WO2009021448A1 - A plant height regulatory gene and uses thereof - Google Patents

A plant height regulatory gene and uses thereof Download PDF

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Publication number
WO2009021448A1
WO2009021448A1 PCT/CN2008/071939 CN2008071939W WO2009021448A1 WO 2009021448 A1 WO2009021448 A1 WO 2009021448A1 CN 2008071939 W CN2008071939 W CN 2008071939W WO 2009021448 A1 WO2009021448 A1 WO 2009021448A1
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Prior art keywords
polynucleotide
plant
polypeptide
elb
gene
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PCT/CN2008/071939
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English (en)
French (fr)
Inventor
Zuhua He
Yingying Zhang
Qun Li
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Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
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Application filed by Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences filed Critical Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
Priority to US12/672,803 priority Critical patent/US8461419B2/en
Priority to AU2008286583A priority patent/AU2008286583B2/en
Priority to JP2010519332A priority patent/JP5323831B2/ja
Priority to EP08783932A priority patent/EP2189474A4/en
Priority to CA2695929A priority patent/CA2695929C/en
Priority to CN200880103067.0A priority patent/CN101932596B/zh
Priority to KR1020107005199A priority patent/KR101246085B1/ko
Priority to UAA201002637A priority patent/UA109249C2/uk
Priority to BRPI0815352-3A2A priority patent/BRPI0815352A2/pt
Publication of WO2009021448A1 publication Critical patent/WO2009021448A1/zh
Priority to US13/893,217 priority patent/US20130247242A1/en

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    • 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
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8225—Leaf-specific, e.g. including petioles, stomata
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8226—Stem-specific, e.g. including tubers, beets
    • 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]
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0077—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with a reduced iron-sulfur protein as one donor (1.14.15)
    • 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

  • the present invention is in the field of genetic technology and botany; more particularly, the present invention relates to genes that regulate plant plant height and their use. Background technique
  • an isolated crop strain regulating polypeptide is provided, the polypeptide being selected from the group consisting of:
  • polypeptide derived from (a) which is formed by substitution, deletion or addition of the amino acid sequence of SEQ ID NO: 3 by one or more amino acid residues, and which has a function of regulating plant strain.
  • polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO: 3.
  • an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of:
  • polynucleotide encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO: 3.
  • sequence of the polynucleotide has the nucleotide sequence shown in SEQ ID NO: 2; or has SEQ ID NO:
  • a vector comprising the polynucleotide is provided.
  • a genetically engineered host cell comprising the vector is provided.
  • a plant comprising the polynucleotide is provided.
  • a method of making the plant comprising transferring the polynucleotide into a plant is provided.
  • the method includes:
  • step (1) (2) contacting a plant cell or tissue or organ with Agrobacterium in step (1) to transfer the polynucleotide into a plant cell and integrate it into the chromosome of the plant cell;
  • step (3) Regenerating the plant cell or tissue or organ in step (3).
  • a seventh aspect of the invention there is provided a method of preparing a plant, the plant into which the polynucleotide is transferred and the non-transformation The gene plants are crossed to obtain hybrid progeny comprising the polynucleotide.
  • a method for preparing the polypeptide comprising:
  • polypeptide or polynucleotide encoding the polypeptide is provided for
  • a substance is prepared to adjust the plant height, volume, tiller, yield, flower size or seed size of the crop.
  • a method of regulating crop plant height, volume, tillering, yield, flower size or seed size comprising: regulating the expression or activity of a crop plant height regulating gene in a crop.
  • an agonist or antagonist of the crop strain regulating polypeptide or a gene encoding the same is provided.
  • a plant stem-leaf-specific expression promoter is provided, wherein the promoter is selected from the group consisting of: (1) a polynucleoside having the nucleotide sequence of SEQ ID NO: Acid
  • a polynucleotide which is capable of hybridizing to a (1) defined polynucleotide sequence under stringent conditions and which has a function of directing expression of a gene of interest in a plant stem or leaf;
  • the promoter is provided for directing expression of a gene of interest in a plant stem or leaf.
  • a construct is provided, the construct comprising the plant stem-leaf-specific expression promoter.
  • the downstream of the plant stem-leaf-specific expression promoter contains at least one multiple cloning site (eg, a cleavage site) that initiates specific expression of the stem and leaf of the plant Sub-operable linkage for insertion of the gene of interest.
  • at least one multiple cloning site eg, a cleavage site
  • the construct is an expression vector.
  • the construct contains the following operably linked elements: the promoter and the gene of interest.
  • the gene of interest is a foreign gene.
  • the gene of interest is a structural gene.
  • the gene of interest encodes a protein having a specific function.
  • the gene of interest is located downstream of the promoter, and the interval from the promoter is less than
  • 2000 bp (preferably, less than l OOObp; more preferably, less than 500 bp; most preferably, less than 300 bp).
  • Figure 1 shows a top view of wild-type Arabidopsis thaliana (WT) and ELb (Em-like b) overexpressing transgenic Arabidopsis plants (ELb-OE).
  • WT wild-type Arabidopsis thaliana
  • ELb Em-like b
  • ELa overexpresses transgenic Arabidopsis plants
  • OsEm overexpressing transgenic Arabidopsis plants
  • Figure 2 shows wild-type Arabidopsis ELb
  • FIG. 1 Flower organs and seed growth of mustard plants and wild-type Arabidopsis plants (WT) Comparison chart.
  • Figure 4 shows the tissue-specific expression of the Gb reporter gene initiated by the ELb promoter. Among them, the area pointed by the arrow is the area showing blue.
  • Figure 5 shows a comparison of the growth status of wild type rice plants (TP309) and ELb overexpressing rice plants (ELb-OE).
  • Figure 6 shows the statistical values of the plant height of wild type rice plants (TP309) and ELb overexpressing rice plants (ELb-OE).
  • Figure 7 shows the statistics of the effective number of tillers of wild type rice plants (TP309) and ELb overexpressing rice plants (ELb-OE).
  • Figure 8 shows a comparison of the individual plant weights of wild type rice plants (TP309) and ELb overexpressing rice plants (ELb-OE). detailed description
  • crop includes, but is not limited to, gramineous plants, cruciferous plants, woody plants, and the like. More preferably, the gramineous plants include, but are not limited to: rice, wheat, barley, corn, sorghum, etc.; or the genus of the genus, including but not limited to: Arabidopsis thaliana.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment;).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • isolated crop strain regulating polypeptide means that the ELb protein is substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. . Those skilled in the art will be able to purify the ELb protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product, either a chemically synthesized product, or produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells;).
  • the polypeptide of the invention may be glycosylated according to the host used in the recombinant production protocol, or may be non-glycosylated. Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the ELb protein.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the ELb protein of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be (1) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (11) a polypeptide having a substituent group in one or more amino acid residues, or (111) a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, for example Polyethylene glycol) fusion of the formed polypeptide, or (IV) additional amino acid sequence fused to the polypeptide A polypeptide formed by a sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein;).
  • a sequence such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein;
  • the term "ELb protein” refers to a polypeptide having the sequence of SEQ ID NO: 3 having ELb protein activity.
  • the term also encompasses variant forms of the sequence of SEQ ID NO: 3 having the same function as the ELb protein. These variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, still more preferably 1 -8 or 1-5) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually within 20, preferably within 10, more preferably The ground is less than 5 amino acids.
  • the function of the protein is usually not altered.
  • the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of ELb proteins.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing to ELb protein DNA under high or low stringency conditions, And a polypeptide or protein obtained using an antiserum against an ELb protein.
  • the invention also provides other polypeptides, such as fusion proteins comprising an ELb protein or a fragment thereof.
  • the present invention also encompasses soluble fragments of the ELb protein.
  • the fragment has at least about 20 contiguous amino acids of the ELb protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
  • a contiguous amino acid typically at least about 20 contiguous amino acids of the ELb protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
  • the invention also provides analogs of ELb proteins or polypeptides.
  • the difference between these analogs and the natural ELb protein may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, y-amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derived forms of the polypeptide, such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • ELb protein conservative variant polypeptide means up to 20, preferably up to 10, more preferably up to 5, optimally up to 3 compared to the amino acid sequence of SEQ ID NO: 3. Amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the present invention also provides a polynucleotide sequence encoding the ELb protein of the present invention or a conservative variant polypeptide thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: 2 or may be a degenerate variant.
  • degenerate variant in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 3 but differs from the coding region sequence set forth in SEQ ID NO: 2.
  • Polynucleotides encoding the mature polypeptide of SEQ ID NO: 3 include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optional additional coding sequences; And non-coding sequences.
  • polynucleotide encoding a polypeptide may be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising an additional coding and/or non-coding sequence.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization. Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum
  • Hybridization occurs only when the identity between the two sequences is at least 80% or more, preferably at least 90% or more, more preferably 95% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 3.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding ELb proteins.
  • the ELb gene of the present invention is preferably obtained from Arabidopsis thaliana, it is highly homologous to other Arabidopsis thaliana ELb genes (e.g., having more than 60%, such as 70%, 80%, 85%, 90%). Other genes, 95%, or even 98% sequence identity) are also contemplated by the present invention. Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
  • the full-length nucleotide sequence of the ELb protein of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first combining a plurality of small segments and then connecting them.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or ELb protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant ELb protein by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally there are the following steps:
  • the ELb protein polynucleotide sequence can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors well known in the art. In general, any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Expression vectors containing the ELb protein encoding DNA sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • Expression vectors also include ribosome binding sites and transcription terminators for translation initiation.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or kanamycin or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or kanamycin or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, Electroporation, liposome packaging, etc.
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, rice immature embryo transformation method and the like.
  • Agrobacterium transformation or gene gun transformation such as leaf disc method, rice immature embryo transformation method and the like.
  • transformed plant cells, tissues or organs plants can be regenerated by conventional methods to obtain plants having altered traits.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cells used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method;), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption Chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Recombinant ELb proteins have many uses. For example, for screening antibodies, polypeptides or other ligands that promote or counteract the function of ELb proteins. Screening a polypeptide library with the expressed recombinant ELb protein can be used to find valuable polypeptide molecules that inhibit or stimulate the function of the ELb protein.
  • a part or all of the polynucleotide of the present invention can be immobilized as a probe on a nucroarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis of genes in tissues.
  • the transcription product of ELb protein can also be detected by RNA-polymerase chain reaction (RT-PCR) in vitro amplification using ELb protein-specific primers.
