WO2015081787A1 - 调控稻类株型的基因及其应用 - Google Patents

调控稻类株型的基因及其应用 Download PDF

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WO2015081787A1
WO2015081787A1 PCT/CN2014/091766 CN2014091766W WO2015081787A1 WO 2015081787 A1 WO2015081787 A1 WO 2015081787A1 CN 2014091766 W CN2014091766 W CN 2014091766W WO 2015081787 A1 WO2015081787 A1 WO 2015081787A1
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plant
rice
plants
gene
resistance
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French (fr)
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李来庚
桂金山
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中国科学院上海生命科学研究院
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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)
    • C12N15/8241—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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • 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 invention belongs to the field of biotechnology and breeding, and in particular, the invention relates to a gene for regulating rice plant type and application thereof.
  • Rice is the main food variety and cash crop of the Asian population.
  • the increase of rice yield is of great significance for solving the food problem. How to increase the production of food to a greater extent has become an important topic in plant science research. It is also attracting attention for the rapid acquisition of high-quality, high-yield and high-resistance new rice materials using good biotechnology.
  • Rice leaf dip angle usually refers to the angle between leaves and plant stems and is an important component of rice plant type.
  • the size of the leaf dip angle, especially the dip angle of the flag leaf, has a great influence on the photosynthetic production of rice. Therefore, the vertical blade with a small leaf inclination angle is an important part of the ideal plant type.
  • Previous studies have shown that leaf dip development is regulated by molecular levels and hormones, especially brassinolides. For example, genetic defects in rice that participate in brassinosteroid synthesis or signal transduction can change the size of rice leaf dip.
  • cloned leaf dip-regulated genes have LC1 and LC2.
  • Moderate curling of leaves is an important part of rice plant type breeding and has important value in the cultivation of high-yield rice varieties.
  • the leaves of the currently cultivated super rice varieties have a certain degree of curl. Twelve leaf-rolling loci have been found in rice so far, but only seven cloned leaf-rolling genes, namely OsAGO7, SLL1, ACL1, Roc5, CFL1, RL14 and SRL1.
  • the leaf curl caused by these leaf-rolling genes is more than 50% (the excessive curling of the leaves reduces the light-receiving area, which is not conducive to high yield), and even the near-cylinder curl, etc., due to these effects, affecting the breeding of these genes. application.
  • an isolated OsREM4.1 protein or a gene encoding the same characterized in that the polypeptide or its encoding gene is used to modulate the plant type of rice.
  • the plant type comprises plant height, leaf inclination, leaf curl, or a combination thereof.
  • polypeptide or its encoding gene is also used in one or more of the following applications:
  • the OsREM4.1 protein is selected from the group consisting of:
  • amino acid sequence having a homology of ⁇ 95% (preferably ⁇ 98%, more preferably ⁇ 99%) to the amino acid sequence shown in SEQ ID NO.: 2, having a regulation of rice plant height, and/or Leaf dip, and/or leaf curl, and/or yield of the polypeptide.
  • the gene encoding the OsREM4.1 protein is selected from the group consisting of:
  • (C) a polynucleotide having a nucleotide sequence homologous to the sequence of SEQ ID NO.: 1 ⁇ 95% (preferably ⁇ 98%, more preferably ⁇ 99%);
  • (E) A polynucleotide complementary to the polynucleotide of any of (A) to (D).
  • a method of modulating a rice plant type comprising the steps of increasing or decreasing the expression or activity of an OsREM4.1 protein or a gene encoding the same in a rice plant.
  • the plant type comprises plant height, leaf inclination, leaf curl, or a combination thereof.
  • the method is to increase the expression or activity of OsREM4.1.
  • the method is to introduce an exogenous OsREM4.1 encoding gene into a rice plant.
  • the method comprises the steps of:
  • step (b) contacting the plant cell or tissue or organ with the Agrobacterium in step (a) such that the polynucleotide sequence encoding the gene encoding the OsREM4.1 protein is transferred into the plant cell and integrated into the chromosome of the plant cell;
  • step (d) regenerating the plant cell or tissue or organ in step (c) into a plant.
  • the method is also used for one or more of the following uses:
  • the drought resistance performance refers to a drought resistance stress test (10 days or 15 days) performed by 20% (w/v) PEG 4000.
  • the salt-tolerant property means that the salt tolerance stress test (7-12 days) performed at 70-150 mM NaCl can survive.
  • a method of improving a rice plant comprising the steps of:
  • the method further comprises one or more of the following steps:
  • the measurement steps can be performed separately, in combination, sequentially, before and after, and simultaneously.
  • the measured drought resistance is carried out at 20% (w/v) PEG 4000.
  • the measured salt resistance is carried out at 70-150 mM NaCl.
  • the method comprises performing the six measurements of (4a)-(4f) to select plants having predetermined properties.
  • Figure 1 shows the expression analysis of OsREM4.1 in different tissues and different developmental stages. Among them, QRT-PCR analysis showed the expression analysis of OsREM4.1 in various tissues in rice materials at 1 month and 2 months (R: root, S: stalk, LS: leaf sheath, LB: leaf, LV: vein) , LIV: Pulse.)
  • Figure 2 shows the expression pattern of OsREM4.1 in various tissues and organs.
  • the promoter of OsREM4.1 drives GUS reporter gene to transform rice, and GUS staining is performed on the expression of OsREM4.1 in various tissues and organs (Fig. 2A: leaves; Fig. 2B: root; Fig. 2C: leaf sheath and young stems) Cross-cut; Figure 2D: cross section of young stem section).
  • Figure 3 shows the response of OsREM4.1 to phytohormones and abiotic stresses. among them,
  • Figure 3A shows the response of OsREM4.1 to phytohormones (C: control, BL: brassinolide, GA: gibberellin, ABA: abscisic acid, 2,4-D: 2,4-dichlorophenoxyacetic acid) , kinetin Kinetin).
  • Figure 3B shows the response of OsREM4.1 to abiotic stress (C: control, PEG: polyethylene glycol treatment, NaCl: sodium chloride treatment, drought (Drought: treatment), cold stress (Cold) treatment).
  • FIG. 4 shows the phenotypic analysis of OsREM4.1 transgenic rice grown in the field. among them:
  • Figure 4A shows the plant morphology of the grain filling stage, wild type and OsREM4.1 transgenic rice, from left to right: wild type (left), OsREM4.1 overexpression (middle) and OsREM4.1 RNAi (right).
  • Figure 4B shows the analysis of the flag leaf dip angle of wild-type and OsREM4.1 transgenic rice, from left to right: wild type (left), OsREM4.1 overexpression (middle) and OsREM4.1 RNAi (right).
  • Figure 4C shows the curl cross-cut analysis of flag leaves of wild-type and OsREM4.1 transgenic rice, from top to bottom: wild type (top), OsREM4.1 overexpression (middle) and OsREM4.1 RNAi (bottom) .
  • FIG. 5 shows the observation of toluidine blue staining of wild-type and OsREM4.1 transgenic rice sections. among them:
  • Figure 5A shows cell elongation in the second internode section of rice, from left to right: wild type (left), OsREM4.1 overexpression (middle), and OsREM4.1 RNAi (right).
  • Figure 5B shows the development of phloem and xylem in the vascular bundle observed by rice sheaths from left to right: wild type (left), OsREM4.1 overexpression (middle) and OsREM4.1 RNAi (right).
  • Figure 6 shows the subcellular localization of rice OsREM4.1. Among them, the subcellular localization of rice GFP-OsREM4.1 was observed in leaf epidermal cells using tobacco transient expression system.
  • Fig. 7 is a view showing the structure of a pHB carrier in Example 4 of the present invention.
  • FIG 8 shows that OsREM4.1 can improve the drought resistance and salt tolerance of plants.
  • the inventors discovered for the first time that the gene OsREM4.1 and its encoded protein can effectively regulate plant plant type, and in particular can regulate various plant type traits such as rice plant height, leaf dip angle and leaf curl degree. .
  • the gene can also regulate the tolerance of abiotic stress in rice. Stress-resistant stress resistance (such as resistance to salt or drought stress), such as moderately up-regulated expression of the gene, can be obtained tolerant to abiotic stress, plant height reduction, leaf flattening and leaf dip angle.
  • Stress-resistant stress resistance such as resistance to salt or drought stress
  • moderately up-regulated expression of the gene can be obtained tolerant to abiotic stress, plant height reduction, leaf flattening and leaf dip angle.
  • the present invention has been completed on this basis.
  • the gene OsREM4.1 and its encoded protein provided by the invention have great application prospects in rice improvement, such as breeding materials for dense planting, lodging resistance and crops with high stress resistance, thereby further increasing crop yield.
