WO2014166012A1 - 植物株型相关蛋白及其编码基因与应用 - Google Patents

植物株型相关蛋白及其编码基因与应用 Download PDF

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WO2014166012A1
WO2014166012A1 PCT/CN2013/000406 CN2013000406W WO2014166012A1 WO 2014166012 A1 WO2014166012 A1 WO 2014166012A1 CN 2013000406 W CN2013000406 W CN 2013000406W WO 2014166012 A1 WO2014166012 A1 WO 2014166012A1
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plant
sequence
gene
protein
transgenic
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PCT/CN2013/000406
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English (en)
French (fr)
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李付广
杨作仁
张朝军
王玉芬
武芝侠
刘传亮
张雪妍
王晔
李凤莲
王倩华
秦文强
孔德培
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中国农业科学院棉花研究所
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Priority to EP13881828.1A priority Critical patent/EP2963117B1/en
Priority to PCT/CN2013/000406 priority patent/WO2014166012A1/zh
Priority to US14/782,976 priority patent/US10041085B2/en
Publication of WO2014166012A1 publication Critical patent/WO2014166012A1/zh

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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)
    • C12N15/8241—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
    • C12N15/8266—Abscission; Dehiscence; Senescence
    • 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
    • C12N15/8291—Hormone-influenced development
    • C12N15/8298—Brassinosteroids
    • 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 genetic engineering, and relates to a plant-type-related protein and a gene encoding the same, and a plant dwarf-related protein derived from cotton, and a coding gene and application thereof.
  • Plant type refers to a set of characteristics related to the yield capacity of crop varieties or the arrangement of plants in space, that is, the growth phase.
  • Ideal plant type also known as ideal type (Ideotype) refers to an ideal plant type consisting of traits that are beneficial to plant photosynthesis, growth and grain yield, which can maximize the utilization of light energy in the group. , increase biological output and increase economic coefficient.
  • Plant height is not only a determinant of crop plant type, but also an important agronomic trait that determines yield.
  • plant height is promoted to change from natural growth to target growth, guiding the growth and development of plants and the appearance of the appearance is more conducive to high yield, high quality, low consumption and high efficiency.
  • people have increased the yield of crop dwarfing.
  • a large number of experimental studies have been carried out. The results show that the plant can be increased by appropriately dwarfing the plant and increasing the population structure.
  • Cotton is one of the most important economic crops in China. Cotton is a perennial woody plant that has been domesticated for a long time and turned into an annual crop, retaining the infinite growth habit of woody plants. The cotton plant is tall, with lush foliage, often causing shady in the field, poor ventilation and light transmission, a large number of bud bells falling off, and easy to fall, cotton bolls are susceptible to infection by bacteria, forming rotten bells and deadlock flowers, seriously affecting cotton yield and quality.
  • the south of China is dominated by hybrid cotton, and the growth of light, heat and water during the growth period of cotton is more vigorous, the growth of vegetative growth is more vigorous, the plant type is more difficult to control, and high, large and empty groups are prone to occur, resulting in reduced production.
  • cotton plant height is reduced, fruit branches are shortened, and leaves are reduced mainly by cultivation techniques.
  • vegetative bodies By controlling the growth of vegetative bodies, promoting the growth of reproductive organs, in order to maintain the ventilation and light transmission of cotton fields, obtain more acres of acres, adjust the economic coefficient of cotton, and achieve higher yields.
  • rice and wheat are realized by dwarf varieties, while cotton is strong through cultivation means.
  • the dwarfing that is, the regulation of fertilizer and water, the reduction of nutrient supply, the suppression of top-end advantages, the reduction of safety and control, etc., require a large amount of labor and fossil energy, which increases the cost of cotton production.
  • the protein provided by the present invention is a protein of the following (a) or (b) or (c) or (d) or (e):
  • a label as shown in Table 1 may be attached to the amino terminus or the carboxy terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  • the protein in (b) or (c) or (d) or (e) above may be artificially synthesized, or may be synthesized by first synthesizing the encoded gene.
  • the gene encoding the protein in (b) or (c) or (d) or (e) above may be obtained by deleting a codon of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and / Alternatively, a missense mutation of one or several base pairs is made, and/or a coding sequence for the tag shown in Table 1 is ligated at its 5' end and/or 3' end.
  • a gene encoding the GhPGD1 protein shown is also within the scope of the present invention, and the gene is named ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene.
  • the gene may specifically be a DNA molecule as follows 1) or 2) or 3) or 4) or 5) or 6) or 7) or 8):
  • the coding region is the DNA molecule of sequence 2 in the sequence listing from nucleotides 133 to 1704 of the 5' end;
  • the coding region is the DNA molecule of sequence 2 in the sequence listing from nucleotides 133 to 1707 at the 5' end;
  • an anthracene molecule having more than 80% homology to a defined DNA sequence of 1) or 2) or 3) or 4) and encoding a plant-type-related protein;
  • a purine molecule that hybridizes under stringent conditions to a DNA sequence defined by 1) or 2) or 3) or 4) and encodes a protein associated with plant brassinosteroid inactivation;
  • a DNA molecule that has more than 80% homology to the defined DNA sequence of 1) or 2) or 3) or 4) and encodes a protein associated with the inactivation of the plant brassinolide.
  • the above stringent conditions can be carried out in a solution of 6XSSC, 0.5% SDS at 65 ° C, and then washed once with 2XSSC, 0.1% SDS and 1XSSC, 0.1% SDS.
  • a recombinant expression vector, expression cassette, transgenic cell line or recombinant strain containing the ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene is within the scope of the present invention.
  • a recombinant expression vector containing the gene can be constructed using an existing plant expression vector.
  • the plant expression vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment and the like.
  • the plant expression vector may further comprise a 3' untranslated region of the foreign gene, ie, comprising a polyadenylation signal and any other purine fragment involved in mRNA processing or gene expression.
  • the polyadenylation signal can direct polyadenylation to the 3' end of the mRNA precursor.
  • any enhanced promoter or constitutive promoter may be added before the transcription initiation nucleotide, and they may be used alone or in combination with other plant promoters;
  • an enhancer including a translation enhancer or a transcriptional increase, may also be used. Hadron, but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.
  • the sources of the translational control signals and initiation codons are broad and may be natural or synthetic.
  • the translation initiation region can be from a transcription initiation region or a structural gene.
  • the plant expression vector used can be processed, such as a gene encoding a color-changing enzyme or luminescent compound that can be expressed in plants, and a resistant antibiotic marker. Or anti-chemical reagents, etc.
  • the recombinant expression vector may specifically be a recombinant plasmid obtained by inserting the gene into the multiple cloning site of the vector PCAMBIA2300.
  • the recombinant expression vector may specifically be a recombinant plasmid obtained by inserting an expression cassette of the gene into the multiple cloning site of the vector PCAMBIA2300.
  • expression of the gene was initiated by the 35S promoter and expression of the ⁇ 3 ⁇ 4 3 ⁇ 43 7 gene was terminated by the nos terminator.
  • the 35S promoter is specifically shown as Sequence 4 of the Sequence Listing.
  • the nos terminator is specifically shown in sequence 5 of the sequence listing.
  • the recombinant expression vector may specifically be the following recombinant plasmid: the vector PCAMBIA2300 is used as a backbone, and the 35S promoter is inserted between the Hi ndl ll and Xbal cleavage sites, and the Xbal and Sad cleavage sites are inserted.
  • the nos terminator was inserted between the GhPGD1 fiber, Sac l and EcoRI cleavage sites.
  • the GhPGD l protein, the gene, the expression cassette, the recombinant expression vector, the transgenic cell line or the recombinant strain can be used to improve the plant type.
  • the modified plant plant type is specifically embodied to dwarf the plant.
  • the plant is a monocot or a dicot.
  • the dicotyledonous plant may be cotton, and specifically may be the cotton variety "China Cotton Institute 24".
  • the dicotyledonous plant may be Arabidopsis thaliana, specifically the Colombian ecotype Arabidopsis thaliana.
  • the GhPGD l protein, the gene, the expression cassette, the recombinant expression vector, the transgenic cell line or the recombinant strain can be used to grow a transgenic plant.
  • the transgenic plant is specifically a plant having a dwarf phenotype.
  • the plant is a monocot or a dicot.
  • the dicotyledonous plant may be cotton, and specifically may be the cotton variety "Zhongmian Institute 24".
  • the dicotyledonous plant may be Arabidopsis thaliana, specifically the Colombian ecotype Arabidopsis thaliana.
  • the present invention also contemplates a method of cultivating a transgenic plant by introducing the GhPGD1 gene into a plant of interest to obtain a transgenic plant having a plant height less than the plant of interest.
  • An expression vector carrying the gene can be obtained by using a Ti plasmid, a Ri plasmid, a plant viral vector, direct DNA transformation, Conventional biological methods such as microinjection, conductance, Agrobacterium-mediated transformation of plant cells or tissues, and transformation of plant tissues into plants.
  • the GhPGD1 gene can be specifically introduced into the plant of interest through the recombinant expression vector.
  • the plant of interest is a monocot or a dicot.
  • the dicotyledonous plant may be cotton, and specifically may be the cotton variety "China Cotton Institute 24".
