WO2014205616A1 - Utilisations de protéine uch320 et gène codant cette protéine par ajustement et contrôle de la croissance et du développement de plantes - Google Patents

Utilisations de protéine uch320 et gène codant cette protéine par ajustement et contrôle de la croissance et du développement de plantes Download PDF

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WO2014205616A1
WO2014205616A1 PCT/CN2013/001674 CN2013001674W WO2014205616A1 WO 2014205616 A1 WO2014205616 A1 WO 2014205616A1 CN 2013001674 W CN2013001674 W CN 2013001674W WO 2014205616 A1 WO2014205616 A1 WO 2014205616A1
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seed
sequence
seq
plant
protein
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王东辉
白书农
许智宏
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北京大学
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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
    • A01N37/46—N-acyl derivatives
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/40—Liliopsida [monocotyledons]
    • A01N65/44—Poaceae or Gramineae [Grass family], e.g. bamboo, lemon grass or citronella grass
    • 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

  • UCH320 protein and its application in regulating plant growth and development
  • the invention belongs to the technical field of plant molecular biology, and relates to the application of a UCH320 protein and a coding gene thereof for regulating plant growth and development.
  • heterosis is based on a combination of two different parents.
  • in order to further explore its potential based on existing applications in addition to the need to strengthen the research on the mechanism of heterosis formation, there is an urgent need for effective methods to create infertile traits in order to effectively expand the screening and excellent combinations of hybrid combinations. Application in production.
  • male sterility traits widely used in breeding work and production are mostly derived from natural mutations and their trait-transferred strains.
  • the source of male sterility traits is very limited and is a serious constraint factor for expanding screening of hybrid combinations, especially applications.
  • all innovations with application potential are protected by intellectual property rights. Therefore, the search for new ideas and methods for artificially controlling the size and yield of crop seeds with independent intellectual property rights has become an unavoidable and urgent problem to be solved in countries and regions that want to grasp the initiative of exploring the potential application of heterosis. one.
  • the genetic engineering method has a temple point compared with the traditional method: the breeding cycle is shortened, the fertility is relatively stable, the environmental impact is small, the genotype is less dependent, and the environmental pollution is less.
  • the UCH320 protein is as follows (a) or (b):
  • the growth and development of the plant can be embodied in at least one of the following 1) -6):
  • the above application is embodied in that the expression of the UCH320 protein in the plant is low, the seed seed setting rate of the plant is lower, and/or the seed weight is lighter, and/or the seed volume is smaller, and/or the seed.
  • UCH320 protein or its coding gene designated as t/CH?20 gene
  • t/CH?20 gene The use of the UCH320 protein or its coding gene (designated as t/CH?20 gene) in the selection of plant varieties with increased seed yield or breakage is also within the scope of the present invention.
  • the increase or decrease in the seed yield is embodied in at least one of the following I) -VI):
  • V the seed grain width is broadened or narrowed
  • the plant having a higher expression level of UCH320 protein needs to be hybridized as a parent.
  • the selected plant species are reduced in seed set rate, and/or reduced in seed weight, and/or reduced in seed volume, and/or reduced in seed length, and/or narrowed in seed width, and/or single ear.
  • the plant having a lower expression level of UCH320 protein is required to be hybridized as a parent.
  • the method for cultivating a transgenic plant provided by the present invention may specifically be as follows (A) or (B):
  • a method of cultivating a transgenic plant having increased seed yield comprising the steps of:
  • step b) obtaining a transgenic plant having an increased seed yield compared to the plant of interest obtained from the transgenic plant obtained in step a);
  • step c) inhibiting expression of the gene encoding the UCH320 protein in the plant of interest to obtain a transgenic plant; d) obtaining a transgenic plant having reduced seed yield compared to the plant of interest obtained from the transgenic plant obtained in step c).