  • salt stress refers to the phenomenon that when a plant grows in a soil or a water body containing a high concentration of salt, its growth and development are inhibited or even died.
  • Salts that cause salt stress include, but are not limited to, sodium chloride, sodium sulfate, sodium carbonate or sodium bicarbonate.
  • drought stress refers to the phenomenon that when plants grow in water-deficient soil or other arid environments, their growth and development are inhibited and even die.
  • the terms "gene of the invention”, “OsREM4.1 gene”, “gene of the regulatory strain of the invention” are used interchangeably and refer to the OsREM4.1 gene derived from rice and variants thereof.
  • a typical CDS nucleotide coding sequence of the OsREM4.1 gene of the present invention is shown in SEQ ID NO.: 1, and a typical genomic sequence is shown in SEQ ID NO.: 3.
  • polypeptide of the invention As used herein, the terms “polypeptide of the invention”, “protein of the invention”, “OsREM4.1 polypeptide”, “OsREM4.1 protein”, “polypeptide of a regulatory plant of the invention”, “adjusted plant type of the invention” Proteins are used interchangeably and refer to OsREM4.1 proteins derived from rice and variants thereof.
  • a typical amino acid sequence of a polypeptide of the invention is set forth in SEQ ID NO.: 2.
  • the present invention also encompasses having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95) with a preferred gene sequence of the present invention (e.g., SEQ ID NO.: 1 or 3). More than %, most preferably 98% or more, such as 99%) homologous nucleic acid, the nucleic acid can also effectively regulate rice seed set rate, grain weight, yield and the like.
  • homologous nucleic acid e.g., SEQ ID NO.: 1 or 3
  • homologous nucleic acid e.g., SEQ ID NO.: 1 or 3
  • homologous nucleic acid e.g., SEQ ID NO.: 1 or 3
  • homologous nucleic acid e.g., SEQ ID NO.: 1 or 3
  • homologous nucleic acid e.g., SEQ ID NO.: 1 or 3
  • homologous nucleic acid e.g., SEQ ID NO.: 1
  • the nucleotide sequence of SEQ ID NO.: 1 may be substituted, deleted or added one or more to generate a derivative sequence of SEQ ID NO.: 1, due to the codon Degeneracy, even if the homology to SEQ ID NO.: 1 is low, the amino acid sequence shown as SEQ ID NO.: 2 can be substantially encoded.
  • the meaning of "the nucleotide sequence in SEQ ID NO.: 1 is substituted, deleted or added with at least one nucleotide-derived sequence” also includes under moderately stringent conditions, more preferably under highly stringent conditions.
  • variants include, but are limited to, a number of nucleotide deletions (usually 1-90, preferably 1-60, more preferably 1-20, optimally 1-10) , inserts and/or substitutions, and adding a few at 5' and / or 3' ends (usually within 60, preferably within 30, more preferably within 10, optimally within 5) ) nucleotides.
  • the genomic sequence encoding the protein of the invention e.g., SEQ ID NO.: 3
  • the gene provided in the examples of the present invention is derived from rice, it is derived from other similar OsREM4.1 having certain homology (conservative) to the sequence of the present invention (preferably, the sequence is as shown in SEQ ID NO.: 1) of a plant (especially a plant belonging to the same family or genus of rice)
  • Gene sequences are also included within the scope of the invention, as long as one skilled in the art can readily isolate the sequences from other plants as described in the present application after reading the present application.
  • REM4.1 homologous proteins from other rice or other gramineous plants.
  • the present invention also relates to an OsREM4.1 polypeptide and a variant thereof for regulating rice seed setting rate.
  • the amino acid sequence of the polypeptide is shown in SEQ ID NO.: 2.
  • the polypeptide of the present invention is capable of effectively regulating the plant type.
  • the present invention also includes 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more, most preferably, the sequence shown by SEQ ID NO.: 2 of the present invention. More than 98%, such as 99%) homologous polypeptides or proteins having the same or similar function.
  • the “identical or similar function” mainly refers to "modulating rice plant type”.
  • the term also refers to traits that are regulated or improved by regulation of plant type, such as yield, stress resistance, and the like.
  • the polypeptide variant is an amino acid sequence as shown in SEQ ID NO.: 2, after several (usually 1-60, preferably 1-30, more preferably 1-20) , optimally 1-10) a derivative sequence obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually 20 or less, preferably 10 or less) at the C-terminus and/or the N-terminus More preferably, it is 5 or less amino acids.
  • SEQ ID NO.: 2 after several (usually 1-60, preferably 1-30, more preferably 1-20) , optimally 1-10) a derivative sequence obtained by substituting, deleting or adding at least one amino acid, and adding one or several (usually 20 or less, preferably 10 or less) at the C-terminus and/or the N-terminus More preferably, it is 5 or less amino acids.
  • the function of the protein is usually not altered, and the addition of one or several amino acids at the C-terminus and/or N-terminus generally does not alter the function of the protein.
  • the invention also includes analogs of the proteins of the invention.
  • the difference between these analogs and the natural SEQ ID NO.: 2 may be a difference in amino acid sequence, or may be a difference in the modification form which does not affect the sequence, or both.
  • Analogs of these proteins 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 be biological. Analogs also include analogs having residues other than the native L-amino acid (such as D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (such as beta, gamma-amino acids). It should be understood that the proteins of the present invention are not limited to the representative proteins exemplified above.
  • Modifications include: chemically derived forms of the protein in vivo or in vitro such as acetate or carboxylation. Modifications also include glycosylation, such as those that undergo glycosylation in protein synthesis and processing. Such modification can be accomplished by exposing the protein to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine.
  • the invention also provides recombinant vectors for OsREM4.1 protein expression.
  • the promoter of the recombinant vector comprises a multiple cloning site or at least one cleavage site downstream.
  • the nucleotide sequence of the gene of interest is ligated into a suitable multiple cloning site or cleavage site to operably link the sequence to a promoter.
  • the recombinant vector comprises (from the 5' to 3' direction): a promoter, a foreign sequence (gene of interest), and a terminator.
  • the recombinant vector may further comprise an element selected from the group consisting of: a 3' polynucleotideization signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancer; or operator.
  • a 3' polynucleotideization signal a non-translated nucleic acid sequence
  • a transport and targeting nucleic acid sequence a resistance selection marker (dihydrofolate reductase, Neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); enhancer; or operator.
  • the present invention also provides a vector for inhibiting the expression of OsREM4.1 protein, which inhibits the expression of OsREM4.1 protein in plant cells by a conventional RNAi technique.
  • the expression vector can be a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, a mammalian cell virus or other vector.
  • any plasmid and vector can be employed as long as it is capable of replication and stabilization in the host.
  • expression vectors containing the genes of the present invention can construct expression vectors containing the genes of the present invention using well known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • any of the enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S may be added before the transcription initiation nucleotide. Promoters, Ubiquitin gene promoters (pUbi), etc., which can be used alone or in combination with other promoters.
  • Vectors comprising exogenous sequences can be used to transform a suitable host cell such that the host expresses the protein.
  • the host cell may be a prokaryotic cell such as Escherichia coli, Streptomyces, Agrobacterium: or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. It will be apparent to one of ordinary skill in the art how to select an appropriate vector and host cell. Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote (such as E. coli), it can be treated with the CaCl 2 method or by electroporation.
  • 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, immature embryo transformation method, flower bud soaking method and the like.
  • Agrobacterium transformation or gene gun transformation such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • plants can be regenerated by conventional methods to obtain transgenic plants.
  • a method for preparing a transgenic plant is to transfer a vector carrying a promoter and an exogenous sequence (both operably linked) to Agrobacterium, and the Agrobacterium will further comprise a promoter and an exogenous sequence.
  • the vector fragment is integrated into the chromosome of the plant.
  • the plant expression vector used can be processed, such as a gene (GUS gene, GFP gene, luciferase) which expresses an enzyme or a luminescent compound which can produce a color change in a plant.
  • GUS gene GFP gene, luciferase
  • Genes, etc. resistant antibiotic markers (gentamicin markers, kanamycin markers, etc.) or anti-chemical marker genes (such as herbicide resistance genes). From the safety of transgenic plants, the transformed plants can be directly screened by adversity without any selectable marker genes.
  • the present invention also provides a method of regulating rice plant type comprising the steps of increasing or decreasing the expression or activity of an OsREM4.1 polypeptide or a gene encoding the same in rice.
  • the method comprises the steps of: (a) providing an Agrobacterium carrying an expression vector comprising a sequence expressing OsREM4.1 or a coding sequence which inhibits expression of an OsREM4.1 polypeptide (exogenous (b) contacting the plant cell or tissue or organ with the Agrobacterium in step (a), thereby transferring the foreign gene into the plant cell and integrating it into the chromosome of the plant cell; (c) selecting Plant cells, or tissues, or organs that are transferred to a foreign gene; (d) regenerate plant cells, or tissues, or organs in step (c) into plants.
