WO2011127744A1 - 与植物株型相关的蛋白ipa1及其编码基因与应用 - Google Patents

与植物株型相关的蛋白ipa1及其编码基因与应用 Download PDF

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WO2011127744A1
WO2011127744A1 PCT/CN2011/000558 CN2011000558W WO2011127744A1 WO 2011127744 A1 WO2011127744 A1 WO 2011127744A1 CN 2011000558 W CN2011000558 W CN 2011000558W WO 2011127744 A1 WO2011127744 A1 WO 2011127744A1
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plant
gene
sequence
protein
rice
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PCT/CN2011/000558
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李家洋
钱前
王永红
矫永庆
薛大伟
刘贵富
王静
董国军
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中国科学院遗传与发育生物学研究所
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Priority to US13/635,052 priority Critical patent/US9309527B2/en
Publication of WO2011127744A1 publication Critical patent/WO2011127744A1/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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • 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 relates to the field of plant genetic engineering technology, in particular to the protein IPA1 and its coding gene and application related to plant plant type. Background technique
  • the plant type of rice includes traits such as tiller number, tiller angle, panicle type and plant height.
  • Good plant type is a key factor in increasing rice yield.
  • Most of the cultivars currently used in production are dwarf varieties containing semi-dwarf genes. Compared with the traditional high-stalk varieties, the dwarf varieties have more advantages, thus setting off the first "green revolution" and greatly increasing the yield of rice.
  • the inherent shortcomings of dwarf varieties limit their further increase in yield, including the disadvantages of more inefficient tillers, smaller ears, too high leaf area index, severe leaf shading and decreased canopy photosynthesis.
  • the breeding experts of the International Rice Research Institute proposed the concept of new plant type of rice. The main characteristics of the new plant type are less tiller and no ineffective tiller; Large, large number of grains per spike; thick stems, lodging resistance.
  • the number of tillers in rice is an important agronomic trait in rice production.
  • the number of effective tillers per unit area determines the number of panicles, and the number of panicles is one of the three key factors determining rice yield per unit area. Therefore, it is of great production significance to reasonably control the occurrence of rice tillers and minimize ineffective tillers.
  • the number of kernels is another important factor in determining rice yield.
  • the typical feature of most high-yield varieties currently used in production is the significant increase in the number of kernels per spike.
  • the reason for the increase in the number of grains per spike is mainly due to the large number of branches and secondary branches of the upper part of the ear, and the high density of grain.
  • Increasing the number of kernels is important for cultivating high-yield varieties.
  • 1000-grain weight is the third decisive factor in rice yield, which directly reflects the dry matter accumulation and grain filling of rice grains, and is closely related to grain size.
  • the lodging resistance has always been an area that rice breeders value. Although it cannot directly increase the yield of rice, it is very important for the stability of yield and is a limiting factor for further increase in yield. Compared with the traditional high-stalk varieties, the dwarf varieties can increase the plant's lodging resistance by lowering the plant height, thus ensuring the stable yield of rice and making the yield increase possible. Therefore, further increasing the lodging resistance of plants is a prerequisite for higher yield of rice. In this case, it is the goal of the breeder to change the traits of the stalks and breed stronger and more lodging resistant.
  • the protein associated with the plant plant type provided by the present invention is derived from rice (Orj ⁇ a ⁇ a L.) and is a protein of the following 1) or 2):
  • a label as shown in Table 1 may be attached to the amino terminus or carboxy terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the Sequence Listing.
  • the IPA1 in the above 2) can be artificially synthesized, or the coding gene can be synthesized first, and then biological expression can be obtained.
  • the coding gene of IPA1 in the above 2) can be deleted or added with a codon of one or several amino acid residues in the DNA sequence shown in the sequence 2 of the sequence 2 from the 124th to the 1377th base of the 5' end. And/or performing a missense mutation of one or several base pairs, and/or obtaining the coding sequence of the tag shown in Table 1 at its 5' end and/or 3' end.
  • the gene encoding the plant type may specifically be the gene according to any one of the following 1) -5):
  • nucleotide sequence of the genomic DNA is as shown in the sequence of Table 3 from the 5' end of positions 1-7229;
  • Sequence 2 in the sequence listing consists of 1624 bases, and its open reading frame (ORF) is from the 5' end of the 124th The instruction is located at base 1377 and encodes the IPA1 protein having the amino acid sequence of SEQ ID NO:1 in the sequence listing.
  • the above stringent conditions may be to hybridize and wash the membrane at 65 ° C in a DNA or RNA hybridization experiment using a solution of O.lxSSPE (or O.lxSSC), 0.1% SDS.
  • a primer pair that amplifies the above-mentioned /3 ⁇ 4 / gene full length or any fragment thereof is also within the scope of the present invention.
  • An expression cassette, a recombinant vector, a transgenic cell line, and a recombinant strain containing the above-described gene encoding a plant-type-related protein are also within the scope of the present invention.
  • a recombinant expression vector containing the H3 ⁇ 4 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, such as pCAMBIA3301, pCAMBIA1300, pBI121, pBin1, pCAMBIA230K pCAMBIA1301-UbiN or other derivative plant expression vectors.
  • any of the enhanced, constitutive, tissue-specific or inducible promoters such as the cauliflower mosaic virus (CAMV) 35S promoter, can be added before the transcription initiation nucleotide.
  • the promoters can also be used, including translation Enhancers or transcription-enhancing hands, these enhancer regions may be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence.
  • the source of the translational control signal and the start codon are broad, either natural or synthetic.
  • the translation initiation region can be derived from a transcription initiation region or a structural gene.
  • the plant expression vector used may 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 anti-tuberculosis genes).
