JPH11504202A - Control of flower induction in plants and its use - Google Patents

Abstract

  (57) [Summary] An Id gene that regulates flowering in corn plants is described. Maize nucleic acid is similar to the nucleic acid of the gene encoding zinc finger regulatory protein in animals. Methods for isolating or preparing other regulatory protein genes in plants and their use are disclosed.

Description

DETAILED DESCRIPTION OF THE INVENTION                Control of flower induction in plants and its use Background of the Invention   Higher plants have a life cycle consisting of vegetative growth and subsequent periods of reproductive development You. Reproduction in angiosperms is a developmental process that begins with flower induction (evoking action) . This is a set of meristems (a set of meristems that give rise to most plant parts above the roots). Spores) stop producing leaves and begin producing flowers. Bernier, G .; (1 988), control of flower arousal and morphogenesis. Ann.Rev.Plant.Physiol.Plant Mole c. Biol. 39: 175-219. However, the molecular and genetic mechanisms that induce plant flowering Little is known about Him.   Further information about regulatory elements in plants is greatly needed. This Increasing knowledge of these elements allows genes to develop in plants Our understanding of the underlying mechanisms that lead to inflammation will deepen significantly.Summary of the Invention   The present invention relates to an Id gene or a part thereof showing the Id gene function of a corn plant. Identifying and providing isolated DNA. The invention further provides an Id or id^★Conflict RNA encoded by the DNA of the gene and portions thereof, and antisense ( (Complementary) DNA and / or RNA or portions thereof. a) Phase higher than 50% Under conditions of homosexuality or moderate stringency, zinc in the Id gene Hybridizes to the finger region, or b) shows 70% or more homology, Alternatively, under moderate stringency conditions, it hybridizes to the Id gene and A nucleic acid referred to as an Id homolog or its equivalent, which exhibits Id-type (start of reproductive phase) function Are also included in the present invention. In addition, regulatory genes in other plants can be detected and Also included are nucleic acid probes and primers for amplification and / or amplification. Therefore, The DNA of the present invention contains the Id gene or a part thereof, or all or a part of SEQ ID NO: 1. Or the homologous DNA.   The present invention further provides a plant-derived Id protein comprising a polypeptide described herein. Includes polypeptides that are part of the protein or Id protein. From all plant species Id proteins or homologs that exhibit similar regulatory functions (reproduction induction) are encompassed by the present invention. Included and as used herein is encompassed by the term Id protein. Amino acid sequence showing 80% or more homology to the amino acid sequence described in this specification Is considered a homologous polypeptide.   In another aspect, the invention provides an anti-binding agent that binds to a polypeptide described herein. About the body. Such antibodies are used to map sites of regulatory activity in plants Can be done. A fusion containing an Id protein and an additional peptide (e.g., a protein tag) Synthetic proteins also detect sites of Id protein / protein interactions in plants Can be used to   In a further aspect, the present invention provides a method for selecting a transition from a trophic stage to a flowering stage. To produce a plant having a different time period. Applicants believe that an active Ac transposable element When present, causes plants to stop vegetative growth and flower earlier than other id variants. New allele (id^★) Was prepared. As shown herein, Id with active Ac factor^★/ id^★Plants have fewer vegetative nodes Show and id without Ac factor^★/ id^★Plant or plant encoding the id allele Blooms earlier.   The present invention provides a novel id gene encoding a product that alters flowering induction in plants. 4.2 kb Id SacI plasmid from maize chromosome 1 The partial nucleotide sequence of the fragment. Articles of high stringency DNA that hybridizes to the SacI fragment or a portion thereof, and RNAs transcribed from any of the above DNAs or corresponding RNAs are also included. Preferred Alternatively, the DNA is the DNA shown in FIG. 4 (SEQ ID NO: 3).   In another aspect, the present invention relates to methods and plants for producing novel id alleles. Methods are provided for detecting other Id alleles or other regulatory genes in. Id inheritance Offspring homologs can be identified throughout the plant kingdom, including multicellular and unicellular algae.   Yet another aspect of the present invention relates to plants, seeds, plant tissue cultures, and / or plants. In the subsequent growth of a part of the plant, the timing of Or an exogenously introduced Id allele or DNA containing a portion of an Id allele. Provided are plants, seeds, plant tissue cultures, and plant parts.   The present invention also relates to a transgenic plant in which the timing of flower arousal has been modified. I do. Naturally occurring plants or wild-type (self- Transgenic plants are provided that are shorter than the (live) plants. Or the flowering Plants that are slow or do not flower are provided. As used herein The term "transgenic plant" refers to a wild-type (native) plant or a known mutation. Addition of either DNA or RNA that is not present in the body or naturally occurring DNA Plants containing a target or reverse copy, and introduced as described herein Plants, as well as any of the above, which result in plants with altered flower arousal time And plants containing the modifications of In one embodiment, such a transgene Transgenic plants are transgenic plants that are angiosperms (both monocotyledonous and dicotyledonous). Includes transgenic plants. The transgenic plant is a tiger into which the DNA has been introduced. Transgenic plants and seeds, vegetative propagation, cells, tissue culture or protopes And their progeny produced from last cultures and the like.   The transgenic plant of the present invention contains all of the proteins essential for the flower evoking action. Or DNA that encodes a part of a plant, and when it is present in a plant cell , The arousal effect of the modified flower (stops vegetative growth earlier than non-transformed plants of the same variety) And the onset of flowering, or later flowering or lack of flower induction) I will. DNA is a sense or antisequence that encodes a protein required for flower induction. Modified forms of exogenous DNA or proteins necessary for the induction of flowers in the sense orientation. Exogenous DNA that has been modified in such a manner as to be loaded. Responsible for starting flowering Specific or targeted mutagenesis of the plant's endogenous DNA may also result in altered flowers Leads to the induction of Exogenous DNA encoding a modified protein required for flower elicitation And the endogenous DNA required for the evoking effect of a flower mutated by specific mutagenesis The sequences contain substitutions, deletions or additions of at least one nucleotide. And the corresponding wild-type protein by at least one amino acid residue And a different wild-type (naturally occurring) DNA Is different. (As used herein, the term "nucleotide" refers to "nucleic acid. "When Used interchangeably. ) Genetic element (e.g., having an active Ac sequence Insertion of a Ds sequence without a Ds) is particularly useful.   Exogenous DNA can be obtained by known methods (e.g.,AgrobacteriumMediated transformation, micro Microprojectile bombardment, micro By injection or electroporation (see below) Is introduced into the plant cells of the target plant. Introduced exogenous DNA or specific alterations Such cells, which carry the endogenous Id DNA mutated by the mutagenesis, can induce flower induction Can be used for the regeneration of transgenic plants modified from No live or seedless asexual reproduction means (i.e., cuttings, tissue culture, etc.) It provides a source of additional plants.   The present invention also provides a plant having an altered flower induction time, Has exogenous DNA or RNA present in the resulting plant, and modified flower development Or construct containing DNA or RNA useful for producing recombinant plants A method of producing a product. Exogenous DN encoding a protein required for flower induction A or RNA (e.g., Id DNA in sense orientation) or proteins required for flower development Exogenous DNA that has been modified in such a way as to encode a modified form of Id^★Seeds produced by plants containing (DNA) are also a subject of the present invention. You.   Studies described herein may utilize Id genes, genomic sequences, or portions thereof. Make it work. This is determined by the applicant and plays a role in inducing flowering of the plant. Has an important role. This gene is used in monocotyledonous plants, especially the most commercially valuable It is derived from maize, one of the plants. The poly-encoded by this gene Peptides switch from vegetative growth to reproductive organ development in maize Is a regulatory protein that causes In addition, as in many other plants, tow In sorghum, the effect of this protein is the onset of senescence in these plants Is shown.   