  • the invention also provides a method of engineering rice comprising the steps of increasing or decreasing the expression level or activity of an OsREM4.1 polypeptide in rice.
  • promoter or “promoter region (domain) refers to a nucleic acid sequence that accurately and efficiently initiates the transcriptional function of a gene, directing the transcription of the gene nucleic acid sequence into mRNA, which is typically found in the coding sequence of the gene of interest. Upstream (5' end), in general, the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the present invention also provides a promoter derived from a rice plant type-related gene derived from the OsREM4.1 gene of rice.
  • a preferred promoter has the nucleotide sequence set forth in SEQ ID NO.: 4.
  • the present invention also encompasses having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, more preferably 95% or more with the preferred promoter sequence (SEQ ID NO.: 4) of the present invention. Most preferably, the nucleic acid of 98% or more, such as 99%) homology. "Homology” refers to a similar level (ie, sequence similarity or identity) between two or more nucleic acids, as a percentage of the same position.
  • OsREM4.1 gene derived from rice is provided in the examples of the present invention, it is derived from other similar plants (especially plants belonging to the same family or genus of rice) and the OsREM4.1 gene of the present invention. Genes having certain homology (conservative) are also included in the scope of the present invention as long as they are The skilled artisan can conveniently isolate the gene from other plants based on the information provided herein after reading the present application.
  • exogenous or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a gene sequence of interest is generally not naturally occurring, the promoter is foreign to the gene of interest. A particular sequence is “exogenous” to the cell or organism into which it is inserted.
  • the plant which can be used as a transgenic receptor is not particularly limited, and representative examples include, but are not limited to, rice, Arabidopsis, tobacco, fruit trees and the like.
  • the present invention provides a novel OsREM4.1 protein and its coding gene OsREM4.1, which are involved in the regulation of rice plant type. Regulation of the expression of the protein and gene can obtain rice with important plant type traits such as plant height, leaf dip angle and leaf curl.
  • OsREM4.1 protein and its coding gene OsREM4.1 can not only regulate a variety of plant type traits, but also regulate the resistance to abiotic stress tolerance.
  • the present invention provides novel pathways for improving plants. For example, a moderately up-regulated expression of the OsREM4.1 gene can be obtained from a rice material that is resistant to abiotic stress, reduced plant height, leaf spreading and leaf dip angle, and high lodging resistance.
  • a test method for transient expression of tobacco leaves is carried out in accordance with Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of limited transformed plants. Nature Protocols 1: 2019-2025.
  • RNA from different developmental stages and different tissues of rice was extracted with an RNA extraction kit (EASYspin Plant RNA Rapid Extraction Kit, HF109-01, http://www.yph-bio.com).
  • Total RNA was reverse transcribed into cDNA using PrimeScriptTM 1st Strand cDNA Synthesis Ki t (TAKARA) and Oligo dT primers.
  • TAKARA PrimeScriptTM 1st Strand cDNA Synthesis Ki t
  • OsREM4.1 The real-time quantitative RT-PCR analysis of OsREM4.1 was carried out using the synthesized cDNA as a template, and the rice actin gene (OsACT1, Os03g0718100) was used as the internal reference gene.
  • the amplification primers of OsREM4.1 are as follows:
  • the inventors In order to study the tissue expression of the OsREM4.1 gene, the inventors cloned a 1311 bp fragment upstream of the ATG of the OsREM4.1 gene. They were subcloned into pCAMBIA1301 (purchased from CAMBIA). After verification, it was transferred to the conventional Agrobacterium strain EHA105 and transferred to Rice Zhonghua 11 (purchased from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences).
  • GUS activity analysis was performed on the transgenic positive material.
  • the tissue was first treated with acetone (about 10 min, 4 ° C), and the residual acetone in the tissue was washed away with 100 mM NaPO 4 buffer (pH 7.0), and incubated at 37 ° C with GUS color developing solution. time. Finally, the reaction was stopped with 75% ethanol and the color of the chloroplast was removed.
  • GUS chromogenic solution is 100 mM NaPO4 (pH 7.0), 10 mM EDTA, 2 mM X-gluc (5-bromo-4-chloro-3-indolyl- ⁇ -glucuronic acid), 5 mM K 4 Fe (CN) 6 , 5m MK 3 Fe(CN) 6 and 0.2% Triton X-100.
  • OsREM4.1 promoter GUS activity analysis showed that OsREM4.1 can detect GUS signal in roots, stems (nodes and internodes), leaves, and sheaths. Among the leaves, OsREM4.1 has the strongest signal in the stomata; in the nodes, internodes, and sheaths, the GUS signal is the strongest in the vascular bundle.
  • Rice is a wild type rice: Zhonghua 11.
  • Each hormone treatment concentration was 10 ⁇ M brassinolide, 100 ⁇ M gibberellin, 100 ⁇ M abscisic acid, 10 ⁇ M 2,4-D, 100 ⁇ M kinetin.
  • the treatment concentrations of each stress factor were: 20% PEG 6000 (polyethylene glycol treatment), 200Mm NaCl (sodium chloride), drought treatment was to put the seedlings in the air for drought stress, and cold stress treatment was set at 4 °C. .
  • Figure 3A shows that the OsREM4.1 gene is more specifically induced by ABA than the control group, and the rice seedlings treated with brassinolide, gibberellin, abscisic acid, 2,4-D and kinetin.
  • Figure 3B shows that stress factors such as PEG, NaCl, drought and cold stress can significantly induce high OsREM4.1 gene. expression.
  • Example 4 Increases or decreases the effect of REM4.1 on plant type
  • a sense overexpression vector the full length sequence of OsREM4.1 (951 bp, corresponding to positions 1-951 of SEQ ID NO.: 1 encoding a polypeptide of SEQ ID NO.: 2 of 316 aa in length)
  • the BamHI and XbaI restriction sites were introduced on both sides, and ligated to the T-vector (purchased from TAKARA) for enzyme digestion and sequencing.
  • pHB vector HyCAMmycin between XhoI in pCAMBIA1301 was replaced with Bar gene by pCAMBIA1301 (purchased from CAMBIA); GUS gene between NcoI and BstEII in pCAMBIA1301 was replaced with Hygromycin resistance gene; in pCAMBIA1301
  • the conventional 2X35S promoter and the rbcS polyA terminator were inserted between the multiple cloning sites EcoRI and HindIII to obtain the vector pHB, and the structure thereof is shown in FIG.
  • RNAi vector is constructed as follows:
  • the OsREM4.1 RNAi vector was constructed by selecting the 361 bp CDS fragment from OsREM4.1.
  • the OsREM4.1 RNAi vector with the hairpin loop was inserted into the restriction enzyme sites HindIII and SacI with the above pHB vector as the backbone.
  • 35S OsREM4.1-RNAi vector.
  • CDS fragment used to construct the OsREM4.1 RNAi vector is as follows:
  • the Agrobacterium-mediated transformation was carried out in accordance with the method described by Hie et al., 1994.
  • Transgenic regulation of OsREM4.1 expression can regulate plant type traits such as plant height, leaf dip angle and leaf curl.
  • Overexpression of OsREM4.1 significantly reduced rice plant height, reduced leaf dip and reduced leaf curl.
  • RNAi reduced the expression of OsREM4.1, which significantly increased leaf dip and increased leaf curl.
  • Fixation Take the plant material in FAA fixative (50% ethanol, 5% acetic acid, 5% formaldehyde), on ice Vacuum the material until it has sunk into the bottom of the bottle; replace with fresh fixative at 4 ° C overnight (12 to 16 hours).
  • FAA fixative 50% ethanol, 5% acetic acid, 5% formaldehyde
  • Dip wax 25% xylene / 75% ethanol, 50% xylene / 50% ethanol and 75% xylene / 25% ethanol were treated at room temperature for 0.5 hours, 100% xylene at room temperature for 2 times each time 1 Hour, 50% xylene / 50% paraffin at 60 ° C overnight, change 100% paraffin (60 ° C) twice a day, repeat 3 days.
  • pCAMBIA1300-GFP The artificially synthesized 35S-GFP-Nos fragment with HindIII and EcoRI sites at both ends was digested with HindIII/EcoRI and inserted into the conventional commercial vector pCAMBIA1300 (purchased from The multiple cloning site of CAMBIA, HindIII and EcoRI, was constructed into a pCAMBIA1300-GFP vector.
  • pCAMBIA1300-GFP as a backbone, the full-length sequence of OsREM4.1 (951 bp) was inserted between the restriction sites XbaI and BamHI and fused to the GFP sequence to construct pCAMBIA1300-GFP-OsREM4.1.