  • the recombinant expression vector may be a recombinant expression vector obtained by inserting the above-described gene encoding a plant-type-related protein between the multiple cloning sites of the plant expression vector PCAMBIA1300.
  • Another object of the invention is to provide a method of growing a transgenic plant.
  • the method for cultivating a transgenic plant is to introduce the above-mentioned coding gene IPA1 or genomic DNA of a plant-type-related protein into a plant to obtain a transgenic plant; and the transgenic plant is compared with the target plant,
  • the stems are thick, the number of branches of the ears increases, and the number of grains per ear increases.
  • the coding gene IPAI of the plant-type-related protein is introduced into the plant of interest through the above recombinant expression vector.
  • the plant expression vector carrying the gene encoding the plant type-related protein of the present invention can be transformed into a conventional biological method such as a sputum plasmid, a Ri plasmid, a plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated transformation or the like.
  • a sputum plasmid a Ri plasmid
  • a plant viral vector direct DNA transformation, microinjection, conductance, Agrobacterium-mediated transformation or the like.
  • the transformed plant host (the plant of interest) is a dicot Or a monocotyledonous plant, preferably rice, more preferably Nipponbare.
  • the interference vector is introduced into the plant of interest to obtain a transgenic plant; the transgenic plant has significantly increased tillering, the stem becomes thinner, and the number of primary stems and the number of spikes are significantly reduced compared with the target plant;
  • the vector is a recombinant vector obtained by sequentially inserting the nucleotide sequence shown by the sequence 4 in the sequence listing and the nucleotide sequence shown by the sequence 5 in the sequence listing into the feyrfll and wl sites of the pTCK303 vector and between the el and the sites.
  • the plant of interest may be a dicotyledonous plant or a monocotyledonous plant, preferably rice, and the rice is preferably day 22.
  • Sequence 4 is the 1014 bp to 1623 bp fragment of SEQ ID NO: 2
  • SEQ ID NO: 5 is the reverse complement of SEQ ID NO: 4. Sequence-wide alignment analysis of sequence 4 and sequence 5 confirmed that there are no other homologous sequences in the rice genome.
  • the present invention separates a pleiotropic gene H3 ⁇ 4 which can simultaneously control the number of tillers, stems and ears by means of map-based cloning and verifies the function of the gene through transgenic functional complementation experiments.
  • the experiment proves that after the gene protected by the present invention is overexpressed in rice, the rice tiller is reduced, the stem is thick, the number of ears is increased, and the number of grains is increased; and the gene protected by the present invention is inactivated or reduced in rice.
  • the plant height decreased, the tiller increased, the stem became thinner, the number of branches and the number of spikes decreased, indicating that the gene can control the plant type of rice. Therefore, the /3 ⁇ 4 gene provides a powerful means for molecular marker-assisted breeding and the use of genetic engineering methods to cultivate new plant type rice varieties, thereby further increasing rice yield, and has important theoretical significance and great application potential.
  • Figure 1 The phenotype of rice less tiller material sputum (SNJ) and conventional japonica rice variety TN1.
  • Figure 2 is a map clone of the IPA1 gene, Figure 2a and Figure 2b are QTL analysis and localization maps using BC 2 F 2 ;
  • Figure 2c is a fine mapping map; the numbers below the markers represent recombinant individuals;
  • Figure 2d predicted within 78kb Gene, the arrow represents the predicted gene;
  • Figure 2e shows the structure of the cloned IPA1 gene of the present invention, the white hollow box represents the 5' and 3' untranslated regions, the black square represents the exon, and the middle horizontal line represents the intron.
  • the red asterisk represents the miRNA156 target site.
  • the base change above the box represents a base mutation that occurs in the minority material.
  • the number in parentheses represents the position of the base that has changed.
  • FIG. 3 The cDNA and protein sequence of the IPA1 gene.
  • the blue nucleotides represent the 5' and 3' non-coding regions.
  • the underlined protein sequence represents the SBP domain, the red asterisk represents the miRNA156 target site, and the red letter represents the Nucleotide mutations that occur in the ruthenium material and the amino acid changes that result therefrom.
  • Figure 4 gIPAl vector map and functional complementation test of transgenic rice phenotype and agronomic traits statistics Figure 4a gIPAl gene map; Figure 4b gIPAl transgenic rice phenotype; Figure 4c by RT-PCR detection of IPA1 expression; Figure 4d gIPAl transgenic material Statistical comparison of related agronomic traits, T test, single star representation is significant; double star representation is extremely significant; Nipponbare in Figures 4b, 4c and 4d is a wild type control, gIPAl is a transgenic plant.
  • Figure 5 Comparison of RNA interference GM phenotype and related agronomic traits, Figure 5a RNA interference of transgenic water
  • Figure 5b shows the expression of IPA1 in transgenic plants by RT-PCR
  • Figure 5c Comparison of agronomic traits of transgenic rice with RNA interference, T test, double star representation is extremely significant
  • Figure 5a, Figure 5b and Figure 5c Day 22 is a non-transgenic control and RNAi is a transgenic plant.
  • rice was cultivated as follows: (1) Field cultivation of rice materials: Rice seeds were soaked in water for 2 days, transferred to a culture at 37 ° C for 3 days, and then the white seeds were seeded on the seedbed. The seedlings are transplanted and the rice seedlings are transplanted into the paddy fields at the 4-leaf stage.
  • the seeds of the rice i Oryza satiD scorpion, the thick stalk, the large stalk, and the seeds of the conventional indica rice variety TN1 were cultivated according to the above-mentioned field cultivation methods, and the morphology of the mature plants is shown in Fig. 1. The leaves are then taken for DNA extraction.