Corn requires more rainfall than wheat and most corn Sorghum cultivars require a long growing season. The work described herein also Others that allow corn to grow and require a long growing season before flowering Latitude Degree-dependent plants can grow in geographical areas with short growing seasons. Therefore, The plant can be induced to flower and seed before the first frost. Similarly, Induction of flowering is postponed for short-term plants growing in areas with long-term warm climates Can be done. As a result of excess vegetative mass and carbohydrates, These plants may produce more and / or larger flowers, and Many seeds can be produced. Or the plant can even be prevented from flowering, Therefore, nutritious silage biomass can be provided.   In another aspect, the invention relates to the production of a hybrid of corn and sorghum. And means for eliminating the need for tassel removal.BRIEF DESCRIPTION OF THE FIGURES   Figure 1 shows the location of the indeterminate and Bz2 (bronze grain pigmentation) genes and 1 is a map of chromosome 1 showing the site of transposon insertion of Ds2.   FIG. 2 shows a genomic sequence containing the DNA of the Id gene (SEQ ID NO: 1).   3A-3F show the genomic sequence and the deduced amino acid sequences a, b, and c of FIG. These are SEQ ID NOs: 4, 5, and 2), respectively. Ds2 transposon insertion Occurs at 168.   FIG. 4 shows a conserved model of a 4.2 kb SacI fragment containing part of the Id gene. It is a chief restriction map. Ds2 transposon insertion location and restriction sites NsiI and S The genomic sequence with acI (SEQ ID NO: 3) is shown.   FIG. 5 shows the polypeptide sequence encoded by SEQ ID NO: 3 (SEQ ID NO: 4) . (ORFs that represent part of the protein sequence are also homologous to zinc finger proteins. It is a part that shows sex. )   FIG. 6 shows the maize Id gene ORF and known zinc fingerta of eukaryotic species. This is a comparison with protein.   FIG. 7 shows the frameshift produced by excision of Ds2 derived from the ORF of the Id gene. 2 shows two null mutants (id-X1 and id-X2) resulting from.   8A-8B show transgenic monocotyledonous plants (FIG.8A) or transgenic For the production of dicotyledonous plants (Figure 8B), a weak promoter was fused with the Id cDNA and 1 shows a schematic diagram of an Id antisense construct that delays flowering of a line that blooms during the period.   9A-9B show transgenic monocotyledonous plants (FIG.9A) or transgenic For the production of dicotyledonous plants (FIG.9B), a constitutive promoter was fused with the Id cDNA, Shows a schematic of an Id sense construct that induces early flowering to a late flowering line.   10A-10B show transgenic monocotyledonous plants (FIG.10A) or transgenic plants. The drought-inducible promoter was replaced with Id cDNA for the production of Schematic of an Id antisense construct that fuses and delays flowering in response to drought Show.   11A-11D show transgenic plants that did not or delayed flowering. GAL4 binding sites (GB) were converted to monocotyledonous plants (Figure 11A) or dicotyledonous plants (Figure 11B) the Id antisense construct fused to the Id cDNA, and the GAL4 gene. Strong promoter (CaMV 35) in monocots (FIG.11C) or dicots (FIG.11D) Shown is a schematic of an Id antisense construct fused to (S) or a weak promoter.Detailed description of the invention   During reproductive growth, the plant enters a program of flower development that reaches fertilization and then Produces offspring. Aging may or may not occur subsequently is there. Maize plants (or their relatives, sorghum) usually have a certain number of Produce vegetative structures (e.g., leaves), and then reproductive structures (flowers), and eventually plant Is programmed to undergo aging. However, the indeterminat of the Id gene Maize (Zea mays) plants homozygous for the e (id) mutation Deficits in gram execution and exhibits several developmental phenotypes: 1) Nutrition The transition from to reproductive is prolonged vegetative phase, resulting in a long (or indeterminate) lifespan Plants (i.e., they bloom much slower than regular plants, or 2) The vegetative phase extends to the reproductive phase of development and Causes abnormal flower development; that is, female flowers (ears) exhibit nutritional properties and Are usually sterile, and male flowers (thighs) are tissues that have the characteristics of floral tissue Can undergo full developmental reversion to generate new developing branches. In the latter case, flowers End-differentiated cells, including other tissues, redifferentiate into vegetative tissues and undergo proliferative growth To resume. Singleton, W.R., J. Heredity, 37: 61-64 (1946); Galinat, W.C. and N aylor, A.W. (1951) Am. J. Bot. 38: 38-47. These phenotypes are associated with the normal Id gene. Function inhibits vegetative growth during certain periods of the plant life cycle, and To signal the start of Lack of Id functionality has created this transition No, and causes prolonged nutritional development.   Thus, normal Id function is the transition from nutrition to reproduction (i.e., In other words, it is important in the induction or evocation of flowers. Genetic data and molecules Data show that Id genes are regenerated from vegetative development in maize and other plants. Encoding regulatory proteins that play a key role in switching to development Suggests. Understanding the mechanism of this regulation is what internal control Or designed to flower and to produce seeds independent of environmental effects Provides the basis for producing specialized plants. In fact, use of homologs of the Id gene The same mechanism used in non-seed plants (e.g., algae) Spore production can be controlled.   The term "Id" refers to the normal (wild-type) gene of maize; "Refers to an altered (mutant) form of the Id gene. Starting the reproductive phase in plants Isolated plant DNA encoding the causing polypeptide can be genomic DNA or cDNA. possible. The DNA included in the present invention is derived from monocotyledonous plants (grass plants); What is done is the Id gene from corn.   Applicants correct by inserting the 1.3 kb transposable element Dissociation (Ds) into the Id gene. Novel alleles of id mutations have been created that disrupt the normal function of the Id gene. Next In the mutant id gene (id^★Clones containing a part of Isolated by the technique of spawn tagging. Hake et al., EMBO J., 8: 15-22 (1989). : Federoff et al. (1984) PNAS 81: 3825-3829. Gene (id^★And Id) some preliminary Sequence analysis shows that Id is homologous to a class of transcription factors found in all eukaryotes Indicates that a region is included.   Transposable elements are transposable pieces of DN that move throughout the genome of an organism. A. It is excised from one site, and another site (on the same chromosome or Inserted on a different chromosome). Transfer of transposable elements can be mutated or stained Other gene expression can be affected because it can cause rearrangement of the body.   Transposons Ac and Ds are related transposable elements present in maize. Make up a family.Mobile DNA M. Howe and D. Berg eds, Washington: ASM p During ress, Fedoroff, N. (1989) Maize Transposable Elements. Ac is another site Its own translocation or Ds (either on the same chromosome or on a different chromosome) Can be promoted. Ds cannot move if Ac is not present in the same cell . Ac is an autonomous transposable element, and Ds is a nonautonomous element of the same family. is there.   Insertion of Ds at a gene locus results in a mutation at that locus. For example, The C locus in corn kernels produces the factors necessary for purple pigment synthesis. Insertion of the Ds element in the locus inactivates the gene and renders the grain colorless. But, This mutation is unstable. In the presence of active Ac factor, Ds Translocated from the locus, resulting in a colored cell sector, resulting in a purple A grain with spots.   Applicants have used novel derivatives of the Ds transposable element Ds2 to create novel mutants of the Id gene. Was prepared. This results in excision of Ds2 from a nearby gene on chromosome 1 (active Ac And the subsequent insertion of Ds2 into the Id gene,^★ Was prepared.   After several generations of outcrossing and backcrossing,^★With an active Ac factor or Introduced into the genetic background without accompanying. Data from these experiments indicate that the active Ac factor Id with^★/ id^★Plants have no Ac^★/ id^★As strict as plants or Id plants Have no phenotype; that is, they have more vegetative nodes Fewer and faster flowering. This result indicates that the Ac factor is id during growth.^★Conflict Predicted when mediating somatic excision of Ds2 factor from a gene. Resection I Restores the function of d and results in a partial restoration of normal development. In addition, These plants have distinct sectoring patterns (ie, positive juxtaposition to mutant tissue). Observations that they do not show sharp boundaries of normal tissue) suggest that Id acts non-cell autonomously. And suggest. This result indicates whether the Id gene product is itself a diffusible factor. , Or modulate the production of a diffusable factor.   