Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
  • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• This invention is in the field of plant molecular biology and relates to compositions and methods for modifying a plant's flowering time or vernalization requirements.
• plants In order to maximize reproductive success, plants have evolved complex mechanisms to ensure that flowering occurs under favorable conditions. Analysis of late flowering mutants and ecotypes in Arabidopsis has revealed that such mechanisms are based upon several genetic pathways which may contain 80 or more genes (Martinez-Zapater and Somerville, (1990) Plant Physiol. 92:770-776; Koornneef et al. (1991 ) Mol. Gen. Genet. 229:57-66; EM Meyerwitz and CR Somerville Eds (1994) Arabidopsis pp 403-433 Cold Spring Harbor Laboratory Press, New York). Together these loci co-ordinate flowering time with environmental variables (e.g. day-length, temperature, light quality, and nutrient availability) and with the developmental stage of the plant.
• environmental variables e.g. day-length, temperature, light quality, and nutrient availability
• FLOWERING LOCUS C FLC (also known as FLOWERING LOCUS F, FLF) and FRIGIDA (FRI)
• FLOWERING LOCUS F FLC
• FRIGIDA FRI
• FLC FLC gene has recently been cloned and shown to encode a MADS box protein (Sheldon C. et al., 1999, Plant Cell 1 1 :445-458; Michaels S. and Amasino, R., 1999, Plant Cell 11 :949-956). Dominant alleles and overexpression of FLC have been reported to delay flowering, while null flc mutants are early flowering (Lee et al., (1994) Plant J. 6:903-909; Michaels and Amasino, (1999) Plant Cell 11 :949-956; Sheldon et al., (1999; Proc. Natl. Acad. Sci. 97:3753-3758). Thus, FLC acts to prevent premature flowering.
• transcription factors that regulate flowering time or vernalization requirements of plants. These transcription factors could therefore be useful to manipulate flowering characteristics of a plant.
• the present invention relates to a transgenic plant comprising a recombinant polynucleotide.
• the nucleotide sequence further comprises a promoter operably linked to the nucleotide sequence. The promoter may be a constitutive or inducible or tissue-active.
• the present invention relates to a method for altering a plant's flowering time or vernalization requirements.
• the nucleotide sequence further comprises a promoter operably linked to the nucleotide sequence. The promoter may be a constitutive or inducible or tissue-active.
• the present invention relates to another method for altering a plant trait associated with flowering time or the plant's vernalization requirements.
• the present invention is yet another method for altering a plant's flowering time or vernalization requirements.
• Figure 1 provides a table of exemplary polynucleotide and polypeptide sequences of the invention.
• the table includes from left to right for each sequence: the SEQ ID No., the internal code reference number, whether the sequence is a polynucleotide or polypeptide sequence, and identification of any conserved domains for the polypeptide sequences.
• Figure 2 provides a table of sequences that are homologous to the sequences provided in the Sequence Listing.
• the table includes from left to right: the SEQ ID No., the internal code reference number, the unique Genbank sequence ID No. (NID), the probability that the comparison was generated by chance (P-value), and the species from which the homologous gene was identified.
• NID unique Genbank sequence ID No.
• P-value the probability that the comparison was generated by chance
• a "recombinant polynucleotide” is a nucleotide sequence comprising a gene coding sequence or a fragment thereof (comprising at least 18 consecutive nucleotides, preferably at least 30 consecutive nucleotides, and more preferably at least 50 consecutive nucleotides). Additionally, the polynucleotide may comprise a promoter, an intron, an enhancer region, a polyadenylation site, a translation initiation site, 5' or 3' untranslated regions, a reporter gene, a selectable marker or the like. The polynucleotide may comprise single stranded or double stranded DNA or RNA. The polynucleotide may comprise modified bases or a modified backbone.
• the polynucleotide may be genomic, a transcript (such as an mRNA) or a processed nucleotide sequence (such as a cDNA).
• the polynucleotide may comprise a sequence in either sense or antisense orientations.
• a “recombinant polynucleotide” is a polynucleotide that is not in its native state, e.g., the polynucleotide is comprised of a nucleotide sequence not found in nature or the polynucleotide is separated from nucleotide sequences with which it typically is in proximity or is next to nucleotide sequences with which it typically is not in proximity.
• a "recombinant polypeptide” is a polypeptide derived from the translation of a recombinant polynucleotide or is more enriched in a cell than the polypeptide in its natural state in a wild type cell, e.g. more than 5% enriched, more than 10% enriched or more than 20% enriched and is not the result of a natural response of a wild type plant or is separated from other components with which it is typically associated with in a cell.
• a "transgenic plant” may refer to a plant that contains genetic material not normally found in a wild type plant of the same species, or in a naturally occurring variety or in a cultivar, and which has been introduced into the plant by human manipulation.
• a transgenic plant is a plant that may contain an expression vector or cassette.
• the expression cassette comprises a gene coding sequence and allows for the expression of the gene coding sequence.
• the expression cassette may be introduced into a plant by transformation or by breeding after transformation of a parent plant.
• a transgenic plant refers to a whole plant as well as to a plant part, such as seed, fruit, leaf, or root, plant tissue, plant cells, protoplasts or any other plant material, and progeny thereof.
• altered expression in reference to polynucleotide or polypeptide expression refers to an expression pattern in the transgenic plant that is different from the expression pattern in the wild type plant or a reference; for example, by expression in a cell type other than a cell type in which the sequence is expressed in the wild type plant, or by expression at a time other than at the time the sequence is expressed in the wild type plant, or by a response to different inducible agents, such as hormones or environmental signals, or at different expression levels (either higher or lower) compared with those found in a wild type plant.
• the term also refers to lowering the levels of expression to below the detection level or completely abolishing expression.
• the resulting expression pattern may be transient or stable, constitutive or inducible.
• TF transcription factor
• a “transcription factor” refers to a polynucleotide or polypeptide that controls the expression of a gene or genes either directly by binding to one or more nucleotide sequences associated with a gene coding sequence or indirectly by affecting the level or activity of other polypeptides that do bind directly or indirectly to one or more nucleotide sequences associated with a gene coding sequence.
• a TF in this definition, includes any polypeptide that can activate or repress transcription of a single gene or a number of genes. This polypeptide group includes, but is not limited to, DNA binding proteins, protein kinases, protein phosphatases, GTP-binding proteins and receptors.