  • the improved CTAB method (Mou Z, He Y, Dai Y, et al. Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology.
  • the present invention first constructs a population using the scorpion and TN1. Further, QTL analysis and localization were performed using the BC 2 F 2 individual of the isolated population. The results of the localization indicated that 1 was initially located on chromosome 8, between the two markers RM149 and RM1345, and the genetic distances were 2.1 cM and 1.8 cM, respectively (Fig. 2a and Fig. 2b).
  • Candidate gene prediction was performed on the 78 kb region and sequencing alignment was performed between TN1 and oligosaccharide materials. As a result, it was found that in the aborted material, the gene (LOC-0s08g39890) had a point mutation of C to A on the third exon. Nipponbare, Zhonghua 11 and other varieties have no mutations in this region. An in-depth analysis found that this mutation is located at the target site of miRNA156 and may therefore affect the regulation of this gene by miRNA156 (Fig. 2e). Based on the above information, the gene was identified as a candidate gene. This candidate gene has a corresponding full-length cDNA sequence AK107191 in the K0ME database. Protein sequence analysis indicated that the candidate gene contained a conserved SBP (SQUAMOSA promoter binding protein) domain (Fig. -3). Example 2. Acquisition and detection of transgenic plants
  • the 1st to 1566th positions are the first exon
  • the 1567th to 3996th are the first intron
  • the 3997th to 4130th are the 2nd intron.
  • the 4131th to 4232th positions are the second intron
  • the 4233th to 4903th are the 3rd exons.
  • the nucleotide sequence of the cDNA corresponding to the genomic DNA shown in Sequence 3 from 1 bp to 7229 bp is shown in SEQ ID NO: 2 in the Sequence Listing.
  • the sequence 2 consists of 1624 bases, and its open reading frame (ORF) is from the 124th to the 1377th base of the 5' end, and encodes the IPA1 protein having the amino acid sequence of the sequence 1 in the sequence listing.
  • the final genomic DNA fragment containing the full-length gene obtained in step 1) was inserted between the /7 1 and Xba I restriction sites of the vector pCAMBIA1300 (purchased from Cambia) to obtain the recombinant expression vector gIPAl (Fig. 4a).
  • the card carrier is constructed correctly.
  • the plasmid gIPAl was transferred to the AAgrobacterium tumefaciens strain EHA105 by electroporation (the public can be obtained from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.
  • the non-patent literature describing this material is Lin H, Wang R, Qian Q, Et al. DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell 2009, 21, 1512-1525.), a recombinant Agrobacterium strain containing the recombinant plasmid gIPAl was screened.
  • the Japanese callus was infested with a recombinant Agrobacterium strain containing the recombinant plasmid g, and cultured for 3 days at 25 ° C in the dark, and the resistant callus and transgenic plants were screened on a selection medium containing 50 mg/L hygromycin. .
  • the hygromycin-resistant plants were smelted in a cool place, and then transplanted into a paddy field, and the obtained transgenic plants were T. generation.
  • Harvest T The seeds of the plants were cultivated according to the above-mentioned field cultivation method, and the transgenic plants of g/3 ⁇ 47 were obtained by conventional molecular detection.
  • the total RNA of the transgenic plants and the control Nipponbare plants was extracted with TRIZ0L (purchased from Ir itrogen), and reverse transcription was carried out using a reverse transcription kit (purchased from Promega) to obtain cDNA.
  • the expression of the J 3 ⁇ 47 gene was detected by PCR using the primers IPA1RT1F and IPA1RT1R (the amplified fragment sequence was the 681 bp to 1362 bp fragment from the 5th end of the sequence 2).
  • the Ubiquitin gene was amplified using the primers UbiRTF and UbiRTR as internal standards, and the primer sequences are shown in Table 3. The results show that in transgenic plants? The amount of expression increased (Fig. 4c).
  • Plant type statistics were performed on transgenic plants of transgenic g/3 ⁇ 47, Nipponbare control plants, and empty vector controls, and 10 individual plants were counted for each material. The results are shown in Fig. 4b and Fig. 4d (Fig. 4b and Fig. 4d, the hollow vector control is consistent with the Nipponbare control phenotype, omitted from the figure), and the ⁇ /3 ⁇ 4 transgenic plants were compared with the Nipponbare control plants and the empty vector control.
  • the number of tillers decreased (average from 11.9 to 6.7), the number of primary branches increased significantly (average from 10.8 to 15.5), and the number of secondary branches increased significantly (average from 21.
  • Example 3 Obtaining and detecting of transgenic plants
  • K0ME full-length cDNA clone (No.: AK107191) was used as a template (purchased from Genome Resource Center, National Institute of Agrobiological Science, Japan), and separately (J uses the primer pair RNAi lF/RNAi lR and primer pair R Ai2F in Table 4). /RNAi2R is subjected to PCR amplification, and the obtained product is subjected to sequencing.
  • the nucleotide sequences of the gene fragments amplified by the two pairs of primers respectively are sequence 4 and sequence 5 in the sequence listing.
  • the front and rear primers of R Ail are respectively added with BaiMl and ⁇ joints, and the front and rear primers 5 of R Ai2 are respectively added with a joint.
  • Sequence 4 is the 1014 bp to 1623 bp fragment of SEQ ID NO: 2
  • SEQ ID NO: 5 is the reverse complement of SEQ ID NO: 4. Sequence 4 and sequence 5 were confirmed by genome-wide alignment analysis to have no other homologous sequences in the rice genome.