  • the invention also relates to a method of improving a crop comprising modulating the expression or activity of an ELb gene or a homolog thereof in said plant. Promoting or inhibiting the expression or activity of ELb depends primarily on the improved traits desired for the crop. When it is necessary to reduce crop height and volume, increase crop tillering and yield, it can be achieved by increasing the expression or activity of ELb in crops; when it is necessary to increase the size of crops, increase seed size, or increase crop height or volume, This is achieved by inhibiting the expression or activity of the plant height regulating gene in the crop.
  • the plant can be overexpressed by transferring an expression construct carrying the ELb encoding gene; or the expression of the ELb gene or its homologous gene can be enhanced by driving with a strong promoter; or by an enhancer (such as rice wax gene) An intron, the first intron of the Actm gene, etc.) enhances the expression of the ELb gene.
  • Strong promoters suitable for use in the methods of the invention include, but are not limited to, the 35s promoter, the Ubi promoter of rice, maize, and the like.
  • RNAi RNA interference
  • knockout knockout
  • a method for obtaining a plant having high ELb expression is as follows:
  • step (1) (2) contacting the plant cell or tissue or organ with the Agrobacterium in step (1), thereby transferring the ELb protein DNA coding sequence into the plant cell and integrating into the chromosome of the plant cell;
  • the method can be carried out by any suitable conventional means including reagents, temperature, pressure conditions and the like.
  • the invention also encompasses antagonists or agonists of the ELb protein or its encoding gene. Since the antagonist or agonist of ELb can modulate the activity or expression of ELb, the antagonist or agonist of ELb can also modulate the crop by affecting ELb. Plant height, volume, tillering, yield, flower size or seed size, etc., to achieve the purpose of trait improvement.
  • the antagonist of ELb refers to any substance which can reduce the activity of ELb, lower the stability of ELb, down-regulate the expression of ELb, reduce the effective action time of ELb, or inhibit the transcription and translation of ELb, and these substances can be used in the present invention.
  • a substance that increases the size of the flower increases the size of the seed, or increases the plant height or volume of the crop.
  • the agonist of ELb refers to any substance which can increase the activity of ELb, maintain the stability of ELb, promote ELb expression, prolong the effective action time of ELb, or promote transcription and translation of ELb, and these substances can be used in the present invention. It is useful as a material for reducing crop plant height and volume and increasing crop tillering and yield.
  • an ELb gene having a genomic sequence as set forth in SEQ ID NO: 1, wherein the open reading frame (ORF) is located at 61-353, 1363-1585, 1690-1943, 2028- At 2414, 2559-2979, the full-length cDNA (SEQ ID NO: 1) is 1578 bp encoding a protein of 525 amino acids (SEQ ID NO: 3).
  • the ELb gene can provide a new way for the improvement of plant height, volume, yield, tillering, etc., and thus has great application prospects. Plant stem and leaf specific expression promoter and its directed gene expression
  • a “promoter” or “promoter region (domain;)” refers to a nucleic acid sequence that is normally present upstream (5') of a coding sequence of a gene of interest, capable of directing transcription of the nucleic acid sequence into mRNA. .
  • the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the promoter or promoter region includes a variant of a promoter which may be a naturally occurring allelic variant or a non-naturally occurring variant. The variants include substitution variants, deletion variants, and insertion variants.
  • tissue-specific promoter is also referred to as an "organ-specific promoter", and under the control of such promoters, genes are often expressed only in certain specific organs or tissues.
  • operably linked refers to the spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is "operably linked" to the nucleic acid sequence.
  • the mRNA is at least 2 times higher in a certain tissue or organ than preferably in other tissues or organs, preferably at least 5 times higher, more preferably at least 10 times higher, more preferably at least 25 times higher, more preferably at least 50 times, more preferably At least 100-fold higher levels are expressed, more preferably at least 1000-fold higher, and the promoter is considered tissue or organ specific.
  • the invention provides a promoter, wherein the promoter is selected from the group consisting of:
  • a polynucleotide which is capable of hybridizing to a (1) defined polynucleotide sequence under stringent conditions and which has a function of directing expression of a gene of interest in a plant stem or leaf.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) only at least 80% identity between the two sequences Preferably, the hybridization occurs at least 90% or more, more preferably 95% or more.
  • the hybridizable polynucleotide also has a function of directing the specific expression of the gene of interest in the stems and leaves of the plant.
  • Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. Accordingly, the present invention also relates to hybridization to the nucleotide sequence set forth in SEQ ID NO: 13 and having at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 80 between the two sequences. %, preferably at least 85%, better A polynucleotide that is at least 90% (eg, 95%, 96%, 97%, 98%, or 99%) identical.
  • the promoter of the present invention is plant tissue or organ specific, and more particularly, it is plant stem and leaf specific.
  • the inventors have found that under the guidance of the promoter of the present invention, the ELb gene or the GUS gene can be specifically expressed in the stems and leaves of plants, but not in other tissues and organs.
  • the promoter of the present invention may be operably linked to a gene of interest which may be foreign to the promoter
  • the gene of interest may generally be any nucleic acid sequence (preferably a structural nucleic acid sequence;), preferably encoding a protein having a specific function, such as certain proteins for controlling the growth of stems and leaves of plants, more specifically
  • the plant strain of the invention is a high-associated protein.
  • the promoter of the present invention may also be operably linked to a modified gene sequence of interest which is exogenous (heterologous) relative to the promoter.
  • the gene of interest can be modified to produce a variety of desirable properties.
  • the gene of interest can be modified to increase expression, to alter post-translational modifications (such as phosphorylation sites), to transport translation products out of the cell, to improve protein stability, to insert or delete cellular signals, and the like.
  • promoters and genes of interest can be designed to downregulate specific genes. This is typically accomplished by ligating the promoter to the sequence of genes of interest, which is directed in antisense orientation. Those of ordinary skill in the art are familiar with such antisense techniques. Any nucleic acid sequence can be modulated in this manner.
  • the recombinant vector generally comprises an operably linked (usually from 5' to 3' direction): a promoter that directs transcription of the gene of interest, and a gene of interest.
  • the recombinant vector may also include a 3 'transcription terminator, a 3 'polynucleotide signal, other non-translated nucleic acid sequences, a transport and targeting nucleic acid sequence, a resistance selection marker, an enhancer or an operator.
  • the gene of interest is located downstream of the plant stem-leaf-specific expression promoter and is less than 2000 bp from the promoter (preferably, less than 1000 bp; more preferably, less than 500 bp; most preferably, less than 300bp).
  • the recombinant vector may contain one or more other promoters in addition to the promoter of the present invention.
  • Such other promoters are, for example, tissue-specific, constitutive or inducible.
  • a vector comprising the appropriate promoter and gene of interest described above can be used to transform a suitable host cell to enable expression of the protein.
  • the main advantages of the invention are:
  • the genes of the present invention can be used to reduce plant height after being transferred to crops for dwarf breeding; for example, for different crops on grass crops.
  • the expression level the plant height is reduced to different degrees, the effective tillering is increased, and the yield is increased.
  • the promoter of the plant plant height regulating gene is isolated for the first time, and the promoter is specifically expressed by the stems and leaves of the plant, so that it can be suitably used for regulating the specific expression of the target protein in the stems and leaves of the plant.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention. The experimental methods in the following examples that do not specify the specific conditions are usually in accordance with conventional conditions.
  • Arabidopsis thaliana the ecological type is Columbia (Col-0).
  • T-DNA insertion mutant ELa (SALK-016089).
  • Taipei 309 (Oryza sativa L.ssp Japonica.cvTaipei309, TP309).
  • Agrobacterium tumefaciens GV3101 (see Narasimhulu, SB et al, Gelvin. 1996. Early transcription of Agrobacterium t-DNA genes in tobacco and maize. Plant Cell 8: 873-886), EHA105 (see Hood, EE et al, 1993) , New Agrobacterium helper plasmids for gene transfer to plants. Transgen. Res. 2:208-218).
  • Plasmid vector
  • pBluescript SK (pSK) : Purchased from Invitrogen.
  • pCambial300S was purchased from CAMBIA.
  • pBHOl.l purchased from Invitrogen.
  • pGEM-T Easy carrier purchased from Promega.
  • RNAi Vector 1300RS purchased from Arkansas State University, USA.
  • T 4 DNA ligase various nucleic acid restriction enzymes and: ⁇ DNA polymerase was purchased from MBI Ferment, TaKaRa,
  • a tapping recovery kit for DNA fragments was purchased from Omega. Reverse Transcription System was purchased from GIBCOBRL. The nucleic acid molecular weight standard is MBI product. pGEM-Teasy carrier was purchased from
  • the seeds are surface-treated (70% ethanol, 30 seconds; washed 4 times with sterile water) and deep (7% sodium hypochlorite,
  • the Arabidopsis thaliana transformation was carried out by spraying method, and the 4-5 week old plants with good growth condition were taken (the main moss was cut one week before the transformation, which was favorable for the lateral moss extraction to produce more flower buds and improve the transformation efficiency).
  • Agrobacterium containing the transgenic vector was grown to OD 6 at 28 ⁇ . . [2.0, 4,000 rpm centrifuged 10 mm, the cell pellet was suspended in freshly prepared transformant (1/2 MS liquid medium containing 5% sucrose, 0.02% Silwet L-77) to a final concentration of OD 6 . . "0.6-0.8. Prior to conversion, the pollinated flowers and pods are removed and the soil is allowed to absorb enough water.
  • the transformation When the transformation, the bacterial liquid was evenly sprayed on Arabidopsis, and the leaves were dropped on the leaves, and the plants were covered with black plastic bags. Keep humidity and stay away from light overnight. After 24 hours, the plants were transferred to normal conditions for growth. After 7 days (d), the transformation was repeated once as described above. After the seeds were mature, the plants were mixed and collected in a paper bag, and placed in a desiccator for 7 days and then threshed. The T1 seed was sterilized and seeded on l/2x MS medium containing 50 g/ml Kan or hygromycin, placed at 4 ° C for 72 h, and then cultured under normal light.
  • Rice 307T seeds are dehulled, soaked in 70% ethanol for 1 minute, 20% (v/v), soaked with sodium hypochlorite for 20-30 minutes, shaken continuously, rinsed 5-6 times with sterile water.
  • the sterile filter paper was blotted, sowed in MSD medium to induce callus, and 26 ⁇ dark culture.
