bit scores of 341.5, 394.6, 313.3, 221 and 439.9 respectively.
[0244]An HMM was made based on SEQ ID NOs 556, 560, 562 and 563, aligned in FIG. 15. When fit to the HMM, SEQ ID NOs 556, 558, 560, 562 and 563 gave HMM bit scores of 752.9, 1053.3, 1101.1, 1086.7 and 1497.2 respectively.
[0245]An HMM was made based on SEQ ID NOs 473, 501, 505, 507, 550, 552, 553 and 554, aligned in FIG. 14. When fit to the HMM, SEQ ID NOs 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 550, 552, 553 and 554 gave HMM bit scores of 780.9, 741.5, 764.3, 763.5, 765.6, 765.6, 742.3, 762.7, 752.1, 749, 754.7, 743.2, 752.4, 750.9, 790, 752.4, 778.3, 779.4, 760.8, 762.6, 753.3, 769.5, 764.3, 771.5, 739.8, 738, 763.6, 778.3, 774.7, 778.1, 729.7, 752.4, 752.1, 739.5, 746.7, 755.2, 758.6, 741.9, 761.6, 762.7, 769, 757.8, 761, 748.9, 724.3, 751.2, 743.1, 752.1, 739.8, 734.6, 757, 754.7, 708.8, 757.8, 713.2, 739.6, 740, 758, 744.9, 779.4, 788.8, 764.8 and 762.7 respectively.
[0246]An HMM was made based on SEQ ID NOs 464, 467, 469, 470 and 471, aligned in FIG. 13. When fit to the HMM, SEQ ID NOs 464, 465, 467, 469, 470 and 471 gave HMM bit scores of 762.8, 576.5, 828.8, 877.5, 646 and 686.8 respectively.
[0247]An HMM was made based on SEQ ID NOs 450, 458, 460, 461 and 462, aligned in FIG. 12. When fit to the HMM, SEQ ID NOs 450, 452, 453, 454, 455, 456, 458, 460, 461 and 462 gave HMM bit scores of 988.2, 908.6, 806.2, 801.5, 812.6, 813.9, 950.1, 781.2, 811.8 and 901.6 respectively.
[0248]An HMM was made based on SEQ ID NOs 436, 443, 445, 447, 448 and 609 aligned in FIG. 11. When fit to the HMM, SEQ ID NOs 436, 438, 439, 440, 441, 443, 445, 447, 448 and 609 gave HMM bit scores of 996.1, 713.20, 719.9, 960.8, 715.6, 1098.4, 1205, 1205, 1199.1 and 688.7 respectively.
[0249]An HMM was made based on SEQ ID NOs 401, 427, 429, 431, 433 and 434, aligned in FIG. 10. When fit to the HMM, SEQ ID NOs 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 424, 425, 427, 429, 431, 433 and 434 gave HMM bit scores of 478.9, 464.8, 477.4, 479.6, 458.6, 472.1, 477.9, 470.5, 452.3, 469.7, 470.4, 478.7, 477.4, 481, 481.2, 479.6, 477.9, 474 and 485 respectively.
[0250]Useful polypeptides of the invention include each of the lead and corresponding functional homolog sequences shown in the Figures and/or the Sequence Listing, as well as polypeptides belonging to the corresponding consensus sequence families as delineated by HMMs. In different embodiments, consensus sequence families have HMM bit score lower limits as about 50%, 60%, 70%, 80%, 90%, or 95% of any of the HMM bit scores of the family members presented in this application. In some embodiments the lower HMM bit score limits correspond approximately to the HMM bit score of any of the family members disclosed in this application. A sequence that has an HMM bit score of 20 means that it has a 95% likelihood of belonging to the consensus sequence defined by a particular HMM. Alternative HMM bit scores that are useful for the current invention are 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450 and 500.
[0251]The present invention further encompasses nucleotides that encode the above described polypeptides, as well as the complements thereof, and including alternatives thereof based upon the degeneracy of the genetic code.
[0252]The invention being thus described, it will be apparent to one of ordinary skill in the art that various modifications of the materials and methods for practicing the invention can be made. Such modifications are to be considered within the scope of the invention as defined by the following claims.