  • RNAilF/RNAilR The product amplified by primer pair RNAilF/RNAilR was digested with BaiMI and ⁇ / ⁇ , and inserted into the vector pTCK303 carrying the Ubiquitin promoter (publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, documented The non-patent literature is Wang Z, Chen C, Xu Y, et al. A practical vector for efficient knockdown of gene expression in rice ( Oryza sativa L. ) . Plant Mol. Instruction manual
  • the interference vector IPAl ⁇ m i was transferred into the Agrobacterium robacier/i/z? tumefaciens strain EHA105 by electroporation, and the recombinant Agrobacterium strain containing the interference vector / ⁇ J-RNAi was screened.
  • the mature seeds of the material containing the point mutation gene ipal 22 were shelled and sterilized, and inoculated into the medium for inducing callus. After 3 weeks of culture, the callus was grown from the scutellum, and the growth was vigorous and the color was yellow. Relatively loose embryogenic callus, used as a receptor for transformation.
  • Rice 22 calli were infested with recombinant Agrobacterium strains containing the interference vector / 3 ⁇ 4 RNAi. After 3 days of culture at 25 ° C in the dark, resistant callus and transgenic plants were screened on selection medium containing 50 mg/L hygromycin. The hygromycin-resistant plants were smelted in a cool place, and then transplanted into the paddy field. The obtained transgenic plants were T? generations, and the transgenic seeds collected were planted to obtain the transgenic plants of the trans-/3 ⁇ 4 R Ai. Plant.
  • the empty vector PTCK303 was transformed into day 22 according to the method of obtaining a transgenic plant transfected with 3 ⁇ 4i -RNAi, and an empty vector control plant was obtained.
  • RNA of the plants of the transgenic plants and the control plants was extracted using TRIZ0L (purchased from Invitrogen), and reverse transcription was carried out using a reverse transcription kit (purchased from Promega) to obtain cDNA.
  • the primers IPA1RT1F and IPA1RT1R were used for PCR to detect the expression of the IPA1 gene.
  • the Ubiquitin gene was amplified using UbiRTF and UbiRTR as internal standards, and the primer sequences are shown in Table 3. The results showed that the expression of / was reduced in transgenic plants (Fig. 5b).

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Description

说 明 书 与植物株型相关的蛋白 IPA1及其编码基因与应用 技术领域
本发明涉及植物基因工程技术领域，特别涉及与植物株型相关的蛋白 IPA1及其编 码基因与应用。 背景技术
水稻的株型包括分蘖数目、 分蘖角度、 穗型和株高等性状。 良好的株型是提高水 稻产量的关键因素。 当前生产上应用的大部分栽培品种为含有半矮秆基因 的矮秆 品种。 与传统的高秆品种相比， 矮秆品种具有较多的优势， 因此掀起了第一次 "绿色 革命"， 大幅度地提高了水稻的产量。但是， 矮秆品种固有的缺点却限制了其产量的进 一步提高， 这包括无效分蘖较多、 穗子较小、 叶面积指数过高、 叶片遮荫现象严重及 冠层光合作用下降等缺点。 为了克服当前大部分栽培品种增产潜力有限的缺点， 进一 步满足人们对粮食的需求， 国际水稻所的育种学家提出了水稻新株型的概念， 新株型 的主要特点是分蘖少， 没有无效分蘖； 穗子大， 穗粒数多； 茎秆粗壮， 抗倒伏。