  • the endosperm, germ, radicle, etc. were removed, the callus was removed, and subcultured on MSD. Once every 2-3 weeks. If TP309 seeds are used for stripping callus, the medium is NBD.
  • AAM including AS 100
  • the callus was blotted with sterile filter paper (change filter paper;), and transferred to a screening medium containing hygromycin (hygr 0 my Cin , Hyg) to screen for resistant callus (dark culture for about 30 days, intermediate change) Screening medium once).
  • sterile filter paper change filter paper;
  • hygromycin hygr 0 my Cin , Hyg
  • the screened rice callus was transferred to pre-differentiation medium and cultured for 10 days.
  • Pre-differentiation medium MS+ BAP with 0.45% Phytagel 2 mg/L + NAA 1 mg/L + ABA 5 mg/L + Hyg
  • the pre-differentiated rice callus was transferred to a differentiation medium to differentiate into seedlings (light, 15-30 days).
  • Differentiation medium NB+BAP with 0.45% Phytagel 3 mg/L+NAA 0.5 mg/L, pH 5.7, no 2, 4-D.
  • Rooting medium 1/2 MS+Hyg 50 mg/L with 0.45% Phytagel, pH 5.7, no 2, 4-D.
  • p35S ELb vector construction
  • the cDNA coding region sequence of ELb was amplified by RT-PCR. The specific procedure was as follows: Total RNA of Arabidopsis thaliana was isolated using the RNeasy Plant Mini kit (Qiagen) according to the method provided in the instruction, and the isolated total RNA was reverse transcribed to produce ELb cDNA using M-MLV reverse transcriptase (Promega). The cDNA coding region sequence of ELb was amplified by PCR reaction using Takara perobest DNA polymerase using cDNA as a template. Primers used:
  • Upstream (SEQ ID NO: 7): 5 ' -TGAGGATCCAAATAAAATAAAAAG-3 ' (M line part is divided into another ij is Bam l site;);
  • Downstream (SEQ ID NO: 8): 5 '-AAAGTCGACCACACACAAAGCAAA-3 ' (the underlined parts are Ctol sites, respectively).
  • the PCR product was recovered, then digested with ⁇ TM ⁇ and Ctol, recovered and ligated with pSK vector carrying out the same double digestion and recovery process, transformed into E. coli DH5 a, and the correct clone was selected for sequencing.
  • the cDNA coding region of ELb was cut with ⁇ « ⁇ and CM, and the transgenic clone was constructed by transfecting the binary expression vector pCambial300S, which was also digested with BamHl and CM, and transformed into E. coli DH5 ⁇ .
  • the plasmid was extracted and the correct plasmid was identified by restriction enzyme digestion into Agrobacterium GV3101 and ⁇ 105.
  • the plasmid was extracted from Agrobacterium and transferred into DH5 ⁇ , and the plasmid was extracted and verified by enzyme digestion to confirm that the correct plasmid had been transferred into Agrobacterium.
  • Rice transgenic plants were obtained by spray-transformation of Arabidopsis thaliana to produce transgenic plants and Agrobacterium-mediated transformation of rice mature embryo callus.
  • the upstream and downstream primers were designed within 1.4 kb upstream of the translation initiation site of the £Lb gene, and the genomic DNA extracted from the Arabidopsis Columbia wild-type 7-day seedling was used as a template for PCR.
  • the primers are as follows:
  • Upstream (SEQ ID NO: 9): 5 '-CTGCTGCAGACTCTATTTCCA-3 ' (lower ij line is the restriction site Pstl); downstream (SEQ ID NO: 10): 5 '-TTCAGGATCCTTTACTTTTTATTTT-3 Restriction sites
  • the amplification conditions were denaturation at 94 °C for 3 min; then denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min for 35 cycles, and incubation at 72 °C for 10 min. Keep it at 16 °C.
  • the PCR product Pstl/BamHI was digested and ligated into the corresponding site of Pstl/BamHI of pSK vector.
  • the correct clones were sequenced, the correct clones were sequenced, and the £6 promoter region (sequence shown in SEQ ID NO: 13) was cut with Pstl/BamHI, and ligated into the same binary expression by Pstl/BamHI.
  • the vector ⁇ ⁇ ⁇ . ⁇ transferred to E. coli DH5 ⁇ .
  • the plasmid was extracted and the correct plasmid was identified and transferred into Agrobacterium GV3101.
  • the plasmid was extracted from Agrobacterium and transferred into DH5 a, and the plasmid was extracted and verified by enzyme digestion to confirm that the correct plasmid had been transferred into Agrobacterium.
  • the ⁇ ⁇ ⁇ .1 vector was transferred to GV3101 as a negative and positive pair, respectively.
  • a transgenic clone was constructed using the RNAi vector 1300RS, and a sequence of about 500 bp was amplified on the fourth exon of the ELb gene, and the genomic DNA extracted from the Arabidopsis Columbia wild type 7-day seedling was used as a template for PCR.
  • the primers were as follows: Upstream (SEQ ID NO: 1 1): 5 '-AATGGTACCACAAGAAACAA-3 ' (The lower cleavage site is the cleavage site KpnD: downstream (SEQ ID NO: 12): 5 '-TCTAGATTTGAGCTGAAAAAA-3 '( The underline is the enzyme cleavage site Xbal.
  • the amplification conditions are denaturation at 94 °C for 3 min; then denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s for 35 cycles, and incubation at 72 °C for 10 min. Keep it at 16 °C.
  • the PCR product Kpnl/Xbal was digested and ligated into the corresponding site of the pSK vector Kpnl/Xbal. Select the correct clone and sequence, The correct clone was sequenced and the £6 fragment was cut with Kpnl/Xbal, and the binary expression vector 1300RS, which was also digested with Kpnl/Xbal, was transfected into E. coli DH5a.
  • the plasmid was extracted and the correct plasmid was identified by restriction enzyme digestion into Agrobacterium GV3101.
  • the plasmid was extracted from Agrobacterium and transferred into DH5 a, and the plasmid was extracted and verified by enzyme digestion to confirm that the correct plasmid had been transferred into Agrobacterium.
  • the T-DNA insertion mutant of ELa was transformed by spraying to obtain an ELb RNAi transgenic Arabidopsis plant (£ 6 RNAi /eLa) in the context of the T-DNA insertion mutant of ELa.
  • ELa also known as AtEuila
  • ELb also known as AtEuilb
  • the ELb gene coding region genomic DNA is represented by SEQ ID NO: 1; the ELb gene coding region cDNA sequence is represented by SEQ ID NO: 2; and the ELb protein sequence is represented by SEQ ID NO: 3.
  • the ELa gene coding region genome sequence is shown in SEQ ID NO: 4; the ELa gene coding region cDNA sequence is shown in SEQ ID NO: 5; and the ELa protein sequence ⁇ U is shown in SEQ ID NO: 6.
  • Example 2 ELb overexpression transgenic Arabidopsis reduced plant height and volume
  • the present inventors separately prepared transgenic Arabidopsis plants which overexpress Arabidopsis thaliana ELb, ELa and rice OsEm.
  • Transgenic plants with wild-type (wt), overexpressing Arabidopsis ELa (ELa-OE) and rice OsEm were used as controls.
  • Fig. 1 After about 4 weeks of culturing each plant, the growth of the plants was examined, and the results are shown in Fig. 1. As can be seen from the figure, the wild-type Arabidopsis thaliana grows the most; the plants overexpressing Arabidopsis thaliana are significantly smaller than the wild type; while the plants overexpressing Arabidopsis ELa and rice OsEm grow minimally.
  • the inventors measured the plant heights of wild-type, transgenic Arabidopsis thaliana plants overexpressing Arabidopsis thaliana ELb, ELa and rice OsEm, respectively, after about 7 weeks of culture, and their plant height averages were 26.2 cm, 14.2, respectively. Cm, 9.63cm, 3.7cm, therefore,
  • Example 3 ELb RNAi or knockout mutant increases plant height or volume
  • the present inventors prepared Arabidopsis plants in which ELb RNAi transgenic Arabidopsis plants 0 ⁇ 6 ⁇ 4 ⁇ ) and ELb were knocked out (konckout) in the context of T-DNA insertion mutants. Variant. After culturing the two plants for about 10 days, the effects of ELb RNAi or knockout mutants on plant height or volume were observed, and wild type plants of the same culture conditions were used as controls.
  • ELb RNAi plants and wild-type plants The growth status of ELb RNAi plants and wild-type plants is shown in Fig. 2. Compared with wild-type plants, the leaf area of ELb RNAi plants increased significantly, the plant height increased significantly, and the volume increased significantly.
  • ELb knockout mutants compared with wild-type plants, ELb knockout mutants also showed significant increase in leaf area, increase in plant height, and increase in volume.
  • ELb is a gene associated with reducing plant height and volume.
  • Example 4 ELb RNAi or Knockout Mutant Increases Flower and Seed Size
  • the present inventors observed the growth state of flowers and seeds of Arabidopsis plants of ELb RNAi or Arabidopsis plants in which ELb was knocked out, and the flowers and seeds of wild type Arabidopsis were used as controls.
  • the flowering and seed growth status of Arabidopsis plants of ELb RNAi and wild-type Arabidopsis plants are shown in Fig. 3.
  • the Arabidopsis plants of ELb RNAi have significantly increased floral organs and significantly increased seeds.
  • ELb knockout mutants also showed significant flower enlargement and seed enlargement.
  • ELb is a gene associated with reducing flower and seed size.
  • Example 5 ELb promoter initiation tissue-specific expression of the GUS reporter gene
  • the inventors constructed a vector of T p £ ⁇ pra «w r Gf « and transferred it into Agrobacterium to prepare an Arabidopsis gene plant.
  • the expression of the GUS reporter gene in the plants was observed using a conventional GUS reporter gene assay.
  • the GUS reporter gene can be expressed in the vigorous growth of the stem and leaves of the plant, and is not substantially expressed at the root of the plant. Therefore, the ELb gene is a tissue-specific gene.
  • the present inventors prepared an ELb-overexpressing transgenic rice plant (ELb-OE), and observed the effect of ELb overexpression on rice plants, and the wild type rice plant TP309 cultured under the same conditions was used as a control.
  • ELb-OE ELb-overexpressing transgenic rice plant
  • Fig. 5 After about 10 days of culturing the plants, the growth of the plants is shown in Fig. 5. It can be seen that the plant height of the transgenic rice plants overexpressing ELb is significantly lower than that of the wild type. A statistical plot of plant height for overexpressing ELb plants and wild type plants is shown in Figure 6.