• the transcription factor sequence may comprise a whole coding sequence or a fragment or domain of a coding sequence.
• a "fragment or domain", as referred to polypeptides, may be a portion of a polypeptide which performs at least one biological function of the intact polypeptide in substantially the same manner or to a similar extent as does the intact polypeptide.
• a fragment may comprise, for example, a DNA binding domain that binds to a specific DNA promoter region, an activation domain or a domain for protein-protein interactions. Fragments may vary in size from as few as 6 amino acids to the length of the intact polypeptide, but are preferably at least 30 amino acids in length and more preferably at least 60 amino acids in length.
• a fragment refers to any sequence of at least consecutive 18 nucleotides, preferably at least 30 nucleotides, more preferably at least 50, of any of the sequences provided herein.
• Exemplary polynucleotides and polypeptides comprise a sequence provided in the Sequence Listing as SEQ ID No. 1 : G157 (cDNA); SEQ ID No. 2: G157 (protein); SEQ ID No. 3: G859 (cDNA); SEQ ID No. 4: G859 (protein); SEQ ID No. 5: G859.1 (cDNA); SEQ ID No. 6: G859.1 (protein); SEQ ID No. 7: G859.2 (cDNA); SEQ ID No. 8: G859.2 (protein); SEQ ID No. 9: G1842 (cDNA); SEQ ID No. 10: G1842 (protein); SEQ ID No. 11 : G1842.2 (cDNA); SEQ ID No.
• a “conserved domain” refers to a polynucleotide or polypeptide fragment that is more conserved at a sequence level than other fragments when the polynucleotide or polypeptide is compared with homologous genes or proteins from other plants.
• the conserved domain may be 1 ) a localization domain, 2) an activation domain, 3) a repression domain, 4) a dimerization or oligomerization domain, 5) a DNA binding domain or any combination thereof.
• the conserved domain is typically a DNA-binding domain.
• a nucleotide sequence is "operably linked" when it is placed into a functional relationship with another nucleotide sequence.
• a promoter or enhancer is operably linked to a gene coding sequence if the presence of the promoter or enhancer increases the level of expression of the gene coding sequence.
• Trait refers to a physiological, morphological, biochemical or physical characteristic of a plant or particular plant material or cell.
• This characteristic may be visible to the human eye, such as seed or plant size, or be measured by biochemical techniques, such as the protein, starch or oil content of seed or leaves or by the observation of the expression level of genes by employing Northerns, RT PCR, microarray gene expression assays or reporter gene expression systems or be measured by agricultural observations such as stress tolerance, yield or disease resistance.
• Trait modification refers to a detectable difference in a characteristic in a transgenic plant with modified expression of a polynucleotide or polypeptide of the present invention relative to a plant not doing so, such as a wild type plant.
• the trait modification may entail at least a 5% increase or decrease in an observed trait (difference), at least a 10% difference, at least a 20% difference, at least a 30%, at least a 50%, at least a 70%, at least a 100% or a greater difference. It is known that there may be a natural variation in the modified trait. Therefore, the trait modification observed entails a change in the normal distribution of the trait in transgenic plants compared with the distribution observed in wild type plant.
• Trait modifications of particular interest include those to seed (embryo), fruit, root, flower, leaf, stem, shoot, seedling or the like, including: enhanced tolerance to environmental conditions including freezing, chilling, heat, drought, water saturation, radiation and ozone; enhanced resistance to microbial, fungal or viral diseases; resistance to nematodes, decreased herbicide sensitivity, enhanced tolerance of heavy metals (or enhanced ability to take up heavy metals), enhanced growth under poor photoconditions (e.g., low light and/or short day length), or changes in expression levels of genes of interest.
• phenotypes that may be modified relate to the production of plant metabolites, such as variations in the production of taxol, tocopherol, tocotrienol, sterols, phytosterols, vitamins, wax monomers, anti-oxidants, amino acids, lignins, cellulose, tannins, prenyllipids (such as chlorophylls and carotenoids), glucosinolates, and terpenoids, enhanced or compositionally altered protein or oil production (especially in seeds), or modified sugar (insoluble or soluble) and/or starch composition.
• Physical plant characteristics that may be modified include cell development (such as the number of trichomes), fruit and seed size and number, yields of plant parts such as stems, leaves and roots, the stability of the seeds during storage, characteristics of the seed pod (e.g., susceptibility to shattering), root hair length and quantity, intemode distances, or the quality of seed coat.
• Plant growth characteristics that may be modified include growth rate, germination rate of seeds, vigor of plants and seedlings, leaf and flower senescence, male sterility, apomixis, flowering time, flower abscission, rate of nitrogen uptake, biomass or transpiration characteristics, as well as plant architecture characteristics such as apical dominance, branching patterns, number of organs, organ identity, organ shape or size.
• traits relating to modified vernalization requirements or flowering time characteristics such as changes in flowering time in response to day-length, in response to temperature, in response to light quality, nutrient availability, and development stage of the plant, and the like.
• TFs plant transcription factors
• the plant transcription factors may belong to one of the following transcription factor families: the AP2 (APETALA2) domain transcription factor family (Riechmann and Meyerowitz (1998) Biol. Chem. 379:633-646); the MYB transcription factor family (Martin and Paz-Ares, (1997) Trends Genet. 13:67-73); the MADS domain transcription factor family (Riechmann and Meyerowitz (1997) Biol. Chem. 378:1079-1101); the WRKY protein family (Ishiguro and Nakamura (1994) Mol. Gen. Genet. 244:563-571 ); the ankyrin-repeat protein family (Zhang et al.
• the TFs that we have discovered that are implicated in flowering time or vernalization include members of the MADS transcription factor family, the MYB family, the WRKY family, the HLH/MYC family, GLD family, AT-HOOK family, the CAAT family, the bZIP family, and members of zinc coordinating protein families (Z-Dof, Z-CLDSH and Z-CH2H2).
• Figure 1 identifies a SEQ ID No., its corresponding GID number, whether the sequence is a polynucleotide or a polypeptide sequence, and indicates the conserved domains.
• the fragments can be from any region of the sequence, can be of any length up to the length of the sequence, and can be as short as six residues for protein and 18 nucleotides for DNA.