id^★The above experiment, which studied the effect of Ac on flowering of the plant, Flowering time can be quantitatively controlled by the amount of available id gene products. Testify. Wild-type (Id) plants from these lines flower 9-11 weeks after planting. Was. id^★Plants homozygous for 25 weeks after the absence of Ac (at this time The experiment was terminated due to frost) but did not flower. id^★With regard to homozygotes Plants that had and also had Ac flowered at approximately 15-22 weeks. Excision of Ds Occurs in these plants. These resections restore Id function, and Sufficient Id gene product to allow this plant to flower faster than plants without Ac Produce Id gene products that are not sufficient to flower as fast as wild-type plants. Bring. The wide range of flowering time is probably due to the timing of Ds excision per plant and And reflect the inherent variability of frequency and frequency. Fedoroff (1989), supra.   Another experiment, id^★The effect of Ac on plants was tested more closely. Ac factor is negative A dose of the effect; that is, one copy of Ac is more than two or more copies of Ac Ds ablation occurs. Fedoroff (1989), supra. id^★The effect of Ac dose on plants^★ Homozygous for and no Ac, one Ac or two per genome It was determined by planting seeds having the above-mentioned Ac factor. Available Id production If the amount of material controls flowering, id containing more than one Ac factor^★One plant Id with an Ac factor of^★Ids that flower later than plants but have no Ac factor^★Plant It was expected that the flowers would bloom earlier. In this experiment, the wild-type control It was carried out under greenhouse conditions in which 13 leaves were formed and then flowered. Id lacking Ac factor^★plant Did not flower even after 24 leaves had emerged. Two or more Ac factors Containing id^★Of the plants, 12.5% flower after having produced 21-23 leaves, while 87.5% of these plants did not flower even after 24 leaves had emerged. versus In contrast, 90% of plants containing one Ac factor flower after 16-24 leaves have emerged did. The result is an id containing one Ac factor^★Plant (with the most Ds excision And thus the plant with the highest amount of Id product) has more than one Ac factor Demonstrates flowering faster than plants (but not as fast as wild-type plants) . This result also indicates that, for example, different doses of Ac alter the frequency of Ds ablation. Changes in the amount of functional Id gene product To Suggest that it can be induced.   By Southern blot analysis using Ds2 factor as a probe, In many outbred offspring, the 4.2 kb SacI fragment^★Same as allele At times it was shown to move. This fragment has the id^★Do not carry alleles It does not exist in plants. This fragment is id^★Segregated with allele This is evidence that this gene is tagged with the Ds2 transposon. You. Separation of this fragment on agarose gel of genomic DNA cut with SacI , And by excision of the gel region containing the fragment, and Subclone into a plasmid vector to sublicense genomic DNA in this region. A blurry was made. Specific clones carrying this factor are Was identified by probing with a Ds2 probe. 60,000 blacks analyzed 1 clone was found to contain a 4.2 kb SacI fragment. Was. By restriction analysis, this recombinant clone was flanked by corn DNA (fla nking) was shown to have a Ds2 fragment: 165 bp for one of the Ds2 elements DNA and 2.8 kb DNA on the other side of this factor (FIG. 4). In this adjacent area Southern blots of DNA from various plants using either as a probe^★ Plants homozygous for the allele contain a single 4.2 kb SacI band That plants containing only normal DNA have a single 2.9 kb SacI fragment showed that. Thus, a 4.2 kb fragment was converted to a 1.3 kb to a 2.9 kb SacI fragment. It is the result of insertion of the Ds2 factor of b. Heterozygous plants contain both bands You.   id^★Further analysis of and other id variants indicates that these variants are normal Id genes Has been demonstrated. This mutation is generally associated with a variant in the Id gene sequence. Insertion or deletion of genetic elements at all sites, or all of the Id gene itself. Or some deletion. Carries the first id allele identified as id-R DNA from the mutant plant is hybridized to any of the adjacent probes. Did not show This is because the original allele is the result of the deletion of the Id gene. Indicates that there is. Another id allele, id-Compeigne, is included in this fragment. Seems to have a 3 kb insertion. These results indicate that Applicants have used i Provides convincing evidence that the d gene was tagged.   Sequence analysis of the DNA immediately adjacent to the Ds2 element of the Id gene revealed that the transposon was inserted. The inserted open reading frame (ORF) was revealed (FIG. 4). This RNA blots probed with adjacent DNA fragments containing the ORF , About 2.0-2.2 kb band, polyA + RNA from apical meristem, and less To a lesser extent, it was evident in mature leaves. Hybridization is Rarely detected in seedling RNA and not detected in root-derived RNA at all Was. This means that this ORF encodes a transcript, and this transcript Shows that it is differentially expressed in tissue.   Analysis of the deduced amino acid sequence encoded by this ORF indicates that this ORF And provide further evidence that it plays an important role in plant development I do. By comparing this ORF with all proteins in the latest database, This ORF is used by many different eukaryotes (humans, mice, frogs (Xenopus), and "Zinc fingers" -like tags identified in Drosophila It is shown to have significant homology with the protein (FIG. 4). Zinc fingerta Proteins are proteins of various eukaryotic transcription factors that utilize zinc-containing DNA binding domains. Known as ras and is an important regulator of development. McKnight, S.M. L. and And K. R. Yamamoto (1992) Transcriptional Regulation. Cold Spring Harbor L aboratory Press, Cold Spring Harbor, New York, Vol. 1, p. 580. Zincfi Nger proteins exert regulatory functions by mediating the transcription of other genes I do.   The results described herein show that the Id gene is a critical point in plant development (ie, Transition from seedling growth to reproductive growth) and that the Id gene is To function by controlling the expression of other plant genes required for development Is shown. It is clearly a "switch", and in corn, other Can be used without affecting the health and viability of the plant. Flower induction). Conversely, mutations in Id alter or inhibit only the induction of flowering In other respects, the mutant grows healthy and well.   Further evidence that the cloned DNA fragment is part of the Id gene Base is the original id^★Incorrect excision of Ds2 factor from alleles results in two new ids Obtained by creating alleles. id^★Unlike these new Alleles no longer respond to Ac; they do not seem to flower at all. Is a mutant. Sequence analysis showed that they were the Id An altered sequence that results in a frameshift in the open reading frame And therefore do not encode the same polypeptide as the Id gene. And   FIG. 7 shows the DNA and amino acid sequences of a part of the normal Id ORF, and the Ds insertion. And its changes as a result of resection. Caused by insertion of a Ds transposon The id-Ds mutation in the id shows an 8 bp target site overlap typical of Ds insertion (underlined). Null mutants (id-X1 and id-X2) are stable, derivative variants of ids resulting from excision of Ds2 Alleles. The id-X1 allele has a 7 bp remaining duplication site and one Has changed residues (from T to A). The id-X2 allele is the same T to A as id-X1 With a 5 bp remaining overlap site with the transposition to The resulting amino acid residue is ORF Inside shows frame shift.   Four. The complete clone carrying the 2 kb SacI fragment was analyzed and the Ds2 factor The complete sequence of the genomic DNA flanking (SEQ ID NO: 1) is provided herein. It was determined using information and analytical methods known to those skilled in the art (FIG. 2). 2930 nucleotchi Sequence contains the DNA of the Id gene. Putative amino acids encoded by this DNA The sequence (SEQ ID NO: 2) is shown in FIGS. 3A-3F as sequence (c).   SEQ ID NO: 1 from nucleotide 1 to at least 1890 (or up to 2150) Transcribed. Nucleotides 176-1600 represent an intron. Ds2 transposon insertion The approximate site of entry is at nucleotide 168. NsiI restriction site and SacI restriction mentioned above The ORF (SEQ ID NO: 3) located between the site and the nucleoside at positions 1-140 in FIGS. 3A-3F. Is represented by (Note: The DNA strand shown in FIG. 5 is complementary to the DNA strand in FIGS. 3A-3F. is there)   Plant fertility directly affects the ability to produce seeds. Therefore, flowering time is controlled. The ability to control is an important factor in the life cycle of a plant. Described herein Genetic studies of maize id mutations require the Id gene to transition to flowering Indicates that it encodes a protein. Use of transposon tagging in books Applicants have isolated the Id gene, especially a part of the zinc finger regulatory region of this gene And characterized. In addition, molecular analysis and eukaryotic regulatory proteins Shows that the polypeptide encoded by this region controls the onset of flowering And possibly from the vegetative growth of gymnosperms and lower plants (including algae) Some of the regulatory Id proteins that also control the transition to reproductive growth (eg, ).   