  • Example 7 ELb overexpression of transgenic rice increased effective tillering and yield
  • the inventors examined the tiller number and yield of ELb overexpressed transgenic rice plants (ELb-OE) and wild type plants TP309 to demonstrate the effect of ELb overexpression on rice tillering and yield.
  • ELb-OE ELb overexpressed transgenic rice plants
  • TP309 wild type plants
  • the number of tillers of the effective tillers was counted after the heading of the cultivated plants, and the statistical results are shown in Fig. 7.
  • the wild type plants have about 10 tillers, while the ELb overexpressed plants have about 20-22 tillers. It can be seen that ELb overexpression can significantly increase the effective number of tillers of plants.

Description

调控植物株高的基因及其应用
技术领域
本发明属于基因技术和植物学领域； 更特别的， 本发明涉及调控植物株高的基因及其应用。 背景技术
当前， 对于农作物高产育种的选育主要集中在株型和穗部性状的改良。 由于品种的改良， 目前已经培育出多种高产农作物品种。
然而， 当前的许多高产作物如水稻高产栽培品种尤其超级杂交稻存在一定的株高过高问题， 导致容易倒伏、 产量潜力受到限制的问题。 这在很大程度上影响了作物产量的进一步提高和高 产品种的推广。
因此， 本领域有必要研究可调节农作物株高的方法， 从而进一步改良农作物的性状， 提高 农作物的产量。 发明内容
本发明的目的在于提供调控植物株高的基因及其应用。
在本发明的第一方面， 提供一种分离的作物株高调节多肽， 该多肽选自下组：
(a) 具有 SEQ ID NO: 3所示氨基酸序列的多肽； 或
(b) 将 SEQ ID NO: 3所示氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添加而形 成的， 且具有调节作物株高功能的由 (a)衍生的多肽。
在另一优选例中， 该多肽是具有 SEQ ID NO: 3所示氨基酸序列的多肽。
在本发明的第二方面， 提供一种分离的多核苷酸， 它包含一核苷酸序列， 该核苷酸序列选 自下组：
(a) 编码所述多肽的多核苷酸； 或
(b) 与多核苷酸 （a) 互补的多核苷酸。
在另一优选例中， 该多核苷酸编码具有 SEQ ID NO: 3所示氨基酸序列的多肽。
在另一优选例中，该多核苷酸的序列具有 SEQ ID NO: 2所示核苷酸序列；或具有 SEQ ID NO:
1所示的核苷酸序列。
在本发明的第三方面， 提供一种载体， 它含有所述的多核苷酸。
在本发明的第四方面， 提供一种遗传工程化的宿主细胞， 它含有所述的载体。
在本发明的第五方面， 提供一种植物， 其包含所述的多核苷酸。
在本发明的第六方面， 提供一种制备所述植物的方法， 其包括将所述的多核苷酸转入植物 中。
在另一优选例中， 所述方法包括：
(1) 提供携带表达载体的农杆菌， 所述的表达载体含有所述的多核苷酸；
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触， 从而使所述的多核苷酸转入植物 细胞， 并且整合到植物细胞的染色体上；
(3) 选择出转入了所述的多核苷酸的植物细胞或组织或器官； 和
(4) 将步骤 (3)中的植物细胞或组织或器官再生成植物。
在本发明的第七方面， 提供一种制备植物的方法， 将转入了所述的多核苷酸的植物与非转 基因植物杂交， 获得包含所述的多核苷酸的杂交后代。
在本发明的第八方面， 提供一种所述多肽的制备方法， 该方法包含：
(a) 在适合表达的条件下， 培养所述的宿主细胞；
(b) 从培养物中分离出所述的多肽。
在本发明的第九方面， 提供所述的多肽或编码该多肽的多核苷酸的用途， 用于
调节作物的株高、 体积、 分蘖、 产量、 花器大小或种子大小； 或
制备调节作物的株高、 体积、 分蘖、 产量、 花器大小或种子大小的物质。
在本发明的第十方面， 提供一种调节作物株高、 体积、 分蘖、 产量、 花器大小或种子大小 的方法， 所述方法包括： 调节作物中作物株高调节基因的表达或活性。
在另一优选例中， 通过提高作物中作物株高调节基因的表达或活性， 降低作物株高、 体积， 增加作物分蘖、 产量； 通过抑制作物中作物株高调节基因的表达或活性， 增加作物花器大小， 增加种子大小， 提高作物株高或体积。
在本发明的第十一方面， 提供一种所述作物株高调节多肽或其编码基因的激动剂或拮抗剂。 在本发明的第十二方面， 提供一种植物茎叶特异性表达启动子， 所述的启动子选自下组： (1) 具有 SEQ ID NO: 13所示的核苷酸序列的多核苷酸；
(2) 在严格条件下能够与 (1)限定的多核苷酸序列杂交且具有指导目的基因在植物茎叶中特异 性表达功能的多核苷酸； 或
(3) 与 SEQ ID NO: 13所示的核苷酸序列具有 70% (优选 80%以上，更优选 90%以上，最优选 95% 以上， 如 98%， 99%)以上相同性且具有指导目的基因在在植物茎叶中特异性表达功能的多核苷酸。
在本发明的第十三方面， 提供所述的启动子用于指导目的基因在植物茎叶中特异性表达。 在本发明的第十四方面， 提供一种构建物， 所述的构建物含有所述的植物茎叶特异性表达 启动子。
在另一优选例中， 所述构建物中， 植物茎叶特异性表达启动子的下游含有至少一个多克隆 位点 (如酶切位点)， 其与所述的植物茎叶特异性表达启动子可操作性连接， 用于插入目的基因。
在另一优选例中， 所述的构建物是表达载体。
在另一优选例中， 所述的构建物含有以下可操作性连接的元件： 所述的启动子和目的基因。 在另一优选例中， 所述的目的基因是外源基因。
在另一优选例中， 所述的目的基因是结构基因。
在另一优选例中， 所述的目的基因可编码具有特定功能的蛋白。
在另一优选例中， 所述的目的基因位于所述启动子的下游， 且与所述启动子的间隔小于
2000bp (优选的， 小于 l OOObp ; 更优选的， 小于 500bp ; 最优选的， 小于 300bp)。
本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易见的。 附图说明
图 1显示了野生型拟南芥 （WT)和 ELb(Em-like b)过表达转基因拟南芥植株 (ELb-OE)的俯视 图。 其中， ELa(Em-like a)过表达转基因拟南芥植株 (ELa-OE)和水稻 OsEm过表达转基因拟南芥 植株 (OsEm-OE)植株作为对照。
图 2显示了野生型拟南芥 ELb
显示了 拟南， RNAi /da 拟南芥植株的生长状况比较图。
图 3 芥植株与野生型拟南芥植株 （WT) 的花器官和种子生长状 况比较图。
图 4显示了 ELb启动子启动 GUS报告基因的组织特异性表达。 其中， 箭头所指处区域为显 示蓝色的区域。
图 5显示了野生型水稻植株 (TP309)和 ELb过表达水稻植株 (ELb-OE) 的生长状况比较。 图 6显示了野生型水稻植株 (TP309)和 ELb过表达水稻植株 (ELb-OE) 的株高的统计值。 图 7显示了野生型水稻植株 (TP309)和 ELb过表达水稻植株 (ELb-OE)的有效分蘖数的统计 值。
图 8显示了野生型水稻植株 (TP309)和 ELb过表达水稻植株 (ELb-OE)的单株粒重的比较。 具体实施方式
本发明人经过广泛的研究， 揭示了一种对于调节作物株高、 体积、 分蘖、 产量、 花器官大 小或种子大小有用的基因 ELb。 提高该基因的表达可降低作物的株高、 体积， 增加作物的有效 分蘖和产量； 降低该基因的表达可增大作物的花器和种子。 在此基础上完成了本发明。 如本文所用， 所述的 "作物"包括但不限于： 禾本科植物、 十字花科植物、 木本科植物等。 更优选的， 所述的禾本科植物包括但不限于： 水稻、 小麦、 大麦、 玉米、 高粱等； 或所述的十 字花科植物包括但不限于： 拟南芥。
如本文所用， "分离的" 是指物质从其原始环境中分离出来 (如果是天然的物质， 原始环境 即是天然环境；)。 如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分离纯化的， 但同样的 多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开， 则为分离纯化的。
如本文所用， "分离的作物株高调节多肽" 、 "分离的 ELb蛋白" 或 "分离的 ELb多肽" 是指 ELb蛋白基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人 员能用标准的蛋白质纯化技术纯化 ELb蛋白。 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产 生单一的主带。
如本文所用， 所述的 "含有" ， "具有 "或 "包括"包括了 "包含"、 "主要由 ... ...构成"、
"基本上由 ... ...构成" 、 和 "由 ... ...构成" ； "主要由 ... ...构成" 、 "基本上由 ... ...构成" 和
"由 ... ...构成" 属于 "含有" 、 "具有" 或 "包括" 的下位概念。
ELb多肽及其用途
本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选的是重组多肽。 本发明的多肽 可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞；)中产生。 根据重组生产方案所用的宿主， 本发明的多肽 可以是糖基化的， 或可以是非糖基化的。 本发明的多肽还可包括或不包括起始的甲硫氨酸残基。
本发明还包括 ELb蛋白的片段、 衍生物和类似物。 如本文所用， 术语 "片段" 、 "衍生物" 和 "类似物" 是指基本上保持本发明的 ELb蛋白相同的生物学功能或活性的多肽。 本发明的多 肽片段、 衍生物或类似物可以是 (1)有一个或多个保守或非保守性氨基酸残基 (优选保守性氨基酸 残基)被取代的多肽， 而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的， 或 (11) 在一个或多个氨基酸残基中具有取代基团的多肽， 或 (111)成熟多肽与另一个化合物 (比如延长多 肽半衰期的化合物， 例如聚乙二醇)融合所形成的多肽， 或 (IV)附加的氨基酸序列融合到此多肽 序列而形成的多肽 (如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列， 或融合蛋 白；)。 根据本文的定义这些片段、 衍生物和类似物属于本领域熟练技术人员公知的范围。