水稻分蘖数目是水稻生产中的一个重要的农艺性状。 单位面积的有效分蘖数决定 了穗数， 而穗数又是决定单位面积上水稻产量三个关键因素之一。 因此， 合理地控制 水稻分蘖的发生， 尽量减少无效分蘖具有重要的生产意义。
穗粒数是决定水稻产量的另外一个重要的因素。 目前生产上应用的大部分高产品 种具有的典型特征就是穗粒数显著增多。 穗粒数增多的原因， 主要是因为穗子上一级 枝梗和二级枝梗数目较多， 籽粒着生密度大。 提高穗粒数对培育高产品种来说十分重 要。 千粒重是水稻产量的第三个决定性因素， 直接反映了水稻籽粒干物质积累和灌浆 的好坏， 并且与籽粒的大小密切相关。
抗倒伏能力一直是水稻育种学家十分重视的方面。 虽然其不能直接提高水稻的产 量， 但它对于产量的稳定具有十分重要的作用， 是产量进一步提高的限制性因素。 与 传统高秆品种相比， 矮秆品种通过降低株高， 提高了植株的抗倒伏能力， 从而保证了 水稻的稳产， 使产量的提高成为可能。 因此， 进一步增加植株的抗倒伏能力， 是水稻 更高产的前提。 在这种情况下， 改变茎秆的性状， 培育更加粗壮， 抗倒伏能力更强的 品种一直是育种学家努力的目标。
国际水稻所新株型的基本特点为少蘖、粗秆和大穗。模拟研究表明， 在热带地区、 干季的条件下， 具有新株型品种的产量可以比当前品种再提高 25%。 因此， 阐明分蘖、 茎秆和穗子发育的遗传基础和分子机理， 对于获得更加高产的品种具有十分重要的意 义。 当前， 尽管人们已经克隆了许多产量相关的基因， 但是能够从整体上***地改变 说 明 书 水稻的株型， 使之产生新株型特点的基因还未见报道。 发明内容
本发明的目的是提供一种与植物株型相关的蛋白及其编码基因。
本发明所提供的与植物株型相关的蛋白，名称为 IPA1，来源于水稻（Orj^a^^ a L. )， 是如下 1) 或 2) 的蛋白质：
1) 由序列表中序列 1所示的氨基酸序列组成的蛋白质；
2)将序列表中序列 1的氨基酸残基序列经过一个或几个氨基酸残基的取代和 /或缺 失和 /或添加与植物株型相关的由 1) 衍生的蛋白质。
为了使 1) 中的 IPA1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 imgf000004_0001
上述 2) 中的 IPA1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 2) 中的 IPA1的编码基因可通过将序列表中序列 2自 5'末端第 124位到第 1377 位碱基所示的 DNA序列中缺失或添加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。
上述与植物株型相关蛋白的编码基因 （命名为/ /基因） 也属于本发明的保护范 围。
与植物株型相关的编码基因具体可为如下 1) -5) 中任一所述的基因：
1)其编码序列如序列表中序列 2的自 5'末端第 124位到第 1377位所示；
2)其核苷酸序列是序列表中的序列 2;
3)其基因组 DNA的核苷酸序列如序列表中的序列 3的自 5'末端第 1-7229位所示；
4)在严格条件下与 1) 或 2) 或 3) 的基因杂交且编码所述蛋白的基因；
5) 与 Π 或 2) 或 3) 的基因具有 90%以上的同源性且编码所述蛋白的基因。
序列表中的序列 2由 1624个碱基组成， 其开放阅读框架 （ORF) 为自 5'末端第 124 说 明 书 位到第 1377位碱基， 编码具有序列表中序列 1的氨基酸序列的 IPA1蛋白。
上述严格条件可为用 O.lxSSPE (或 O.lxSSC), 0.1 %SDS的溶液，在 DNA或者 RNA 杂交实验中 65°C下杂交并洗膜。
扩增上述/ ¾ /基因全长或其任一片段的引物对也属于本发明的保护范围。
含有上述与植物株型相关蛋白的编码基因的表达盒、 重组载体、 转基因细胞系和 重组菌也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 H¾ 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， 如 pCAMBIA3301、 pCAMBIA1300、 pBI121、 pBinl9、 pCAMBIA230K pCAMBIA1301-UbiN或其它衍生植 物表达载体。
使用 IPAl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 （U iquitin) 基因启动子 （pUbi) 等， 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植物表达载体时， 还可使用增强子， 包括翻译 增强子或转录增强手，这些增强子区域可以是 ATG起始密码子或邻界区域起始密码子 等， 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 （如抗除莠剂基因） 等。
所述重组表达载体具体可为在植物表达载体 PCAMBIA1300的多克隆位点间***上 述与植物株型相关蛋白的编码基因得到的重组表达载体。
本发明的另一个目的是提供一种培育转基因植物的方法。
本发明所提供的培育转基因植物的方法，是将上述与植物株型相关蛋白的编码基因 IPA1或基因组 DNA导入植物中， 得到转基因植物； 所述转基因植物与所述目的植物相 比， 分蘖»、 茎杆粗壮、 穗子枝梗数增多， 穗粒数增多。
所述与植物株型相关蛋白的编码基因 IPAI 是通过上述重组表达载体导入目的植 物中的。
携带有本发明的与植物株型相关蛋白编码基因 的植物表达载体可通过 Ή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 生物学方法转化到植物细胞或组织中。 被转化的植物宿主 （目的植物） 为双子叶植物 或单子叶植株， 优选是水稻， 更优选是日本晴。