Using the DNA provided by the present invention, flowering similar in function to the Id gene Homologous nucleic acids from other plant species that encode the relevant regulatory genes can be isolated. this In context, homology is at least 50% of the overall sequence identity, preferably Id It means greater than 70% identity with the zinc finger portion of the allele. other Identification and isolation of Id-type genes (homologues of Id) of plant species are performed using standard techniques known to those of skill Are achieved by appropriate methods and procedures. See, for example, Sambrook et al., (1989) Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold  See Spring Harbor, NY. An example of this application is given in Example 5 below. Will be issued.   By using these and other similar techniques, one skilled in the art can easily Id genes in different cells and tissues of corn, as well as from other plant species Homologues of the Id allele of can also be isolated. As an example, the Id gene in a plant Preparing a genomic or cDNA library of the species; Or use a part or homolog to generate a genomic or cDNA library Probing; identifying the hybridized sequence; and the hybridized DNA. Can be identified by obtaining the Id gene of the plant. Once identified These genes were then mapped, restricted, and cloned. Can be done. In particular, the zinc finger region or a fragment thereof Especially useful as probes due to the conserved homology to the link finger region. It is fruitful. A short fragment of about 20 bp in length is used for other zinc finger regions. Can be used to hybridize to   Hybridization refers to Ausubel et al. (1994) Current Protocols in Molecu lar Biology, Addendum 26, John Wiley & Sons, Inc. , New York, NY According to the method, a complementary polynucleotide is obtained under conditions of moderate stringency. Use DNA and / or RNA in hybridization assays to detect Means that   Other zinc finger proteins that regulate events other than the onset of flowering are corn May be present in sorghum and other plants. Regulatory genes offer some potential To show, seed germination, plant height and morphology, leaf number, and fruit ripening You can control. Isolation and isolation of these genes and the genes responsible for initiating the reproductive phase of plants And characterization are generally very significant in flower production, food production, and crop production. Important and worthwhile. Such zinc finger genes in plants Prepares a genomic or cDNA library of a plant species; Under conditions appropriate for hybridization of complementary DNA to identify the amplified DNA, All or part of the Id gene described herein (eg, SEQ ID NO: 1) or Probe genomic or cDNA libraries with homologues; To isolate the hybridized DNA and obtain the zinc finger gene for the plant. Can be identified. Next, a restriction map was created for the zinc finger gene. Produced, sequenced, and cloned.   The present invention also provides various means (transposons, site-specific mutations and random Includes mutations and changes using engineered nucleotide substitutions, deletions or additions. Nucleic acids and polypeptides having a structure altered by (but not limited to) Provide the peptide.   Transformers create Id gene alleles with altered functions in plants The poson method involves the following steps: Ds transposon or another non-autonomous transposition Inserting the factor into the Id gene, and then Ac transposon or another autologous Excision of Ds transposon using a regulatory transposable element The process of making inside.   Further methods for generating alleles of Id genes with altered functions in plants Examples include in vitro techniques using molecular genetic techniques (eg, site-directed mutagenesis). To change the molecular structure of the Id gene, and then insert the altered Id gene into the plant. To produce an altered Id allele in the plant.   These technologies perform dramatically different functions than the corresponding naturally occurring proteins. The indicated Id homolog can be generated. For example, identification of amino acid residues using site-specific mutation Id alleles can be created that encode the replacement of a functional gene (their Which amino acids are needed to produce a gene product that induces a reproductive response in plants). Can be determined. Similarly, by manipulating the Id allele, new functions (eg, For example, it is possible to produce a protein that has earlier flowering induction than that of a naturally occurring plant. Can be made.   Multiple varieties of maize whose flowering time has evolved widely depending on the growing environmental conditions Shi exists. In particular, the length of the day (determined by latitude) depends on when the plant Determine when. The Id gene is open in all of these maize mutants. Flower timing determinant, and flowering time correlates to specific mutations in Id gene products I can do it. In fact, the Id gene may be a major determinant of flower evoking.   Alter the Id gene or its homologue and introduce it into maize plants The flowering time of certain types of corn can be changed. How the parent strain grew as a result At different latitudes, the corn plant can grow. Therefore, the operation Of the identified Id gene for other characteristics (eg, high yield and disease resistance) Maize that can be incorporated into maize lines and grow at many different latitudes Lines can be created. Id using antisense constructs or cosuppression (see below) Reducing protein levels can delay flowering time, while Increasing levels of Id or earlier production of this protein (different Its increase by the promoter-linked Id gene) causes plants to flower earlier. Can be induced to do so. In addition, sense control under the control of different inducible promoters Or, by placing the antisense Id gene, it can be used for specific environmental conditions. Or when chemicals are applied, it may be possible to control the flowering time.   Cosuppression is the overexpression of an endogenous gene or transgene (transgene). Say. Here, an excess copy of this gene is Resulting in equivalent silencing of the component, thereby resulting in reduced expression of the feature Or resulting in excluded expression. See, for example, Jorgensen et al., U.S. Patent No. 5,034,323. No. 5,283,184. Transgene is introduced in the sense direction And full-length sequences or endogenous sequences intended to be suppressed It need not be completely homologous.   Expression of the endogenous gene is also determined by DNA complementary to the strand that transcribes the endogenous gene. Incorporation of oligonucleotides having a sequence identical or homologous to the sequence of the strand Can be suppressed through. Antisense oligonucleotides are derived from endogenous genes Nucleic acid that provides RNA that binds stably to the transcribed RNA and therefore blocks translation Includes specific sequences of otides. See Shewmaker et al., U.S. Patent No. 5,107,065. .   Flowering using other oligonucleotides of the present invention called "ribozymes" May be inhibited or arrested. Antisense and binds to RNA, DNA, or protein Unlike other oligonucleotides that work with ribozymes, the target RNA (eg, Catalytic RNA that can bind to and specifically cleave Is a molecule. Ribozymes designed to cleave at specific sites It can inactivate RNA molecules. Thus, the reduction of Id products is due to the endonuclease mode. (Endonucleolytic manner) to specifically cleave the transcript of the endogenous Id gene Can be achieved by introducing a DNA encoding the ribozyme designed in (1).   Among the known classes of ribozymes, group I introns and hammerheads Doribozymes are useful candidates for conversion for targeted cleavage of Id transcripts . Because they have a short (4 to 12 bases) recognition sequence; However, other types of ribozymes can be developed for site-specific cleavage of Id mRNA. Cec h, T.  R. (1988) J. Amer. Amer. Med. Assoc. See 260: 3030-3034.   The above strategy for delaying or completely destroying flowering is Relies on the use of chisense and similar techniques. "Dominant negative (domina nt-negative) alternative strategies based on the use of muteins Can be Makes the regulatory protein non-functional, but the mutant protein Certain types of mutations allow regulatory proteins to compete for quality and target Can be introduced. Such competition by non-functional proteins may Means that the activity of the wild-type protein can be suppressed using overexpression of gin Dominant negative mutation in Kufinger transcription factor is activated / silenced By deleting the main while retaining the DNA binding zinc finger domain Has been constructed in Drosophila and human cells. Overexpressed Mutant proteins are therefore wild-type by binding unproductively to DNA targets. Compete with proteins. O'Neill, E .; M. Et al., (1995) Proc. Nat'l. Acad. Sci. US A 92: 6557-6561. In plants, dominant negative strategies are Has been successfully used in regulatory proteins. Boylan, M. Et al., (1994) Plant Cell 6 : 449-460; Rieping, M. Et al. (1994) Plant Cell 6: 1087-1098; and Hemerly, A. See (1995) EMBO J, 14: 3925-3936.   