在本发明中， 术语 " ELb蛋白"指具有 ELb蛋白活性的 SEQ ID NO: 3序列的多肽。 该术语 还包括具有与 ELb蛋白相同功能的、 SEQ ID NO:3序列的变异形式。 这些变异形式包括 (但并不 限于）： 若干个 (通常为 1-50个， 较佳地 1-30个， 更佳地 1-20个， 最佳地 1-10个， 还更佳如 1-8 个或 1-5个)氨基酸的缺失、 插入和 /或取代， 以及在 C末端和 /或 N末端添加一个或数个 (通常为 20个以内， 较佳地为 10个以内， 更佳地为 5个以内)氨基酸。 例如， 在本领域中， 用性能相近 或相似的氨基酸进行取代时， 通常不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添 加一个或数个氨基酸通常也不会改变蛋白质的功能。 该术语还包括 ELb蛋白的活性片段和活性 衍生物。
多肽的变异形式包括： 同源序列、 保守性变异体、 等位变异体、 天然突变体、 诱导突变体、 在高或低的严紧度条件下能与 ELb蛋白 DNA 杂交的 DNA所编码的蛋白、 以及利用抗 ELb蛋 白的抗血清获得的多肽或蛋白。 本发明还提供了其他多肽， 如包含 ELb蛋白或其片段的融合蛋 白。 除了几乎全长的多肽外， 本发明还包括了 ELb蛋白的可溶性片段。 通常， 该片段具有 ELb 蛋白序列的至少约 20个连续氨基酸， 通常至少约 30个连续氨基酸， 较佳地至少约 50个连续氨 基酸， 更佳地至少约 80个连续氨基酸， 最佳地至少约 100个连续氨基酸。
本发明还提供 ELb蛋白或多肽的类似物。 这些类似物与天然 ELb蛋白的差别可以是氨基酸 序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者兼而有之。 这些多肽包括天然 或诱导的遗传变异体。 诱导变异体可以通过各种技术得到， 如通过辐射或暴露于诱变剂而产生 随机诱变， 还可通过定点诱变法或其他已知分子生物学的技术。 类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨基酸)的类似物， 以及具有非天然存在的或合成的氨基酸 (如 β、 y -氨基 酸)的类似物。 应理解， 本发明的多肽并不限于上述例举的代表性的多肽。
修饰 (通常不改变一级结构)形式包括： 体内或体外的多肽的化学衍生形式如乙酰化或羧基 化。 修饰还包括糖基化。 修饰形式还包括具有磷酸化氨基酸残基 (如磷酸酪氨酸， 磷酸丝氨酸， 磷酸苏氨酸)的序列。 还包括被修饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中， " ELb蛋白保守性变异多肽" 指与 SEQ ID NO: 3的氨基酸序列相比， 有至多 20个， 较佳地至多 10个， 更佳地至多 5个， 最佳地至多 3个氨基酸被性质相似或相近的氨基 酸所替换而形成多肽。 这些保守性变异多肽最好根据表 1进行氨基酸替换而产生。
Figure imgf000005_0001
Figure imgf000006_0001
本发明还提供了编码本发明 ELb蛋白或其保守性变异多肽的多核苷酸序列。
本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、 基因组 DNA或 人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或非编码链。 编码成熟 多肽的编码区序列可以与 SEQ ID NO: 2所示的编码区序列相同或者是简并的变异体。如本文所 用， "简并的变异体" 在本发明中是指编码具有 SEQ ID NO: 3的蛋白质， 但与 SEQ ID NO: 2 所示的编码区序列有差别的核酸序列。
编码 SEQ ID NO: 3的成熟多肽的多核苷酸包括： 只编码成熟多肽的编码序列； 成熟多肽的 编码序列和各种附加编码序列； 成熟多肽的编码序列 (和任选的附加编码序列；)以及非编码序列。
术语 "编码多肽的多核苷酸" 可以是包括编码此多肽的多核苷酸， 也可以是还包括附加编 码和 /或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体， 其编码与本发明有相同的氨基酸序列的多肽或多肽 的片段、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的 变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和插入变异体。 如本领域所知的， 等 位变异体是一个多核苷酸的替换形式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不 会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少 70%， 更佳地 至少 80%相同性的多核苷酸。 本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多 核苷酸。 在本发明中， "严格条件" 是指 :( 1)在较低离子强度和较高温度下的杂交和洗脱， 如 0.2 X SSC , 0.1%SDS , 60 °C ; 或 (2)杂交时加有变性剂， 如 50%(v/v)甲酰胺， 0.1%小牛血清
/0.1%Ficoll, 42°C等； 或 (3)仅在两条序列之间的相同性至少在 80%以上， 较好至少 90%以上， 更好是 95%以上时才发生杂交。 并且， 可杂交的多核苷酸编码的多肽与 SEQ ID NO: 3所示的成 熟多肽有相同的生物学功能和活性。
本发明还涉及与上述的序列杂交的核酸片段。 如本文所用， "核酸片段" 的长度至少含 15 个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最好是至少 100个核苷酸以上。 核酸片段可用于核酸的扩增技术 (如 PCR)以确定和 /或分离编码 ELb蛋白的多聚核苷酸。
应理解， 虽然本发明的 ELb基因优选获自拟南芥， 但是获自其它植物的与拟南芥 ELb基因 高度同源 (如具有 60%以上， 如 70%、 80%、 85%、 90%、 95%、 甚至 98%序列相同性）的其它基 因也在本发明考虑的范围之内。比对序列相同性的方法和工具也是本领域周知的，例如 BLAST。
本发明的 ELb蛋白核苷酸全长序列或其片段通常可以用 PCR扩增法、重组法或人工合成的 方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序列， 尤其是开放阅读框序列 来设计引物， 并用市售的 cDNA库或按本领域技术人员已知的常规方法所制备的 cDNA库作为 模板， 扩增而得有关序列。 当序列较长时， 常常需要进行两次或多次 PCR扩增， 然后再将各次 扩增出的片段按正确次序拼接在一起。 一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常是将其克隆入 载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通常， 通过先合 成多个小片段， 然后再进行连接可获得序列很长的片段。
目前， 已经可以完全通过化学合成来得到编码本发明蛋白 (或其片段， 或其衍生物)的 DNA 序列。 然后可将该 DNA序列引入本领域中已知的各种现有的 DNA分子 (或如载体)和细胞中。 此外， 还可通过化学合成将突变引入本发明蛋白序列中。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或 ELb蛋白编码序列经 基因工程产生的宿主细胞， 以及经重组技术产生本发明所述多肽的方法。
通过常规的重组 DNA技术 (Science, 1984； 224： 1431) , 可利用本发明的多聚核苷酸序列 可用来表达或生产重组的 ELb蛋白。 一般来说有以下步骤：
(1) .用本发明的编码 ELb蛋白的多核苷酸 (或变异体)， 或用含有该多核苷酸的重组表达载体 转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养宿主细胞；
(3).从培养基或细胞中分离、 纯化蛋白质。
本发明中， ELb蛋白多核苷酸序列可插入到重组表达载体中。 术语 "重组表达载体" 指本 领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒或其他载体。 总 之， 只要能在宿主体内复制和稳定， 任何质粒和载体都可以用。 表达载体的一个重要特征是通 常含有复制起点、 启动子、 标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含 ELb蛋白编码 DNA序列和合适的转录 /翻译控 制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体内重组技术等。 所 述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指导 mRNA合成。 表达载体还包 括翻译起始用的核糖体结合位点和转录终止子。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转化的宿主细胞 的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗性以及绿色荧光蛋白 (GFP) , 或用 于大肠杆菌的卡那霉素或氨苄青霉素抗性。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主 细胞， 以使其能够表达蛋白质。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核 细胞， 如植物细胞。 代表性例子有： 大肠杆菌， 链霉菌属、 农杆菌； 真菌细胞如酵母； 植物细 胞等。
本发明的多核苷酸在高等真核细胞中表达时， 如果在载体中插入增强子序列时将会使转录 得到增强。 增强子是 DNA的顺式作用因子， 通常大约有 10到 300个碱基对， 作用于启动子以 增强基因的转录。