• Exemplary fragments of the DNA sequences are as follows: 1-50, 51-100, 101-200, 201-218, 218-300, 301-450 and 450-600; and exemplary fragments of proteins are as follows 1-50, 51-100, 101-200, 201-206, 206-250, 251-300.
• the numbers may be measured from either 5' or 3' end of the DNA.
• the fragment location is determined from the N-terminus or C-terminus of the protein and may include adjacent amino acid sequences, such as for example for SEQ ID No. 2 an additional 10, 20, 40, 60 or 100 amino acids in either N-terminal or C-terminal direction of the described fragments.
• the identified polypeptide fragments may be linked to fragments or sequences derived from other transcription factors so as to generate additional novel sequences, such as by employing the methods described in Short, PCT publication WO9827230, entitled “Methods and Compositions for Polypeptide Engineering” or in Patten et al., PCT publication W09923236, entitled “Method of DNA Shuffling” or in Minshull and Stemmer, US Patent No.
• the identified fragment may be linked to a transcription activation domain.
• a transcription activation domain assists in initiating transcription from a DNA binding site.
• a common feature of some activation domains is that they are designed to form amphiphilic alpha helices with excess positive or negative charge (Giniger and Ptashne (1987) Nature 330:670-672, Gill and Ptashne (1987) Cell 51 :121-126, Estruch et al (1994) Nucl.
• Acids Res. 22:3983-3989 examples include the transcription activation region of VP16 or GAL4 ( Moore et al. (1998) Proc. Natl. Acad. Sci. USA 95: 376-381 ; and Aoyama et al. (1995) Plant Cell 7:1773-1785), peptides derived from bacterial sequences (Ma and Ptashne (1987) Cell 51 ; 1 13-119) and synthetic peptides (Giniger and Ptashne, supra).
• the isolated polynucleotides and polypeptides may be used to modify plant development, physiology or biochemistry such that the modified plants have a trait advantage over wild type plants.
• the identified polynucleotide fragments are also useful as nucleic acid probes and primers.
• a nucleic acid probe is useful in hybridization protocols, including protocols for microarray experiments. Primers may be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA strand by a DNA polymerase enzyme.
• Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods.
• PCR polymerase chain reaction
• Homologous sequences to those provided in the Sequence Listing derived from Arabidopsis thaliana or from other plants may be used to modify a plant trait.
• Homologous sequences may be derived from any plant including monocots and dicots and in particular agriculturally important plant species, including but not limited to, crops such as soybean, wheat, corn, potato, cotton, rice, oilseed rape (including canola), sunflower, alfalfa, sugarcane and turf; or fruits and vegetables, such as banana, blackberry, blueberry, strawberry, and raspberry, cantaloupe, carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion, papaya, peas, peppers, pineapple, spinach, squash, sweet corn, tobacco, tomato, watermelon, rosaceous fruits (such as apple, peach, pear, cherry and plum) and vegetable brassicas (such as broccoli, cabbage, cauliflower, brussel sprouts and kohlrabi).
• crops such as soybean, wheat,
• Other crops, fruits and vegetables whose phenotype may be changed include barley, currant, avocado, citrus fruits such as oranges, lemons, grapefruit and tangerines, artichoke, cherries, nuts such as the walnut and peanut, endive, leek, roots, such as arrowroot, beet, cassava, turnip, radish, yam, sweet potato and beans.
• the homologs may also be derived from woody species, such pine, poplar and eucalyptus.
• substitutions, deletions and insertions introduced into the sequences provided in the Sequence Listing are also envisioned by the invention. Such sequence modifications can be engineered into a sequence by site-directed mutagenesis (Wu (ed.) Meth. Enzymol. (1993) vol. 217, Academic Press). Amino acid substitutions are typically of single residues; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. In preferred embodiments, deletions or insertions are made in adjacent pairs, e.g., a deletion of two residues or insertion of two residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a sequence. The mutations that are made in the polynucleotide encoding the transcription factor should not place the sequence out of reading frame and should not create complementary regions that could produce secondary mRNA structure.
• substitutions are those in which at least one residue in the amino acid sequence has been removed and a different residue inserted in its place. Such substitutions may be conservative with little effect on the function of the gene, for example by substituting alanines for serines, arginines for lysines, glutamate for aspartate and the like.
• substitutions which are not conservative are expected to produce the greatest changes in protein properties will be those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
• a hydrophilic residue e.
• homologous sequence may encompass a polypeptide sequence that is modified by chemical or enzymatic means.
• the homologous sequence may be a sequence modified by lipids, sugars, peptides, organic or inorganic compounds, by the use of modified amino acids or the like. Protein modification techniques are illustrated in
• Homologous sequences also may mean two sequences having a substantial percentage of sequence identity after alignment as determined by using sequence analysis programs for database searching and sequence alignment and comparison available, for example, from the Wisconsin Package Version 10.0, such as BLAST, FASTA, PILEUP,
• FINDPATTERNS or the like (GCG, Madision, Wl).
• Public sequence databases such as GenBank, EMBL, Swiss-Prot and PIR or private sequence databases such as PhytoSeq (Incyte Pharmaceuticals, Palo Alto, CA) may be searched. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (1981 ) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and
• sequence comparisons between two (or more) polynucleotides or polypeptides are typically performed by comparing sequences of the two sequences over a comparison window to identify and compare local regions of sequence similarity.
• the comparison window may be a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150 contiguous positions.
• Transcription factors that are homologs of the disclosed sequences will typically share at least 40% amino acid sequence identity. More closely related TFs may share at least 50%, 60%, 65%, 70%, 75% or 80% sequence identity with the disclosed sequences. Factors that are most closely related to the disclosed sequences share at least 85%, 90% or 95% sequence identity.
• the sequences will typically share at least 40% nucleotide sequence identity, preferably at least 50%, 60%, 70% or 80% sequence identity, and more preferably 85%, 90%, 95% or 97% sequence identity.
• the degeneracy of the genetic code enables major variations in the nucleotide sequence of a polynucleotide while maintaining the amino acid sequence of the encoded protein.
• stringent conditions are selected to be about 5°C to 20°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
• Nucleic acid molecules that hybridize under stringent conditions will typically hybridize to a probe based on either the entire cDNA or selected portions of the cDNA under wash conditions of 0.2x SSC to 2.0 x SSC, 0.1 % SDS at 50-65° C, for example 0.2 x SSC, 0.1 % SDS at 65° C. For detecting less closely related homologs washes may be performed at 50° C.