A dominant negative mutation in the Id protein is detected in the zinc finger domain (inferred). Contains only the sequence coding for the specific DNA binding domain) and the activation domain Can be constructed by using an truncated version of the Id gene that lacks This end Where the truncated gene is introduced into corn plants under the control of a strong promoter Corn plants that have significantly delayed flowering or are unable to flower Bring things. Thus, the truncated dominant negative Id gene is referred to herein as In all of the constructs used to delay flowering described in It can be replaced by the sense Id gene.   The dominant-negative Id gene approach has been used in crops other than corn. When manipulating delayed flowering, it has advantages over antisense constructs. A Antisense strategies are based on the initial part or all of the Id gene from each crop species. Cloning then relies on expression of these genes in the reverse orientation. Antisense Suppression depends on the expression of the complementary nucleotide sequence. Complementary sequences vary by crop species Become. In contrast, dominant negative strategies involve the functional It depends only on conservation and its target site. Overall, this is the nucleotide sequence The requirements are much less stringent than the preservation. Regulatory genes encoding transcription factors Some known examples show similar mechanisms when expressed in a very wide variety of plant species. Perform noh. For example, Lloyd, A .; M. Et al., (1992) Science 258: 1773-1775; Irish,  V. F. And Y. T. See Yamamoto (1995) Plant Cell 7: 1635-1644. This Functional preservation of the type of maize Id gene indicates that the dominant negative type It means that it can work in crop species as well. This includes other cereal species and And it can certainly be expected to work in all monocotyledonous plants.   For applications on dicotyledons, dicotyledonous species (eg, tobacco or First isolated closer Id homologue from Arabidopsis And as described above (all sequences except zinc finger DNA binding domain) It is advantageous to construct a dominant negative derivative) possible. The dominant negative Id of this dicotyledonous plant type Can be used for plants. Therefore, the application of dominant negative technology to a wide range of crops Use can be achieved without having to clone the Id gene from each crop.   Introducing the nucleic acids and constructs of the present invention using any suitable technique to induce flower induction Can be produced. Gramineae like corn For plants, microprojectile bombardment (eg, Sanfor d, J. C. Et al., US Pat. No. 5,100,792 (1992)) may be used. In this embodiment In this case, the nucleotide construct or the vector containing this construct is And then targeted via high speed ballistic penetration Introduced into the tissue (cells). The vector is located outside the plant cell in which the vector has been introduced. It can be any vector that expresses the causative DNA. The transformed cells are then plant Cultivated under conditions suitable for the regeneration of, resulting in the production of transgenic plants. Transgenic plants harboring this construct can be produced in a variety of ways (appropriate phenotypic mapping). (Eg, antibiotic or herbicide resistance), or naturally occurring plants (Including, but not limited to, visual observation of the timing of flower induction compared to) Is tested for the desired phenotype.   Other known methods include Agrobacterium-mediated transformation (e.g., Smith, R. et al. H. La U.S. Pat.No. 5,164,310 (1992)), electroporation (e.g., Cal. vin, N. U.S. Pat.No. 5,098,843 (1992)). (For example, Kasuiya, T. Et al., U.S. Pat.No. 5,013,660 (1991)) or poly Introduction using agents such as ethylene glycol (e.g., Golds, T. et al. Et al. (1993) B iotechnology, 11: 95-97). Generally, plant cells are various Vectors (e.g., viral vectors, episomal vectors, Ti plasmid vectors) , Etc.) according to well-known procedures. Nucleic acids in plant cells Is not important to the present invention.   A transcription initiation region may provide for constitutive or regulated expression. Plant smell Many promoters are available that are both functional and functional. An exemplary promoter Are octopine synthase promoter, nopaline synthase promoter, Reflower mosaic virus (35S) promoter, Scrophularis mosaic virus (FMV) promoter, heat shock promoter, ribulose-1,6-bisphosphate (R UBP) carboxylase small subunit (ssu), tissue-specific promoter Etc. are included. The regulatory region is used for physical stimulation (e.g., RUBP carboxylase ssu If present), may be reactive to differentiation signals, or metabolites . The time and level of expression in the sense or antisense direction is produced It can have a clear effect on the phenotype. Therefore, the selected promoter is In conjunction with the direction of the incoming DNA, determine the effect of the introduced gene.   When the transgenic plant of the present invention is grown under the same conditions, it induces flowers. Is faster than the induction of flowers of the same varieties of plants that do not contain foreign nucleic acids as described above. Exogenous nucleic acids that alter the timing of the induction of E. coli. Or transgenic When plants are grown under the same conditions, the induction of flowers will include the foreign nucleic acids described above. Not delayed or inhibited compared to the induction of flowers in plants of the same variety As such, it includes foreign nucleic acids that alter the timing of flower induction.   Further, the present invention provides a method for producing a transgenic plant in which the flowering induction time is changed. Methods. In this method, the presence of a plant in a plant cell Introducing a foreign nucleic acid that causes a change in the induction time of the growth, and including the foreign nucleic acid. Plant cells under conditions appropriate for the growth of the plant cells, thereby Producing a plant whose induction time has been changed. The raw material to which this method can be applied Objects include angiosperms (monocots and dicots), gymnosperms, and spores. Include vegetatively propagated plants, and algae.   Transgenic plants containing the Id recombinant construct can be transformed cells, tissues Alternatively, it can be regenerated from plant parts by methods known to those skilled in the art. The plant parts are It is intended to include any part of a plant that can give rise to a regenerated plant. Therefore The present invention relates to one or more cells, tissues (particularly meristems and / or Embryo tissue), protoplast, epicotyl, hypocotyl, cotyledon, cotyledonary node, flower Includes flour, ovules, stems, roots, leaves and the like. Plants can also be regenerated from explants. The method varies according to the species of the plant.   Seeds are transferred from the regenerated plant or between the regenerated plant and a suitable plant of the same species. Distribution can be obtained. Alternatively, the plant can regenerate the plant parts, By culturing under appropriate conditions, vegetative propagation can be achieved.   Isolated and purified Id or id protein or polypeptide, and it These epitopic fragments localize the site of Id regulation (localiz ation) and to analyze developmental pathways in plants. Can be used for For example, an antibody that specifically binds to the Id protein Whether the protein is expressed in particular cells or tissues of the plant, and It can be used to determine when it is expressed. This information tells Id that flowering And how it acts to alter flowering guidance pathways Can be used to determine.   The antibodies of the present invention may be polyclonal, monoclonal, or antibody fragments. And the term “antibody” can be a polyclonal antibody, a monoclonal antibody, And antibody fragments. The antibodies of the present invention can be isolated Raised against recombinant or recombinant Id or id proteins or polypeptides obtain. The preparation of the immunizing antigen and the production of antibodies are performed using any suitable technique. obtain. Various methods have been described (e.g., Harlow, E. And D. Lane (1988) An tibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold  Spring Harbor, NY; Ausubel et al. (1994) Current Protocols in Molecular Biol. ogy, Vol. 2, Chapter 11 (Addendum 27), John Wiley & Sons: New York, NY).   Whether the antibody of the present invention can be labeled by any physical or chemical means, Alternatively, a secondary antibody that binds to the primary antibody can be labeled. The label is UV light during the reaction Or an enzyme that is assayed by the addition of a substrate that releases a product that absorbs visible light Or a radioactive material, chromophore, or fluorescent dye. E. Ha rlow and D. Lane (1988) supra.   