本领域一般技术人员都清楚如何选择适当的载体、 启动子、 增强子和宿主细胞。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如 大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤 在本领域众所周知。 另一种方法是使用 MgCl2。 如果需要， 转化也可用电穿孔的方法进行。 当 宿主是真核生物， 可选用如下的 DNA转染方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 转化植物也可使用农杆菌转化或基因枪转化等方法， 例如叶盘法、 水 稻幼胚转化法等。 对于转化的植物细胞、 组织或器官可以用常规方法再生成植株， 从而获得性 状发生改变的植物。
获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据所用的宿主细 胞， 培养中所用的培养基可选自各种常规培养基。 在适于宿主细胞生长的条件下进行培养。 当 宿主细胞生长到适当的细胞密度后， 用合适的方法 (如温度转换或化学诱导)诱导选择的启动子， 将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。 这些方法是本 领域技术人员所熟知的。 这些方法的例子包括但并不限于： 常规的复性处理、 用蛋白沉淀剂处 理 (盐析方法；)、 离心、 渗透破菌、 超处理、 超离心、 分子筛层析 (凝胶过滤)、 吸附层析、 离子交 换层析、 高效液相层析 (HPLC)和其它各种液相层析技术及这些方法的结合。
重组的 ELb蛋白有多方面的用途。 例如用于筛选促进或对抗 ELb蛋白功能的抗体、 多肽或 其它配体。用表达的重组 ELb蛋白筛选多肽库可用于寻找有价值的能抑制或刺激 ELb蛋白功能 的多肽分子。
本发明的多核苷酸的一部分或全部可作为探针固定在微阵列 (nncroarray)或 DNA芯片 (又称 为 "基因芯片"）上， 用于分析组织中基因的差异表达分析。 用 ELb蛋白特异的引物进行 RNA- 聚合酶链反应 (RT-PCR)体外扩增也可检测 ELb蛋白的转录产物。
本发明还涉及一种改良作物的方法， 该方法包括调节所述植物中 ELb基因或其同源基因的 表达或活性。 促进还是抑制 ELb的表达或活性主要取决于作物所需改良的性状而定。 当需要降 低作物株高、 体积， 增加作物分蘖、 产量时， 可通过提高作物中 ELb的表达或活性来实现； 当 需要增加作物花器大小， 增加种子大小， 或提高作物株高或体积时， 可通过抑制作物中作物株 高调节基因的表达或活性来实现。
增加 ELb基因或其同源基因表达的方法是本领域周知的。 例如， 可通过转入携带 ELb编码 基因的表达构建物使植株过表达 ELb ; 或可通过用强启动子驱动从而增强 ELb基因或其同源基 因的表达； 或者通过增强子 (如水稻 waxy基因第一内含子、 Actm基因第一内含子等)来增强该 ELb基因的表达。 适用于本发明方法的强启动子包括但不限于： 35s启动子、 水稻、 玉米的 Ubi 启动子等。
抑制 ELb基因或其同源基因表达的方法也是本领域周知的。例如，可通过 RNA干扰 (RNAi) 或基因沉默 (敲除；)的技术来实现。
作为本发明的一种优选方式， 获得 ELb高表达的植株的方法如下：
(1) 提供携带表达载体的农杆菌， 所述的表达载体含有 ELb蛋白的 DNA编码序列；
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触，从而使该 ELb蛋白 DNA编码序列 转入植物细胞， 并且整合到植物细胞的染色体上；
(3) 选择出转入所述 ELb蛋白 DNA编码序列的植物细胞或组织； 和
(4) 将步骤 (3)中的植物细胞或组织再生成植株。
其中， 可采用任何适当的常规手段， 包括试剂、 温度、 压力条件等来实施此方法。
本发明还包括 ELb蛋白或其编码基因的拮抗剂或激动剂。 由于 ELb的拮抗剂或激动剂可调 节 ELb的活性或表达， 因此， 所述的 ELb的拮抗剂或激动剂也可通过对 ELb的影响来调节作物 株高、 体积、 分蘖、 产量、 花器大小或种子大小等， 从而达到性状改良的目的。
所述的 ELb的拮抗剂是指任何可降低 ELb的活性、 降低 ELb的稳定性、 下调 ELb的表达、 减少 ELb有效作用时间、 或抑制 ELb的转录和翻译的物质， 这些物质均可用于本发明， 作为增 加作物花器大小， 增加种子大小， 或提高作物株高或体积有用的物质。
所述的 ELb的激动剂是指任何可提高 ELb的活性、 维持 ELb的稳定性、 促进 ELb表达、 延长 ELb有效作用时间、 或促进 ELb的转录和翻译的物质， 这些物质均可用于本发明， 作为降 低作物株高、 体积， 增加作物分蘖、 产量有用的物质。
在本发明的一个实例中， 提供了一种 ELb基因， 其基因组序列如 SEQ ID NO: 1所示， 其中 开放阅读框 (ORF) 位于第 61-353 , 1363-1585 , 1690-1943 , 2028-2414, 2559-2979位，全长 cDNA (SEQ ID NO: 1) 为 1578bp，编码一个含有 525个氨基酸的蛋白质 （SEQ ID NO: 3) 。所述的 ELb 基因可以为作物的株高、 体积、 产量、 分蘖等方面的改良提供新的途径， 因而具有巨大的应用 前景。 植物茎叶特异性表达启动子及其指导的基因表达
如本文所用， 所述的 "启动子"或 "启动子区 (域；) "是指一种核酸序列， 其通常存在于目的 基因编码序列的上游 (5')， 能够引导核酸序列转录为 mRNA。 一般地， 启动子或启动子区提供 RNA聚合酶和正确起始转录所必需的其它因子的识别位点。 在本文中， 所述的启动子或启动子 区包括启动子的变异体， 该变异体可以是天然发生的等位变异体或非天然发生的变异体。 所述 变异体包括取代变异体、 缺失变异体和插入变异体。
如本文所用， "组织特异性启动子 " 又称 "器官特异性启动子" ， 在这类启动子调控下， 基因往往只在某些特定的器官或组织部位表达。
如本文所用， 所述的 "可操作性连接" 是指两个或多个核酸区域或核酸序列的功能性的空 间排列。 例如： 启动子区被置于相对于目的基因核酸序列的特定位置， 使得核酸序列的转录受 到该启动子区域的引导， 从而， 启动子区域被 "可操作地连接" 到该核酸序列上。
通常， 如果在某组织或器官中 mRNA以比在其它组织或器官中高至少 2倍， 优选至少高 5 倍， 更优选至少高 10倍， 更优选至少高 25倍， 更优选至少 50倍， 更优选至少高 100倍水平被 表达， 更优选至少高 1000倍水平被表达， 则该启动子被认为是组织或器官特异性的。
本发明提供一种启动子， 所述的启动子选自下组：
(1) 具有 SEQ ID NO: 13所示的核苷酸序列的多核苷酸； 或
(2) 在严格条件下能够与 (1)限定的多核苷酸序列杂交且具有指导目的基因在植物茎叶中特异性 表达功能的多核苷酸。
在本发明中， "严格条件" 是指： （1)在较低离子强度和较高温度下的杂交和洗脱， 如 0.2 X SSC, 0.1%SDS , 60 °C ;或 (2)杂交时加有变性剂，如 50%(v/v)甲酰胺， 0.1%小牛血清 /0.1%Ficoll， 42°C等； 或 (3)仅在两条序列之间的相同性至少在 80%以上， 较好至少 90%以上， 更好是 95%以 上时才发生杂交。 并且， 可杂交的多核苷酸也具有指导目的基因在植物茎叶中特异性表达的功 能。
多核苷酸的杂交是本领域技术人员熟知的技术， 特定的一对核酸的杂交特性指示它们的相 似性或同一性。 因此， 本发明还涉及与 SEQ ID NO: 13所示的核苷酸序列杂交且两个序列之间具 有至少 50%， 较佳地至少 60%， 更佳地至少 70%， 更佳地至少 80%， 更佳地至少 85%， 更佳 地至少 90% (例如 95%、 96%、 97%、 98%、 或 99%) 相同性的多核苷酸。
本发明的启动子是植物组织或器官特异性的， 更特别的， 其是植物茎叶特异性的。 在本发 明的实例中， 本发明人发现， 在本发明的启动子的指导下， 可以使 ELb基因或 GUS基因特异地 在植物茎叶中表达， 而在其它组织、 器官中基本上不表达。
本发明的启动子可以被可操作地连接到目的基因上， 该目的基因相对于启动子可以是外源
(异源；)的。 所述的目的基因通常可以是任何核酸序列 (优选结构性核酸序列；)， 所述的目的基因优 选编码具有特定功能的蛋白， 例如某些对于控制植物茎叶生长的蛋白， 更具体地如本发明的植 物株高相关蛋白。
本发明的启动子还可以被可操作地连接到被改进的目的基因序列上， 该目的基因相对于启动子 是外源 (异源)的。 所述的目的基因可以被改进来产生各种期望的特性。 例如， 目的基因可以被改进来 增加表达量， 改变翻译后的修饰 (如磷酸化位点)， 将翻译产物转运到细胞外， 改善蛋白的稳定性， 插 入或删除细胞信号等。
此外， 启动子和目的基因可以设计成下调特定基因。 这一般是通过将启动子连接到目的基因序 列上来实现， 该序列以反义反向被引导。 本领域的普通技术人员熟悉这种反义技术。 任何核酸序列 可以以这种方式被调节。
任何一种前述的启动子和目的基因序列可被包含在构建物中， 更具体地如重组载体中。 所述的重组载体一般包括可操作性连接的 (通常从 5 '到 3 '方向）： 引导目的基因转录的启动 子， 和目的基因。 如果需要， 所述的重组载体还可以包括 3 '转录终止子， 3 '多聚核苷酸化信号， 其它非翻译核酸序列， 转运和靶向核酸序列、 抗性选择标记、 增强子或操作子。
通常， 所述的目的基因位于所述植物茎叶特异性表达启动子的下游， 且与所述启动子的间隔 小于 2000bp (优选的， 小于 lOOObp ; 更优选的， 小于 500bp ; 最优选的， 小于 300bp)。
重组载体中除了含有本发明的启动子， 还可含有一种或多种其它启动子。 所述的其它启动 子例如是： 组织特异性的、 组成型的或诱导型的。
包含上述适当的启动子和目的基因的载体， 可以用于转化适当的宿主细胞， 以使其能够表 达蛋白质。 本发明的主要优点在于：
(1) 首次分离得到一种新的作物株高调节基因， 该基因具有调节作物株高、 体积、 分蘖、 产 量、 花器大小或种子大小的作用， 因而可极好地应用于作物品种的改良。
(2) 适当的矮杆和有效分蘖的增加是高产品种育种的理想株型，本发明的基因转入作物后可 降低作物株高， 用于矮化育种； 例如在禾本科作物上通过不同的表达水平， 对品种进行不同程 度株高的降低、 增加有效分蘖、 提高产量。
(3) 首次分离得到所述的作物株高调节基因的启动子， 该启动子是植物茎叶特异性表达的， 从而可良好地用于调控目的蛋白在植物茎叶中特异性表达。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明本发明而不用 于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件如
Sambrook等人， 分子克隆： 实验室指南 (New York: Cold Spring Harbor Laboratory Press , 2001) 或 PCR引物： 实验室指南 (Carl W. Dieffenbach和 Gabriela S. Devksler eds.， Cold Spring Harbor Laboratory Press, 1995) 中所述的条件， 或按照制造厂商所建议的条件。 材料