[0253]The following references are cited in the Specification. Each of the references from the patent and periodical literature cited herein is hereby expressly incorporated in its entirety by such citation.
REFERENCES
[0000]- (1) Koomneef, M., Alonso-Blanco, C., Peeters, A. J. M., and Soppe, W. (1998) Annu. Rev. Plant. Physiol. Mol. Biol., 49: 345-370.
- (2) Zhang et al. (2004) Plant Physiol. 135:615.
- (3) Walbot, V. (1985) Trends Genet., 1:165-169.
- (4) Gamer, W. W. and Allard, H. A. (1920) J. Agric. Res., 18:553-606.
- (5) Bernier, G (1988) Annu. Rev. Plant Physiol. Plant Mol. Biol., 39:175-219.
- (6) Millar, A. J. (1999) New Phytol., 14:175-197.
- (7) Battey, N. H. (2000) J. Exp. Bot., 51:1769-1780.
- (8) Samach, A. and Coupland, G. (2000) Bioessays, 22:38-47.
- (9) Salomon et al. (1984) EMBO. J. 3:141.
- (10) Herrera-Estrella et al. (1983) EMBO J. 2:987.
- (11) Escudero et al. (1996) Plant J 10:355.
- (12) Ishida et al. (1996) Nature Biotechnology 14:745.
- (13) May et al. (1995) Bio/Technology 13:486)
- (14) Armaleo et al. (1990) Current Genetics 17:97.
- (15) P. Tijessen, “Hybridization with Nucleic Acid Probes” In Laboratory Techniques in Biochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993 by Elsevier, Amsterdam.
- (16) Bonner et al., (1973) J. Mol. Biol. 81:123.
- (17) Xu et al. (1995) Plant Mol. Biol. 27:237.
- (18) Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989, New York.
- (19) Shizuya et al. (1992) Proc. Natl. Acad. Sci. USA, 89: 8794-8797.
- (20) Hamilton et al. (1996) Proc. Natl. Acad. Sci. USA, 93: 9975-9979.
- (21) Burke et al. (1987) Science, 236:806-812.
- (22) Sternberg N. et al. (1990) Proc Natl Acad Sci USA., 87:103-7.
- (23) Bradshaw et al. (1995) Nucl Acids Res, 23: 4850-4856.
- (24) Frischauf et al. (1983) J. Mol Biol, 170: 827-842.
- (25) Huynh et al., Glover N. Mex. (ed) DNA Cloning: A practical Approach, Vol. 1 Oxford: IRL Press (1985).
- (26) Walden et al. (1990) Mol Cell Biol 1: 175-194.
- (27) Fromm et al. (1989) The Plant Cell 1:977-984.
- (28) Jordano, et al. (1989) Plant Cell, 1:855-866.
- (29) Bustos, et al. (1989) Plant Cell, 1:839-854.
- (30) Green, et al. (1988) EMBO J. 7, 4035-4044.
- (31) Meier, et al. (1991) Plant Cell, 3, 309-316.
- (32) Zhang, et al. (1996) Plant Physiology 110: 1069-1079.
- (33) Lam et al. (1989) Proc. Natl. Acad. Sci. USA 86:7890-7894.
- (34) Conkling et al. (1990) Plant Physiol. 93:1203-1211.
- (35) Bustos et al. (1989) Plant Cell 1(9):839-853.
- (36) Riggs et al. (1989) Plant Cell 1(6):609-621.
- (37) Baerson et al. (1993) Plant Mol Biol, 22(2):255-267.
- (38) Slocombe et al. (1994 Plant Physiol 104(4):167-176).
- (39) Chen et al. (1986) Proc Natl Acad Sci USA 83:8560-8564.
- (40) Hong et al. (1997) Plant Mol Biol 34(3):549-555.
- (41) Zheng et al. (1993) Mol. Cell. Biol. 13:5829-5842.
- (42) Urao (1996) Plant Mol. Biol., 32:571-57.
- (43) Conceicao (1994) Plant, 5:493-505.
- (44) Sheridan (1996) Genetics, 142:1009-1020.
- (45) Abler, Plant Mol. Biol., 22:10131-1038 (1993).
- (46) Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778.
- (47) Fejes et al., (1990) Plant Mol. Biol. 15:921-932.