The isolated polypeptides of the present invention also exhibit protein / protein interactions. Can be used for detection and analysis. Fusion proteins for this purpose are: An Id DNA encoding a functional Id polypeptide is converted to a different polypeptide (Id polypeptide). Polypeptides that are not related or homologous to the peptide) By fusion with foreign DNA to be loaded. The resulting fusion tamper The protein is a prokaryotic cell (e.g., E. coli), isolated, labeled, and Antibodies to detect Id allele binding sites and Id / protein interactions And can be used in essentially the same way. Ron and Dressler (1992) Biotech 13: 866-69. See Smith and Johnson (1988) Gene 67: 31-40;   Maize strains adapted to temperate regions (temperate latitudes) When planted, it blooms before maturity due to shorter daylength. Success Pre-ripening flowering results in severe yield loss. Salamini, F. (1985) Breeding Stra tegies for Maize Production Improvement in the Tropics. Brandolini, A. You And Salamini, F. Ed., Food and Agriculture Organization of U. N. , Istituto A gronomico Per L'Oltremare, Firenze, Italy. One skilled in the art will appreciate that the cloned Id gene Can be used to solve this problem. Id gene is weak Transgenics inserted in antisense direction under the control of motor Maize plants can be created (FIG. 8A). Weak promoters used to develop During this time, it should be constitutively active, at least in the shoot apical meristem. Id As it appears to be non-cell autonomous, the exact details of the promoter's site of action are unnecessary. An example of a weak promoter useful in this application is the weak promoter in corn. Nopaline synthase (nos) derived from T-DNA that has been shown to be constitutive It is a promoter. Callis et al. (1987) Genes Dev. 1: 1183-1200. Another promotion Ter is a cyclin promoter derived from maize. Cyclin Cell division proteins found in plants, animals, and yeast. plant Cyclin transcripts have meristem tissue and cells growing at low levels It is expressed in tissues but not elsewhere. Renaudin et al. (1994) PNAS 91: 7375-7379. The cyclin promoter is available from Applicants' cyclin 1b or Using the full-length cDNA clone of Clin III as a probe, standard isolation and Flanking from a maize genomic library using cloning and cloning techniques It is easily isolated by removing the upstream genomic sequence. Sambrook et al., Supra; Fr eeling and Walbot, see above. Those skilled in the art will recognize that Other weaknesses having the properties necessary to practice this embodiment of the invention are contemplated. Recognizes a different promoter.   An example of a useful construct for the above application is illustrated in FIG. 8A. The cDNA for the Id gene is , Downstream of the promoter in an antisense orientation. ADH1 intron is RN A Required for stability, and the 3 'end of the nos gene is an efficient polyadenylate Is added to guarantee the optimization. Callis et al. (1987) supra. DNA is The bar gene for resistance to the herbicide Basta by simple standard methods Used as a replacement marker, it is introduced into corn plants. Gordon-Kamm et al. (19 90) Plant Cell 2: 603-618; Freeling and Walbot (1993) supra.   Any construct that expresses foreign DNA in a plant cell into which the foreign DNA has been introduced, or A vector (for example, a pMON530 vector having a 35S promoter) can be used . Another useful vector or construct of the present invention is in varieties that bloom early, Weak promoters were introduced into the pMON530 vector in the antisense orientation to delay flowering. Foreign DNA encoding the Id protein inserted downstream of the protein.   Similar constructs are available for other cereals (e.g., rice, barley, and other monocots) Can be used for For antisense applications, first a homogeneous cD from the species to be modified NA may need to be isolated. The Corn Id clone is used for this purpose Id homologs from cDNA libraries of other cereal species using as probes for It is recognized that screening can be performed. Then Id homology for the manipulated species The product can be inserted as a replacement for the corn Id gene in the construct of FIG. 8A.   The same technique can be extended to dicots as well. Some of these crops Delaying the flower season has similar benefits to those described for corn, That is, a longer vegetative growth period resulting in a higher yield of fruits and seeds obtain. Gottschalk and Wolff (1983) Induced Mutations in Plant Breeding, Springer-Verlag, Berlin, Heidelberg. In addition, some dicots are mainly The products of vegetative growth are beneficial (e.g., spinach, tobacco, etc.) and In these plants, prolonged vegetative growth is associated with higher and more efficient yields of the product. Produces a harvest. Antisense constructs use Id homologs isolated from these species Thus, the transgenic plant can be designed as shown in FIG. It can be made by DNA transformation (preferably using Agrobacterium transformation technology). Used, but transformations by other standard techniques are also used). Lycett, G. W. And D. Grierson (1990) Genetic Engineering of Crop Plants, Butterworths , London; Setlow, J. K. (1994) Genetic Engineering Principles and Methods, Volume 16, Plenum Press, New York.   Corn varieties that are adapted to the tropics have extremes when grown in temperate regions. Flowering late at the edge (Salamini, supra), reaching a height of 15-20 feet at flowering, It has 30 leaves (compared to about 20 leaves for the average temperate variety). This is Not only is it inconvenient to handle and harvest, but also the plants are harmed by late season frost. Susceptible. Strategies for inducing early flowering in these plants Gee inserted the cloned Id gene in the sense orientation below the constitutive promoter (Figure 9A). Introducing this gene early in the nutritional development of these varieties It is to make it manifest. Strong or weak promoters (e.g., CaMV3 5S (strong) promoter or nos (weak) promoter), and both Also function in corn. Callis et al. (1987), supra. Building for this purpose Products and transformation methods are compatible with the antisense applications described above, except for the orientation of the Id gene. And the transformation method used in the above.   This technique is generally used with the same constructs or constructs that are similar in principle. It has been recognized that it can be adapted for other cereal species and for monocots. You. In fact, homologs of Id may not be required for early expression. why If the corn Id gene product is suitable for other monocotyledonous plants (including cereals) In order to promote early flowering.   In another embodiment of the present invention, the early flowering of the dicotyledonous plant is the target plant or By transforming a plant cell with the maize Id gene product or Id homolog Can be provided. Maize genes function efficiently in dicots Homologous genes need to be isolated from the species to be transformed. Maybe not. For example, the corn R and C genes are compatible with the CaMV 35S promoter. It functions in dicotyledon Arabidopsis when expressed under the control of a protein. Lloyd (1992) Science 258: 1773-1775. The construct shown in FIG. Can be used for expression of the Id gene or homologue in T-DNA transformation. Or may be inserted using other standard techniques, such as those already described.   Drought stress causes severe loss of harvest due to injury to plants. obtain. In addition, flowering time can also be affected. When stressed, many plants Blooms before maturity. Drought stress in corn is much lower than in ears. Ear development can occur early, resulting in reduced or no yield. These problems Some are alleviated, when the entire flowering time of the plant is delayed during the drought period obtain. This delay can allow plants to grow vegetatively for longer periods than normal. You. Thus, the plant can recover from the drought injury before flowering. The Id gene Although this is in the antisense orientation as described above in plants, Alternatively, if introduced in combination with a drought-inducible promoter, Can be used. Any drought-inducible promoter can be used. For example, RAB- 17 gene promoters (this is likely due to drought and other stresses) Which are induced as a result of regulation by the plant hormone ABA). Vilard ell et al. (1990) Plant Mol. Biol. 14: 423-432. A second type of professional that can be used The motor is a corn hsp70 heat shock promoter (this is a 37 ° C to 42 ° C high Induced in response to temperature). Callis et al. (1988) Plant Physiol. 88: 965-968 .   Vectors or constructs useful for producing plants responsive to environmental effects include: Exogenous DNA encoding the Id protein is transferred in the pMON530 vector in the antisense orientation. Inserted downstream of a drought-inducible promoter to delay flowering in response to drought It is produced by having Some constructs for this purpose are shown in Figure 10A. Show.   This technique also uses the same or similar constructs as in FIG. If so, using the homolog of the Id gene for the particular cereal to be transformed, Generally, it can be extended to monocotyledonous plants (including other cereals).   The extension of this technology to dicotyledonous crops requires proper drought to function in dicotyledonous plants. One inducible promoter can be used. Arabidopsis Atmyb2 Promo Is a common ABA-responsive, drought- and stress-inducible promoter. Can be used. Urao et al. (1993) Plant Cell 5: 1529-1539. Soybean heat shock A motor can also be used. Schoffl et al. (1989) Mol. Gen. Genet. 217: 246-25 3. A construct containing such a promoter is shown in FIG. 10B. This application is Crop to be manipulated, rather than the corn Id gene, as it depends on It may be necessary to use homologs of the Id gene from the species.   