  • Example 7 The material in Example 7 was an OsREM4.1 overexpressing transgenic line (4.10X) and an OsREM4.1 RNAi transgenic line (4.1 RNAi) against the indica variety Zhonghua 11 (ZH11). (Agrobacterium-mediated transformation, see Hiei et al., 1994).
  • the location is located at Shanghai Songjiang Wuyi Farm.
  • the planting area was designed as follows: the plant spacing was 20cm ⁇ 20cm, and 6 lines were inserted in each row, 72 plants were planted in each line, and the distance between the lines was 40cm.
  • the water, fertilizer management and pesticide spraying after cultivation are managed by farm workers.
  • the above experimental results indicate that the plant type and yield-related traits of the OsREM4.1 overexpressing transgenic lines are significantly altered. For example, the plant height is reduced, the leaf inclination angle is reduced, the ear length is long, and the flag leaf length is shortened. In contrast, in the OsREM4.1 RNAi transgenic lines, the leaf dip angle increased and the 1000-grain weight increased.
  • OsREM4.1 was expressed in various tissues of rice and in various stages of growth and development (Fig. 1, Fig. 2), and the gene was affected by phytohormone abscisic acid (ABA) and non- Biological stress (PEG, drought, salt, cold) induced high expression (Fig. 3). The results indicate that OsREM4.1 is involved in plant hormone responses and responses to stress environmental factors.
  • OsREM4.1 is involved in the regulation of cell elongation and phloem and xylem development in vascular tissues.
  • up-regulation of the expression of this gene can inhibit cell elongation, inhibit xylem development, and promote phloem development; down-regulation of expression of this gene is just the opposite ( Figure 5).
  • Example 8 OsREM4.1 transgenic rice has improved drought resistance and salt tolerance
  • the overexpressing transgenic lines (4.1OX), wild strains (ZH11) and OsREM4.1 RNAi transgenic lines (4.1 RNAi) of OsREM4.1 transgenic rice in Example 7 were treated with drought (20% PEG4000) and salt. Treatment with stress (70 mM and 140 mM NaCl) observed drought and salt tolerance phenotypes.
  • the seeds of the above rice were soaked in room temperature tap water for 3 days, and germinated at 37 ° C for 2 days. After germination, spot it on a 96-well plate. Hydroponics was carried out in an artificial climate chamber under the conditions of 12 hours of light per day, 28 ° C during the day, and 25 ° C at night.
  • OsREM4.1 gene or its encoded protein or polypeptide can enhance the resistance of plants to drought stress.
  • the improvement in resistance is shown as a control plant (wild type ZH11 or OsREM4.1) Expression of down-regulated plants 4.1 RNAi) compared to: OsREM4.1 expressing plants (4.1 OX) were not affected or affected by growth under water deficit conditions, or were able to survive under more arid conditions.
  • Salt tolerance stress experiments indicate that the OsREM4.1 gene or its encoded protein or polypeptide enhances plant resistance to salt stress.
  • the increase in resistance was compared to the control plants (wild-type ZH11 or OsREM4.1 expression down-regulated 4.1 RNAi): growth of OsREM4.1 expressing plants (4.1 OX) in the presence of high concentrations of salt was not affected or affected The degree of influence is reduced or it can survive at higher salt concentrations.

Abstract

提供了调控稻类株型的基因及其应用。具体地，提供了一种能够调控水稻株型的OsREM4.1蛋白及其编码基因，所述蛋白及其编码基因不仅能够调控水稻等植物的株高、叶倾角，叶片卷曲度，还可用于提高水稻产量、改善植株的抗倒伏性、抗旱性能、抗盐性能、抗冷性能等各种不同方面。还提供了用OsREM4.1蛋白及其编码基因对水稻等植物进行改良的方法。

Description

调控稻类株型的基因及其应用 技术领域
本发明属于生物技术和育种领域，具体地，本发明涉及一种调控稻类株型的基因及其应用。
背景技术
粮食问题是一个世界范围内的问题，随着人口的迅速增加和可用耕地面积的日益减少，尽快提高粮食产量已成为各国的重要战略措施。水稻(Oryza sativa)是亚洲人群的主要粮食品种和经济作物，水稻产量的提高对解决粮食问题具有重要意义。如何更大限度的增加粮食的产量成为植物科学研究的重要课题。对优良基因利用生物技术手段快速获得优质高产高抗的新型水稻材料也备受关注。
半矮秆水稻的推广引起了上世纪60年代水稻生产的“绿色革命”，实现了水稻单产大幅度提高。研究发现，该半矮秆水稻是由sd-1基因缺陷，导致赤霉素合成缺陷，引起茎秆伸长受阻所致。尽管在过去十多年里，发现了一大批与矮化相关的位点或基因，但是这些基因由于没有被克隆或育种应用价值不大而未能在育种上应用。因此，sd-1基因仍然是现代水稻杂交育种中重要的矮源，并被广泛的使用。但是，这种狭窄的遗传背景和单一基因的广泛利用，可能会影响粮食安全和水稻产量的进一步提高。因此，筛选和鉴定新的具有育种利用价值的半矮秆基因是水稻研究的重要课题。