本发明的又一目的在于提供一种培育转基因植物的方法。 该方法是将干扰载体导 入目的植物中， 得到转基因植物； 所述转基因植物与所述目的植物相比， 分蘖显著增 多， 茎杆变细， 一级枝梗数和穗粒数显著减少； 所述干扰载体是将序列表中序列 4所示 的核苷酸序列和序列表中序列 5所示的核苷酸序列依次*** pTCK303载体的feyrfll和 wl位点以及 el和 位点之间得到的重组载体。该目的植物可以是为双子叶植物 或单子叶植株， 优选是水稻， 所述水稻优选是日 22。
序列 4是序列 2第 1014bp到 1623bp片段， 序列 5是序列 4的反向互补序列。 序 列 4和序列 5经全基因组比对分析确说认在水稻基因组中无其它同源序列。
本发明通过图位克隆的方法， 分离了一个可以同时控制分蘖数目， 茎秆和穗子发 育的多效性基因 H¾ 并且通过转基因功能互书补实验验证了该基因的功能。
实验证明： 将本发明保护的基因在水稻中过量表达后， 水稻分蘖减少、茎杆粗壮、 穗子枝梗数增多， 穗粒数增多； 而将本发明保护的基因在水稻中失活或降低活性后， 株高降 低， 分蘖增多， 茎杆变细， 枝梗数和穗粒数减少， 说明该基因可控制水稻的株型。 因 此， / ¾ 基因为分子标记辅助育种及利用基因工程的方法培育新株型水稻品种， 从而 进一步提高水稻的产量提供了有力的手段， 具有重要的理论意义和巨大的应用潜力。 附图说明
图 1 水稻少分蘖材料少蘖粳 (SNJ)和常规籼稻品种 TN1的表型。
图 2 IPA1基因的图位克隆， 图 2a和图 2b是利用 BC2F2进行 QTL分析和定位图； 图 2c是精细定位图谱； 标记下面的数字代表重组的个体； 图 2d 78kb范围内预测的基 因， 箭头代表预测的基因； 图 2e本发明克隆的 IPA1基因的结构示意图， 白色空心的 方框代表 5 ' 和 3' 非翻译区， 黑色方框代表外显子， 中间横线代表内含子， 红色星号 代表 miRNA156靶位点。 方框上面的碱基变化代表在少蘖粳材料中所发生的碱基突变。 括号内的数字代表发生变化的碱基的位置。
图 3 IPA1基因的 cDNA和蛋白序列图， 蓝色的核苷酸代表 5' 和 3 ' 非编码区， 下 划线所标注的蛋白序列代表 SBP结构域，红色星号代表 miRNA156靶位点，红色字母代 表在少蘖粳材料中所发生的核苷酸突变以及由此引发的氨基酸变化。
图 4 gIPAl 载体图谱及功能互补试验的转基因水稻表型及农艺性状统计， 图 4a gIPAl基因图谱； 图 4b gIPAl转基因水稻表型； 图 4c通过 RT- PCR检测 IPA1 的表达 量； 图 4d gIPAl转基因材料相关农艺性状的统计比较， T检验， 单星代表显著； 双星 代表极显著； 图 4b、 图 4c和图 4d中的日本晴是野生型对照， gIPAl是转基因植株。
图 5 RNA干扰的转基因水稻表型及相关农艺性状比较， 图 5a RNA干扰的转基因水 说 明 书 稻表型； 图 5b通过 RT-PCR检测转基因植株中 IPA1的表达量； 图 5c RNA干扰的转基 因水稻相关农艺性状比较， T检验， 双星代表极显著； 图 5a、 图 5b和图 5c中的日 22 是非转基因对照， RNAi是转基因植株。 具体实施方式
下面结合具体实施例对本发明作进一步说明， 但本发明并不限于以下实施例。 下述实施例中， 如无特殊说明， 均为常规方法。
下述实施例中水稻是按照如下方法栽培得到的： （1) 水稻材料的田间栽培： 水稻 种子在水中浸种 2天后， 移入 37°C培养间催芽 3天， 然后将露白的种子播在苗床上进 行育秧， 到 4叶期时将水稻秧苗移栽入水田。
实施例 1、 基因的发现
将水稻 i Oryza satiD 少蘖、 粗杆、 大穗的材料少蘖粳的种子以及常规籼稻 品种 TN1的种子分别按照上述田间栽培方法进行栽培， 成熟植株的形态如图 1所示。 然后取叶片进行 DNA的提取。
水稻基因组 DNA的提取：
采用改进的 CTAB方法 (Mou Z, He Y， Dai Y, et al. Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology. The Plant Cell. 2000, 12, 405-418. ) 从水稻叶片中提取基因组 DNA。 取 100 mg水稻叶片， 经液氮冷冻， 在直径 5cm的小研钵中磨成粉状， 转移到 1. 5 ml 离心管里提取 DNA, 获得的 DNA沉淀溶解于 100 μΐ MQ Η20中。
按照如下步骤进行图位克隆：
1、 / 7基因的初步定位。
为了分离 / 基因， 本发明首先利用少蘖粳和 TN1构建定位群体。进而， 利用该 分离群体的 BC2F2个体进行 QTL分析和定位。 定位结果表明， 1初步定位在第 8染 色体上， 处于 RM149和 RM1345两个标记之间， 遗传距离分别为 2. 1 cM和 1. 8 cM (图 2a和图 2b)。
2、 /基因的精细定位
为了进一步縮小目的基因的界定区域， 从 BC2F2分离群体中， 选取了 5500个株型 近于 TN1的单株进行了精细定位。 同时， 利用已经公布的 93- 11和日本晴在定位区间 内籼粳稻亚种间基因组序列， 寻找差异位点， 开发新的 STS和 SSR标记。 在进行精细 定位时，首先用标记 RM149和 RM1345筛选与目的基因之间发生交换的单株，然后用新 的分子标记对这些交换单株继续进行筛选， 最后发现分子标记 M3和 M6与目的基因紧 密连锁， 与目的基因之间分别有 5个和 3个交换单株。 最终， 被精确定位在分子 标记 M4和 M5之间约 78kb的范围之内 （图 2c和图 2d)。
3、 候选基因的鉴定和序列分析
对该 78kb区域进行候选基因预测并在 TN1和少蘖粳材料间进行测序比对。结果发 现， 在少蘖粳材料中， 基因 (LOC— 0s08g39890)在第 3个外显子上发生了一个 C到 A的点突变。 而日本晴， 中花 11等品种在该区域均无突变。 深入的分析发现， 该 突变位位于 miRNA156的靶位点， 因此可能影响了 miRNA156对该基因的调控（图 2e)。 综合以上信息， 该基因确定为候选基因。 该候选基因在 K0ME 数据库内有对应的全长 cDNA序列 AK107191。 蛋白序列分析表明， 该候选基因含有保守的 SBP (SQUAMOSA promoter binding protein) 结构域 (图说 - 3 )。 实施例 2、 转基因植物的获得及其检测 书