Maize plants that completely lack flowering (i.e., knock out tow) Sorghum plants are a special use. Non-flowering corn plants are nutritionally And produce large biomass that can be harvested for silage purposes. But, When the Id gene is completely knocked out for silage production purposes, Transgenic plants never flower and are unable to produce hybrid seeds. No.   One of the present invention for producing hybrid seeds of transgenic corn The method described above involves the control of a regulatory sequence, called the GAL4 binding site, in the antisense orientation. To produce a transgenic plant having the Id gene under your control. The result As a result, the antisense Id gene is not expressed without the GAL4 protein. GAL4 Is a transcription factor derived from yeast, which is a plant like tobacco (Ma, J. (1988) Nature  334: 631-633) and corn (McCarty, D. (1991) Cell 66: 895-905) It is shown to be. This is for genes containing a GAL4 binding site in the promoter. Activates transcription.   In this embodiment, the silent antisense Id gene and GAL4 binding site Transgenic inbreds containing the GAL4 gene Below (to delay flowering due to corn growth at low latitudes) or high Under the promoter (to eliminate flowering for silage production) Bred with another transgenic inbred that expresses it. Each similar The hybrids flower normally. However, hybrids express antisense Id and And flowering depends on the promoter used to drive the GAL4 gene , Delayed or gone Similar modifications can be made using the appropriate Id homolog to other It can also be done for plants (either monocotyledonous or dicotyledonous).   Constructs using the GAL4 binding site are shown in FIGS. 11A, 11B, 11C and 11D. like this In corn, the inbred comprising the construct shown in FIG. With an inbred that contains the construct In the resulting hybrid, GAL4 Flowering is delayed when the gene is under the control of CaMV35S (P35s). But GAL4 remains High if the gene is under the control of the nos (Pnos) or cyclin (Pcyclin) promoter. In brid, flowering is only slightly delayed. Illustration of dicotyledonous plant Crossing a plant containing the construct shown in FIG. 11C with a plant containing the construct shown in FIG. 11D By doing so, a similar result is obtained.   The above application is an illustration of the use of antisense Id constructs. Any suitable construct (E.g., dominant negative type Id gene) It will be appreciated by those skilled in the art that it can be replaced with an antisense construct.   The Id gene was isolated from corn, but homologs of Id were found in other cereals (grain cro ps) and most likely in all other plants high. Applicants have determined that the near relatives (clones) of Id as determined by sequence homology. se relative) is also present in dicots. This in other species If their homologs are also important in controlling flowering time, many agriculturally important cereals Can be operated at the time of flowering. Uses the compositions and methods described herein Those skilled in the art will recognize other cereals (e.g., sorghum, rye, wheat, etc.) and other Use known procedures to alter the onset of the reproduction phase of commercially important plants in I can do it.   For example, the modification of the flowering time is the time of fruit ripening, the time of flower production (production) Used to affect seed size and quality, latitude at which varieties can grow, etc. Can be done. Flowering time starts at different times when flowering occurs in different parts of the same plant Can be adjusted as follows. The present invention also provides a hybrid of corn and sorghum. Means for eliminating the need for tassel removal in the preparation of sardines. Id is the cell itself Although it does not appear to act in a rhythmic fashion, the Id signal is localized to certain areas of the plant, Thus, Id is transcribed or Id mRNA is expressed in some regions of the plant. Must be activated to induce flower development in each of these areas It may not seem. Both corn and sorghum have male vase at the top of the plant. A government officer (Yuho) is produced. Female organs occur in the lower part of the axilla. Tissue characteristics to which Id gene is bound Different Female in other parts of the plant through the use of selective or other selective promoters While selectively inducing reproductive development of the reproductive organs, It is possible to inhibit or prevent production. Alternatively, after normal flower induction, male Sexual reproductive organ development may be inhibited, or pollen producing tissue or cells Combine sense production with the formation of pollen-producing cells (eg, carpet cells) This can be induced to revert to the vegetative phase.   Another application of this technology is in crops harvested as leaves (e.g., lettuce, cabbage, Increase the vegetative period of spinach, maize (and thus the leaves produced This, and thereby delay flowering. To increase the yield of their crops. Therefore, any plant (angiosperms, naked (Including cotyledons, monocots, and dicots) can be used in accordance with the present invention. . Plants of interest include cereals (e.g., wheat, barley, corn, sorghum , Triticale, etc.); other commercially beneficial crops (sunflower, soy, safflower, yellow Fruits (eg apricot, orange, apple, abo) Vegetables (eg carrots, lettuce, tomatoes, broccoli, etc.); trees Woody species (eg, poplar, pine, oak, etc.); and ornamental flowers ( For example, clematis, roses, chrysanthemums, tulips, etc.) are included.   The following examples are provided to illustrate the invention and to isolate and identify the Id gene. Describe certain aspects of the invention to provide a description of the methods used to . The examples should not be construed as limiting the invention in any way.   All references in this application for materials and methods are incorporated herein by reference. Used.                                 Example 1 Transposon tagging:   Plants are grown under normal field conditions at Uplands Farm Agricultural Field Station (Co ld Spring Harbor Laboratory). Was. Use standard corn genetic technology for all mating and analysis procedures did. Freeling, M and Walbot, V. (1993) The Maize Handbook, Springer-Verla g, New York; Gottschalk, W. and Wolff, G. (1983) Induced Mutations in Plant  Breeding, Springer-Verlag, Berlin Heidelberg.   The Id gene is mapped near the grain pigmentation gene (Bz2) on chromosome 1. You. Mutable allele of the Bz2 gene (bz2-m2) is the Ds2 This is the result of insertion of a sposon (Dooner et al. (1985) Mol. Gen. Genetics 200: 240-246). (Ds2 is a defective derivative of the transposable element Ac / Ds family, and It can only be translocated in the presence of an Ac-factor providing ze. ) Id is close to bz2-m2 And the fact that the Ac / Ds element preferentially translocates to the linked site Applicants then selected for id mutants from embryo revertants in the bz2-m2 population. Was. That is, complete, resulting from embryo removal of the Ds2 factor (i.e., from bz2-m2 to Bz2) By selecting for the purple kernel, the Ds2 factor is inserted elsewhere F1 population was created. One id mutant was detected from the F2 population of these revertants. Isolated from the 600 families examined, and id^★I named it. id^★And the known pair of id A cross with an allele (eg, id-R) will result in an allele with an id mutation on chromosome 1. And confirmed.                                 Example 2 DNA analysis:   All molecular biology procedures are essentially as described in Sambrook, J. et al. (1989) Molecular Cloni. ng-A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). Solution of corn DNA and RNA The method for the analysis was performed according to Freeling, M. and Walbot, V. (1993) supra.   For Southern blot analysis, 2-4 mg of corn DNA extracted from leaves was used. , Restriction with SacI and electrophoresis on a 1% agarose gel. Transferred to Lurose membrane. For Ds2 probing, use the Ds2 transposon An internal 108 bp fragment was isolated from the plasmid carrying this part of Ds2 and Using the restriction enzymes BamHI and EcoRI. This fragment can be Purified from a loin gel, and radioisotope-containing nucleotides (32P-dATP and 3 2P-dGTP) was replaced with random primed labeling using a kit from Boehringer-Mannheim ( ra ndom primed labeling). Marked Using the fragment, a standard formamide hive containing 10% dextran sulfate Southern blot was probed using the re-dialysis solution.   To isolate a 4.2 kb SacI fragment that hybridizes to Ds2, a single 100 mg of DNA from plants is digested with SacI and on a 1% low melting point agarose gel For electrophoresis. The region of the gel between 4 kb and 5 kb (marked by side markers) Excised from the gel and purify the DNA contained in this fragment. Made. The DNA was ligated into the plasmid vector pLITMUS29 (New England Biolabs). (T4 DNA ligase, New England Biolabs). This plasmid vector is self- To prevent ligation, cut with SacI and phosphatase (shrimp alkaline) Phosphatase, U.S. Biochemical) treated to remove the 5 'phosphate group . The recombinant plasmid is transferred into E. coli DH10B cells by electroporation. Transformed and plated on L agar plates containing 100 μg / ml ampicillin . Approximately 60,000 ampicillin-resistant colonies are grown on plates and then replicated. Mosquitoes were transferred to a nitrocellulose membrane. Lyse the colonies on the filter, Then, those DNAs were immobilized on the membrane. Which colonies hive to Ds2 factor Filter the filters to determine if you have a recombinant plasmid to rediscover. (Hake et al. (1989) EMBO J., 8: 15-22). ). One out of 60,000 colonies screened has Ds2 factor Was found. By restriction analysis of this recombinant plasmid, About 2.9 kb genomic DNA on one side of the 1.3 kb Ds2 element and 165 bp on the other side. Genomic DNA revealed. Sequence analysis of adjacent DNA segments is performed using plasmid vectors. Use primers that hybridize to sequences within the promoter and within the Ds2 factor itself It was carried out by doing. Double-stranded plasmid using dideoxy chain termination sequencing DNA was sequenced.                                 