  • the seed yield increase may be embodied by at least one of the following bl) - b6): bl) an increase in seed seed setting rate;
  • the seed yield breakage may specifically be embodied in at least one of the following dl) - d6): dl) a seed seed set rate is lowered;
  • the gene encoding the UCH320 protein may be the DNA molecule according to any one of (1) to (4) below:
  • the coding sequence is a DNA molecule of the sequence 2 in the sequence table from the 102' to the 791th nucleotide at the 5' end;
  • the above rigorous bovine can be hybridized at 65 °C with a solution of 6xSSC, 0.5% SDS, and then washed once with 2xSSC, 0.1% SDS and lxSSC, 0.1% SDS.
  • sequence 2 consists of 974 nucleotides, which is the cDNA sequence of the t/CH?2 asleep, wherein the 102-791 is the coding sequence (ORF); the sequence 2 is encoded by the sequence 1 in the sequence listing.
  • Protein, Sequence 1 consists of 229 amino acid residues.
  • the gene encoding the UCH320 protein can be introduced into the plant of interest through a recombinant expression vector containing the gene encoding the protein.
  • the recombinant 3 ⁇ 4 ⁇ 4 vector can be constructed using existing plant expression.
  • the plant 3 ⁇ 4 ⁇ 4 vector includes a dual Agrobacterium vector and a vector which can be used for plant microprojectile bombardment, etc., such as pGreen0029, pCAMBIA3301 pCAMBIA1300 pBI121, pBinl9 pCAMBIA2301 pCAMBIA1301-Ubi or other derivatized expression vector.
  • the plant expression vector may further comprise a 3' untranslated region of a foreign gene, i.e., a DNA fragment comprising a polyadenylic acid signal and which may be otherwise involved in mRNA processing or gene expression.
  • the polyadenylic acid signal can be added to the 3' end of the mRNA precursor.
  • an enhanced, constitutive, tissue-specific or inducible promoter may be added before the transcription initiation nucleotide, for example, cauliflower mosaic virus (CAMV) 35S is activated.
  • CAMV cauliflower mosaic virus
  • Enhancers including translational enhancers or transcriptional enhancers, may be ATG start codons or contiguous regions ⁇ 1 ⁇ 2 start codons, etc., but must be identical to the coding sequence of the vines to ensure proper translation of the 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 recombinant expression vector used can be processed, such as a gene encoding a protease or a luminescent compound which can be produced in plants, and a resistant antibiotic marker.
  • anti-chemical agents have genes. It is also possible to selectively label the genes without adding ftf and directly screen the transformed plants by stress.
  • the promoter that initiates transcription of the t/CH?2 gene in the recombinant expression vector is an Actin promoter.
  • the recombinant expression vector is a recombinant plasmid obtained by inserting the t/CH?203 gene into a multiple cloning site of the pCAM23A vector; in one embodiment of the invention, the multiple cloning position
  • the points are specifically Xbfl l and / L
  • a method of reducing the expression of the t/CH?203 in the plant of interest can be a cocoa.
  • the expression of the gene encoding the UCH320 protein in the plant of interest is specifically achieved by transferring a DNA fragment represented by the following formula (I) into the plant of interest:
  • the SEQ is the nucleotides 14 to 268 of SEQ ID NO: 3 in the sequence listing;
  • the sequence reversed by the SEQ is inversely complementary to the sequence forward of the SEQ;
  • the core of the DNA fragment represented by the formula (I) is listed as positions 14 to 730 of the sequence 3 in the sequence listing.
  • Sequence 3 consists of 748 nucleotides.
  • the 14th-268th bit is the forward sequence of a fragment of the t/CH?2 cause (corresponding to the SEQ forward in the above formula (1), which is consistent with the 534-788 position of the sequence 2 in the sequence listing )
  • position 269481 corresponds to X in the above formula (1)
  • positions 482-736 are the inverse of a fragment of the t/CH?20 gene.
  • the sequence (corresponding to SEQ in the above formula (1), which is the reverse complement of the 534th to the 788th position of the sequence 2 in the sequence listing).