水稻叶倾角通常是指叶片和植株茎秆之间的夹角，是构成水稻株型的重要组成部分。叶倾角的大小，特别是剑叶倾角的大小对水稻的光合生产量有很大影响。因此，叶倾角变小的直立型叶片是理想株型塑造的重要内容。已有的研究表明，叶倾角发育受到分子水平和激素，特别是油菜素内酯等多方面的调控。如：水稻中参与油菜素内酯合成或信号转导的基因缺陷会改变水稻叶倾角的大小。目前，克隆的叶倾角调控基因有LC1和LC2。研究表明，lc1-D突变体叶倾角变大是由于叶夹角处的细胞伸长造成；lc2突变体叶倾角变大是由于叶片和叶鞘连接处近轴面上表皮细胞的分裂增强所致。
叶片的适度卷曲是水稻株型育种中的重要组成部分，在高产水稻品种的培育中具有重要的价值。如：当前栽培的超级稻品种的叶片均具有一定程度的卷曲。迄今在水稻中已发现了12个卷叶基因座，但是，克隆的卷叶基因只有7个，即OsAGO7、SLL1、ACL1、Roc5、CFL1、RL14和SRL1。总体而言，这些卷叶基因造成的叶片卷曲度均超过50％(叶片过分卷曲会减少受光面积，不利于高产)，甚至近筒状卷曲等，由于这些效应，影响了这些基因在育种上的应用。
然而，目前对调控植物株型的基因和机理了解甚少。因此，本领域迫切需要开发可用于调控植物株型，进而对植物进行改良的方法。
发明内容
本发明的目的就是提供一种可用于调控植物的蛋白及其编码基因，以及所述蛋白和基因在植物改良中的应用。
在本发明的第一方面，提供了一种一种分离的OsREM4.1蛋白或其编码基因的用途，其特征在于，所述多肽或其编码基因用于调节稻类的株型。
在本发明的一个优选例中，所述的株型包括株高、叶倾角，叶片卷曲度、或其组合。
在本发明的另一个优选例中，所述的多肽或其编码基因还用于以下一种或多种用途：
(a)调节稻类的产量；
(b)提高植株的抗倒伏性；
(c)改善植株的抗旱性能；
(d)改善植株的抗盐性能；
(e)改善植物的抗冷性能。
在本发明的另一个优选例中，所述的OsREM4.1蛋白任选自下组：
(i)具有SEQ ID NO.:2所示氨基酸序列的多肽；
(ii)将如SEQ ID NO.:2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而形成的，具有调节水稻株高，和/或叶倾角，和/或叶片卷曲度，和/或产量功能的、由(i)衍生的多肽；或
(i i i)氨基酸序列与SEQ ID NO.:2所示氨基酸序列的同源性≥95％(较佳地≥98％，更佳地≥99％)，具有调节水稻株高，和/或叶倾角，和/或叶片卷曲度，和/或产量的多肽。
在本发明的另一个优选例中，所述的OsREM4.1蛋白的编码基因任选自下组：
(A)编码如SEQ ID NO.:2所示多肽的多核苷酸；
(B)序列如SEQ ID NO.:1所示的多核苷酸；
(C)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)的多核苷酸；
(D)在SEQ ID NO.:1所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
(E)与(A)-(D)任一所述的多核苷酸互补的多核苷酸。
在本发明的第二方面，提供了一种调控稻类株型的方法，包括步骤：提高或降低稻类植物中OsREM4.1蛋白或其编码基因的表达或活性。
在另一优选例中，所述的株型包括株高、叶倾角，叶片卷曲度、或其组合。
在另一优选例中，所述的方法是提高OsREM4.1的表达或活性。
在另一优选例中，所述的方法是将外源OsREM4.1编码基因导入稻类植物。
较佳地，所述方法包括步骤：
(a)提供携带表达载体的农杆菌，所述的表达载体含有表达OsREM4.1蛋白的 编码基因的多核苷酸序列；
(b)将植物细胞或组织或器官与步骤(a)中的农杆菌接触，从而使表达OsREM4.1蛋白的编码基因的多核苷酸序列转入植物细胞，并且整合到植物细胞的染色体上；
(c)选择已转入表达OsREM4.1蛋白编码基因的多核苷酸序列的植物细胞或组织或器官；
(d)将步骤(c)中的植物细胞或组织或器官再生为植株。
在本发明的一个优选例中，所述方法还用于以下一种或多种用途：
(a)调节稻类的产量；
(b)提高植株的抗倒伏性；
(c)改善植株的抗旱性能；
(d)改善植株的抗盐性能；
(e)改善植物的抗冷性能。
在另一优选例中，所述的抗旱性能指在20％(w/v)PEG4000进行的抗旱胁迫实验(10天或15天)能够存活。
在另一优选例中，所述的抗盐性能指在70－150mM NaCl进行的抗盐胁迫实验(7-12天)能够存活。在本发明的第三方面，提供了一种改良稻类植物的方法，包括步骤：
(1)将外源OsREM4.1导入待改良的稻类植物的植物细胞，从而使外源OsREM4.1蛋白的编码基因整合到植物细胞的染色体上；
(2)选择已转入外源OsREM4.1蛋白的编码基因的植物细胞；
(3)将步骤(2)中的植物细胞再生为植株。
在本发明的一个优选例中，所述方法还包括以下一个或多个步骤：
(4a)测量所述植株的株型；
(4b)测量所述植株的产量；
(4c)测量所述植株的抗倒伏性；
(4d)测量所述植株的抗旱性能；
(4e)测量所述植株的抗盐性能；
(4f)测量所述植株的抗冷性能；
其中各测量步骤可分别、组合、依次、前后、同时进行。
在另一优选例中，所述的测量抗旱性能在20％(w/v)PEG4000下进行。
在另一优选例中，所述的测量抗盐性能在70－150mM NaCl下进行。
在本发明的另一个优选例中，所述方法包括进行(4a)-(4f)所述的6项测量，从而挑选出具有预定性能的植株。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一赘述。
附图说明
下列附图用于说明本发明的具体实施方案，而不用于限定由权利要求书所界定的本发明范围。
图1显示了OsREM4.1在不同组织及不同发育时期的表达分析。其中，QRT-PCR分析显示了1个月和2个月水稻材料中OsREM4.1在各个组织中的表达分析情况(R：根，S：茎秆，LS：叶鞘，LB：叶片，LV：叶脉，LIV：脉间)。
图2显示了OsREM4.1在各组织器官中的表达模式。其中，OsREM4.1的启动子驱动GUS报告基因转化水稻后，对OsREM4.1在各组织器官中的表达做了GUS染色(图2A：叶片；图2B：根；图2C：叶鞘及幼茎茎间横切；图2D：幼茎节部横切)。
图3显示了OsREM4.1对植物激素和非生物逆境胁迫的响应。其中，
图3A显示了OsREM4.1对植物激素的响应(C:对照，BL:油菜素内酯，GA：赤霉素，ABA:脱落酸，2,4-D:2,4-二氯苯氧乙酸，激动素Kinetin)。
图3B显示了OsREM4.1对非生物逆境胁迫的响应(C:对照，PEG:聚乙二醇处理，NaCl：氯化钠处理，干旱(Drought:)处理，冷胁迫(Cold)处理)。
图4显示了大田种植OsREM4.1转基因水稻的表型分析。其中：
图4A显示了灌浆期、野生型以及OsREM4.1转基因水稻的植株形态，由左到右依次为：野生型(左)、OsREM4.1过表达(中)和OsREM4.1 RNAi(右)。
图4B显示了野生型以及OsREM4.1转基因水稻的剑叶叶倾角分析，由左到右依次为：野生型(左)、OsREM4.1过表达(中)和OsREM4.1 RNAi(右)。
图4C显示了野生型以及OsREM4.1转基因水稻的剑叶叶片卷曲横切分析，由上到下依次为：野生型(上)、OsREM4.1过表达(中)和OsREM4.1 RNAi(下)。
图5显示了野生型和OsREM4.1转基因水稻切片甲苯胺蓝染色观察。其中：
图5A显示了水稻第二节间纵切观察细胞伸长，由左到右依次为：野生型(左)、OsREM4.1过表达(中)和OsREM4.1 RNAi(右)。
图5B显示了水稻叶鞘横切观察维管束中韧皮部和木质部的发育，由左到右依次为：野生型(左)、OsREM4.1过表达(中)和OsREM4.1 RNAi(右)。
图6显示了水稻OsREM4.1的亚细胞定位。其中，采用烟草瞬时表达系统，在叶片表皮细胞中观察水稻GFP-OsREM4.1的亚细胞定位。
图7显示了本发明实施例4中pHB载体的结构示意图。
图8显示了OsREM4.1可提高植株的抗干旱性和耐盐性。
具体实施方式
本发明人通过广泛而深入的研究，首次意外发现，基因OsREM4.1及其编码蛋白能够有效调控植物株型，尤其能调控水稻株高、叶倾角及叶片卷曲度等多种重要的株型性状。此外，本发明人还发现，该基因还可调控水稻的耐受非生物逆境 胁迫的抗逆性状(如对盐或干旱胁迫的抗性)，如适度上调表达该基因，可获得耐受非生物逆境胁迫、株高降低、叶片平展及叶倾角变小的水稻株型材料。在此基础上完成了本发明。本发明提供的基因OsREM4.1及其编码蛋白在稻类改良中有巨大的应用前景，如培育密植、抗倒伏和具有高抗逆性的作物的育种材料，从而为进一步增加作物的产量。
术语
如本文所用，盐胁迫是指植物在含有高浓度盐分的土壤或者水体中生长时，其生长发育受到抑制，甚至死亡的现象。造成盐属胁迫的盐类包括(但不限于)：氯化钠、硫酸钠、碳酸钠或碳酸氢钠。
如本文所用，干旱胁迫是指植物在缺水的土壤或者其它干旱环境中生长时，其生长发育受到抑制，甚至死亡的现象。
如本文所用，术语“本发明基因”、“OsREM4.1基因”、“本发明调节株型的基因”可以互换使用，都是指来源于水稻的OsREM4.1基因及其变体。本发明的OsREM4.1基因典型的CDS核苷酸编码序列如SEQ ID NO.:1所示，典型的基因组序列如SEQ ID NO.:3所示。
如本文所用，术语“本发明多肽”、“本发明蛋白”、“OsREM4.1多肽”、“OsREM4.1蛋白”、“本发明的调节株型的多肽”、“本发明的调节株型的蛋白”可以互换使用，都是指来源于水稻的OsREM4.1蛋白及其变体。本发明多肽的一种典型的氨基酸序列如SEQ ID NO.:2所示。