  • the dicotyledonous plant may be Arabidopsis thaliana, specifically the Colombian ecotype Arabidopsis thaliana.
  • the present invention also contemplates a method of cultivating a transgenic plant by overexpressing said GhPGD1 in a plant of interest to obtain a transgenic plant having a plant height less than said plant of interest.
  • Said "overexpression of said GhPGD1 gene in a plant of interest” can be achieved by introducing said gene into a plant of interest or by promoting expression of said gene possessed by the plant of interest (eg by introducing a promoter or enhancer to facilitate said Gene expression).
  • the plant of interest is a monocot or a dicot.
  • the dicotyledonous plant may be cotton, and specifically may be the cotton variety "Zhongmian Institute 24".
  • the dicotyledonous plant may be Arabidopsis thaliana, specifically the Colombian ecotype Arabidopsis thaliana.
  • Brass inostero i ds is a plant-specific sterol hormone that is widely found in plants and regulates various aspects of plant growth and development, including vegetative growth, reproductive growth, germination, aging, and Multiple biotic and abiotic stress responses.
  • Very low concentration (nmo l/L) of brassinolide exhibits extremely high physiological activity and is therefore considered to be the sixth class of plant hormones following auxin, gibberellin, cytokinin, abscisic acid and ethylene. .
  • the present invention also contemplates a method of cultivating a transgenic plant by overexpressing said GhPGD1 ⁇ in a plant of interest to obtain a brassinosteroid-deficient transgenic plant.
  • the brassinolide-deficient type is embodied in that the transgenic plant has at least one of the following phenotypes compared to the plant of interest: 1 hypocotyl shortening; 2 plant height reduction; 3 petiole and/or leaf sheath shortening; Flowering delay; 5 life cycle extension; 6 phenotype with photomorphogenesis in dark conditions.
  • Said "overexpression of said GhPGD1 fiber in a plant of interest” is achieved by introducing said gene into a plant of interest or by promoting expression of said GhPGD1 gene possessed by the plant of interest (eg by introducing a promoter or enhancer to facilitate said Gene expression).
  • the plant of interest is a monocot or a dicot.
  • the dicotyledonous plant may be cotton, and specifically may be the cotton variety "Zhongmian Institute 24".
  • the dicotyledonous plant may be Arabidopsis thaliana, specifically the Colombian ecotype Arabidopsis thaliana.
  • the present invention also protects a method for shaping a plant shape by locally applying a brassinolide to a plant (only promoting growth of a spray site) to grow the plant into a desired shape; the plant is any of the above a transgenic plant obtained by the method, a selfed progeny of the transgenic plant, the Hybrid progeny of the transgenic plant or backcross progeny of the transgenic plant.
  • Figure 1 shows the phenotypic comparison of the cotton variety "Zhongmian 24" with the mutant pagodal.
  • Figure 2 shows the response of the mutant and cotton variety "Zhongmian 24" to brassinolide.
  • Figure 3 shows the photomorphogenesis of the mutant / ⁇ and the cotton variety "Zhongmian 24".
  • Figure 4 shows the shaping of cotton by topical application of brassinolide.
  • Figure 5 shows the relative expression of the cotton variety "Zhongmian 24" and the mutant pagodal ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene.
  • Figure 6 shows a phenotypic comparison between the ecotype Arabidopsis thaliana and transgenic Arabidopsis thaliana.
  • Figure 7 shows the relative expression of the ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene in the ecotype Arabidopsis thaliana and transgenic Arabidopsis.
  • Figure 8 shows the growth status of the mutant and cotton variety "Zhongmian Institute 24" in Henan in mid-October.
  • Figure 9 shows the phenotypic comparison of cotton variety "Zhongmian Institute 24" with genetically modified cotton.
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • Cotton variety "China Cotton Institute 24" (indicated by WT): A variety of Gossypiim hirsutum, selected by the Chinese Academy of Agricultural Sciences for cotton research, can be purchased from China Cotton Seeds or other seed companies.
  • Colombian ecotype Arabidopsis thaliana (Col-0): purchased from ABRC (Arabidopsis Biological
  • Plant expression vector PCAMBIA2300 (referred to as vector pCAMBIA2300): purchased from Cambia (http://www. cambia. org/ daisy/ cambia/585. html ).
  • Agrobacterium strain LBA4404 purchased from clontech.
  • Example 1 Obtainment of cotton GhPGD1 protein and its coding gene
  • a mutant dwarfing mutant was obtained by genetically transforming the cotton variety "Zhongmian 24" with an activation tag, which was named mutant / (indicated by /).
  • the phenotype of the cotton variety "Zhongmian 24" (indicated by WT) and the mutant pagodal is shown in Figure 1.
  • the plant height of the mutant pagodal is extremely dwarfed (Fig. 1A).
  • the flower organ is shrunk (Fig. 1B) and the petiole is shortened (Fig. 1C, inverted 4 leaf petiole).
  • T. Generation T.
  • T After planting seeds, 1 ⁇ generation plants were harvested, and the phenotype and separation ratio of the plants were observed. The results showed that the ⁇ generation showed a 1:3 ratio of high and low separation from the seedling stage.
  • the kanamycin smear showed that the dwarf tightening trait was co-segregated with the transgenic resistance marker gene Nptll, indicating that this dwarf tightening trait is Genetic.
  • Further genetic analysis of T 2 plants showed that the ratio of tall and dwarf plants in the 2nd generation plants was still 1:3 (see Table 2), which was consistent with the genetic expression of a pair of single dominant genes, so the dwarf The mutation is a dominant mutation.
  • the mutant/and cotton variety "Zhongmian 24" were identified as follows: Experimental group: The cotton plants in the cotyledon stage were cultured in cotton liquid medium containing 500 ⁇ brassinolide (Sigma) to the five-leaf stage. Hypocotyl length
  • Control group An equal volume of 0.2% aqueous ethanol solution was used instead of brassinolide, and the other experimental groups.
  • the hypocotyl length measurement results of the plants treated in each group (the average of the measurements of each group were 20 plants).
  • Mutant / Hypocotyl elongation after bryolide treatment was 270%, comparable to the length of hypocotyl of the cotton variety "Chongmian 24".
  • the cotton variety "Zhongmian 24" had a 16% elongation of the hypocotyl after treatment with brassinolide.
  • the results showed that brassinolide could restore the dwarf phenotype of the mutant pagodal to the phenotype of the cotton variety "Zhongmian 24", ie the mutant pagodal is a BRs-deficient mutant.
  • the mutant/and cotton variety "Zhongmian Institute 24" were identified as follows: Illumination group: Cotton plants were cultured under continuous light for 2 weeks, photographed and measured for hypocotyl length;
  • the photographs of the plants treated in each group are shown in Fig. 3A, and the results of the hypocotyl length measurement of the plants treated in each group were all the average values of 20 plants. See Fig. 3B.
  • the hypocotyl of the cotton variety "Zhongmian 24” was significantly elongated and did not exhibit photomorphogenesis.
  • the elongation of the mutant/hypocotyls was inhibited compared to the cotton variety "Zhongmian 24", which showed co-morphogenesis of cotyledon opening and hook opening.
  • the mutant pagodal is a BRs-deficient mutant.
  • the dwarf condensed phenotype is co-segregated with T-DNA, using hiTAIL-PCR (Yao-Guang Liu et al, High-effic i ency thermal asymmetric interlaced PCR for ampl i ficat ion of unknown flanking sequences.
  • Yao- Guang Liu and Yuanl ing Chen The method of BioTechniques Vol. 43, No. 5: pp 649-656 (Nov 2007) amplifies the flanking sequence of the T-DNA insertion site.
  • TAIL-PCR also known as thermal asymmetric interlaced PCR
  • thermal asymmetric interlaced PCR is a technique that can effectively separate It is known that the unknown sequence adjacent to the DNA sequence is simple and easy to perform, and the reaction is efficient and sensitive, and the target fragment can be obtained in a short time, which is a very suitable technique in molecular biology research.
  • Professor Liu Yaoguang has made great improvements to the TAIL-PCR method and created a new method of hiTAIL-PCR, which has achieved good amplification effects in many species such as rice, Arabidopsis, and insects.
  • RB-1, RB-2 and RB-3 were designed based on the known T-DNA border sequences using the genomic ⁇ of the mutant pagodal as a template, and three rounds of PCR reactions were performed.
  • RB-1 5' - CGTGACTGGGAAAACCCTGGCGTT- 3';
  • RB-3 5' - GAAGAGGCCCGCACCGATCGCCCTT- 3'.
  • the nested primers were paired with random primers and anchor primers to carry out a 25 ul reaction system, with specific reference to Yao-Guang Liu et al.
  • the product was separated on a 1% agarose gel, and a specific band was recovered using a Promega gel recovery kit, and T-A was cloned according to TAKARA's pMD18-T kit.
  • the reaction system was as follows: DNA fragment 4 ul (25 ng/ul), T carrier lul, Solution I 5 ul, total volume 10 ul.
  • the cells were transformed into a competent cell of Escherichia coli DH5 ⁇ by a water bath at 16 °C for 1 hour, and positive clones were selected on LB plates containing ampicillin, and positive clones were selected for sequencing.