  • the DNA fragment represented by the formula (I) is transferred into the plant of interest in the form of the iliiRNA interference expression vector; and the transcription of the DNA fragment represented by the formula (I) is initiated on the interference expression vector.
  • the interference expression vector is a recombinant plasmid obtained by inserting the [/CH32 Kanin RNA interference sequence (SEQ ID NO: 3) at the multiple cloning site of the pCAM23A vector; more specifically, the user;
  • the interference expression vector is prepared according to the method comprising the following steps: digesting the DNA fragment shown by the sequence 3 in the sequence table with i Sal I, and recovering the gel and the idZbo I (Xbfll and ⁇ el are the same tail enzyme) and The double-cleaved pCAM23A vector backbone large fragment was ligated to obtain the interference vector.
  • the recombinant expression vector carrying the t/CH?20 gene is described as a t/CH?2 Kanin; an MRNA interference expression vector is introduced.
  • the plant of interest may specifically be: transforming plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant viral vector, direct DNA transformation, microinjection, conductance, Agrobacterium-mediated transformation, and transformation Plant tissue is grown into plants.
  • the plant may be a monocot or a dicot.
  • the plant is specifically a monocotyledonous rice, such as the rice variety Zhonghua 11.
  • the seed setting rate described in each of the above applications or methods is the percentage of the total number of grains in the total number of grains (the number of real grains + the number of empty grains) (Reference) Zhang Yi, Shen Fucheng. Rice The relationship between the weighing rate and the counting rate. Miscellaneous 3 ⁇ 4/ ⁇ , 2006, 21(2): 64-68").
  • a DNA fragment represented by the following formula (I) is also within the scope of the present invention:
  • the SEQ is the nucleotides 14 to 268 of SEQ ID NO: 3 in the sequence listing;
  • sequence of SEQ is inversely complementary to the sequence forward of the SEQ;
  • the nucleoside bribe of the 3 ⁇ 41» ⁇ fragment is specifically the 14th-736th position of the sequence 3 in the sequence listing.
  • a recombinant vector, recombinant strain, expression cassette or transgenic cell line containing the DNA fragment is also protected by the present invention. Scope.
  • the recombinant vector may be either a recombinant expression vector or a recombinant cloning vector.
  • the promoter for initiating transcription of the RNA interference sequence in the recombinant expression vector is an Actin promoter, specifically, a recombinant expression vector is inserted at a multiple cloning site of the PCAM23A vector.
  • the DNA fragment shown in Fig. 3 was ligated to a large fragment of the pCAM23A vector backbone ligated with bal (Xtol and ⁇ el is the same tail enzyme) and /1 to obtain the RNA interference expression vector.
  • the resulting transgenic plants cultivated by the method of cultivating transgenic plants as described above are also within the scope of protection of the present invention.
  • Figure 1 is a structural map of the pUCCRNAi interference vector.
  • Fig. 2 shows the results of PCR identification of the transgenic rice of the partial transgenic ARNAi expression vector pCAM23A-[/CH?20 in Example 1.
  • swimming 3 ⁇ 4M is the DNA molecular weight standard, and the bands are 5000, 3000, 2000, 1000, 750, 500, 300, 200 bp from large to small; lanes 1-12 are positive plants.
  • Figure 3 is a PCR identification result of a control plant partially transferred into the pCAM23A empty vector in Example 1.
  • swimming 3 ⁇ 4M is the DNA standard, and each band is 5000, 3000, 2000, 1000, 750, 500, 300, 200 bp from large to small ; lanes 1-10 are positive plants.
  • Fig. 4 is a rice phenotype of rice of each genetic material of t/CH?2 in Example 2.
  • wt3 ⁇ 4 ⁇ untransformed wild-type rice cultivar Zhonghua No. 11; 320R13-1-1 and 320R28-13 are two transgenic rice plants with positive expression of the Ti ⁇ ARNAi expression vector pCAM23A-[/CH?20 .