- (48) Lubberstedt et al. (1994) Plant Physiol. 104:997-1006.
- (49) Luan et al. (1992) Plant Cell 4:971-981.
- (50) Matsuoka et al. (1993) Proc Natl Acad. Sci. USA 90:9586-9590.
- (51) Cerdan et al. (1997) Plant Mol. Biol. 33:245-255.
- (52) Truernit et al. (1995) Planta 196:564-570.
- (53) Perriman, et al. (1995) Proc. Natl. Acad. Sci. USA, 92(13):6175-6179.
- (54) de Feyter and Gaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “Expressing Ribozymes in Plants”, Edited by Turner, P. C, Humana Press Inc., Totowa, N.J.
- (55) Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev., 7:187-195.
- (56) Hyrup et al. (1996) Bioorgan. Med. Chem., 4: 5-23.
- (57) Vissenberg et al. (2005) Plant Cell Physiol 46:192.
- (58) Husebye et al. (2002) Plant Physiol 128:1180.
- (59) Plesch et al. (2001) Plant J 28:455.
- (60) Griesbach (1987) Plant Sci. 50:69-77.
- (61) Fromm et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824.
- (62) Paszkowski et al. (1984) EMBO J. 3:2717.
- (63) Klein et al. (1987) Nature 327:773.
- (64) Willmitzer, L. (1993) Transgenic Plants. In: iotechnology, A Multi-Volume Comprehensive treatise (H. J. Rehm, G. Reed, A. Püler, P. Stadler, eds., Vol. 2, 627-659, VCH Weinheim-New York-Base1-Cambridge).
- (65) Crit. Rev. Plant. Sci. 4:1-46.
- (66) Fromm et al. (1990) Biotechnology 8:833-844.
- (67) Cho et al. (2000) Planta 210:195-204.
- (68) Brootghaerts et al. (2005) Nature 433:629-633.
- (69) Lincoln et al. (1998) Plant Mol. Biol. Rep. 16:1-4.
- (70) Lacomme et al. (2001), “Genetically Engineered Viruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOS Scientific Publishers, Ltd. Oxford, UK.
- (71) Parenicova, L., Folter, S. D., Kieffer, M., Horner, D. S., Favalli, C., Busscher, J., Cook, H. E., Ingram, R. M., Kater, M. M., Davies, B., Angenent, G. C., and Colombo, L. (2003) Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: new openings to the MADS world. Plant Cell 15:1538-1551.
- (72) Jofuku, K. D., der Boer, B. G. W., Van Montagu, M., and Okamuro, J. K. (1994) Control of Arabidopsis flower and seed development by the homeotic gene APETAL2. Plant Cell 6: 1211-1225.
- (73) Umemura, Y., Ishiduka, Y., Yamamoto, R., and Esaka, M. (2004) The D of domain, a zinc finger DNA-binding domain conserved only in higher plants, truly functions as a Cys2/Cys2 Zn finger domain. Plant J. 37: 741-749.
- (74) Sugano, S., Andronis, C., Green, R. M., Wang, Z., and Tobin, E. M. (1998) Protein kinase CK2 interacts with and phosphorylates the Arabidopsis circadian clock-associated 1 protein. Proc. Natl. Acad. Sci. USA 95: 11020-11025.
- (75) Neer, E. J., Schmidt, C. J., Nambudripad, R., and Smith, T. F. (1994) The ncient regulatory-protein family of WD-repeat proteins. Nature 371: 297-300.
- (76) Wang, G., Kong, H., Sun, Y., Zhang, X., Zhang, W., Altman, N., dePamphilis, C. W., and Ma, H. (2004) Genome-wide analysis of the cyclin family in Arabidopsis and comparative phylogenetic analysis of plant cyclin-like proteins. Plant Physiol. 135: 1084-1099.
- (77) Serikawa, K. A., Zambryski, P. C. (1997) Domain exchanges between KNAT3 and KNAT1 suggest specificity of the kn1-like homeodomains requires sequences outside of the third helix and N-terminal arm of the homeodomain. Plant J 11:863-869.
- (78) Crouzet, M. and Begueret J. (1980) A new mutant form of the ribosomal protein L21 in the fungus Podospora anserina: identification of the structural gene for this protein. Mol Gen Genet. 180(1):177-83.