本发明还包括与本发明的优选基因序列(如SEQ ID NO.:1或3)具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的核酸，所述核酸也能有效地调节水稻的结实率、粒重、产量等性状。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。在本文中，所述基因的变体可以通过插入或删除调控区域，进行随机或定点突变等来获得。
在本发明中，以CDS序列为例，SEQ ID NO.:l中的核苷酸序列可以经过取代、缺失或添加一个或多个，生成SEQ ID NO.:l的衍生序列，由于密码子的简并性，即使与SEQ ID NO.:l的同源性较低，也能基本编码出如SEQ ID NO.:2所示的氨基酸序列。另外，“在SEQ ID NO.:l中的核苷酸序列经过取代、缺失或添加至少一个核苷酸衍生序列”的含义还包括能在中度严谨条件下，更佳的在高度严谨条件下与SEQ ID NO.:l所示的核苷酸序列杂交的核苷酸序列。这些变异形式包括(但并小限于)：若干个(通常为1-90个，较佳地1-60个，更佳地1-20个，最佳地1-10个)核苷酸的缺失、插入和/或取代，以及在5'和/或3'端添加数个(通常为60个以内，较佳地为30个以内，更佳地为10个以内，最佳地为5个以内)核苷酸。同样地，编码本发明蛋白的基因组序列(如SEQ ID NO.:3)也可如CDS序列同样进行修饰和突变。
应理解，尽管本发明的实例中提供的基因来源于水稻，但是来源于其它类似 的植物(尤其是与水稻属于同一科或属的植物)的、与本发明的序列(优选地，序列如SEQ ID NO.:1所示)具有一定同源性(保守性)的OsREM4.1基因序列，也包括在本发明的范围内，只要本领域技术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该序列。例如来自其他稻类、或其他禾本科植物的REM4.1同源蛋白。
本发明还涉及一种调节水稻结实率的OsREM4.1多肽及其变体，在本发明的一个优选例中，所述多肽的氨基酸序列如SEQ ID NO.:2所示。本发明的多肽能够有效调节株型。
本发明还包括与本发明的SEQ ID NO.:2所示序列具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的具有相同或相似功能的多肽或蛋白。
所述“相同或相似功能”主要是指“调节水稻株型”。此外，该术语也指出通过调节株型进而调控或改善的性状，如产量、抗逆性等。
本发明中，所述的多肽变体是如SEQ ID NO.:2所示的氨基酸序列，经过若干个(通常为1-60个，较佳地1-30个，更佳地1-20个，最佳地1-10个)取代、缺失或添加至少一个氨基酸所得的衍生序列，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，在所述蛋白中，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，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。这些保守性变异最好根据表A进行替换而产生。
表A
Figure PCTCN2014091766-appb-000001
Figure PCTCN2014091766-appb-000002
本发明还包括本发明蛋白的类似物。这些类似物与天然SEQ ID NO.:2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。这些蛋白的类似物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了生物学的技术。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、γ-氨基酸)的类似物。应理解，本发明的蛋白并不限于上述例举的代表性的蛋白。
修饰(通常不改变一级结构)形式包括：体内或体外蛋白的化学衍生形式如乙酸化或羧基化。修饰还包括糖基化，如那些在蛋白质合成和加工中进行糖基化修饰。这种修饰可以通过将蛋白暴露于进行糖基化的酶(如哺乳动物的糖基化酶或去糖基化酶)而完成。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。
本发明还提供了用于OsREM4.1蛋白表达的重组载体。作为一种优选的方式，重组载体的启动子下游包含多克隆位点或至少一个酶切位点。当需要表达目的基因时，将目的基因的核苷酸序列连接入适合的多克隆位点或酶切位点内，从而将所述序列与启动子可操作地连接。作为另一种优选方式，所述的重组载体包括(从5'到3'方向)：启动子，外源序列(目的基因)，和终止子。如果需要，所述的重组载体还可以包括选自下组的元件：3'多聚核苷酸化信号；非翻译核酸序列；转运和靶向核酸序列；抗性选择标记(二氢叶酸还原酶、新霉素抗性、潮霉素抗性以及绿色荧光蛋白等)；增强子；或操作子。
本发明还提供了用于抑制OsREM4.1蛋白表达的载体，通过常规的RNAi技术对植物细胞中的OsREM4.1蛋白的表达进行抑制。
制备重组载体的方法是本领域普通技术人员所熟知的。表达载体可以是细菌质粒、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，只要其能够在宿主体内复制和稳定，任何质粒和载体都是可以被采用的。
本领域普通技术人员可以使用熟知的方法构建含有本发明所述的基因的表达载体。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。使用本发明的基因构建重组表达载体时，可在其转录起始核苷酸前加上任何一种增强型、组成型、组织特异型或诱导型启动子，如花椰菜花叶病毒(CAMV)35S启动子、泛素(Ubiquitin)基因启动子(pUbi)等，它们可单独使用或与其它的启动子结合使用。
包括外源序列的载体可以用于转化适当的宿主细胞，以使宿主表达蛋白质。宿主细胞可以是原核细胞，如大肠杆菌，链霉菌属、农杆菌：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。本领域一般技术人员都清楚如何 选择适当的载体和宿主细胞。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，可以用CaCl2法处理，也可用电穿孔法进行。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、幼胚转化法、花芽浸泡法等。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，从而获得转基因的植物。
作为本发明的一种优选方式，制备转基因植物的方法是：将携带启动子和外源序列(两者可操作地连接)的载体转入农杆菌，农杆菌再将含启动子和外源序列的载体片段整合到植物的染色体上。
为了便于对转基因植物细胞或植物进行鉴定及筛选，可对所用植物表达载体进行加工，如加入在植物中表达可产生颜色变化的酶或发光化合物的基因(GUS基因、GFP基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除草剂基因)等。从转基因植物的安全性考虑，可不加任何选择性标记基因，直接以逆境筛选转化植株。
本发明还提供了一种调控水稻株型的方法，包括步骤：升高或降低水稻中OsREM4.1多肽或其编码基因的表达或活性。在本发明的一个优选例中，所述方法包括步骤：(a)提供携带表达载体的农杆菌，所述表达载体含有表达OsREM4.1的序列或抑制OsREM4.1多肽表达的编码序列(外源基因)；(b)将植物细胞或组织或器官与步骤(a)中的农杆菌接触，从而使所述外源基因转入植物细胞，并且整合到植物细胞的染色体上；(c)选择已转入外源基因的植物细胞、或组织、或器官；(d)将步骤(c)中的植物细胞、或组织、或器官再生为植株。
本发明还提供了一种改造水稻的方法，包括步骤：升高或降低水稻中OsREM4.1多肽的表达水平或活性。
如本文所用，术语“启动子”或“启动子区(域)”是指一种准确有效起始基因转录功能的核酸序列，引导基因核酸序列转录为mRNA，其通常存在于目的基因编码序列的上游(5'端)，一般地，启动子或启动子区域提供RNA聚合酶和正确起始转录所必需的其它因子的识别位点。
本发明还提供了一种来自水稻株型相关基因的启动子，所述启动子来源于水稻的OsREM4.1基因。一种优选的启动子的核苷酸序列如SEQ ID NO.:4所示。
本发明还包括与本发明的优选启动子序列(SEQ ID NO.:4)具有50％或以上(优选60％以上，70％以上，80％以上，更优选90％以上，更优选95％以上，最优选98％以上，如99％)同源性的核酸。“同源性”是指按照位置相同的百分比，两条或多条核酸之间的相似水平(即序列相似性或同一性)。
应理解，尽管本发明的实例中提供了来源于水稻的OsREM4.1基因，但是来源于其它类似的植物(尤其是与水稻同属于一科或属的植物)的、与本发明OsREM4.1基因具有一定同源性(保守性)的基因，也包括在本发明的范围内，只要本领域技 术人员在阅读了本申请后根据本申请提供的信息可以方便地从其它植物中分离得到该基因。
如本文所用，“外源的”或“异源的”是指不同来源的两条或多条核酸或蛋白质序列之间的关系。例如，如果启动子与目的基因序列的组合通常不是天然存在的，则启动子对于该目的基因来说是外源的。特定序列对于其所插入的细胞或生物体来说是“外源的”。