  • Fig. 5 is a phenotype of rice grain of each genetic material of t/CH?2 in Example 2.
  • wt3 ⁇ 4 ⁇ untransformed wild-type rice cultivar Zhonghua No. 11; 320R13-6-1 was transformed into RNAi3 ⁇ 43 ⁇ 4 vector pCAM23A-[/CH?20 transgenic rice plants.
  • Fig. 6 is a graph showing the change ratio of t/CH?2 of rice seed weight in each of the genetic materials in Example 2.
  • the wild type rice variety Zhonghua No. 11 showing the untransformed gene;
  • 24 "320R-" are 24 transgenic rice plants of the T ⁇ ifARNAi vector pCAM23A-[/CH?20 which were positive for the identification of Example 1.
  • Fig. 7 is the seed setting rate of rice seed of each genetic material t/CH?2 in Example 2.
  • the wild rice variety Zhonghua No. 11 which showed no transgenic gene; 24 "320R-" were 24 transgenic rice plants which were positively transformed into the RNAi expression vector pCAM23A-[/CH?20.
  • Figure 8 is a PCR identification result of the transgenic rice in which the partial expression was transferred to the recombinant expression vector pCAM23A--?20 in Example 4.
  • swimming 3 ⁇ 4M is the DNA molecular weight standard, and the bands are 5000, 3000, 2000, 1000, 750, 500, 300, 200 bp from large to small ; lanes 1-12 are the plants with positive identification (the position indicated by the arrow is for the purpose) Lanes; Lane 13 is a wild-type rice variety Zhonghua No. 11 that has not been transgenic.
  • Figure 9 is a real-time quantitative PCR assay for the t/CH?20 gene in transgenic rice transformed into the recombinant expression vector pCAM23A-320 in Example 4.
  • Fig. 10 is a graph showing the earning type of rice in the t/CH?2 due to various genetic materials in Example 4.
  • the right ear is a wild-type rice variety Zhonghua No. 11 which is not transgenic; the left ear is the transgenic rice plant 320 which is positively transferred to the recombinant vector PCAM23A-320. -39 and 320-40.
  • Figure 11 is a graph showing the phenotype, grain length, grain thickness and grain width of rice grains of each genetic material of t/CH?20 gene in Example 4.
  • A is the grain phenotype, and in the first and second rows, the first row of the upper row is the wild-type rice cultivar Zhonghua No.
  • Figure 12 is a graph showing the 1000-grain weight of rice seeds of each genetic material of t/CH?2 in Example 4. Among them, ZH-11 indicates the wild type rice variety Zhonghua No. 11 which has no transgenic gene.
  • Figure 13 is a graph showing the number of single-leaf seeds of rice in each of the genetic materials of t/CH?2 in Example 4.
  • WT indicates the wild-type rice variety Zhonghua No. 11 which is not transgenic
  • 320 indicates 6 transgenic rice plants which were positively transformed into the recombinant expression vector pCAM23A-320.
  • 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 a conventional biochemical reagent store. In the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged.
  • pUCCRNAi interference vector Obtained from Professor Chu Chengcai, Institute of Genetics, Chinese Academy of Sciences, recorded in "Yan Peiqiang, Bai Xianquan, Wan Xiuqing, etc. Application of RNAi Technology to Cultivate Anti-TMV Virus Transgenic Tobacco. Inheritance, 2007, 29(8): 1018-1022" .
  • the recognition sites for the restriction enzymes Spe I and Bgl ⁇ are located upstream of the intron, and the recognition sites for the restriction enzymes BamH I and Xba I are located downstream of the intron.
  • the structural map of the pUCCRNAi interference vector is shown in Figure 1.
  • pCAM23A carrier Beijing Dingguo Changsheng Biotechnology Co., Ltd. It is described in "Chi Zhengchang. Rice meiosis gene OsSGOl function research and analysis. Yangzhou University, 2010, Master thesis” in the article.