• the hybridization probe is conjugated with a detectable label such as a radioactive label, and the probe is preferably of at least 20 nucleotides in length.
• a detectable label such as a radioactive label
• the labeled probe derived from the Arabidopsis nucleotide sequence may be hybridized to a plant cDNA or genomic library and the hybridization signal detected using means known in the art.
• the hybridizing colony or plaque (depending on the type of library used) is then purified and the cloned sequence contained in that colony or plaque isolated and characterized. Homologs may also be identified by PCR-based techniques, such as inverse PCR or RACE, using degenerate primers. See Ausubel et al. (eds) (1998) Current Protocols in Molecular Biology, John Wiley & Sons. TF homologs may alternatively be obtained by immunoscreening an expression library.
• the polypeptide may be expressed and purified in a heterologous expression system (e.g., E. coli) and used to raise antibodies (monoclonal or polyclonal) specific for the TF.
• Antibodies may also be raised against synthetic peptides derived from TF amino acid sequences. Methods of raising antibodies are well known in the art and are described in Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such antibodies can then be used to screen an expression library produced from the plant from which it is desired to clone the TF homolog, using the methods described above. The selected cDNAs may be confirmed by sequencing and biological activity.
• Any of the identified sequences may be incorporated into a cassette or vector for expression in plants.
• a number of expression vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach, (1989) Methods for Plant Molecular Biology, Academic Press, and Gelvin et al., (1990) Plant Molecular Biology Manual, Kluwer Academic Publishers. Specific examples include those derived from a Ti plasmid of Agrobacterium tumefaciens, as well as those disclosed by Herrera-Estrella, L., et al., (1983) Nature 303: 209, Bevan, M., Nucl. Acids Res.
• Ti-derived plasmids can be transferred into both monocotonous and docotyledonous species using Agrobacterium-mediated transformation (Ishida et al (1996) Nat. Biotechnol. 14:745-50; Barton et al. (1983) Cell 32:1033-1043).
• non-Ti vectors can be used to transfer the DNA into plants and cells by using free DNA delivery techniques.
• free DNA delivery techniques may involve, for example, the use of liposomes, electroporation, microprojectile bombardment, silicon carbide wiskers, and viruses.
• transgenic plants such as wheat, rice (Christou, P., (1991 ) Bio/Technology 9: 957-962) and corn (Gordon-Kamm, W., (1990) Plant Cell 2: 603-618) can be produced.
• An immature embryo can also be a good target tissue for monocots for direct DNA delivery techniques by using the particle gun (Weeks, T. et al., (1993) Plant Physiol.
• plant transformation vectors include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
• plant transformation vectors typically also contain a promoter (e.g., a regulatory region controlling inducible or constitutive, environmentally-or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.
• constitutive plant promoters which may be useful for expressing the TF sequence include: the cauliflower mosaic virus (CaMV) 35S promoter, which confers constitutive, high-level expression in most plant tissues (see, e.g., Odel et al., (1985) Nature 313:810); the nopaline synthase promoter (An et al., (1988) Plant Physiol. 88:547); and the octopine synthase promoter (Fromm et al., (1989) Plant Cell 1 : 977).
• CaMV cauliflower mosaic virus
• a variety of plant gene promoters that regulate gene expression in response to environmental, hormonal, chemical, developmental signals, and in a tissue-active manner can be used for expression of the TFs in plants, as illustrated by seed-specific promoters (such as the napin, phaseolin or DC3 promoter described in US Pat. No. 5,773,697), root-specific promoters, such as those disclosed in US Patent Nos. 5,618,988, 5,837,848 and 5,905,186; fruit-specific promoters that are active during fruit ripening (such as the dm 1 promoter (US Pat. No. 5,783,393), or the 2A11 promoter (US Pat. No.
• seed-specific promoters such as the napin, phaseolin or DC3 promoter described in US Pat. No. 5,773,697
• root-specific promoters such as those disclosed in US Patent Nos. 5,618,988, 5,837,848 and 5,905,186
• fruit-specific promoters that are active during fruit ripening such as the d
• Plant expression vectors may also include RNA processing signals that may be positioned within, upstream or downstream of the coding sequence.
• the expression vectors may include additional regulatory sequences from the 3'-untranslated region of plant genes, e.g., a 3' terminator region to increase mRNA stability of the mRNA, such as the Pl-ll terminator region of potato or the octopine or nopaline synthase 3' terminator regions.
• plant expression vectors may also include dominant selectable marker genes to allow for the ready selection of transformants.
• Such genes include those encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide resistance genes (e.g., phosphinothricin acetyltransferase).
• antibiotic resistance genes e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin
• herbicide resistance genes e.g., phosphinothricin acetyltransferase
• a reduction of TF expression in a transgenic plant to modifiy a plant trait may be obtained by introducing into plants antisense constructs based on the TF cDNA.
• the TF cDNA is arranged in reverse orientation relative to the promoter sequence in the expression vector.
• the introduced sequence need not be the full length TF cDNA or gene, and need not be identical to the TF cDNA or a gene found in the plant type to be transformed. Generally, however, where the introduced sequence is of shorter length, a higher degree of homology to the native TF sequence will be needed for effective antisense suppression.
• the introduced antisense sequence in the vector will be at least 30 nucleotides in length, and improved antisense suppression will typically be observed as the length of the antisense sequence increases.
• the length of the antisense sequence in the vector will be greater than 100 nucleotides.
• ribozyme sequences within antisense RNAs may be used to confer RNA cleaving activity on the antisense RNA, such that endogenous mRNA molecules that bind to the antisense RNA are cleaved, which in turn leads to an enhanced antisense inhibition of endogenous gene expression.
• RNA encoded by the TF cDNA may also be used to obtain co-suppression of the endogenous TF gene in the manner described in U.S. Patent No. 5,231 ,020 to Jorgensen.
• Such co-suppression also termed sense suppression
• the suppressive efficiency will be enhanced as (1 ) the introduced sequence is lengthened and (2) the sequence similarity between the introduced sequence and the endogenous TF gene is increased.
• Vectors expressing an untranslatable form of the TF mRNA may also be used to suppress the expression of endogenous TF activity to modify a trait.
• Methods for producing such constructs are described in U.S. Patent No. 5,583,021 to Dougherty et al.