1.1 植物材料
拟南芥 (Arabidopsisthaliana), 生态型为 Columbia(Col-0)。
T-DNA 插入突变体： ELa (SALK— 016089)。
水稻品种： 台北 309 (Oryza sativaL.ssp Japonica.cvTaipei309， TP309)。
1.2 菌种和质粒载体
农杆菌 (Agrobacterium tumefaciens): GV3101 (参见 Narasimhulu, S.B等， Gelvin. 1996. Early transcription of Agrobacterium t-DNA genes in tobacco and maize. Plant Cell 8:873-886) 、 EHA105 (参见 Hood, E.E.等， 1993 , New Agrobacterium helper plasmids for gene transfer to plants. Transgen. Res.2:208-218)。
质粒载体：
pBluescript SK (pSK) ： 购自 Invitrogen公司。
pCambial300S: pCambial300RS购自 CAMBIA公司。
pBHOl.l: 购自 Invitrogen公司。
pGEM-T Easy 载体： 购自 Promega公司。
RNAi载体 1300RS: 购自美国 Arkansas州立大学。
1.3 试剂和酶
T4DNA连接酶、 各种核酸限制性内切酶和： Γα DNA 聚合酶购自 MBI Ferment, TaKaRa 、
New England Biolabs、 Promega。 DNA 片段的割胶回收试剂盒购自 Omega。 Reverse Transcription System购自 GIBCOBRL。 核酸分子量标准为 MBI产品。 pGEM-Teasy 载体购自
Promega(Madison, USA)。 （a-32P)dCTP购自亚辉生物工程公司（北京）。 反转录试剂盒采用 Superscript First-Strand Synthesis System for RT-PCR(#11904-018, Invitrogen)系统。其它常规化学 试剂均为进口原装、进口分装或国产分析纯。各种脱氧核苷酸引物由上海 Sangon 合成。 DNA 序 列由上海基康， 上海英俊公司测定， 序列分析用 Genedoc, DNAStar等软件完成。 方法
1. 拟南芥种植、 转化
拟南芥无菌培养时， 种子经表面 (70%乙醇， 30秒； 无菌水冲洗 4次)和深层 (7%次氯酸钠，
10分钟； 无菌水冲洗 3次)消毒后，播于 1/2 MS (1/2 X Murashige and Skoog basal medium, 0.8% 琼脂粉， pH 5.8)固体培养基上， 4Ό放置 72 h， 然后转入 22Ό培养。 一周后， 将幼苗移栽于浸 透营养液 (3 g/10 L 花无缺， 上海永通化工有限公司）的人工土壤 (蛭石、黑土和珍珠岩为 3:1:0.5) 中， 然后转入人工气候室在光周期为 14/10(L/D)中培养。
拟南芥转化采用喷洒法， 取生长状况良好的 4-5周龄植株 (转化前一周剪去主苔， 利于侧苔 抽出产生更多的花苞， 提高转化效率)。 将含有转基因载体的农杆菌于 28Ό培养至 OD6。。《2.0， 4,000 rpm 离心 10 mm, 菌体沉淀悬浮于新鲜配制的转化液 (1/2 MS 液体培养基含 5%蔗糖， 0.02% SilwetL-77)中， 至终浓度 OD6。。《0.6-0.8。 转化前， 将已授粉花以及果荚去除， 并使土 壤吸足水。 转化时将菌液均匀喷洒拟南芥， 至叶片上有液滴落下， 将植物用黑色的塑料袋覆盖 保持湿度， 避光过夜。 24小时候后将植物转移到正常条件下生长。 7天 (d)后按上述方法重复转 化 1次。 待种子成熟后将植株混合采收于纸袋中， 在干燥器中放置 7 d后脱粒。 T1代种子消毒 后播种在含有 50 g/ml Kan或者潮霉素的 l/2x MS培养基上， 4°C放置 72 h，然后正常光照培养。
2 农杆菌介导的水稻成熟胚愈伤的基因转化
(1) 水稻成熟胚愈伤的诱导
水稻 307T种子去壳， 70%乙醇浸泡 1分钟， 20%(v/v)， 次氯酸钠浸泡 20-30分钟， 并不停 摇晃， 用无菌水漂洗 5-6次。 无菌滤纸吸干， 播于 MSD培养基诱导愈伤， 26Ό暗培养。 一周后， 剔去胚乳、 胚芽、 胚根等， 剥取愈伤组织， 并继代于 MSD上暗培养。 每 2-3周继代一次。 如用 TP309种子剥取愈伤， 培养基用 NBD。
(2) 准备转化用菌液
第一天上午： 从 -70 °〇保存的菌种中挑少许于 5ml YEB(Rif 20 mg/L+ Kan 50mg/L)液体培养 基， 28 °C振荡培养过夜。
第二天上午： 从含有农杆菌的 YEB培养液中吸取 l-2ml， 转入 25-50ml AB(20mg/L Rif + 50mg/L Kan)液体培养基中培养， 28 °C培养 4小时左右至 OD6。。=0.5左右。
(3) 共培养
第二天下午： 测 OD6。。值， 菌液离心 5,000rpm， 15分钟， 菌体沉淀悬浮于 AAM (含 AS 100) 至菌液 OD6。。=0.4-0.6。 将菌液倒入装有水稻愈伤的三角瓶中， 使愈伤浸泡其中 20分钟， 并不时 摇晃； 用无菌纸吸干菌液 （或用吸管吸干菌液)， 把已浸泡过的愈伤组织转移到垫有一层无菌滤 纸的含 2.5% Phytagel的 NBD(AS IOO)培养基上共培养 2-3天。每皿再加 1ml AAM(+AS 100)培养 液充分湿润无菌滤纸。
(4) 筛选
将愈伤组织用无菌滤纸吸干 (换一次滤纸；)， 转至含潮霉素 (hygr0myCin, Hyg)的筛选培养基上 筛选抗性愈伤 (暗培养 30天左右， 中间换一次筛选培养基)。
筛选培养基：
第一次筛选用： 含 0.26%Phytagel MS + Timentin 100 mg/L + Hyg 30mg/L;
第二次筛选用： 含 0.26%Phytagel NBD + Timentin 100 mg/L + Hyg 50mg/L;
第三次筛选用： 含 0.26%Phytagel MS + Timentin 100 mg/L + Hyg 50mg/L。
(5) 预分化
将筛选的水稻愈伤转至预分化培养基， 培养 10天。
预分化培养基： 含 0.45%Phytagel 的 MS+ BAP 2 mg/L + NAA 1 mg/L +ABA 5 mg/L + Hyg
50mg/L, pH 5.7, 无 2,4-二氯苯氧基乙酸 (2, 4-D)。
(6) 分化
将经过预分化的水稻愈伤转至分化培养基上分化成苗 (光照， 15-30天)。
分化培养基： 含 0.45%Phytagel 的 NB+BAP 3 mg/L+NAA 0.5mg/L, pH 5.7, 无 2, 4-D。
(7) 生根
将绿色小苗转至生根培养基上长根。
生根培养基： 含 0.45%Phytagel 的 1/2 MS+Hyg 50mg/L, pH 5.7， 无 2, 4-D。
3. 载体构建
p35S:: ELb 载体构建 利用 RT-PCR的方法扩增 ELb的 cDNA编码区序列。具体步骤如下：使用 RNeasy Plant Mini kit(Qiagen)，按照说明书提供的方法分离拟南芥的总 RNA，分离到的总 RNA使用 M-MLV reverse transcriptase (Promega)进行反转录产生 ELb的 cDNA。使用 Takara perobest DNA聚合酶以 cDNA 作为模板进行 PCR反应扩增 ELb的 cDNA编码区序列。 所用引物：
上游（SEQ ID NO: 7)： 5 ' -TGAGGATCCAAATAAAATAAAAAG-3 ' (M线部分分另 ij为 Bam l 位点；)；
下游 (SEQ ID NO: 8)： 5 ' -AAAGTCGACCACACACAAAGCAAA-3 ' (划线部分分别为 Ctol位点）。
PCR 条件： 94°C变性 3 min; 94°C变性 30 s， 58 °C复性 30 s， 72°C延伸 2min扩增 35个循环； 72 °C保温 10 min。 16 °C保温。
PCR产物回收， 然后用 ί™ΗΙ和 Ctol进行双酶切， 回收并与进行了同样双酶切与回收过程 的 pSK载体连接， 转化大肠杆菌 DH5 a， 选酶切正确的克隆测序。 测序正确后， 再用 α«ίΗΙ 禾口 CM 切下 ELb的 cDNA编码区序列， 连入同样经 BamHl和 CM双酶切的双元表达载体 pCambial300S构建转基因克隆， 转大肠杆菌 DH5 α。 提质粒， 酶切鉴定正确的质粒转入农杆菌 GV3101和 ΕΗΑ105。 从农杆菌中提取质粒再转入 DH5 α中， 提取质粒并且酶切验证， 确定正确 的质粒已经转入农杆菌中。 分别采用喷洒法转化拟南芥产生转基因植株和农杆菌介导的水稻成 熟胚愈伤的基因转化获得水稻转基因植株。
p ELb promoter: :GUS载体构建
根据 Genbank上检索到的序列 （GenelD: 832559)， 在 £Lb 基因翻译起始位点上游 1.4kb范 围内设计上下游引物，以拟南芥 Columbia 野生型 7天小苗提取的基因组 DNA为模板进行 PCR。 引物如下：
上游 (SEQ ID NO: 9)： 5 ' -CTGCTGCAGACTCTATTTCCA-3 ' (下戈 ij线处为酶切位点 Pstl) ; 下游（SEQ ID NO: 10)： 5 '-TTCAGGATCCTTTACTTTTTATTTT-3 下戈 ij线为酶切位点
BamHI)。
扩增条件为 94°C变性 3min; 然后 94°C变性 30s， 58 °C退火 30s， 72°C延伸 2min 扩增 35个 循环； 72 °C保温 10 min。 16 °C保温。
PCR产物 Pstl/BamHI 酶切后连入 pSK载体的 Pstl/BamHI 相应位点。 选酶切正确的克隆测 序， 测序正确的克隆再用 Pstl/BamHI切下 £ 6启动子区域 (序列如 SEQ ID NO: 13所示）， 连入 同样经 Pstl/BamHI双酶切的双元表达载体 ρΒΙ ΙΟ Ι . Ι , 转大肠杆菌 DH5 α。 提质粒， 酶切鉴定正 确的质粒转入农杆菌 GV3101。 从农杆菌中提取质粒再转入 DH5 a中， 提取质粒并且酶切验证， 确定正确的质粒已经转入农杆菌中。 同时将 ρΒΙ ΙΟ Ι .1载体转入 GV3101分别作为阴性和阳性对 昭。
ELb在 EIM的 T-DNA插入突变体背景下的 RNAi转基因植株构建