  • the sequencing results showed that a 1.5 kb flanking sequence of the T-DNA insertion site was obtained.
  • BAC library of the existing pagodal in the laboratory was screened by PCR.
  • PCR reaction system 25ul, of which lOXBuffer 2.5ul, dNTP Mixture (lOuM) 2ul, Ex Taq (5u/ul) 0.5ul, BAC library plasmid lul (125ng), upstream primer (lOuM) lul, downstream primer (lOuM) lul, ddH 2 0 is added to 25ul.
  • Reaction conditions pre-denaturation at 94 ° C for 5 min; 94 ° C for 30 s, 58 ° C for 30 s, 72 ° Clmin, 30 cycles; 72 ° C extension for 5 min. Five positive clones were screened and three of them were sequenced.
  • the sequencing results showed that the T-DNA was inserted upstream of a gene promoter.
  • the nucleotide sequence of the gene was as shown in sequence 3 in the sequence listing.
  • GhPAGODAl ⁇ M (referred to as i3 ⁇ 4 3 ⁇ 4y?7 gene).
  • the coding region sequences in the ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene are as follows: Sequence 3 of the Sequence Listing from nucleotides 1 to 279 at the 5' end, nucleotides 1135 to 1356, nucleotides 1457 to 1703 , nucleotides 1849 to 2216, nucleotides 2356 to 2814.
  • the cDNA of the GhPGD1 gene is shown in Table 2 (1800 bp), and its open reading frame is the sequence of the sequence 2 from the 5' end of the 133th to 1707th nucleotide (1575bp).
  • the GhPGD1 gene encodes the GhPGD1 protein (composed of 524 amino acid residues) shown in SEQ ID NO: 1.
  • the dwarfed phenotype of the mutant pagodal may be due to overexpression of the GhPGD1 gene.
  • Extract mutant pagodal and cotton separately The total RNA of the cultivar "Zhongmian 24" was reverse transcribed into cDNA.
  • Real-time PCR was performed using primer pairs consisting of qpgdl-S and qpgdl-A to identify the expression of ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene.
  • the cotton housekeeping gene Hi stone 3 was used as an internal reference gene, and the primer pair consisting of Hi stone3-S and Hi stone3-A was subjected to Real- ime PCR.
  • the relative expression level of GhPGD1 gene in cotton variety "Zhongmian 24" is 1.
  • the relative expression of GhPGD1 gene in mutant pagodal is shown in Fig. 5.
  • the expression level of GhPGD1 gene in mutant pagodal was 30 times higher than that of cotton variety "Zhongmian 24". Therefore, it was confirmed that the dominant dwarf phenotype of the mutant pagodal was caused by overexpression of the GhPGD1 gene downstream of the T-DNA insertion region.
  • a primer pair consisting of pgd l-s and pgd l-a was used for PCR amplification to obtain a PCR amplification product.
  • step 3 The PCR amplification product of step 2 was digested with restriction endonucleases Xbal and Sac l, and the digested product was recovered.
  • the recombinant plasmid pCAMBIA2300-35S-nos was digested with restriction endonuclease Xbal and Sac l to recover a vector backbone of about 10 kb.
  • the vector pCAMBIA2300 was used as the backbone, and the 35S promoter shown in SEQ ID NO: 4 of the sequence listing was inserted between the Hindl ll and Xbal cleavage sites, and the Sac l and EcoRI cleavage sites were used. The nos terminator shown in sequence 5 of the sequence listing is inserted.
  • the recombinant plasmid pCAMBIA-i3 ⁇ 43 ⁇ 43 ⁇ 4?7 was introduced into Agrobacterium strain LBA4404 to obtain recombinant Agrobacterium.
  • step 2 The recombinant Agrobacterium obtained in step 1 is transformed into Colombian ecotype Arabidopsis by inflorescence infection method.
  • the specific steps are as follows:
  • Arabidopsis seeds were sterilized for 10 min with an aqueous solution containing 0.01% by volume of Triton X-100 and lOg/lOOmL NaCIO, and then washed 6 times with sterile water in a clean bench.
  • step (2) Seeding the seed of step (1) into MS medium containing 3.0g/100mL sucrose and 0.8g/100mL agar powder for 3-4 days after being vernalized in artificial climate chamber (22°C, 70% relative humidity, The light intensity was 150 ⁇ 1 m - 2 s - 12 h light / 12 h dark) and cultured for 1 week.
  • step (3) Move the seedlings of step (2) to the culture soil (the equal mass of mixed charcoal soil and vermiculite, and place the flowerpots containing the culture soil in a plastic box with water before the seedlings to make the water pass the flowers.
  • the small hole at the bottom of the basin is infiltrated, and the culture soil in the flower pot can be moved after being soaked, and the seedling is covered with plastic wrap for 4 days, and the film is uncovered for 4 weeks (22 ° C, 70% relative).
  • Humidity, light intensity is 150 ⁇ 1 m— 2 s—12h light/12h darkness.
  • step 1 (4) recombinant obtained in step 1 with Agrobacterium bacterial cell suspension (sucrose concentration of 50g / L, containing 200uL / L silwet-77, other solutes and their concentrations with MS medium) was suspended to give 0D 6. .
  • a cell suspension with nm 0.8.
  • step (3) Soak the whole inflorescence of the plant of step (3) to the bacterial suspension obtained in step (4) for 45 s, remove the plants and store them in the dark for 24 hours, and then culture the plants for 1 week (22 ° C, 70%). Relative humidity, light intensity is 150 ⁇ m— 2 s—12h light/12h darkness, and 1 ⁇ generation seeds are harvested.
  • the 1 ⁇ generation seed was sown in MS medium containing 50 mg/L kanamycin and cultured normally to obtain a 1 ⁇ generation plant. (7) Self-crossing the 1 ⁇ generation plants and harvesting 1 ⁇ generation seeds.
  • the recombinant plasmid pCAMmIA-GhPGD1 was replaced with the recombinant plasmid pCAMBIA2300-35S-nos, and the same as step 2, the transduced vector Arabidopsis thaliana was obtained.
  • T 3 generation seeds (20 per line) of 5 homozygous transgenic lines (strain 1, line 2, line 3, line 4 and line 5) were randomly selected and sown in soil. Normal culture (22 ° C, 70% relative humidity, light intensity 150 ⁇ 1 m - 2 s - 12 h light / 12 h dark), counted from the start of sowing, photographed 30 days later, measured plant height and detected gene expression.
  • the photo of the plant is shown in Figure 6.
  • the average plant height of line 1 was 25.11 ⁇ 1.54 cm.
  • the average plant height of line 2 was 19.78 ⁇ 2.05 cm.
  • the average plant height of line 3 was 12.78 ⁇ 1.39 cm.
  • the average plant height of line 4 was 6.33 ⁇ 1.2 cm.
  • the average plant height of line 5 was 3.83 ⁇ 0.71 cm.
  • the average plant height of the transgenic vector Arabidopsis thaliana was 31.89 ⁇ 2.15 cm.
  • the average plant height of the Colombian ecotype Arabidopsis thaliana is 32.17 ⁇ 1.46 cm.
  • RNA of each plant leaf was extracted and reverse transcribed into cDNA.
  • real-time PCR was performed using primer pairs consisting of qpgdl-S and qpgdl-A to identify the expression of GhPGD1 gene.
  • the actinl gene was used as an internal reference gene, and the primer pair consisting of actinl-S and actinl-A was used for Real-time PCR.
  • the relative expression levels of the genes in each line are shown in Figure 7. In the ecotype Arabidopsis thaliana and the transgenic vector Arabidopsis thaliana, there is no LY3 ⁇ 43 ⁇ 43 ⁇ 4?7 gene expression. The degree of gene expression varies among individual transgenic lines.
  • qpgdl-S 5' - CATTGGAAGAAAATCTATGGTGC - 3'; qpgdl-A: 5' - ATGATGAGCCCATTTTTCGC-3'.
  • the mutant/and cotton variety "Zhongmian Institute 24" were identified as follows: Cotton plants were planted in the Anyang field during the sowing period, and normal field management was carried out. The phenotype was observed and photographed in early October. The photo is shown in Figure 8. Most of the leaves of the cotton variety "Zhongmian 24" are yellow and withered, while the mutant / leaves are still green and the apical tissue can continue to grow. The results showed that overexpression of the ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene delayed plant senescence and prolonged the life cycle of the plant. Experimental Example 4. Acquisition of genetically modified cotton
  • the cotton seedlings of the cotton variety "Zhongmian 24" which grow 4-5 pieces of true leaves are used as rootstocks, and the regenerated seedlings are grafted and cultured in the greenhouse (culture conditions are: 14h light/10h dark; daytime 28-35°) C, light intensity is 150 ⁇ 1 m - 2 s - night 25-28 ° C), harvest 1 ⁇ generation seeds.
  • Seeding 1 ⁇ generation seeds cultured in the greenhouse (culture conditions: 14h light/10h dark; daytime 28-35°C, light intensity 150 ⁇ 1 m— 2 s—night 25-28°C), Kanamycin Identification by smear, screening positive plants, selfing the 1 ⁇ generation plants, and harvesting 1 ⁇ generation seeds.