在本发明中，还提供对植物进行改良的方法，其中可作为转基因受体植物没有特别限制，代表性例子包括(但并不限于)：水稻、拟南芥、烟草、果树等。
本发明的主要优点：
(1)本发明提供了一种新颖的OsREM4.1蛋白及其编码基因OsREM4.1，它们参与了水稻株型的调控。调控该蛋白和基因的表达能获得具有不同株高、叶倾角及叶片卷曲度等重要株型性状的水稻。
(2)OsREM4.1蛋白及其编码基因OsREM4.1不仅可调控多种株型性状，而且可调控耐受非生物逆境胁迫的抗逆性状。(3)本发明提供了新颖的改良植物的途径。例如，适度上调表达OsREM4.1基因，可获得耐受非生物逆境胁迫、株高降低、叶片平展及叶倾角变小的密植、抗倒伏的高育种利用价值的水稻材料。
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：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。
通用方法：
农杆菌介导的转化的试验方法，参照Hiei Y,Ohta S,Komari T,Kumashiro T(1994)Efficient transformation of rice(Oryza sativa L.)mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA.Plant Journal 6:271-282进行。
烟草叶片瞬时表达的试验方法，参照Sparkes IA,Runions J,Kearns A,Hawes C(2006)Rapid,transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants.Nature Protocols 1:2019-2025进行。
实施例1 OsREM4.1基因表达分析
水稻不同发育时期、不同组织的总RNA用RNA提取试剂盒(EASYspin植物RNA快速提取试剂盒，HF109-01，http://www.yph-bio.com)进行抽提。总RNA用PrimeScriptTM 1st Strand cDNA Synthesis Ki t(TAKARA)和Ol igo dT引物进行反转录合成cDNA。
以合成的cDNA为模板对OsREM4.1进行实时定量RT-PCR分析，并以水稻肌动蛋白基因(OsACT1,Os03g0718100)作为内参基因。
其中OsREM4.1的扩增引物如下：
4.1RT F:5'-CTCAAGAAGTACGAGAGGAAG-3',(SEQ ID NO:6)
4.1RT R:5'-GTGGATACAACACTAAAACGA-3'(SEQ ID NO:7)
结果如图1所示。所检测的一月龄和二月龄的水稻，在各组织中均能检测到OsREM4.1基因的表达，说明该基因表达没有组织特异性。就相对表达量而言，OsREM4.1在叶鞘中表达量最高。
实施例2 OsREM4.1启动子克隆、载体构建、转化及GUS活性分析
为了研究OsREM4.1基因的组织表达情况，发明人克隆了OsREM4.1基因ATG上游1311bp片段。并将它们亚克隆到pCAMBIA1301(购自CAMBIA公司)。验证后转入常规的农杆菌EHA105菌株，并转入水稻中花11(购自中国农业科学院作物科学研究所)中。
对转基因阳性材料进行GUS活性分析，组织首先用丙酮处理(约10min，4℃)，用100mM NaPO4buffer(pH 7.0)洗掉组织中残留的丙酮，用GUS显色液于37℃保温适当的时间。最后，用75％的乙醇终止反应和脱掉叶绿体的颜色。其中，GUS显色液为100mM NaPO4(pH 7.0)、10mM EDTA、2mM X-gluc(5-溴-4-氯-3-吲哚基-β-葡糖醛酸)、5mM K4Fe(CN)6、5m MK3Fe(CN)6和0.2％Triton X-100。
结果如图2所示。OsREM4.1启动子GUS活性分析显示，OsREM4.1在根、茎(节和节间)、叶片、叶鞘中均能检测到GUS信号。在叶片中，OsREM4.1在气孔中信号最强；在节、节间和叶鞘中，GUS信号在维管束中最强。
实施例3植物激素及非生物逆境胁迫处理
为了研究OsREM4.1基因对植物激素和非生物逆境胁迫的响应，发明人用各种植物激素和非生物逆境胁迫处理生长一周的水稻小苗6小时。水稻为野生型水稻：中花11。
各激素处理浓度分别为10μM油菜素内酯、100μM赤霉素、100μM脱落酸、10μM 2,4-D,100μM激动素。
各胁迫因子处理浓度分别是：20％PEG 6000(聚乙二醇处理)，200Mm NaCl(氯化钠)，干旱处理是将小苗置于空气中干旱胁迫，冷胁迫处理是设定温度为4℃。
结果如图3所示。图3A显示,油菜素内酯、赤霉素、脱落酸、2,4-D和激动素处理的水稻小苗与对照组相比，OsREM4.1基因较特异性的受ABA诱导高表达。图3B显示,PEG、NaCl、干旱和冷胁迫等逆境因子均能显著诱导OsREM4.1基因高 表达。
实施例4提高或降低REM4.1对株型的影响
4.1正反义表达载体的构建
正义过表达载体的构建：在OsREM4.1的全长序列(951bp，对应于SEQ ID NO.:1中第1-951位,该序列编码长度为316aa的如SEQ ID NO.:2所示多肽)在两侧引入BamHI和XbaI酶切位点，并连接到T-载体(购自TAKARA公司)上，分别进行酶切和测序验证。
pHB载体的构建：以pCAMBIA1301(购自CAMBIA公司)为骨架，将pCAMBIA1301中XhoI之间的Hygromycin替换为Bar基因；将pCAMBIA1301中NcoI和BstEII之间的GUS基因替换为Hygromycin抗性基因；在pCAMBIA1301中多克隆位点EcoRI和HindIII之间插入常规的2X35S启动子和rbcS polyA终止子，从而得到载体pHB，其结构如图7所示。
以使用上述方法构建的pHB载体为骨架，将OsREM的全长序列(951bp)插入到酶切位点BamHI和XbaI之间，构建成2×35S:OsREM4.1-OX载体。
其中，RNAi载体的构建方式如下：
以OsREM4.1中选取361bp的CDS片段构建OsREM4.1 RNAi载体，以上述pHB载体为骨架，将带发卡环的OsREM4.1 RNAi组件插入到酶切位点HindIII和SacI之间，构建成2×35S:OsREM4.1-RNAi载体。
其中，用于构建OsREM4.1 RNAi载体的CDS片段如下：
CCATCCCGTCCCCTCGCCGCGCCCACCTCGCGCTCCCCGCCCCCGGCGACGTGTCGTCGGCGGGCGGCGGCCACGGCGACGAGGTGTCGGTGGGGCAGGTGAAGAAGGAGGAGGTGGAGTCCAAGATCGCCGCGTGGCAGATCGCCGAGGTCGCCAAGGTCAACAACCGCTTCAAGCGCGAGGAGGTCGTCATCAATGGCTGGGAGGGCGACCAGGTCGAGAAGGCCAACGCCTGGCTCAAGAAGTACGAGAGGAAGCTGGAGGAGAAGAGGGCCAAGGCGATGGAGAAGGCGCAGAACGAGGTGGCGAAGGCGCGGCGGAAGGCGGAGGAGAAGCGGGCGTCGGCGGAGGCGAAGAGGGG(SEQ ID NO.:5)。
4.2转基因植株
农杆菌介导的转化方式参照Hie i et al.,1994所述方法进行。
结果如图4所示。转基因调控OsREM4.1的表达，可调节水稻的株高、叶倾角和叶片卷曲度等株型性状。过表达OsREM4.1能显著降低水稻株高，减小叶倾角和降低叶片卷曲度。RNAi降低OsREM4.1的表达，能显著增加叶倾角和提高叶片卷曲度。
实施例5石蜡包埋和组织切片
1.固定：取植物材料置于FAA固定液(50％乙醇,5％乙酸,5％甲醛)中，冰上 抽真空至材料沉入瓶底；换新鲜的固定液，4℃过夜(12～16小时)。
2.脱水：50％、60％、70％、85％、95％的乙醇4℃依次各1小时；无水乙醇于室温换液3次每次1小时，每一步均需多次轻摇。
3.浸蜡：25％二甲苯/75％乙醇、50％二甲苯/50％乙醇和75％二甲苯/25％乙醇在室温各处理0.5小时，100％二甲苯室温换液2次每次1小时，50％二甲苯/50％石蜡60℃过夜，换100％石蜡(60℃)每日2次，重复3天。
4.包埋：将新融化的石蜡倾倒于事先用氯仿处理的包埋框，将材料置入包埋框的石蜡中，平放入冷水，完全凝固后晾干，保存于4℃备用。
5.制片：将新的载玻片置于预热至42℃的烤片机上，加1500μl DEPC-H2O覆盖均匀。修好的蜡块在切片机上切成7-10μm厚的蜡带，用镊子将切出的蜡带漂浮在DEPC-H2O上，1-2min待蜡带展平后用Kimwipe尽量吸走多余的水，置于42℃展片台展片过夜使切片粘牢。
6.染色：用0.1％的甲苯胺蓝染色30分钟，烘干后脱蜡并封片。
结果如图5所示。野生型和OsREM4.1转基因水稻第二节间纵切观察显示，与野生型(A)相比较OsREM4.1过表达(B)节间细胞的伸长明显受到抑制，而OsREM4.1 RNAi(C)节间细胞的伸长明显受到促进。
野生型和OsREM4.1转基因水稻叶鞘横切观察显示，OsREM4.1过表达水稻木质部导管细胞的扩展明显受到抑制，而韧皮部细胞数目则明显增加。OsREM4.1 RNAi水稻的维管束中韧皮部和木质部的发育与野生型水稻相比，无明显差别。
实施例6 OsREM4.1亚细胞定位