• such constructs are made by introducing a premature stop codon into the TF gene.
• a plant trait may be modified by gene silencing using double-strand RNA (Sharp (1999) Genes and Development 13: 139-141). This approach, whereby a vector is prepared in which a cDNA or gene is arranged in duplicated fashion and is capable of generating upon expression a double stranded RNA molecule with a hairpin structure. This procedure has been used to modify gene activity in plants (Chuang and Meyerowitz (1999) Proc. Natl. Acad. Sci. 97:4985-9490).
• Another method for abolishing the expression of a gene is by insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens. After generating the insertion mutants, the mutants can be screened to identify those containing the insertion in a TF gene. Mutants containing a single mutation event at the desired gene may be crossed to generate homozygous plants for the mutation (Koncz et al. (1992) Methods in Arabidopsis Research. World Scientific).
• a plant trait may also be modified by using the cre-lox system (for example, as described in US Pat. No. 5,658,772).
• a plant genome may be modified to include first and second lox sites that are then contacted with a Cre recombinase. If the lox sites are in the same orientation, the intervening DNA sequence between the two sites is excised. If the lox sites are in the opposite orientation, the intervening sequence is inverted.
• polynucleotides and polypeptides of this invention may also be expressed in a plant in the absence of an expression cassette by manipulating the activity or expression level of the endogenous gene by other means.
• T-DNA activation tagging ectopically expressing a gene by T-DNA activation tagging (lchikawa et al., (1997) Nature 390 698-701 , Kakimoto et al., (1996) Science 274: 982-985).
• This method entails transforming a plant with a gene tag containing multiple transcriptional enhancers and once the tag has inserted into the genome, expression of a flanking gene coding sequence becomes deregulated.
• the transcriptional machinery in a plant may be modified so as to increase transcription levels of a polynucleotide of the invention (See PCT Publications WO9606166 and WO 9853057 which describe the modification of the DNA binding specificity of zinc finger proteins by changing particular amino acids in the DNA binding motif).
• the transgenic plant may also comprise the machinery necessary for expressing or altering the activity of a polypeptide encoded by an endogenous gene, for example by altering the phosphorylation state of the polypeptide to maintain it in an activated state.
• an expression cassette comprising a polynucleotide encoding a TF gene of this invention
• standard techniques may be used to introduce the polynucleotide into a plant in order to modify a trait of the plant.
• the plant may be any higher plant, including gymnosperms, monocotyledonous and dicotyledenous plants.
• Suitable protocols are available for Leguminosae (alfalfa, soybean, clover, etc.), Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage, radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.), Solanaceae (potato, tomato, tobacco, peppers, etc.), and various other crops. See protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture -Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833- 839; and Vasil et al. (1990) Bio/Technology 8:429-434.
• Transformation and regeneration of both monocotyledonous and dicotyledonous plant cells is now routine, and the selection of the most appropriate transformation technique will be determined by the practitioner.
• the choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence.
• plants are preferably selected using a dominant selectable marker incorporated into the transformation vector.
• a dominant selectable marker incorporated into the transformation vector.
• such a marker will confer antibiotic or herbicide resistance on the transformed plants, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic or herbicide.
• the modifed trait may be any of those traits described above. Additionally, to confirm that the modified trait is due to changes in expression levels or activity of the polypeptide or polynucleotide of the invention may be determined by analyzing mRNA expression using Northern blots, RT-PCR or microarrays, or protein expression using immunoblots or Western blots or gel shift assays.
• genes of the present invention relate to their potential roles in plant flowering time or the vernalization response.
• Most modern crop varieties are the result of extensive breeding programs and many generations of backcrossing may be required to introduce desired traits. Systems that accelerate flowering could have valuable applications in such programs since they allow much faster generation times. Additionally, in some instances, a faster generation time might allow additional harvests of a crop to be made within a given growing season.
• transformation systems for tree species such as oil palm, aspen, pine and eucalyptus
• forest biotechnology is a growing area of interest.
• inducible promoters By regulating the expression of flowering-time controlling genes, using inducible promoters, flowering could potentially be triggered as desired (for example, by application of a chemical inducer). This would allow, for example, flowering to be synchronized across a crop and facilitate more efficient harvesting.
• inducible systems could be used to tune the flowering of crop varieties to different latitudes. At present, species such as soybean and cotton are available as a series of maturity groups that are suitable for different latitudes on the basis of their flowering time (which is governed by day-length).
• a system in which flowering could be chemically controlled would allow a single high-yielding northern maturity group to be grown at any latitude. In southern regions such plants could be grown for longer, thereby increasing yields, before flowering was induced. In more northern areas, the induction would be used to ensure that the crop flowers prior to the first winter frosts.
• the existence of a series of maturity groups for different latitudes represents a major barrier to the introduction of new valuable traits.
• Strawberry has a well-defined perennial cycle of flower initiation, dormancy, chilling, crop growth and runner production.
• the plants flower in early spring, and fruit is produced in May or June. Following fruiting, runners are generated that carry plantlets which take root. The plants then remain dormant all through the late summer and autumn. Flowering cannot be repeated until the following spring after the plants have received a winter cold treatment.
• a system which bypasses this vernalization requirement, might permit a second autumn crop of strawberries to be harvested in addition to the spring crop.
• genes themselves, their regulatory regions could also be of value. If the promoters of these genes are responsive to low temperatures they could be incorporated into expression systems for regulation of genes that confer tolerance to freezing. Such genes would then be up regulated specifically at the time required, thereby minimizing any toxic effects that result from their constitutive expression.
• a transcription factor coding provided by the present invention may also be used to identify exogenous or endogenous molecules that may affect expression of the transcription ⁇ factors and may affect flowering time.
• These molecules may include organic or inorganic compounds.
• the method may entail first placing the molecule in contact with a plant or plant cell.
• the molecule may be introduced by topical administration, such as spraying or soaking of a plant, and then the molecule's effect on the expression or activity of the TF polypeptide or the expression of the polynucleotide monitored. Changes in the expression of the TF polypeptide may be monitored by use of polyclonal or monoclonal antibodies, gel electrophoresis or the like.