一、 转基因植物的获得
1、 重组表达载体的构建
1 )基因的克隆
利用表 2中的 gIPAl lF/glPAl lR引物组合， 从日本晴 （公众可从中国科学院遗传 与发育生物学研究所获得， 记载过该材料的非专利文献是 Lin H， Wang R, Qian Q, et al. DWARF27, an iron-containing protein required for the biosynthesi s of strigolactones, regulates rice t iller bud outgrowth. Plant Cell 2009, 21, 1512-1525. ) 基因组 DNA中扩增出了包含 i7¾7基因的 DNA预备片段（测序表明， 预备 片段的核苷酸序列如序列表中序列 3所示）。 将该预备片段通过 Kpn I和 ¾a I酶切并 进行回收， 得到包含全长 的最终基因组 DNA片段（核苷酸序列如序列表中序列 3 的自 5' 端第 1-7229位所示）。
序列 3第 lbp到 7229bp所示的基因组 DNA中，第 1118位到 1566位为第 1个外显 子， 第 1567位到 3996位为第 1个内含子， 第 3997位到 4130位为第 2个外显子， 第 4131到 4232位为第 2个内含子， 第 4233位到 4903位为第 3个外显子。
序列 3第 lbp到 7229bp所示的基因组 DNA对应的 cDNA的核苷酸序列如序列表中 序列 2所示。 序列 2由 1624个碱基组成， 其开放阅读框架 （ORF ) 为自 5'末端第 124 位到第 1377位碱基， 编码具有序列表中序列 1的氨基酸序列的 IPA1蛋白。
表 2、 引物序列
Figure imgf000008_0002
gIPAl lF引物的 5 ' 端加
Figure imgf000008_0001
l酶切位点， gIPAl lR引物的 5 ' 端加 ^ I酶切位 说 明 书 点。
2 )构建表达载体
将步骤 1 )得到的包含全长雇基因的最终基因组 DNA片段***载体 pCAMBIA1300 (购自 Cambia 公司）的 /7 1和 Xba I酶切位点之间， 得到重组表达载体 gIPAl (图 4a)， 经验证载体构建正确。
2、 转基因植物的获得
将质粒 gIPAl通过电击的方法转入农杆菌 iAgrobacterium tumefaciens) 株系 EHA105 (公众可从中国科学院遗传与发育生物学研究所获得， 记载过该材料的非专利 文献是 Lin H, Wang R, Qian Q, et al. DWARF27, an iron-containing protein required for the biosynthesis of strigolactones, regulates rice tiller bud outgrowth. Plant Cell 2009, 21, 1512-1525. ) 中， 筛选得到含有重组质粒 gIPAl 的重组农杆 菌株。
用含有重组质粒 g謂的重组农杆菌菌株侵染日本晴愈伤组织，在黑暗处 25°C培 养 3天后， 在含有 50 mg/L潮霉素的选择培养基上筛选抗性愈伤和转基因植株。 将潮 霉素抗性植株在阴凉处炼苗， 后移栽到水田中， 获得的转基因植株为 T。代。 收获 T。代 植株的种子，按照上述田间栽培的方法进行栽培，并通过常规分子检测，获得转 g/ ¾7 的 ^代转基因植株。
按照获得转？ PA1的 T,代转基因植株的方法，将空载体 pCAMBIA1300转化日本晴， 得到空载体对照植株。
二、 转基因植物的株型检测
1、 通过 RT-PCR检测 / /¾7基因的表达量
利用 TRIZ0L (购自 Ir itrogen公司）提取转基因植株和对照日本晴植株的植物 总 RNA，并利用反转录试剂盒（购自 Promega公司）进行反转录， 得到 cDNA。利用引物 IPA1RT1F和 IPA1RT1R进行 PCR检测 J ¾7基因的表达（扩增片段序列为序列 2的自 5， 端第 681bp到 1362bp片段）。 利用引物 UbiRTF和 UbiRTR扩增 Ubiquitin基因作为内 标， 引物序列如表 3。 结果显示转基因植株中 ？表达量增加 （图 4c)。
表 3、 引物序列
引物名称 引物序列（5' -3 ' )
IPA1RT1F CGGTCGACTAGCTGCATCTGTTGG
IPA1RT1R CATCGTGTTGCTGGTTTGGTCGAAG
UbiRTF CCCTCCACCTCGTCCTCAG
UbiRTR AGATAACAACGGAAGCATAAAAGTC 说 明 书
2、 转基因植物的株型检测
对转 g/ ¾7的 ^代转基因植株、 日本晴对照植株和空载体对照进行株型的统计， 每种材料统计 10个单株。 结果如图 4b和图 4d (图 4b和图 4d中空载体对照与日本晴 对照表型一致， 图中省略）所示， 转 §/ ¾ 的1\代转基因植株与日本晴对照植株以及 空载体对照相比， 其分蘖数目降低 （平均从 11. 9个降到 6. 7个）， 一级枝梗数显著增 多（平均从 10. 8个增加到 15. 5个），二级枝梗数显著增多（平均从 21. 9个增加到 25. 6 个），茎杆加粗（第 3节直径平均从 0. 35厘米增加到 0. 47厘米），穗粒数显著增多（平 均从 117. 1粒增加到 135. 7粒）。 实施例 3、 转基因植物的获得及其检测
一、 转基因植物的获得
1、 干扰片段的获得
利用 K0ME全长 cDNA克隆 （编号为： AK107191 ) 为模板（从日本 Genome Resource Center, National Institute of Agrobiological Science购买)， 分另 (J用表 4中的 引物对 RNAi lF/RNAi lR和引物对 R Ai2F/RNAi2R进行 PCR扩增，获得的产物进行测序。 两对引物分别扩增得到的基因片段的核苷酸序列如序列表中序列 4和序列 5。
表 4、 引物序列
Figure imgf000010_0001
R Ail的前后引物 5' 端分别加 BaiMl和 ΚριΛ接头， R Ai2的前后引物 5, 端分别 加 和 接头。