使用 RNAi载体 1300RS构建转基因克隆， 在 ELb 基因的第四外显子上扩增约 500bp的序 歹 U， 以拟南芥 Columbia 野生型 7天小苗提取的基因组 DNA为模板进行 PCR。 引物如下： 上游 （SEQ ID NO: 1 1) : 5 ' -AATGGTACCACAAGAAACAA-3 ' (下戈 ί线处为酶切位点 KpnD： 下游 （SEQ ID NO: 12) : 5 ' -TCTAGATTTGAGCTGAAAAAA-3 '(下划线为酶切位点 Xbal)。 扩增条件为 94°C变性 3min; 然后 94°C变性 30s， 50°C退火 30s， 72 °C延伸 30s 扩增 35 个 循环； 72 °C保温 10 min。 16 °C保温。
PCR产物 Kpnl/Xbal酶切后连入 pSK载体 Kpnl/ Xbal 相应位点。 选酶切正确的克隆测序， 测序正确的克隆再用 Kpnl/ Xbal切下 £ 6部分片断， 同样经 Kpnl/ Xbal双酶切的双元表达载体 1300RS , 转大肠杆菌 DH5 a。 提质粒， 酶切鉴定正确的质粒转入农杆菌 GV3101。 从农杆菌中 提取质粒再转入 DH5 a中， 提取质粒并且酶切验证， 确定正确的质粒已经转入农杆菌中。
采用喷洒法转化 ELa的 T-DNA插入突变体， 从而获得在 ELa的 T-DNA插入突变体背景下 ELb RNAi转基因拟南芥植株 (£ 6 RNAi /eLa)。 实施例 基因的克隆
本发明人通过拟南芥基因组序列的搜索和生物信息学的研究，发现 2个 P450单加氧酶 714A1 与 714A2基因， 初步预计它们参与赤霉素控制的植物生长发育， 本发明人将它们命名为 ELa (也 可称为 AtEuila)和 ELb (也可称为 AtEuilb)。
其中， ELb基因编码区基因组序列 (genomic DNA)如 SEQ ID NO: 1所示； ELb基因编码区 cDNA序列如 SEQ ID NO: 2所示； ELb蛋白序列如 SEQ ID NO: 3所示。 ELa基因编码区基因组 序列如 SEQ ID NO: 4所示； ELa基因编码区 cDNA序列如 SEQ ID NO: 5所示； ELa蛋白序歹 U 如 SEQ ID NO: 6所示。 实施例 2 ELb过表达转基因拟南芥降低株高与体积
本实施例中， 本发明人分别制备了可过表达拟南芥 ELb、 ELa和水稻 OsEm的转基因拟南芥 植株。 以野生型 (wt)、 过表达拟南芥 ELa(ELa-OE)和水稻 OsEm 的转基因植株为对照。
在培养各植株约 4周后， 检测植株的生长状况， 结果见图 1。 由该图可见， 野生型的拟南芥 生长得最大； 过表达拟南芥 的植株比野生型明显小； 而过表达拟南芥 ELa和水稻 OsEm的 植株生长得最小。
本发明人在培养各植株约 7周后分别测量了野生型、 过表达拟南芥 ELb、 ELa和水稻 OsEm 的转基因拟南芥植株的株高， 它们的株高平均值依次是 26.2cm， 14.2cm, 9.63cm, 3.7cm, 因此，
ELb过表达转基因拟南芥的株高明显低于野生型。 实施例 3 ELb RNAi或敲除突变体增加植株高度或体积
本实施例中， 本发明人制备了在 的 T-DNA插入突变体背景下 ELb RNAi转基因拟南芥 植株 0^6 ^4 Λ^α)， 和 ELb被敲除 (konckout)的拟南芥植株变异体。 培养两种植株约 10天后， 观察 ELb RNAi或敲除突变体对于株高或体积的影响， 以相同培养条件的野生型植株为对照。
ELb RNAi的植株与野生型植株的生长状况见图 2所示， 与野生型植株相比， ELb RNAi的 植株的叶片面积明显增加， 株高明显增加， 体积明显增加。
另外， 与野生型植株相比， ELb敲除突变体也存在明显的叶片面积增加、 株高增加、 体积 增加现象。
由此可见， 降低或沉默 ELb的表达可增加植株高度和体积。 也即 ELb是一种与降低植株高 度和体积相关的基因。 实施例 4 ELb RNAi或敲除突变体增加花器与种子大小
本实施例中， 本发明人观察了 ELb RNAi的拟南芥植株或 ELb被敲除的拟南芥植株的花器 和种子的生长状况， 以野生型拟南芥的花器和种子为对照。 ELb RNAi的拟南芥植株与野生型拟南芥植株的花器和种子生长状况如图 3所示， 与野生型 相比， ELb RNAi的拟南芥植株的花器明显增大， 种子明显增大。
另外， 与野生型植株相比， ELb敲除突变体也存在明显的花器增大， 种子增大现象。
由此可见， 降低或沉默 ELb的表达可增加植株的花器和种子。 也即 ELb是一种与减小花器 和种子大小相关的基因。 实施例 5 ELb启动子启动 GUS报告基因的组织特异性表达
本实施例中， 本发明人构建 T p £ ^ pra«w r Gf«载体， 转入农杆菌后制备拟南芥基因植 株。 利用常规的 GUS报告基因检测法观察植株中 GUS报告基因的表达情况。
结果如图 4所示， 可见 GUS报告基因可在植株的茎、 叶生长旺盛的部位表达， 在植株的根 部基本上没有表达。 因此， ELb基因是一种组织特异性基因。 实施例 6 ELb过表达转基因水稻降低株高
本实施例中， 本发明人制备了 ELb过表达的转基因水稻植株 (ELb-OE)， 观察 ELb过表达对 于水稻植株的影响， 以相同条件下培养的野生型水稻植株 TP309作为对照。
在培养植株约 1 10天后， 植株的生长情况如图 5所示， 可见 ELb过表达的转基因水稻植株 的株高明显低于野生型。 对于过表达 ELb植株和野生型植株的株高的统计图见图 6所示。 实施例 7 ELb过表达转基因水稻增加有效分蘖与产量
本实施例中， 本发明人检测了 ELb过表达的转基因水稻植株 (ELb-OE)和野生型植株 TP309 的分蘖数与产量， 以论证 ELb过表达对于水稻分蘖与产量的影响。
在培养植株抽穗后对有效分蘖进行分蘖数计数， 统计结果如图 7所示。 野生型植株的分蘖 数约 10个， 而 ELb过表达植株的分蘖数为约 20-22个。 可见 ELb过表达可明显增加植株的有 效分蘖数。
在植株成熟后， 本发明人还统计了 ELb过表达植株和野生型植株的产量情况。 结果如图 8 所示， 可见 ELb过表达的转基因水稻植株的单株粒重明显增加。 实施例 8 ELb蛋白变异体的功能
用一编码序列替换/ 载体中的 b cDNA编码区， 所述编码序列编码的蛋白具有 SEQ ID NO: 3类似的序列， 不同处仅在于第 522位为 Leu的 (ELb野生型蛋白中为 lie；)。 同前述 方法将该载体转化农杆菌， 制备转基因植物。 结果显示， 该转基因植株的株高比野生型降低。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被单独引用作为 参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本领域技术人员可以对本发明 作各种改动或修改， 这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种分离的作物株高调节多肽， 其特征在于， 该多肽选自下组：
(a) 具有 SEQ ID NO: 3所示氨基酸序列的多肽； 或
(b) 将 SEQ ID NO: 3所示氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添加而形 成的， 且具有调节作物株高功能的由 (a)衍生的多肽。
2. 如权利要求 1所述的多肽， 其特征在于， 该多肽是具有 SEQ ID NO: 3所示氨基酸序列的 多肽。
3. 一种分离的多核苷酸， 其特征在于， 它包含一核苷酸序列， 该核苷酸序列选自下组： (a) 编码如权利要求 1所述多肽的多核苷酸； 或
(b) 与多核苷酸 （a) 互补的多核苷酸。
4. 如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸编码具有 SEQ ID NO: 3所示氨 基酸序列的多肽。
5. 如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸：
(i) 具有 SEQ ID NO: 2所示的核苷酸序列； 或
(11) 具有 SEQ ID NO: 1所示的核苷酸序列。
6. 一种载体， 其特征在于， 它含有权利要求 3-5中任一项所述的多核苷酸。
7. 一种遗传工程化的宿主细胞， 其特征在于， 它含有权利要求 5所述的载体。
8. 一种植物， 其包含权利要求 3-5中任一项所述的多核苷酸。
9. 一种制备植物的方法， 其包括将权利要求 3-5中任一项所述的多核苷酸转入植物中。
10. 如权利要求 9所述的方法， 其特征在于， 所述方法包括：
(1) 提供携带表达载体的农杆菌， 所述的表达载体含有权利要求 3-5任一所述的多核苷酸；
(2) 将植物细胞或组织或器官与步骤 (1)中的农杆菌接触，从而使权利要求 3-5任一所述的多 核苷酸转入植物细胞， 并且整合到植物细胞的染色体上；
(3) 选择出转入了权利要求 3-5任一所述的多核苷酸的植物细胞或组织或器官； 和
(4) 将步骤 (3)中的植物细胞或组织或器官再生成植物。
11. 一种制备植物的方法， 其特征在于， 将转入了权利要求 3-5中任一项所述的多核苷酸的 植物与非转基因植物杂交， 获得包含权利要求 3-5中任一项所述的多核苷酸的杂交后代。
12. 一种权利要求 1所述的多肽的制备方法， 其特征在于， 该方法包含：
(a) 在适合表达的条件下， 培养权利要求 7所述的宿主细胞；
(b) 从培养物中分离出权利要求 1所述的多肽。
13. 权利要求 1-2任一项所述的多肽或编码该多肽的多核苷酸的用途， 其特征在于， 用于 调节作物的株高、 体积、 分蘖、 产量、 花器大小或种子大小； 或
制备调节作物的株高、 体积、 分蘖、 产量、 花器大小或种子大小的物质。
14. 一种调节作物株高、 体积、 分蘖、 产量、 花器大小或种子大小的方法， 其特征在于， 所 述方法包括： 调节作物中作物株高调节基因的表达或活性。
15. —种权利要求 1所述的作物株高调节多肽或其编码基因的激动剂或拮抗剂。
16. 一种植物茎叶特异性表达启动子， 其特征在于， 所述的启动子选自下组：
(1) 具有 SEQ ID NO: 13所示的核苷酸序列的多核苷酸；
(2) 在严格条件下能够与 (1)限定的多核苷酸序列杂交且具有指导目的基因在植物茎叶中特异 性表达功能的多核苷酸； 或
(3) 与 SEQ ID NO: 13所示的核苷酸序列具有 70%以上相同性且具有指导目的基因在在植物茎叶 中特异性表达功能的多核苷酸。
17. 权利要求 16所述的启动子的用途， 其特征在于， 所述的启动子用于指导目的基因在植 物茎叶中特异性表达。
18. 一种构建物， 其特征在于， 所述的构建物含有权利要求 16所述的植物茎叶特异性表达 启动子。
19. 如权利要求 18所述的构建物， 其特征在于， 所述的构建物含有以下可操作性连接的元 件：
权利要求 16所述的启动子； 和
目的基因。
PCT/CN2008/071939 2007-08-10 2008-08-11 A plant height regulatory gene and uses thereof WO2009021448A1 (en)

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