• Changes in the expression of the corresponding polynucleotide sequence may be detected by use of microarrays, Northerns or any other technique for monitoring changes in mRNA expression. These techniques are exemplified in Ausubel et al. (eds) Current Protocols in Molecular Biology, John Wiley & Sons (1998). Such changes in the expression levels may be correlated with modified plant traits and thus identified molecules may be useful for soaking or spraying on fruit, vegetable and grain crops to modify traits in plants.
• the transcription factors may also be employed to identify promoter sequences with which they may interact. After identifying a promoter sequence, interactions between the transcription factor and the promoter sequence may be modified by changing specific nucleotides in the promoter sequence or specific amino acids in the transcription factor that interact with the promoter sequence to alter a plant trait. Typically, transcription factor DNA binding sites are identified by gel shift assays. After identifying the promoter regions, the promoter region sequences may be employed in double-stranded DNA arrays to identify molecules that affect the interactions of the TFs with their promoters (Bulyk et al. (1999) Nature Biotechnology 17:573-577).
• the identified transcription factors are also useful to identify proteins that modify the activity of the transcription factor. Such modification may occur by covalent modification, such as by phosphorylation, or by protein-protein (homo or-heteropolymer) interactions. Any method suitable for detecting protein-protein interactions may be employed. Among the methods that may be employed are co-immunoprecipitation, cross-linking and co-purification through gradients or chromatographic columns, and the two-hybrid yeast system. The two-hybrid system detects protein interactions in vivo and is described in Chien, et al., (1991 ), Proc. Natl. Acad. Sci. USA, 88, 9578-9582 and is commercially available from Clontech (Palo Alto, Calif.).
• plasmids are constructed that encode two hybrid proteins: one consists of the DNA-binding domain of a transcription activator protein fused to the TF polypeptide and the other consists of the transcription activator protein's activation domain fused to an unknown protein that is encoded by a cDNA that has been recombined into the plasmid as part of a cDNA library.
• the DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene.
• the library plasmids responsible for reporter gene expression are isolated and sequenced to identify the proteins encoded by the library plasmids. After identifying proteins that interact with the transcription factors, assays for compounds that interfere with the TF protein-protein interactions may be preformed.
• G157 refers to SEQ ID Nos 1 and 2
• G859 refers to SEQ ID Nos 1 and 2
• G1842 refers to SEQ ID Nos. 9-16
• G1843 refers to SEQ ID Nos. 17 and 18, G1844 refers to SEQ ID Nos. 19-22
• G861 refers to SEQ ID Nos. 23-26 and FLC or G1759 refers to SEQ ID Nos. 27, 28.
• Putative transcription factor sequences (genomic or ESTs) related to known transcription factors were identified in the Arabidopsis thaliana GenBank database using the tblastn sequence analysis program using default parameters and a P-value cutoff threshold of -4 or -5 or lower, depending on the length of the query sequence. Putative transcription factor sequence hits were then screened to identify those containing particular sequence strings. If the sequence hits contained such sequence strings, the sequences were confirmed as transcription factors.
• BAC F22K20 GenBank accession AC002291
• An 872bp cDNA clone for G157 was identified among clones isolated from a library derived from leaf mRNA.
• the encoded protein was 196 amino acids in length, and shared 62% overall amino acid sequence identity with FLC, and 82% identity within the MADS DNA binding domain.
• G157 is also related to G859, G1842, G1843, and G1844 that map together as a tightly linked cluster, at the bottom of chromosome V, that occupies approximately 22 kb and spans three adjacent clones, MXK3, F1505, and MQN23 (GenBank accession numbers AB019236, AB026633, and AB013395, respectively).
• G859, G1842, G1843, and G1844 are all arranged in the same orientation.
• G859, G1842, G1843, and G1844 were likely created by a duplication event; this could have allowed their divergence into different aspects of gene regulation. Their physical proximity suggests that they may act as a unit controlled via common regulatory elements.
• G1842, G1843, and G1844 are displayed in Table 1.
• the table shows percent amino acid sequence identity and, in parentheses, the sequence identity percentages when conservative amino acid substitutions are considered.
• the MADS domains of the proteins encoded by G859, G1842, G1843, and G1844 are highly conserved with those of FLC and G157: these proteins share from 75% to 91 % of amino acid sequence identity, depending on the pair-wise comparison as shown below. When conservative amino substitutions are made, the MADS domains of these proteins are 88%-99% identical to each other (shown in parentheses).
• Amino acid residue 30 of FLC and by G157, G859, G1842, G1843, and G1844 is an acidic residue (E or D) whereas, in all other Arabidopsis MADS domain proteins so far identified, that position is occupied by a positively charged lysine residue.
• the crystal structure of the human SRF MADS domain bound to DNA has shown that lysine residue (which is also conserved in yeast MCM1 and human MEF2A proteins) to contact the phosphate backbone of the DNA target site (Pellegrini et al., (1995) Nature 376:490-498).
• MADS domain proteins with an acidic residue at position 30 may be particularly useful in modifying plant flowering time and vernalization response.
• the transcripts from these genes were analyzed by 3' RACE (Rapid Amplification cDNA Ends) and corresponding cDNAs were isolated by RT-PCR from mixed samples of Arabidopsis tissue (Columbia ecotype). During this analysis, it was found that G859, G1842 and G1844 transcripts exist in multiple alternatively spliced forms.
• Reverse transcriptase PCR was done using gene specific primers within the coding region for each sequence identified. Where possible, the primers were designed near the 3' region of each coding sequence initially identified.
• Total RNA was isolated from plant tissue tissue and extracted using CTAB. Once extracted total RNA was normalized in concentration across all the tissue types to ensure that the PCR reaction for each tissue received the same amount of cDNA template using the 28S band as reference. Poly A+ was purified using a modified protocol from the Qiagen Oligotex kit batch protocol.
• cDNA was synthesized using standard protocols. After the first strand cDNA synthesis, primers for Actin 2 were used to normalize the concentration of cDNA across the tissue types. Actin 2 is found to be constitutively expressed in fairly equal levels across the Arabidopsis tissue types.
• cDNA template was mixed with corresponding primers and Taq polymerase.
• Each reaction consisted of 0.2 ul cDNA template, 2ul 10X Tricine buffer, and 16.8 ui water, 5pmol Primer 1 , 5pmol Primer 2, 0.3 ul Taq polymerase, 200uM dNTPs and 8.6 ul water.