  • the promoter located upstream of Xba I on the pCAM23A vector is the Actin promoter.
  • Rice variety Zhonghua No. 11 purchased from the Crop Research Institute of the Chinese Academy of Agricultural Sciences; by the Chinese Academy of Agricultural Sciences Crops Institute in 1979 with Jingfeng No. 5 / Dojipu / Fujin for flower cultivation. It is described in "Ni Yichong. New Rice Variety No. 1 Zhonghua No. 11 . Crop Variety Resources, 1989, 04".
  • the medium involved in the process of obtaining transgenic plants in the following examples is as follows:
  • the amount of AAM is 1/10 of the trace amount of N6, and it is only necessary to dilute the trace of N6 by 10 times.
  • AAM Vitamins Thiamine Hydrochloride VB1 1 mg L 0.1g/L (lOOx)
  • Inositol 100 mg/L lO g/L (lOOx) The brown bottle is stored at 4 degrees, and each time with 100 new ones, the bacteria are added to the medium when the tendon is mixed with the medium.
  • rice differentiation medium indica rice 1L poured in a large test tube, about 20 or so
  • G418 150 mg/L medium was added to the adriamycin 300 mg/L medium at 55 degrees and added to 55 degrees.
  • KT preparation Weigh 100mg of kinetin Kinetin, dissolve it with a small amount of 1M KOH, dilute to 20ml with water. After filtration and sterilization, it was placed in a sterile vial and stored frozen at -20 °C.
  • the t/CH?20 gene involved in the present example is derived from rice (Oryza.saUva., the cDNA sequence thereof is shown in SEQ ID NO: 2 in the sequence listing, and the sequence 2 is composed of 974 nucleotides, of which 102-791
  • the coding sequence (ORF); the sequence 2 encodes the protein shown in SEQ ID NO: 1 in the sequence listing (UCH320 protein), and the sequence 1 consists of 229 amino acid residues.
  • RNAi arch I sequence According to the sequence 2 in the sequence listing, the following RNAi arch I sequence:
  • RNAi-23A320-F 5 '-cc ACT AGT ATG GAG GAT GCT CAT TCC-3 ' (underlined is the recognition sequence of the restriction site Spe I, followed by sequence 534-551 of sequence 2);
  • RNAi-23A320-R 5'-Tc GGA TCC CAC AAC TTT CGA AAG AGC-3 ' (underline is the recognition sequence of the cleavage site B imH I, followed by the reverse of the 771-788 position of sequence 2 Complementary)
  • PCR amplification was carried out using the RNAi-23A320-F and RNAi-23A320-R.
  • the PCR product was digested with restriction endonuclease I and ⁇ mH I and the target fragment was recovered and ligated to the large fragment of the pUCCRNAi vector backbone digested with restriction endonucleases Spe I and Bgl II (Bgl II and ⁇ I). Is the same tail enzyme), and the intermediate plasmid 1 was obtained.
  • the intermediate plasmid 1 was digested with restriction endonucleases BamH I and Xto l to recover large fragments of the backbone.
  • the PCR product (Xbo I and I is the same tail enzyme) digested with restriction endonucleases ⁇ el and BomH I was ligated to obtain IJ intermediate plasmid 2.
  • the restriction fragment was then digested with restriction endonuclease and the intermediate plasmid 2 was digested, and the fragment of interest (748 bp) was recovered and ligated with the large fragment of the pCAM23A vector backbone digested with restriction enzymes Xba I and Sal I (Xba I and Spe I). Is the same tail enzyme), and a recombinant plasmid is obtained.
  • the recombinant plasmid which was sequenced to indicate the cleavage site Xto l of the pCAM23A vector and the DNA fragment shown in SEQ ID NO: 3 in the sequence listing was named pCAM23A-[/CH?20.
  • the promoter for transcription of the DNA fragment shown in SEQ ID NO:3 in the Sequence Listing is the Actin promoter.
  • the sequence 3 consists of 748 nucleotides.