  • Seeding 1 ⁇ generation seeds, cultured in the greenhouse (culture conditions: 14h light / 10h dark; daytime 28-35 °C, light intensity 150 ⁇ 1 m - 2 s - night 25-28 ° C), Kanamycin Identification by smear, screening positive plants.
  • the genomic ⁇ of the leaves of 1 ⁇ generation plants and T 2 generation plants were extracted, and the primer pairs consisting of pgdl-s and pgdl-a were identified by PCR, and the target sequence was about 1.6 kb.
  • the ⁇ 2 generation plants were homozygous transgenic plants, and the plant and its progeny were a homozygous transgenic line. A total of 16 pure and transgenic lines were obtained.
  • the homozygous transgenic plants ( ⁇ 2 generation plants) are selfed, and the 3rd generation seeds are harvested.
  • the recombinant plasmid pCAMBIA2300-35S-nos was used in place of the recombinant plasmid pCPmik-GhPGD1, and in the same manner as in the first step, the transduced vector cotton was obtained.
  • T 3 generation seeds (15 per line) of 4 pure transgenic lines (strain a, line b, line c, line d) were randomly seeded in the greenhouse soil. Observe its traits during the flowering period, measure and photograph.
  • Each transgenic line showed short side branches, shortened internodes, and dwarfed plants.
  • the average plant height of line a was 58.77 ⁇ 2.55 cm.
  • the average plant height of line b was 35.92 ⁇ 2.47 cm.
  • the average plant height of strain c was 29.85 ⁇ 1.99 cm.
  • the average plant height of line d was 21.77 ⁇ 3.03 cm.
  • the average plant height of the empty carrier cotton was 89.46 ⁇ 3.31 cm.
  • the average plant height of Zhongmian 24 is 90.77 ⁇ 3.14 cm.
  • the results indicate that cotton overexpressing the ⁇ 3 ⁇ 4 3 ⁇ 43 ⁇ 4?7 gene The plant height was significantly lower than that of the cotton and the target plants.
  • the present invention discloses a protein derived from cotton, associated with dwarfing of plants and inactivation of brassinolide, and a gene encoding the same.
  • Overexpression of the gene in plants can reduce endogenous brassinolide content, manifested by hypocotyl shortening, plant dwarfing, petiole shortening, internode shortening, leaf dark green and life cycle extension, and still in dark conditions It shows the formation of light form.
  • the use of this gene can improve and shape plant types and delay plant senescence.
  • the gene is dominant, which is of great value for improving plants (especially crops), shortening breeding years, and improving breeding efficiency.
  • the dwarf gene provided by the invention genetically regulates the plant height of the cotton, selects the variety with the appropriate plant height and the ideal plant type, and utilizes the utilization potential of the photothermal resource and the space-time advantage of cotton flowering into the bell to improve the cotton economy.
  • the coefficient can reduce chemical regulation and manual regulation, reduce cotton production costs, and improve the economic benefits of cotton planting.
  • the protein and the coding gene thereof provided by the invention have extremely important application value for improving the yield of crops (fruit trees), improving the ornamental properties of green plants, realizing light planting and simplifying planting, and improving breeding efficiency, in genetic improvement of plants, cultivation and application of new varieties.
  • the aspect has broad application prospects.

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Abstract

本发明公开了一种植物株型相关蛋白及其编码基因与应用。本发明提供的蛋白是如下（a）或（b）或（c）：（a）序列 1 所示的氨基酸序列组成的蛋白质；（b）序列 1 经过氨基酸残基的取代和/或缺失和/或添加且与植物株型和/或植物油菜素内酯失活相关的由序列 1 衍生的蛋白质；（c）与序列 1 具有 80%以上的同源性且与植物株型和/或植物油菜素内酯失活相关的蛋白质。本发明提供的蛋白质及其编码基因对提高作物产量，提高绿化植物观赏性，实现植物轻简化种植，提高育种效率具有极其重要的应用价值，在植物的遗传改良、新品种培育和应用方面具有广阔的应用前景。

Description

植物株型相关蛋白及其编码基因与应用 技术领域
本发明属于生物技术和基因工程领域， 涉及一种植物株型相关蛋白及 其编码基因与应用， 特别是来源于棉花的植物矮化相关蛋白及其编码基因 与应用。
背景技术
株型 （plant type ) ， 是指与作物品种产量能力有关的一组特征或植 物体在空间的排列方式， 即长势长相。 理想株型 （Ideal plant type ) 亦 称为理想型 （Ideotype ) ， 指由有利于植株光合作用、 生长发育和籽粒产 量的性状所组成的理想化株型， 它能最大限度提高群体光能利用率， 增加 生物学产量和提高经济系数等。