1.pCAMBIA1300-GFP的构建：将人工合成的、两端分别带有HindIII和EcoRI位点的35S-GFP-Nos片段，经HindIII/EcoRI酶切后，插入到常规的市售载体pCAMBIA1300(购自CAMBIA公司)的多克隆位点HindIII和EcoRI之间，从而构建成pCAMBIA1300-GFP载体。
以pCAMBIA1300-GFP为骨架，将OsREM4.1的全长序列(951bp)插入到酶切位点XbaI和BamHI之间与GFP序列融合，构建成pCAMBIA1300-GFP-OsREM4.1。
2.烟草叶片观察OsREM4.1蛋白的亚细胞定位
(1)挑取鉴定好的常规农杆菌GV3101(购自Invitrogen公司)单克隆于5ml LB培养基(加入适当的抗生素)中28℃，220rpm摇晃20h；
(2)按1％转接菌液至新培养基中，28℃，220rpm摇晃16-20h；
(3)测菌液600nm OD值，5000rpm 5min沉淀菌液，用注射液(10mM MgCl2+10mM Mes(pH＝5.7)+20umAS(乙酰丁香酮))将沉淀稀释至OD600＝1重悬，在室温下静置3h；
(4)用1ml注射器注射烟草平展叶片的下表面，使注射液在叶片内部漫延至硬币大小；
(5)培养2-3天；
(6)用激光扫描共聚焦显微镜(Confocal laser scanning microscope Zeiss LSM 510 META)进行观察。
结果如图6所示。采用注射本氏烟草(Nicotiana benthamiana)叶片的办法，共聚焦荧光显微镜观察显示，OsREM4.1在烟草叶片下表皮细胞中的绿色荧光信号非常清晰地勾勒了叶片表皮细胞的轮廓，而在表皮细胞的其他细胞器中均检测不到绿色荧光信号，说明OsREM4.1定位于细胞膜。
实施例7大田实验
实施例7中的材料为以粳稻品种中花11(ZH11)为背景的OsREM4.1过表达转基因株系(4.10X)和OsREM4.1 RNAi转基因株系(4.1 RNAi)。(农杆菌介导的转化，参照Hiei et al.,1994进行)。
地点位于上海松江五厍农场。
采用人工育秧，人工单本插秧。种植小区设计为：株行距20cm×20cm，每行单本插6株，每个株系种植72株，株系间距离40cm。栽培后的水、肥管理，农药喷洒均由农场工人统一管理。
实验结果如表1所示。
表1.OsREM4.1转基因水稻大田试验性状统计
Figure PCTCN2014091766-appb-000003
(“**”代表P＜0.01的显著性水平。)
上述实验结果说明了，OsREM4.1过表达转基因株系株型及产量相关的性状发生显著改变。如：株高降低，叶倾角变小，穗长、旗叶长变短等。相反，OsREM4.1 RNAi转基因株系中叶倾角变大，千粒重增加。
统计结果进一步证实，过表达OsREM4.1能获得株高降低、叶倾角变小(叶片直立)的高育种利用价值的水稻材料。
实验结果总结：
1)QRT-PCR和启动子-GUS分析表明，OsREM4.1在水稻各组织以及生长发育的各个时期均存在表达(图1，图2)，并且该基因受植物激素脱落酸(ABA)和非生物逆境(PEG、干旱、盐、冷)诱导高表达(图3)。结果表明，OsREM4.1参与植物激素应答和对逆境环境因子的响应。
2)构建OsREM4.1基因的植物转化载体，应用农杆菌介导的转化技术(Hiei et al.,1994)，调控该基因在水稻体内的表达，获得具有不同表达量的OsREM4.1转基因水稻材料。通过温室(设定参数为：12小时光周期、光照强度200～250μmol·m-2·s-1、温度28±1℃)和大田栽培试验，发现该基因能控制水稻株高，叶倾角及叶片卷曲度等重要的株型性状(图4)。
3)组织切片分析发现，OsREM4.1参与调控细胞的伸长和维管组织中韧皮部和木质部的发育。如：上调表达该基因的表达，可抑制细胞的伸长，抑制木质部的发育，促进韧皮部的发育；下调表达该基因的表达，则恰好相反(图5)。
4)利用OsREM4.1融合GFP(绿色荧光蛋白)构建亚细胞定位载体，农杆菌侵染瞬时转化烟草叶片和PEG介导的原生质体转化均发现，该基因编码的蛋白特异并稳固的定位于细胞膜(图6)。通过一系列的截短蛋白片段融合GFP构建亚细胞定位载体，利用PEG介导的原生质体转化发现，该蛋白C-端的氨基酸片段负责该蛋白的细胞膜靶向和定位。
实施例8 OsREM4.1转基因水稻具有改良的的抗干旱性和耐盐性
将实施例7中OsREM4.1转基因水稻的过表达转基因株系(4.1OX)、野生株系(ZH11)和OsREM4.1 RNAi转基因株系(4.1 RNAi)，进行干旱(20％PEG4000)处理、盐胁迫(70mM和140mM NaCl)处理，观察抗旱、耐盐性表型。
方法如下：
将上述水稻的种子，用室温自来水浸种3天，37℃催芽2天。等萌发后，点播于96孔板。在人工气候室中进行水培，培养条件为：每天光照12小时，白天28℃，晚上25℃培养。
待幼苗基本出齐后，将自来水换成常规水稻培养液进行培养。培养14天的幼苗(两叶一心期)，进行抗逆性处理：
(i)用额外添加终浓度为70mM或140mM的NaCl的水稻营养液分别进行盐处理12天和7天；或
(ii)用额外添加终浓度为20％(w/v)PEG4000的水稻营养液进行模拟干旱胁迫处理15天。
处理结束后，换用常规培养液进行恢复处理。恢复处理7天后，结束实验。
结果如图8所示。
抗干旱胁迫实验表明，OsREM4.1基因或其编码的蛋白质或多肽可增强植物对干旱胁迫的抗性。该抗性的提高表现为与对照植物(野生型ZH11或OsREM4.1 表达下调植株4.1 RNAi)相比：OsREM4.1表达植株(4.1OX)在缺水条件下的生长发育未受影响或受影响程度降低、或可在更干旱的条件下存活。
抗盐胁迫实验表明，OsREM4.1基因或其编码的蛋白质或多肽可增强植物对盐胁迫的抗性。该抗性的提高表现为与对照植物(野生型ZH11或OsREM4.1表达下调植株4.1 RNAi)相比：OsREM4.1表达植株(4.1OX)在高浓度盐分存在下的生长发育未受影响或受影响程度降低、或可在更高的盐浓度下存活。
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 (10)

  1. 一种分离的OsREM4.1蛋白或其编码基因的用途，其特征在于，所述多肽或其编码基因用于调节稻类的株型。
  2. 如权利要求1所述的用途，其特征在于，所述的株型包括株高、叶倾角，叶片卷曲度、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述的多肽或其编码基因还用于以下一种或多种用途：
    (a)调节稻类的产量；
    (b)提高植株的抗倒伏性；
    (c)改善植株的抗旱性能；
    (d)改善植株的抗盐性能；
    (e)改善植物的抗冷性能。
  4. 如权利要求1所述的用途，其特征在于，所述的OsREM4.1蛋白任选自下组：
    (i)具有SEQ ID NO.:2所示氨基酸序列的多肽；
    (ii)将如SEQ ID NO.:2所示的氨基酸序列经过一个或几个氨基酸残基的取代、缺失或添加而形成的，具有调节水稻株高，和/或叶倾角，和/或叶片卷曲度，和/或产量功能的、由(i)衍生的多肽；或
    (iii)氨基酸序列与SEQ ID NO.:2所示氨基酸序列的同源性≥95％(较佳地≥98％，更佳地≥99％)，具有调节水稻株高，和/或叶倾角，和/或叶片卷曲度，和/或产量的多肽。
  5. 如权利要求1所述的用途，其特征在于，所述的OsREM4.1蛋白的编码基因任选自下组：
    (A)编码如SEQ ID NO.:2所示多肽的多核苷酸；
    (B)序列如SEQ ID NO.:1所示的多核苷酸；
    (C)核苷酸序列与SEQ ID NO.:1所示序列的同源性≥95％(较佳地≥98％，更佳地≥99％)的多核苷酸；
    (D)在SEQ ID NO.:1所示多核苷酸的5'端和/或3'端截短或添加1-60个(较佳地1-30，更佳地1-10个)核苷酸的多核苷酸；
    (E)与(A)-(D)任一所述的多核苷酸互补的多核苷酸。
  6. 一种调控稻类株型的方法，其特征在于，包括步骤：提高或降低稻类植物中OsREM4.1蛋白或其编码基因的表达或活性。
  7. 如权利要求6所述的方法，其特征在于，所述方法还用于以下一种或多种用途：
    (a)调节稻类的产量；
    (b)提高植株的抗倒伏性；
    (c)改善植株的抗旱性能；
    (d)改善植株的抗盐性能；
    (e)改善植物的抗冷性能。
  8. 一种改良稻类植物的方法，其特征在于，包括步骤：
    (1)将外源OsREM4.1导入待改良的稻类植物的植物细胞，从而使外源OsREM4.1蛋白的编码基因整合到植物细胞的染色体上；
    (2)选择已转入外源OsREM4.1蛋白的编码基因的植物细胞；
    (3)将步骤(2)中的植物细胞再生为植株。
  9. 如权利要求8所述的方法，其特征在于，所述方法还包括以下一个或多个步骤：
    (4a)测量所述植株的株型；
    (4b)测量所述植株的产量；
    (4c)测量所述植株的抗倒伏性；
    (4d)测量所述植株的抗旱性能；
    (4e)测量所述植株的抗盐性能；
    (4f)测量所述植株的抗冷性能；
    其中各测量步骤可分别、组合、依次、前后、或同时进行。
  10. 如权利要求9所述的方法，其特征在于，所述方法包括进行(4a)-(4f)所述的6项测量，从而挑选出具有预定性能的植株。
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