序列 4是序列 2第 1014bp到 1623bp片段， 序列 5是序列 4的反向互补序列。 序 列 4和序列 5经全基因组比对分析确认在水稻基因组中无其它同源序列。
2、 干扰载体的构建
将引物对 RNAilF/RNAilR扩增得到的产物用 BaiMI和 Κρ/ή进行酶切， ***带有 Ubiquitin启动子的载体 pTCK303 (公众可从中国科学院遗传与发育生物学研究所获 得， 记载过该材料的非专利文献是 Wang Z, Chen C, Xu Y, et al. A practical vector for efficient knockdown of gene expression in rice ( Oryza sativa L. ) . Plant Mol. 说 明 书
Biol. Rep. 2004, 22, 409-417. ) 的 Bai l 和 Kpnl 位点， 得到载体 1 ; 将引物对 RNAi2F/RNAi2R扩增得到的产物用 Spe/和 5¾c/进行酶切，***得到载体 1的 I和 位点， 得到重组表达载体/ ¾i-RNAi (即干扰载体 i7¾ - RNAi ), ***片段表达后 形成发夹结构。
3、 转基因植物的获得
将干扰载体 IPAl^m i通过电击的方法转入农杆菌 robacier/i/z? tumefaciens) 株系 EHA105中， 筛选得到含有干扰载体 / ^J-RNAi的重组农杆菌菌株。
将含有点突变基因 ipal的材料日 22的成熟种子脱壳灭菌， 接种到诱导愈伤的培 养基中， 培养 3周后， 从盾片处生长出愈伤组织， 挑选生长旺盛， 颜色浅黄， 比较松 散的胚性愈伤组织， 用作转化的受体。
用含有干扰载体 / ¾ RNAi的重组农杆菌菌株侵染日 22水稻愈伤组织。 黑暗处 25°C培养 3天后， 在含有 50 mg/L潮霉素的选择培养基上筛选抗性愈伤和转基因植株。 将潮霉素抗性植株在阴凉处炼苗， 后移栽到水田中， 获得的转基因植株为 T„代， 将丁。 代收的转基因种子进行种植， 得到转 / ¾ R Ai的 ^代转基因植株。
按照获得转 ¾i -RNAi的 ^代转基因植株的方法， 将空载体 PTCK303转化日 22， 得到空载体对照植株。
二、 转基因植物的检测
1、 通过 RT- PCR检测歴基因的表达量
利用 TRIZ0L (购自 Invitrogen公司）提取转基因植株和对照植株日 22的植物总 RNA,并利用反转录试剂盒（购自 Promega公司）进行反转录， 得到 cDNA。 利用的引物 IPA1RT1F和 IPA1RT1R进行 PCR检测 IPA1基因的表达。 利用 UbiRTF和 UbiRTR扩增 Ubiquitin基因作为内标，引物序列如表 3。 结果显示， 在转基因植株中， / 的表达 量降低 (图 5b)。
2、 转基因植物的株型检测
对转 RNAi的 1\代转基因植株、 日 22对照植株和空载体对照进行株型的统 计， 每种材料统计 10个单株。 结果如图 5a和图 5c (图 5a和图 5c中空载体对照与日 本晴对照表型一致，图中省略）所示，与日 22对照植株和空载体对照相比，转 J/¾i-R Ai 的 ^代转基因植株株高降低（平均从 115. 7厘米降到 91. 2厘米），分蘖数显著增多（平 均从 3. 7个增加到 23. 3个）， 一级枝梗数显著减少 （平均从 15. 2个降到 6. 2个）， 二 级枝梗数显著减少 （平均从 57. 5个降到 9. 7个）， 穗粒数显著减少 （平均从 259. 6粒 降到 54. 6粒） ，茎杆变细 （第二节直径平均从 0. 68厘米降到 0. 29厘米）。

Claims

权 利 要 求 书
1、 一种蛋白， 是如下 1 ) 或 2 ) 的蛋白质：
1 ) 由序列表中序列 1所示的氨基酸序列组成的蛋白质；
2 )将序列表中序列 1的氨基酸残基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 1 ) 衍生的蛋白质。
2、 权利要求 1所述蛋白的编码基因。
3、 根据权利要求 2所述的编码基因， 其特征在于： 所述蛋白的编码基因为如下 1 ) -5 ) 中任一所述的基因：
1 ) 其编码序列如序列表中序列 2的自 5'末端第 124-1377位所示；
2 )其核苷酸序列是序列表中的序列 2;
3 )其基因组 DNA的核苷酸序列如序列表中的序列 3的自 5'末端第 1-7229位所示；
4)在严格条件下与 1 ) 或 2 ) 或 3 ) 的基因杂交且编码权利要求 1所述蛋白的基因；
5 )与 1 )或 2 )或 3 )的基因具有 90%以上的同源性且编码权利要求 1所述蛋白的基 因。
4、 含有权利要求 2或 3所述基因的表达盒、 重组载体、 转基因细胞系或重组菌。
5、根据权利要求 4所述的重组载体， 其特征在于： 所述重组载体为在 pCAMBIA1300 的多克隆位点间***权利要求 2或 3所述基因得到的重组表达载体。
6、一种培育转基因植物的方法，是将权利要求 2或 3所述的编码基因转入目的植物 中， 得到转基因植物； 所述转基因植物与所述目的植物相比， 分蘖减少、 茎杆粗壮、 穗子枝梗数增多， 穗粒数增多。
7、 根据权利要求 6所述的方法， 其特征在于： 权利要求 2或 3所述的编码基因是通 过权利要求 4或 5所述的重组表达载体导入目的植物中。
8、根据权利要求 6或 7所述的方法， 其特征在于： 所述目的植物为双子叶植物或单 子叶植株， 优选是水稻； 所述水稻优选是日本晴。
9、一种培育转基因植物的方法，是将干扰载体导入目的植物中，得到转基因植物； 所述转基因植物与所述目的植物相比， 株高降低， 分蘖增多， 茎杆变细， 枝梗数和穗 粒数减少； 所述干扰载体是将序列表中序列 4所示的核苷酸序列和序列表中序列 5所示 的核苷酸序列依次*** PTCK303载体的 和 A^7l位点以及 e I和 feci位点之间得 到的重组载体。
10、根据权利要求 9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 叶植株， 优选是水稻； 所述水稻优选是日 22。
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