株型中的重要因素之一是株高， 株高不仅是影响作物株型的决定因 素， 也是决定产量的重要农艺性状。 为了提高作物的产量， 促使其由自然 生长向目标生长转变， 引导植株的生长发育及外貌长相朝着更有利于高 产、 优质、 低耗、 高效的方向发展， 近年来人们对作物矮化增产效应进行 了大量试验研究。结果表明通过植株适当矮化，增大群体结构可实现增产。
棉花是我国最重要的经济作物之一。 棉花原为多年生木本植物， 经过 长期驯化， 变为一年生作物， 保留了木本植物无限生长习性。 棉株高大， 枝叶繁茂， 往往造成田间荫蔽， 通风透光不良， 蕾铃大量脱落， 且易倒伏， 棉铃易受病菌感染， 形成烂铃和僵瓣花， 严重影响棉花产量和品质。 我国 南方以杂交棉为主， 且棉花生长时期光、 热、 水同步， 营养生长更加旺盛， 株型更难控制， 易出现高、 大、 空的群体， 造成减产。 塑造理想株型， 协 调好棉株生育和外界环境条件、营养生长和生殖生长、个体和群体的关系， 使棉株有一个适应不同生态区特点的合理的生育进程， 对实现棉花优质高 产至关重要。
棉花生产中， 主要通过栽培技术使棉花株高降低、 果枝缩短、 叶片缩 小。 通过控制营养体生长， 促进生殖器官生长， 以保持棉田的通风透光， 获得较多的亩铃数， 调节棉花的经济系数， 实现较高的产量。 目前我国主 要作物中， 水稻、 小麦是利用矮化品种实现的， 而棉花是通过栽培手段强 制矮化的， 即通过肥水调控、 减少营养供应、 打顶抑制顶端优势、 缩节安 化控等， 需要花费大量劳动力和化石能源， 增加了棉花生产成本。
发明公开
本发明的目的是提供一种植物株型相关蛋白及其编码基因与应用。 本发明的目的是提供一种植物株型相关蛋白及其编码基因与应用。 本发明提供的蛋白， 来源于陆地棉 iGossypiwn hirsutwn) ， 命名为 GhPGDl蛋白， 是如下 （a) 或 （b) 或 （c) 或 （d) 或 （e) 的蛋白质：
(a) 由序列表中序列 1所示的氨基酸序列组成的蛋白质；
(b) 将序列 1的氨基酸序列经过一个或几个氨基酸残基的取代和 /或 缺失和 /或添加且与植物株型相关的由序列 1衍生的蛋白质；
(c)与序列 1的氨基酸序列具有 80%以上的同源性且与植物株型相关 的由序列 1衍生的蛋白质；
(d) 将序列 1的氨基酸序列经过一个或几个氨基酸残基的取代和 /或 缺失和 /或添加且与植物油菜素内酯失活相关的由序列 1衍生的蛋白质； (e)与序列 1的氨基酸序列具有 80%以上的同源性且与植物油菜素内 酯失活相关的由序列 1衍生的蛋白质。
为了使 （a) 中的蛋白质便于纯化， 可在由序列表中序列 1所示的 氨基酸序列组成的蛋白质的氨基末端或羧基末端连接上如表 1 所示 的标签。
表 1 标签的序列
Figure imgf000003_0001
上述 （b) 或 （c) 或 （d) 或 （e) 中的蛋白质可人工合成， 也可先合 成其编码基因， 再进行生物表达得到。 上述 （b) 或 （c) 或 （d) 或 （e) 中的蛋白质的编码基因可通过将序列表中序列 2所示的 DNA序列中缺失一 个或几个氨基酸残基的密码子， 和 /或进行一个或几个碱基对的错义突变， 和 /或在其 5'端和 /或 3'端连上表 1所示的标签的编码序列得到。 编码所示 GhPGDl 蛋白的基因也属于本发明的保护范围， 将该基因命 名为 ί¾ ¾¾?7基因。
所述基因具体可为为如下 1) 或 2) 或 3) 或 4) 或 5) 或 6) 或 7) 或 8) 的 DNA分子:
1) 编码区如序列表中序列 2 自 5 '末端第 133至 1704位核苷酸所示 的 DNA分子；
2) 编码区如序列表中序列 2 自 5 '末端第 133至 1707位核苷酸所示 的 DNA分子；
3) 序列表中序列 2所示的 DNA分子；
4) 序列表中序列 3所示的 DNA分子；
5) 在严格条件下与 1) 或 2) 或 3) 或 4) 限定的 DNA序列杂交且编码 植物株型相关蛋白的匪分子；
6) 与 1) 或 2) 或 3) 或 4) 限定的 DNA序列具有 80%以上的同源性且 编码植物株型相关蛋白的匪分子；
7) 在严格条件下与 1) 或 2) 或 3) 或 4) 限定的 DNA序列杂交且编码 植物油菜素内酯失活相关蛋白的匪分子；
8) 与 1) 或 2) 或 3) 或 4) 限定的 DNA序列具有 80%以上的同源性且 编码植物油菜素内酯失活相关蛋白的 DNA分子。
上述严格条件可为在 6XSSC, 0.5% SDS的溶液中， 在 65°C下杂交， 然后用 2XSSC、 0.1% SDS和 1XSSC、 0.1% SDS各洗膜一次。
含有所述 ί¾ ¾¾?7基因的重组表达载体、 表达盒、 转基因细胞系或重 组菌均属于本发明的保护范围。
可用现有的植物表达载体构建含有所述基因的重组表达载体。 所述植 物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。 所述植 物表达载体还可包含外源基因的 3' 端非翻译区域， 即包含聚腺苷酸信号 和任何其它参与 mRNA加工或基因表达的匪片段。 所述聚腺苷酸信号可 引导聚腺苷酸加入到 mRNA前体的 3' 端。使用所述基因构建重组表达载体 时， 在其转录起始核苷酸前可加上任何一种增强型启动子或组成型启动 子， 它们可单独使用或与其它的植物启动子结合使用； 此外， 使用本发明 的基因构建重组表达载体时， 还可使用增强子， 包括翻译增强子或转录增 强子， 但必需与编码序列的阅读框相同， 以保证整个序列的正确翻译。 所 述翻译控制信号和起始密码子的来源是广泛的， 可以是天然的， 也可以是 合成的。 翻译起始区域可以来自转录起始区域或结构基因。 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， 如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因、 具有抗性的抗生素标记物或是抗化学试剂标记基因等。
所述重组表达载体具体可为在载体 PCAMBIA2300的多克隆位点插入所 述 基因得到的重组质粒。
所述重组表达载体具体可为在载体 PCAMBIA2300的多克隆位点插入所 述 基因的表达盒得到的重组质粒。 所示表达盒中， 由 35S启动子 启动所述 基因的表达，由 nos终止子终止所述 ί¾ ¾¾?7基因的表达。
所述 35S启动子具体如序列表的序列 4所示。 所述 nos终止子具体如 序列表的序列 5所示。
所述重组表达载体具体可为如下重组质粒： 以载体 PCAMBIA2300为骨 架， 在其 Hi ndl l l和 Xbal酶切位点之间插入了所述 35S启动子， Xbal和 Sad酶切位点之间插入了所述 GhPGDl纖， Sac l和 EcoRI酶切位点之间 插入了所述 nos终止子。
所述 GhPGD l蛋白、 所述 基因、 所述表达盒、 重组表达载体、 转基因细胞系或重组菌均可用于改良植物株型。 所述改良植物株型具体体 现为使植株矮化。 所述植物为单子叶植物或双子叶植物。 所述双子叶植物 可为棉花， 具体可为棉花品种"中棉所 24 "。所述双子叶植物可为拟南芥， 具体为哥伦比亚生态型拟南芥。
所述 GhPGD l蛋白、 所述 基因、 所述表达盒、 重组表达载体、 转基因细胞系或重组菌均可用于培育转基因植物。 所述转基因植物具体为 具有矮化表型的植物。 所述植物为单子叶植物或双子叶植物。 所述双子叶 植物可为棉花， 具体可为棉花品种 "中棉所 24 " 。 所述双子叶植物可为拟 南芥， 具体为哥伦比亚生态型拟南芥。
本发明还保护一种培育转基因植物的方法， 是将所述 GhPGDl基因导 入目的植物， 得到株高小于所述目的植物的转基因植物。 携带有所述基因 的表达载体可通过使用 Ti质粒、 Ri质粒、 植物病毒载体、 直接 DNA转化、 显微注射、 电导、 农杆菌介导等常规生物学方法转化植物细胞或组织， 并 将转化的植物组织培育成植株。 所述 GhPGDl基因具体可通过所述重组表 达载体导入所述目的植物。 所述目的植物为单子叶植物或双子叶植物。 所 述双子叶植物可为棉花， 具体可为棉花品种 "中棉所 24 "。 所述双子叶植 物可为拟南芥， 具体为哥伦比亚生态型拟南芥。
本发明还保护一种培育转基因植物的方法， 是在目的植物中过表达所 述 GhPGDl 繊， 得到株高小于所述目的植物的转基因植物。 所述 "在目 的植物中过表达所述 GhPGDl基因" 可通过将所述基因导入目的植物实现 或通过促进目的植物本身具有的所述 基因的表达实现 （如通过引 入启动子或增强子促进所述基因的表达） 。 所述目的植物为单子叶植物或 双子叶植物。所述双子叶植物可为棉花， 具体可为棉花品种"中棉所 24 "。 所述双子叶植物可为拟南芥， 具体为哥伦比亚生态型拟南芥。
油菜素内酯 （Brass inostero i ds，BRs)为一类植物特异的甾醇类激素， 广泛存在于植物体中， 调节植物生长发育的多个方面， 包括营养生长、 生 殖生长、 萌发、 衰老以及对多种生物胁迫和非生物胁迫的响应。 极低浓度 ( nmo l/L ) 的油菜素内酯就表现出极高的生理活性， 因此被认为是继生长 素、 赤霉素、 细胞分裂素、 脱落酸和乙烯之后的第六类植物激素。