• Stepl 93° C for 3 mins
• Step 2 93° C for 30 sec
• Step 3 60-65° C for 1 min
• Step 4 72° C for 2 mins
• Steps 2, 3 and 4 were repeated for 20-35 cycles
• Step 6 4° C The PCR plate was sometimes placed back in the thermocycler to amplify more products for 5-15 more cycles to identify genes that have very low expression.
• the reaction cycle was as follows: Step 2 93° C for 30 sec, Step 3 65° C for 1 min, and Step 4 72° C for 2 ins, repeated for 8 cycles, and Step 4 4° C.
• Eight microliters of PCR product and 1.5 ul of loading dye were loaded on a 1.2% agarose gel for analysis between 21 and 36 cycles. Expression levels of specific transcripts were considered low if they were only detectable after 35 cycles of PCR. Expression levels were considered medium or high depending on the levels of transcript compared with observed transcript levels for actin2.
• PCR was carried out over 25 cycles using primers 5'-GGCATAACCCTTATCGGAGATTTGAAGC-3' (SEQ ID No. 57) and 5'-ACACAAACTCTGATCTTGTCTCCGAAGG-3' (SEQ ID No. 58).
• 25 or 30 cycles of PCR were performed using primers 5'-GCATAACCCTTATCGGAGATTTGAAGCCAT-3' (SEQ ID No. 59) and 5'-AACATTCCTCTCTCATCATCTGTTGCCAGC-3' (SEQ ID No. 60).
• PCR for FLC was performed either with primers 5'-AACGCTTAGTATCTCCGGCGACTTGAAC-3' (SEQ ID No. 51) and 5'-CTCACACGAATAAGGTACAAAGTTCATC-3' (SEQ ID No. 62) over 35 cycles, or 5'-TTAGTATCTCCGGCGACTTGAACCCAAACC-3' (SEQ ID No. 63) and 5'- AGATTCTCAACAAGCTTCAACATGAGTTCG-3' (SEQ ID No. 64) over 30 cycles. Primer specificity was verified by sequencing RT-PCR products. Samples were standardized via 20-
• Example III Construction of Expression Vectors
• the sequence was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region.
• the expression vector was pMEN20 or pMEN65, which are both derived from pMON316 (Sanders et al, (1987) Nucleic Acids Research 15:1543-58) and contain the CaMV 35S promoter to express transgenes.
• pMEN20 and the amplified DNA fragment were digested separately with Sail and Notl restriction enzymes at 37° C for 2 hours. The digestion products were subject to electrophoresis in a 0.8% agarose gel and visualized by ethidium bromide staining.
• the DNA fragments containing the sequence and the linearized plasmid were excised and purified by using a Qiaquick gel extraction kit (Qiagen, CA).
• the fragments of interest were ligated at a ratio of 3:1 (vector to insert).
• Ligation reactions using T4 DNA ligase (New England Biolabs, MA) were carried out at 16° C for 16 hours.
• the ligated DNAs were transformed into competent cells of the E. coli strain DH5alpha by using the heat shock method. The transformations were plated on LB plates containing 50 mg/l spectinomycin (Sigma).
• Plasmid DNA was purified by using Qiaquick Mini Prep kits (Qiagen, CA).
• the vector was used to transform Agrobacterium tumefaciens cells expressing the gene products.
• the stock of Agrobacterium tumefaciens cells for transformation were made as described by Nagel et al. FEMS Microbiol Letts 67: 325-328 (1990).
• Agrobacterium strain GV3101 was grown in 250 ml
• LB medium (Sigma) overnight at 28°C with shaking until an absorbance (A ⁇ oo) of 0.5 - 1.0 was reached.
• Cells were harvested by centrifugation at 4,000 x g for 15 min at 4° C. Cells were then resuspended in 250 ⁇ l chilled buffer (1 mM HEPES, pH adjusted to 7.0 with KOH). Cells were centrifuged again as described above and resuspended in 125 ⁇ l chilled buffer. Cells were then centrifuged and resuspended two more times in the same HEPES buffer as described above at a volume of 100 ⁇ l and 750 ⁇ l, respectively. Resuspended cells were then distributed into 40 ⁇ l aliquots, quickly frozen in liquid nitrogen, and stored at -80° C.
• Agrobacterium cells were transformed with plasmids prepared as described above following the protocol described by Nagel et al. FEMS Microbiol Letts 67: 325-328 (1990). For each DNA construct to be transformed, 50 - 100 ng DNA (generally resuspended in 10 mM
• Tris-HCl, 1 mM EDTA, pH 8.0 was mixed with 40 ⁇ l oi Agrobacterium cells.
• the DNA/cell mixture was then transferred to a chilled cuvette with a 2mm electrode gap and subject to a 2.5 kV charge dissipated at 25 ⁇ F and 200 ⁇ F using a Gene Pulser II apparatus (Bio-Rad). After electroporation, cells were immediately resuspended in 1.0 ml LB and allowed to recover without antibiotic selection for 2 - 4 hours at 28° C in a shaking incubator. After recovery, cells were plated onto selective medium of LB broth containing 100 ⁇ g/ml spectinomycin (Sigma) and incubated for 24-48 hours at 28° C. Single colonies were then picked and inoculated in fresh medium. The integrity of the plasmid construct was verified by PCR amplification and sequence analysis.
• Agrobacterium tumefaciens After transformation of Agrobacterium tumefaciens with plasmid vectors containing the gene, single Agrobacterium colonies were identified, propagated, and used to transform Arabidopsis plants. Briefly, 500 ml cultures of LB medium containing 50 mg/l spectinomycin were inoculated with the colonies and grown at 28° C with shaking for 2 days until an absorbance (A ⁇ o) of > 2.0 is reached.
• Arabidopsis thaliana seeds Prior to transformation, Arabidopsis thaliana seeds (ecotype Columbia) were sown at a density of ⁇ 10 plants per 4" pot onto Pro-Mix BX potting medium (Hummert International) covered with fiberglass mesh (18 mm X 16 mm). Plants were grown under continuous illumination (50-75 ⁇ E/m 2 /sec) at 22-23° C with 65-70% relative humidity. After about 4 weeks, primary inflorescence stems (bolts) are cut off to encourage growth of multiple secondary bolts. After flowering of the mature secondary bolts, plants were prepared for transformation by removal of all siliques and opened flowers.