Example 3 RNA analysis:   Poly-A RNA Northern blot analysis from various corn tissues was analyzed using the Ds2 factor Was performed using the 165 bp genomic DNA region on the right side as a probe. RNA, top Extracted from the meristems (young and old leaves, and root tips), and 1 μg of each poly A + mRNA from the sample was converted to 1.1% agarose containing formaldehyde. Electrophoresed on gel, then transferred to Genescreen nylon membrane. 16 Label the 5 bp fragment as above and probe the blot used.                                 Example 4 Determination of the Id gene sequence from the isolated genomic clone:   Genomic clones were distributed by the dideoxy method as described in Sambrook et al. (Supra). Column decided. The strategy used was called "primer walking". Step Use oligonucleotide primers that hybridize to the To obtain the first sequence data for the end of this fragment. Then, New primers in the sequenced region were synthesized using the sequence data of This Primers are used in an iterative process until the entire fragment is sequenced. For further sequencing of genomic clones. Approximately 200 to 350 bp Read from primer.                                 Example 5 Identification and isolation of regulatory genes from other plant species:   Identify and isolate regulatory genes (homologous to Id genes) in plants of other species In order to obtain a DNA sequence encoding the Id ORF or another fragment of the Id gene, Used as a probe to generate from mRNA from tissues that express the regulatory gene Screened plant cDNA libraries (Sambrook et al. (1989) supra; Freelin g and Walbot (1993) supra). Constructed from mRNA from seed and immature inflorescence tissues The constructed cDNA library is particularly likely to contain these genes. Corn A similar library from sorghum has been identified by Applicants as having Division cycle genes (for example, cdc2 (Colasanti et al. (1991) PNAS, 88: 3377-3381) and And cyclin (Renaudin et al. (1994) PNAS, 91: 7375-7379). Used to obtain by using short DNA probes for these genes . Identifying and isolating clones that hybridize with the radioactive probe; and Sequence analysis is performed by standard methods as described in Sambrook et al. (Supra).Equivalent   Those skilled in the art will recognize many equivalents to the specific embodiments of the invention specifically described herein. Or can be ascertained using only routine experimentation. Such an even Objects are intended to be encompassed by the following claims.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12Q 1/68 C12Q 1/68 A G01N 33/566 G01N 33/566 // C12P 21/00 C12P 21/00 C

Claims (1)

[Claims] 1. An isolated DNA that has the function of regulating flowering induction and contains the Id gene or a part thereof The Id gene contains all or part of SEQ ID NO: 1 or homologous DNA , Isolated DNA. 2. A transposon; while maintaining the flowering induction regulating function; site-directed mutagenesis Onset and random mutagenesis; and nucleotide substitutions, deletions, or additions; The modified DNA related to the DNA according to claim 1, wherein the modified DNA has been modified by modification using any of them. Or part of it. 3. Ds2 translocation factor derived from the adjacent Bz2 gene while retaining the flowering induction regulatory function 2. The method according to claim 1, wherein the modified gene is modified by excision of the offspring and insertion of the transposable element into the Id gene. Modified DNA or a part thereof that is related to the listed DNA. 4. A single gene encoded by the DNA according to any one of claims 1, 2, and 3. Isolated RNA or a part thereof. 5. A polypeptide encoded by the DNA according to any one of claims 1, 2, and 3. Repeptide or a part thereof. 6. An isolated cDNA or a DNA complementary to the DNA according to any one of claims 1, 2, and 3. Is a part of it. 7. 4. A method according to claim 1, 2 or 3 under conditions of moderate stringency. 4. Hybridizing to the described DNA or any of claims 1, 2, and 3 A plant exhibiting at least 50% homology to the zinc finger region of the DNA described in Isolated DNA. 8. An Id gene encoding a polypeptide comprising SEQ ID NO: 2. 9. A plant or part of a plant: (A) the DNA defined in claim 2 or claim 3; (B) an exogenous DNA as defined in any of claims 1, 2 and 3; (C) a DNA comprising an antisense construct of the cDNA defined in claim 6; (D) encoding a dominant negative mutant Id protein; DNA according to, Or a plant or part of a plant. 10. A seed of the plant according to claim 9. 11. A tissue culture of the plant or plant part according to claim 9. 12. The plant is corn or sorghum, or one of the plants The plant or plant according to claim 9, wherein the part is derived from corn or sorghum. Part of. 13. The seed is a corn seed or a sorghum seed. A seed according to claim 1. 14． 12. The tissue of claim 11, wherein the tissue is a corn tissue or a sorghum tissue. 2. The tissue culture according to item 1. 15. 4. The method according to claim 1, wherein the timing of flowering induction is modified. Transgenic plants containing exogenous DNA, parts of transgenic plants , Or transgenic plant cells. 16. The plant is corn or sorghum, or one of the plants 16. The part or plant cell of claim 15, wherein the part or plant cell is derived from corn or sorghum. Plant or plant part. 17． When grown under the same conditions, flower induction is the same without the exogenous DNA The timing of flower induction is modified so as to be earlier than the flower induction of the plant of claim 1, 2, 3. 4. A transgenic plant comprising the exogenous DNA according to any one of 3. and 3. 18. When grown under the same conditions, flower induction is the same without the exogenous DNA Of flower induction so that it is delayed or inhibited compared to flower induction in different plants Comprising the exogenous DNA according to any one of claims 1, 2, and 3, Lancegenic plants. 19. Those producing transgenic plants with altered times of flower induction A method for producing an exogenous DNA according to any one of claims 1, 2, and 3 using a plant cell. Containing the exogenous DNA under conditions suitable for the growth of the plant cells A method comprising maintaining the plant cells. 20. The transgenic plants are in the following groups: angiosperms, gymnosperms, monocotyledons 20. The method of claim 19, wherein the method is selected from the group consisting of: 21. A method for identifying an Id gene in a plant, comprising the following steps: (A) preparing a genomic or cDNA library of a plant; (B) All, part, or phase of the Id gene partially described by SEQ ID NO: 1 Use the same to probe the genomic or cDNA library. Step; (C) identifying the hybridized DNA; and (D) cloning the hybridized DNA to obtain the Id gene; A method comprising: 22. Method for identifying a gene encoding a zinc finger protein in a plant And the following steps: (A) preparing a genomic or cDNA library of a plant; (B) using all or a part or a homolog of the Id gene defined in claim 1; Probing the genomic DNA library or cDNA library; (C) identifying the hybridized DNA; and (D) sequencing the hybridized DNA and determining the zinc finger protein Obtaining an encoding gene; A method comprising: 23. A part or homolog of the Id gene is a zinc finger of the Id protein. 23. The method according to claim 22, wherein the part is encoded. 24. By producing alleles of Id genes with altered functions in plants There are the following steps: (A) inserting a non-autonomous transposable element into the Id gene defined in claim 1 ;and (B) excision of the non-autonomous transposable element by use of an autonomous transposable element, Producing a modified Id allele, A method comprising: 25. By producing alleles of Id genes with altered functions in plants There are the following steps: (A) The molecule of the Id gene defined in claim 1 by using molecular genetic technology. Modifying the structure in vitro; and (B) inserting the modified Id gene into a plant to produce a modified Id allele in the plant Process, A method comprising: 26. An antibody or an antibody that binds to a polypeptide comprising SEQ ID NO: 2 or a portion thereof; Body fragment. 27. All or part or homologs of SEQ ID NO: 2, and not related to SEQ ID NO: 2 An Id fusion protein comprising a polypeptide that is not or homologous. 28. Cleaving the RNA transcript of the DNA according to any one of claims 1, 2, and 3; A ribozyme that inactivates. 29. 2. The method according to claim 1, for the development of a technique for regulating flowering induction in plants. Use of DNA.