本发明还保护一种培育转基因植物的方法， 是在目的植物中过表达所 述 GhPGDl 繊， 得到油菜素内酯缺陷型的转基因植物。 所述油菜素内酯 缺陷型体现为与目的植株相比， 所述转基因植株具有如下表型中的至少一 种： ①下胚轴缩短； ②株高降低； ③叶柄和 /或叶鞘缩短； ④开花延迟； ⑤生命周期延长； ⑥在黑暗条件下具有光形态建成反应的表型。 所述 "在 目的植物中过表达所述 GhPGDl纖"通过将所述 基因导入目的植 物实现或通过促进目的植物本身具有的所述 GhPGDl基因的表达实现 （如 通过引入启动子或增强子促进所述基因的表达） 。 所述目的植物为单子叶 植物或双子叶植物。 所述双子叶植物可为棉花， 具体可为棉花品种 "中棉 所 24 " 。 所述双子叶植物可为拟南芥， 具体为哥伦比亚生态型拟南芥。
本发明还保护一种塑造植物造型的方法， 是对植物局部喷施油菜素内 酯 （仅促进喷施部位生长） ， 使所述植物生长为预期的造型； 所述植物为 以上任一所述方法得到的转基因植物、 所述转基因植物的自交后代、 所述 转基因植物的杂交后代或所述转基因植物的回交后代。
以下结合附图及具体实施例进一步阐述本发明。 以下的实施例便于更 好地理解本发明， 但并不限定本发明。
附图说明
图 1为棉花品种 "中棉所 24" 与突变体 pagodal的表型比较。
图 2为突变体 和棉花品种"中棉所 24"对油菜素内酯的反应。 图 3为突变体/^ 和棉花品种 "中棉所 24" 的光形态建成反应。 图 4为通过局部涂抹油菜素内酯塑造棉花造型。
图 5为棉花品种 "中棉所 24" 与突变体 pagodal中 ί¾ ¾¾?7基因的相 对表达量比较。
图 6为哥伦比亚生态型拟南芥与转基因拟南芥的表型比较。
图 7为哥伦比亚生态型拟南芥与转基因拟南芥中 ί¾ ¾¾?7基因的相对 表达量比较。
图 8为突变体 和棉花品种 "中棉所 24" 10月中旬在河南安 阳的生长状态比较。
图 9为棉花品种 "中棉所 24" 与转基因棉花的表型比较。
实施发明的最佳方式
以下的实施例便于更好地理解本发明， 但并不限定本发明。 下述实施 例中的实验方法， 如无特殊说明， 均为常规方法。 下述实施例中所用的试 验材料， 如无特殊说明， 均为自常规生化试剂商店购买得到的。 以下实施 例中的定量试验， 均设置三次重复实验， 结果取平均值。
棉花品种 "中棉所 24 " (用 WT表示）：为陆地棉（ Gossypiim hirsutum) 的一个品种， 由中国农业科学院棉花研究所选育， 可以从中棉种业或其他 种子公司购买。
哥伦比亚生态型拟南芥（Col- 0)：购自 ABRC(Arabidopsis Biological
Resource Center)。
植物表达载体 PCAMBIA2300 (简称载体 pCAMBIA2300) ： 购自 Cambia (http://www. cambia. org/ daisy/ cambia/585. html ) 。
农杆菌菌株 LBA4404: 购自 clontech。 实施例 1、 棉花 GhPGDl蛋白及其编码基因的获得
一、 棉花矮化紧缩突变体的获得和遗传分析
在利用激活标签进行棉花品种 "中棉所 24"的遗传转化时获得一个植 株矮化紧缩的突变体， 将其命名为突变体/ (用 / 表示） 。 棉花品种 "中棉所 24" (用 WT表示） 与突变体 pagodal的表型比较见图 1， 与棉花品种 "中棉所 24"相比， 突变体 pagodal的株高极端矮化 （图 1A) ， 花器官缩小 （图 1B) ， 叶柄缩短 （图 1C， 倒 4叶叶柄） 。
将棉花品种 "中棉所 24"和突变体 pagodal杂交， 获得 T。代， T。代种 子种植后收获 1\代植株， 观察记录 Ί\代植株的表型及分离比。 结果表明 Ί\代从幼苗期开始就表现出 1:3的高矮分离比， 经卡那霉素涂抹表明矮化 紧缩性状与转基因抗性标记基因 Nptll共分离， 说明这一矮化紧缩性状是 可遗传的。 对 T2代植株进行进一步的遗传分析， Τ2代植株中高杆和矮杆植 株的比例仍为 1:3 (见表 2) ， 符合一对单显性基因控制的遗传表现， 因 此该矮化突变为显性突变。
表 2 1^代群体分离
Figure imgf000008_0001
二、 突变体/ 对油菜素内酯的反应
将突变体/ 和棉花品种 "中棉所 24" 分别进行如下鉴定： 实验组： 将子叶期的棉花植株在含 500ηΜ油菜素内酯 （Sigma) 的棉花 液体培养基培养到五叶期， 测量下胚轴长度；
对照组： 用等体积的 0.2%乙醇水溶液代替油菜素内酯， 其它同实验组。 各组处理的植株的下胚轴长度测量结果 （各组的测量结果均为 20株植 株的平均值） 见图 2。 突变体/ 在油菜素内酯处理后下胚轴伸长了 270%, 与棉花品种 "中棉所 24"下胚轴长度相当。 棉花品种 "中棉所 24" 在油菜素内酯处理后下胚轴仅伸长了 16%。 结果表明， 油菜素内酯可以将 突变体 pagodal的矮化表型恢复为棉花品种 "中棉所 24"的表型， 即突变 体 pagodal是一种 BRs缺陷型突变体。
三、 突变体/ 的光形态建成反应
将突变体/ 和棉花品种 "中棉所 24" 分别进行如下鉴定： 光照组： 将棉花植株在持续光照条件下培养 2周， 拍照并测量下胚轴 长度；
黑暗组： 将棉花植株在持续黑暗条件下培养 2周， 拍照并测量下胚轴 长度。
各组处理的植株的照片见图 3A，各组处理的植株的下胚轴长度测量结 果各组的测量结果均为 20株植株的平均值） 见图 3B。 在黑暗条件下， 棉 花品种 "中棉所 24 " 的下胚轴显著伸长， 未表现出光形态建成反应。 在黑 暗条件下， 与棉花品种 "中棉所 24 "相比， 突变体/ 下胚轴的伸长 受到抑制， 表现出子叶张开和弯钩打开这些光形态建成反应。 进一步说明 突变体 pagodal是一种 BRs缺陷型突变体。
四、 通过局部涂抹油菜素内酯塑造棉花造型
由于油菜素内酯在植物体内不能进行长距离运输， 进行局部涂抹可以 促进涂抹部位的生长而不影响距离较远的部位。 利用 500nM的油菜素内酯 涂抹突变体 幼苗的顶端， 发现油菜素内酯可以明显促进幼苗顶端 生长， 涂抹部位以下则不受影响 （见图 4A) 。 利用 500nM 的油菜素内酯 涂抹突变体/ 幼苗的侧枝， 侧枝明显伸长， 长度甚至超过主茎， 而 主茎部分生长不受影响 （见图 4B ) 。
五、 棉花 GhPGDl蛋白及其编码基因的获得
矮化紧缩表型与 T-DNA共分离，采用 hiTAIL-PCR (Yao-Guang Liu 等， High-effic i ency thermal asymmetric interlaced PCR for ampl i ficat ion of unknown flanking sequences. Yao- Guang Liu and Yuanl ing Chen. BioTechniques Vol. 43， No. 5： pp 649-656 (Nov 2007) 的方法扩增 T-DNA插入位点的侧翼序列。 TAIL-PCR又称热不对称交错 PCR， 这一技术能有效分离与已知 DNA序列邻近的未知序列， 简单易行， 反应高 效灵敏， 能够短时间内获得目标片段， 是分子生物学研究中非常适宜的技 术。 为了提高获得特异的、 长片段目标产物的成功率， 刘耀光教授对 TAIL-PCR方法作了较大改进， 创建了新的 hiTAIL-PCR方法， 该方法在水 稻、 拟南芥、 昆虫等多个物种中都获得了良好地扩增效果。
以突变体 pagodal的基因组匪为模板， 根据已知的 T-DNA边界序列 设计巢式引物 RB-1 、 RB-2和 RB-3 , 进行三轮 PCR反应。 RB-1: 5' - CGTGACTGGGAAAACCCTGGCGTT- 3'； RB-3: 5' - GAAGAGGCCCGCACCGATCGCCCTT- 3'。
巢式引物与随机引物和锚定引物配对进行 25ul的反应体系，具体参照 Yao-Guang Liu 等论文。 三轮巢式 PCR后， 产物经 1%琼脂糖凝胶分离， 采 用 Promega胶回收试剂盒回收特异条带， 按照 TAKARA的 pMD18-T试剂盒 进行 T-A克隆。反应体系如下： DNA片段 4ul( 25ng/ul )， T载体 lul， Solution I 5ul， 总体积 10ul。 16°C水浴连接 1小时， 转化大肠杆菌 DH5 α 的感受 态细胞， 在含有氨苄青霉素的 LB 平板上筛选阳性克隆， 挑选阳性克隆进 行测序。 测序结果表明， 获得了 T-DNA插入位点 1.5kb的旁侧序列。
在 T-DNA和旁侧序列部分设计引物如下：
RB1 5' - CAGATTGTCGTTTCCCGCCTTCAG- 3'，
FL1 5' - TCAGACGAGCAATACTCCACAGCAGG- 3'。
用 PCR池的方法筛选实验室已有的 pagodal 的 BAC文库。 PCR反应体 系 25ul， 其中 lOXBuffer 2.5ul, dNTP Mixture (lOuM) 2ul， Ex Taq (5u/ul) 0.5ul, BAC文库质粒 lul (125ng) ， 上游引物 (lOuM) lul, 下游引物 (lOuM) lul, ddH20补齐至 25ul。 反应条件： 94°C预变性 5min; 94°C30s， 58°C30s， 72°Clmin, 30个循环； 72°C延伸 5min。 筛选到 5个 阳性克隆， 对其中的 3个进行测序， 测序结果表明， T-DNA插入到一个基 因启动子上游， 该基因的核苷酸序列如序列表中序列 3所示， 将其命名为 GhPAGODAl^M (简称 i¾ ¾y?7基因） 。