• the pots were then immersed upside down in the mixture of Agrobacterium infiltration medium as described above for 30 sec, and placed on their sides to allow draining into a 1 ' x
• Seeds collected from the transformation pots were sterilized essentially as follows. Seeds were dispersed into in a solution containing 0.1 % (v/v) Triton X-100 (Sigma) and sterile H 2 0 and washed by shaking the suspension for 20 min. The wash solution was then drained and replaced with fresh wash solution to wash the seeds for 20 min with shaking. After removal of the second wash solution, a solution containing 0.1% (v/v) Triton X-100 and 70% ethanol (Equistar) was added to the seeds and the suspension was shaken for 5 min.
• a solution containing 0.1 % (v/v) Triton X-100 and 30% (v/v) bleach (Clorox) was added to the seeds, and the suspension was shaken for 10 min. After removal of the bleach/detergent solution, seeds were then washed five times in sterile distilled H 2 0. The seeds were stored in the last wash water at 4° C for 2 days in the dark before being plated onto antibiotic selection medium (1 X Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH), 1 X Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin).
• antibiotic selection medium (1 X Murashige and Skoog salts (pH adjusted to 5.7 with 1M KOH), 1 X Gamborg's B-5 vitamins, 0.9% phytagar (Life Technologies), and 50 mg/l kanamycin).
• T2 progeny seeds were germinated on kanamycin as described above and kanamycin resistant seedlings were selected, transferred to soil and analyzed.
• G157 plants ie plants expressing the G157 transgene
• 31 of 40 lines flowered earlier than control plants transformed with a control vector.
• Mean rosette leaf number of early T1 lines was 12.4+/-0.8 whereas control lines had 27+/-1.2 rosette leaves.
• 2 of 40 T1 plants flowered at the same time as controls and 7 of 40 lines were late flowering and produced visible inflorescences 2 to 3 weeks after wild type.
• G1842.7 encodes a 118 amino acid splice variant
• 8/10 T1 plants flowered early (accurate leaf counts not made) and 2/10 were wild type.
• the G1842 splice variants produced comparable effects to the full-length cDNA clone when over-expressed.
• 7/11 flowered early mean total of 6.4+/-0.5 leaves, flower buds visible at 16.0+/-1.6 days
• 2/11 had a wild type flowering time.
• the G1843 T1 plants were dwarfed and showed retarded development of some organs. This suggests that G1843 has unpredicted toxic effects when over-expressed.
• RT-PCR was performed on materials from G157 plants using G157 specific primers at approximately 25 cycles. The highest levels of G157 expression were detected in late flowering individual plants or in samples from pooled seedlings that contained late flowering individuals. Plants that showed only moderate or low levels of overexpression compared to wild type were slightly early flowering or normal.
• G157 transgene expression was also observed in G157 Swedish and Pitztal T1 plants.
• RT-PCR was performed with two early and two late flowering lines in each background. Again, the late flowering lines contained the higher levels of G157 expression.
• the factor appears to affect flowering time in a quantitative manner; a modest level of overexpression triggers early flowering, whereas a larger increase delays flowering.
• over-expression of G 157 or any of the related genes modifies flowering time in plants: a modest level of over-expression triggers early flowering, whereas a larger increase delays flowering.
• Late flowering vernalization responsive ecotypes and mutants have high steady state levels of FLC transcript, which decrease during the promotion of flowering by vernalization (Michaels and Amasino, (1999) Plant Cell 1 1 :949-956; Sheldon et al., (1999) Plant Cell 11 :445-458; Sheldon et al., (2000) Proc. Natl. Acad. Sci. 97: 3735-3758).
• G157 transcript levels show no consistent correlation with the vernalization response in the late flowering Swiss and
• G 157 did not influence FLC levels.
• the effects of vernalization on expression of G861 , G859, G1842, G1843, and G1844 were also examined. Germinating seeds of Columbia, Pitztal, Swiss, constans-1, and fca-9 were vernalized on MS agar plates in a 4°C cold room for 8 weeks, and then transferred to a continuous light growth room. Total tissues from the vernalized seedlings, and freshly sown non-vernalized controls were harvested at 9 days after the transfer. RT-PCR was performed for FLC, G157, G859, G1842, G1843, G1844, and G861 , and actin.
• Example IX Identification of Homologous Sequences
• Homologs from plant species other than Arabidopsis were identified using database sequence search tools, such as the Basic Local Alignment Search Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucl. Acid Res. 25: 3389-3402).
• the tblastx sequence analysis programs were employed using the BLOSUM-62 scoring matrix (Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919).
• the entire NCBI Genbank database was filtered for sequences from all plants except
• Arabidopsis thaliana by selecting all entries in the NCBI Genbank database associated with NCBI taxonomic ID 33090 (Viridiplantae; all plants) and excluding entries associated with taxonomic ID 3701 (Arabidopsis thaliana). These sequences were compared to sequences representing genes of SEQ IDs 1-56 on 9/26/2000 using the Washington University TBLASTX algorithm (version 2.0a19MP). For each gene of SEQ IDs 1-56, individual comparisons were ordered by probability score (P-value), where the score reflects the probability that a particular alignment occurred by chance. For example, a score of 3.6e-40 is 3.6 x 10 "40 . For up to ten species, the gene with the lowest P-value (and therefore the most likely homolog) is listed in Figure 2.
• P-value probability score
• Percentage identity reflects the degree to which two segments of DNA or protein are identical over a particular length.
• the ranges of percent identity between the non-Arabidopsis genes shown in Figure 2 and the Arabidopsis genes in the sequence listing are: SEQ ID No. 1 : 54%- 67%; SEQ ID Nos. 3,5,7: 37%-47%; SEQ ID Nos. 9,11 ,13,15: 54%-62%; SEQ ID No. 17: 62%-71%; SEQ ID Nos. 19, 21 : 50%-67%; SEQ ID Nos. 23,25: 75%-91 %; SEQ ID No. 27: 46%-69%; SEQ ID No.
• Arabidopsis homologs of genes in Table 2 were also identifed using BLAST. These genes are found in the following Arabidopsis BAC sequences, identified by their Genbank sequence NID numbers: 2827698 (G234 homolog), 3241917 (G748 homolog), 2618604 (G994 homolog), 6598548 (G1335 homolog), 7340331 (G736 homolog), 6523051 (G1435 homolog), 6598491 (G208 homolog) and 3172156 (G208 homolog).

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