ί¾ ¾¾?7基因中的编码区序列如下： 序列表的序列 3 自 5' 末端第 1至 第 279位核苷酸、 第 1135至第 1356位核苷酸、 第 1457至第 1703位核苷 酸、 第 1849至第 2216位核苷酸、 第 2356至第 2814位核苷酸。
GhPGDl基因的 cDNA如序列表 2所示 （1800bp) ， 其开放阅读框为序 列表的序列 2 自 5' 末端第 133至 1707位核苷酸 （1575bp) 。
GhPGDl基因编码序列表的序列 1所示的 GhPGDl蛋白 （由 524个氨基 酸残基组成） 。
由于 T-DNA区含有 35S增强子， 因此突变体 pagodal的矮化紧缩表型 可能由于 GhPGDl基因过量表达造成的。 分别提取突变体 pagodal和棉花 品种"中棉所 24 "的叶片总 RNA并反转录成 cDNA，采用 qpgdl-S和 qpgdl-A 组成的引物对进行 Real-t ime PCR， 鉴定 ί¾ ¾¾?7基因的表达量。 以棉花看 家基因 Hi stone 3作为内参基因， 采用 Hi stone3- S和 Hi stone3- A组成的 引物对进行 Real- t ime PCR。
qpgdl-S 5' - CATTGGAAGAAAATCTATGGTGC - 3'；
qpgdl-A 5' -ATGATGAGCCCATTTTTCGC-3'。
Hi stone3-S 5' - TCAAGACTGATTTGCGTTTCCA- 3'；
Hi stone3-A 5' - GCGCAAAGGTTGGTGTCTTC- 3'。
与棉花品种 "中棉所 24 " 中 GhPGDl基因的相对表达量为 1， 突变体 pagodal中 GhPGDl基因的相对表达量见图 5。 突变体 pagodal中 GhPGDl 基因的表达量比棉花品种 "中棉所 24 " 高出 30多倍。 因此可以确定突变 体 pagodal的显性矮化表型是由于 T-DNA插入区下游的 GhPGDl基因过量 表达造成的。 实施例 2、 转基因拟南芥的获得 （ί¾ ¾¾?7基因的过表达）
一、 重组表达载体的构建
1、 提取棉花品种 "中棉所 24 " 的叶片的总 RNA并反转录为 cDNA。
2、 以步骤 1得到的 cDNA为模板， 用 pgd l-s和 pgd l-a组成的引物对 进行 PCR扩增， 得到 PCR扩增产物。
pgdl-s 5' -GCTCTAGAATGGAGGGTGTTTTACAGTGG-3'，
pgdl-a 5' -CGAGCTCTCATGACCCTTGATCTCTTGT-3'。
3、 用限制性内切酶 Xbal和 Sac l双酶切步骤 2的 PCR扩增产物， 回 收酶切产物。
4 、 用 限 制 性 内 切 酶 Xbal 和 Sac l 双 酶切 重 组质粒 pCAMBIA2300-35S-nos , 回收约 10kb的载体骨架。
重组质粒 pCAMBIA2300-35Sios的构建方法： 以载体 pCAMBIA2300为 骨架， 在其 Hindl l l和 Xbal酶切位点之间插入了序列表的序列 4所示的 35S启动子， Sac l和 EcoRI酶切位点之间插入了序列表的序列 5所示的 nos 终止子。
5、 将步骤 3 的酶切产物和步骤 4 的载体骨架连接， 得到重组质粒 C k-GhPGDl。 根据测序结果， 对重组质粒≠m k-GhPGDl进行结构 描述如下： 以载体 PCAMBIA2300为骨架， 在其 Hindlll和 Xbal酶切位点 之间插入了序列表的序列 4所示的 35S启动子， Xbal和 Sacl酶切位点之 间插入了序列表的序列 2 自 5' 末端第 133至 1707位核苷酸所示的双链 DNA分子， Sacl和 EcoRI酶切位点之间插入了序列表的序列 5所示的 nos 终止子。
二、 转基因拟南芥的获得
1、 将重组质粒 pCAMBIA-i¾¾¾?7导入农杆菌菌株 LBA4404, 得到重组 农杆菌。
2、 将步骤 1 得到的重组农杆菌通过花序侵染法转化哥伦比亚生态型 拟南芥， 具体步骤如下：
(1) 用含 0.01% (体积比） Triton X- 100和 lOg/lOOmL NaCIO的水溶 液将拟南芥种子消毒处理 10min，然后在超净工作台中用灭菌水清洗 6次。
(2) 将步骤 (1) 的种子播种到含 3.0g/100mL蔗糖、 0.8g/100mL琼 脂粉的 MS培养基春化 3-4天后置于人工气候室 （22°C、 70%相对湿度， 光 强为 150μπιο1 m— 2 s— 12h光照 /12h黑暗） 培养 1周。
(3)将步骤（2) 的小苗移苗至培养土（等质量混合的草炭土和蛭石， 移苗前将装有培养土的花盆放入有水的塑料箱中， 使水通过花盆底部的小 孔上渗， 待花盆中的培养土湿透后即可移苗） ， 用保鲜膜覆盖培养 4d 后 揭膜，从移苗开始共培养 4周（22°C、 70%相对湿度，光强为 150μπιο1 m— 2 s— 12h光照 /12h黑暗） 。
(4) 将步骤 1得到的重组农杆菌用菌体悬浮液 （蔗糖浓度为 50g/L， 含 200uL/L silwet-77, 有其它溶质及其浓度同 MS 培养基） 悬浮， 得到 0D6。。nm=0.8的菌体悬浮液。
(5) 将步骤 （3) 的植株的整个花序浸泡到步骤 （4) 得到的菌体悬 浮液中 45s， 取出植株并避光保存 24h， 然后将植株正常培养 1周 （22°C、 70%相对湿度， 光强为 150 μπιοΐ m— 2 s— 12h光照 /12h黑暗） ， 收获 1\代 种子。
(6)将 1\代种子播种于含 50mg/L卡那霉素的 MS培养基并正常培养， 得到 1\代植株。 (7) 将1\代植株自交， 收获 1^代种子。
(8) 将^代种子播种于 MS培养基并正常培养， 得到 1^代植株。
(9) 将 1\代植株和 T2代植株分别提取叶片的基因组 DNA， 用 pgdl-s 和 pgdl-a组成的引物对进行 PCR鉴定， 靶序列约为 1.6kb。 对于某一 Ί\ 代植株来说， 如果相应的 Τ2代植株均 PCR鉴定为阳性， 该 Τ2代植株为纯合 的转基因植株， 该植株及其后代为一个纯合的转基因株系。
(10) 将纯合的转基因植株 （Τ2代植株） 自交， 收获 Τ3代种子。
共获得了 60个纯合的转基因株系。
三、 转空载体拟南芥的获得
用重组质粒 pCAMBIA2300-35S-nos代替重组质粒 pCPmik- GhPGDl,其 它同步骤二， 得到转空载体拟南芥。
四、 表型鉴定
随机取 5个纯合的转基因株系 （株系 1、 株系 2、 株系 3、 株系 4和株 系 5) 的 T3代种子 （每个株系 20粒） ， 播种于土壤中并正常培养 （22°C、 70%相对湿度， 光强为 150μπιο1 m— 2 s— 12h光照 /12h黑暗） ， 从播种开始 计天数， 30天后拍照、 测量株高并检测 基因的表达量。 将 20粒转 空载体拟南芥的 T3代种子和 20粒哥伦比亚生态型拟南芥种子作为对照， 进行平行处理。
植株照片见图 6。 株系 1的平均株高为 25.11±1.54厘米。 株系 2的 平均株高为 19.78±2.05厘米。 株系 3的平均株高为 12.78± 1.39厘米。 株系 4的平均株高为 6.33±1.2厘米。 株系 5的平均株高为 3.83±0.71 厘米。 转空载体拟南芥的平均株高为 31.89±2.15厘米。 哥伦比亚生态型 拟南芥的平均株高为 32.17±1.46厘米。
分别提取各个植株叶片的总 RNA并反转录为 cDNA， 以 cDNA为模板， 采用 qpgdl- S和 qpgdl- A组成的引物对进行 Real- time PCR, 鉴定 GhPGDl 基因的表达量。采用 actinl基因作为内参基因，采用 actinl-S和 actinl-A 组成的引物对进行 Real-time PCR。 基因在各个株系中的相对表达 量见图 7。 哥伦比亚生态型拟南芥和转空载体拟南芥中， 没有 ί¾¾¾?7基因 表达。 各个转基因株系中， 基因表达程度不同。
qpgdl-S: 5' - CATTGGAAGAAAATCTATGGTGC - 3'； qpgdl-A: 5' - ATGATGAGCCCATTTTTCGC- 3'。
act inl-S 5' - ACTCTCCCGCTATGTATGTCGC- 3'；
act inl-A 5' - AGAAACCCTCGTAGATTGGCAC- 3'。
以上结果表明， 拟南芥的株高与 基因的表达量负相关， 即 基因的表达量越高， 植株的矮化表型越明显。 实施例 3、 棉花 基因具有延迟衰老作用
将突变体/ 和棉花品种 "中棉所 24 " 分别进行如下鉴定： 在播种期将棉花植株种植到安阳田间， 进行正常的田间管理， 十月上 旬观察表型并拍照。 照片见图 8， 棉花品种 "中棉所 24 " 的叶片大部分发 黄、 枯萎脱落， 而突变体/ 的叶片依然浓绿， 且顶端组织仍能继续 生长。 结果表明， ί¾ ¾¾?7基因超表达后能够延迟植物衰老， 延长植物的生 命周期。 实验例 4、 转基因棉花的获得
一、 转基因棉花的获得
1、 取棉花品种 "中棉所 24 " 的种子， 剥去外壳后， 用 0. 1%的升汞浸 泡消毒 5min， 然后用无菌水冲洗 3-5 次， 播种于无菌苗培养基（MS培养 基 +蔗糖 25g/L+琼脂 6. 5g/L， pH值 7. 0 ) 使其萌发成无菌苗。
2、 在无菌工作台， 用经过酒精灯火焰消毒的手术刀片切取生长 7 天 的无菌苗的下胚轴， 将下胚轴切成 0. 5-0. 8cm的切段， 用实施例 2的步骤 二的 1 的重组农杆菌的菌悬液 （0D6。。nJ =0. 5 ) 浸泡 5分钟， 用滤纸吸干 表面的菌液后置于愈伤诱导培养基 （MS 培养基 +30g/L 葡萄糖 +0. 01mg/L 2, 4-D+O. 05mg/L IAA+0. 05mg/L KT+50mg/L卡那霉素， pH 值 6. 5 ) 上， 暗 培养 48小时。
3、 转接到新的愈伤诱导培养基上， 在光照培养室中培养 2 个月 （培 养条件： 28°C， 16h光照 /8h黑暗， 光强为 150μπιο1 m— 2 s— 每 20天继代 一次） 。
4、 转接到再生苗诱导培养基 （MS 培养基 +30g/L 蔗糖 +0. lmg/L IAA+0. lmg/L 6- BA+50mg/L卡那霉素， pH 值 6. 5 ) 上， 在光照培养室中培 养 3个月 （培养条件： 28°C， 16h光照 /8h黑暗， 光强为 150μπιο1 m— 2 s— 每 20天继代一次； 从 1.5个月开始陆续有胚状体产生） ， 得到再生苗。
5、 将长出 4-5 片真叶的棉花品种 "中棉所 24"的棉苗作为砧木， 将 再生苗嫁接后在温室培养（培养条件为： 14h光照 /10h黑暗；白天 28-35°C， 光强为 150μπιο1 m— 2 s— 夜间 25-28°C ) ， 收获 1\代种子。
6、 播种 1\代种子， 在温室培养 （培养条件为： 14h光照 /10h黑暗； 白天 28-35°C， 光强为 150μπιο1 m— 2 s— 夜间 25-28°C) ， 进行卡纳霉素涂 抹鉴定， 筛选阳性植株， 将1\代植株自交， 收获 1^代种子。
7、 播种 1^代种子， 在温室培养 （培养条件为： 14h光照 /10h黑暗； 白天 28-35°C， 光强为 150μπιο1 m— 2 s— 夜间 25-28°C) ， 进行卡纳霉素涂 抹鉴定， 筛选阳性植株。
8、 分别提取 1\代植株和 T2代植株叶片的基因组匪， 用 pgdl-s和 pgdl-a组成的引物对进行 PCR鉴定， 靶序列约为 1.6kb。 对于某一1\代植 株来说， 如果相应的 T2代植株均 PCR鉴定为阳性， 该 Τ2代植株为纯合的转 基因植株， 该植株及其后代为一个纯合的转基因株系。 共获得 16 个纯和 转基因株系。
9、 将纯合的转基因植株 （Τ2代植株） 自交， 收获 Τ3代种子。
二、 转空载体棉花的获得
用重组质粒 pCAMBIA2300-35S-nos代替重组质粒 pCPmik-GhPGDl,其 它同步骤一， 得到转空载体棉花。
三、 表型鉴定
随机取 4个纯和的转基因株系 （株系 a、 株系 b、 株系 c、 株系 d) 的 T3代种子（每个株系 15粒）播种于日光温室土壤中。在盛花期观察其性状， 测量并拍照。 将 20粒转空载体棉花的 Τ3代种子和 20粒棉花品种 "中棉所 24" 的种子作为对照， 进行平行处理
照片如图 9。 各个转基因株系都表现出侧枝变短、 节间缩短、 植株矮 化。 株系 a的平均株高为 58.77±2.55厘米。 株系 b的平均株高为 35.92士 2.47厘米。 株系 c的平均株高为 29.85±1.99厘米。 株系 d的平均株高为 21.77±3.03厘米。 转空载体棉花的平均株高为 89.46±3.31厘米。 中棉所 24的平均株高为 90.77±3.14厘米。 结果表明超表达 ί¾ ¾¾?7基因的棉花的 株高明显低于转空载体的棉花和目的植株。
工业应用
本发明公开了一种来自于棉花， 与植株矮化以及油菜素内酯失活相关 的蛋白质及其编码基因。 在植物中过表达所述基因可以降低内源油菜素内 酯含量， 表现为下胚轴缩短、 植株矮化、 叶柄缩短、 节间缩短、 叶片暗绿 和生命周期延长， 并且在黑暗条件下仍表现出光形态建成。 利用该基因可 以改良和塑造植物株型， 延缓植物衰老。 所述基因为显性， 这对改良植物 (特别是农作物） 、 缩短育种年限、 提高育种效率具有极其重要的价值。 利用本发明提供的矮化基因从遗传上调控棉花株高， 选育出株高适当、 株 型理想的品种， 有利用充分利用了光热资源生产潜力和棉花开花成铃的时 空优势提高棉花经济系数， 又可减少化学调控和人工调控， 降低棉花生产 成本， 提高植棉经济效益。 本发明提供的蛋白质及其编码基因对提高作物 (果木） 产量， 提高绿化植物观赏性， 实现植物轻简化种植， 提高育种效 率具有极其重要的应用价值， 在植物的遗传改良、 新品种培育和应用方面 具有广阔的应用前景。

Claims

权利要求
1、 一种蛋白， 是如下 （a)或（b)或（c) 或（d)或（e) 的蛋白质： (a) 由序列表中序列 1所示的氨基酸序列组成的蛋白质；
(b)将序列 1的氨基酸序列经过一个或几个氨基酸残基的取代和 /或 缺失和 /或添加且与植物株型相关的由序列 1衍生的蛋白质；
(c)与序列 1的氨基酸序列具有 80%以上的同源性且与植物株型相关 的由序列 1衍生的蛋白质；
(d)将序列 1的氨基酸序列经过一个或几个氨基酸残基的取代和 /或 缺失和 /或添加且与植物油菜素内酯失活相关的由序列 1衍生的蛋白质；
(e)与序列 1的氨基酸序列具有 80%以上的同源性且与植物油菜素内 酯失活相关的由序列 1衍生的蛋白质。
2、 编码权利要求 1所述蛋白质的基因。
3、 根据权利要求 2所述的基因， 其特征在于： 所述基因为如下 1)或 2) 或 3) 或 4) 或 5) 或 6) 或 7) 或 8) 的 DNA分子：
1) 编码区如序列表中序列 2自 5 '末端第 133至 1704位核苷酸所示 的 DNA分子；
2) 编码区如序列表中序列 2自 5 '末端第 133至 1707位核苷酸所示 的 DNA分子；
3) 序列表中序列 2所示的 DNA分子；
4) 序列表中序列 3所示的 DNA分子；
5)在严格条件下与 1) 或 2)或 3)或 4) 限定的 DNA序列杂交且编码 植物株型相关蛋白的 DNA分子；
6) 与 1)或 2)或 3)或 4) 限定的 DNA序列具有 80%以上的同源性且 编码植物株型相关蛋白的 DNA分子；
7)在严格条件下与 1)或 2)或 3)或 4) 限定的 DNA序列杂交且编码 植物油菜素内酯失活相关蛋白的 DNA分子；
8) 与 1)或 2)或 3)或 4) 限定的 DNA序列具有 80%以上的同源性且 编码植物油菜素内酯失活相关蛋白的 DNA分子。
4、 含有权利要求 2或 3所述基因的表达盒、 重组表达载体、 转基因
16
更正页 （细则第 91条） ISA/CN 细胞系或重组菌。
5、 权利要求 1所述蛋白、 权利要求 2或 3所述基因、 权利要求 4所 述表达盒、 重组表达载体、 转基因细胞系或重组菌在改良植物株型中的应 用。
6、 如权利要求 5所述的应用， 其特征在于： 所述改良植物株型体现 为使植株矮化。
7、 如权利要求 5或 6所述的应用， 其特征在于： 所述目的植物为双 子叶植物或单子叶植物。
8、 权利要求 1所述蛋白、 权利要求 2或 3所述基因、 权利要求 4所 述表达盒、 重组表达载体、 转基因细胞系或重组菌在培育转基因植物中的 应用。
9、 如权利要求 7所述的应用， 其特征在于： 所述转基因植物为具有 矮化表型的植物。
10、 如权利要求 8或 9所述的应用， 其特征在于： 所述目的植物为双 子叶植物或单子叶植物。
11、 一种培育转基因植物的方法， 是将权利要求 2或 3所述基因导入 目的植物， 得到株高小于所述目的植物的转基因植物。
12、 如权利要求 11 所述的方法， 其特征在于： 所述目的植物为双子 叶植物或单子叶植物。
13、 一种培育转基因植物的方法， 是在目的植物中过表达权利要求 2 或 3所述基因， 得到株高小于所述目的植物的转基因植物。
14、 如权利要求 13所述的方法， 其特征在于： 所述 "在目的植物中 过表达权利要求 2或 3所述基因"通过将权利要求 2或 3所述基因导入目 的植物实现或通过促进目的植物本身具有的权利要求 2或 3所述基因的表 达实现。
15、 如权利要求 13或 14所述的方法， 其特征在于： 所述目的植物为 双子叶植物或单子叶植物。
16、 一种培育转基因植物的方法， 是在目的植物中过表达权利要求 2 或 3所述基因， 得到油菜素内酯缺陷型的转基因植物。
17、 如权利要求 16所述的方法， 其特征在于： 所述油菜素内酯缺陷
17
更正页 （细则第 91条） ISA/CN 型体现为与目的植株相比， 所述转基因植株具有如下表型中的至少一种： ①下胚轴缩短； ②株高降低； ③叶柄和 /或叶鞘缩短； ④开花延迟； ⑤生 命周期延长； ⑥在黑暗条件下具有光形态建成反应的表型。
18、 如权利要求 16或 17所述的方法， 其特征在于： 所述 "在目的植 物中过表达权利要求 2或 3所述基因"通过将权利要求 2或 3所述基因导 入目的植物实现或通过促进目的植物本身具有的权利要求 2或 3所述基因 的表达实现。
19、 如权利要求 16或 17或 18所述的方法， 其特征在于： 所述目的 植物为双子叶植物或单子叶植物。
20、 一种塑造植物造型的方法， 是对植物局部喷施油菜素内酯， 使所述 植物生长为预期的造型； 所述植物为权利要求 11至 19中任一所述方法得到 的转基因植物、 所述转基因植物的自交后代、 所述转基因植物的杂交后代或 所述转基因植物的回交后代。
18
更正页 （细则第 91条） ISA/CN
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