  • positions 14-268 are the forward sequence of a fragment of the t/CH?20 gene (consistent with positions 534-788 of sequence 2 in the sequence listing), and positions 276-474 are derived from the pUCCRNAi vector.
  • the GA20 intron nucleotide sequence, and positions 482-736 are the reverse sequences of a fragment of the UCH320 gene (reverse complement of positions 534-788 of SEQ ID NO: 2 in the sequence listing).
  • the forward sequence and the reverse sequence are separated by an intron sequence to maintain the stability of the vector; the system transcribes a shRNA with a hairpin structure (haiipin) in a plant cell, triggers RNAi, thereby inhibiting Expression of the target gene.
  • the young ears of Zhonghua No. 11 of the rice variety 12-15 days after flowering were threshed, rinsed with water, soaked in 1-2 with 70% ethanol, and then added with 1% (v/v) Tween20.
  • a 1.25% sodium hypochlorite aqueous solution active chlorine content of 1.25% (w/v) was immersed in 90 for surface sterilization. (Stir well during sterilization) Rinse with sterile water for 3-4 times, drain off the water for later use.
  • Rice immature embryos were extruded on a sterile filter paper with forceps and a scraper on a solid induction medium (NB minimal medium), and callus was induced by dark culture at 26 °C. After about 5-7 days, the callus was peeled off, transferred to freshly prepared subculture medium (NB minimal medium), and subcultured in the same right 5 for co-culture.
  • the dehulled rice mature seeds are first immersed in 1-2 with 70% ethanol, and then immersed in 30% with 30%-40% sodium hypochlorite aqueous solution (active chlorine content 30%-40% (w/v)) for surface sterilization ( It is best to rinse 3-4 times with sterile water, then place the seeds on sterile filter paper and blot them on the mature embryo callus induction medium (NB basic medium), 26°. C dark culture (can be light culture, light culture grows fast). After about 20 days, the callus grown from the mature embryo scutellum was peeled off, transferred to mature embryo subculture medium (NB basic medium), and subcultured under the same conditions. It will be subcultured every two weeks. ⁇ Deuterated culture 4-5 days, color yellowish granular callus co-culture.
  • the E. coli DH5ct strain containing the RNAi expression vector pCAM23A-[/CHJ20 and pCAM23A empty vector containing the first step was inoculated into 5 ml LB (containing kanamycin 50 mg/L) liquid medium, and shaken at 37 ° C, 200 rpm. Cultivate overnight.
  • the recombinant plasmid was extracted according to the plasmid extraction kit of V-GENE.
  • Agrobacterium EHA105 was inoculated into 5 ml of YEP (streptomycin-containing Sm50 mg/L) liquid medium, and the light OD600 value was 0.4 at 28 °C and shaking at 200 ipm.
  • RNAi expression vector pCAM23A-[/CH?20 or P CAM23A empty vector constructed in step 1) Take the plasmid (the RNAi expression vector pCAM23A-[/CH?20 or P CAM23A empty vector constructed in step 1) to 200 ⁇ 1 ⁇ 105 competent state, mix it gently, then transfer it to the electric shock cup and place it on ice.
  • step 2 Recombinant Agrobacterium suspension (at least ensure that there is enough bacterial fluid in contact with the material), 80-100r / min room i3 ⁇ 43 ⁇ 4S 20min. Remove the callus, remove the excess bacterial solution on the sterile filter paper, and transfer it to the solid co-cultivation medium with a layer of sterile filter paper to induce the callus and the side of the medium that is always in close contact with the medium. Still placed down, the callus should be placed neatly, it is best not to stack them with each other, and culture at 25 ° C for 3 days in the dark.
  • the callus after co-cultivation is fully washed with sterile water for 4-6 times, until the washed aqueous solution becomes clear, and then washed with sterile water containing 3 ⁇ 4? of the aglycone cef at a concentration of 300 mg/L. Times, each time 15-20min, use a sterile filter paper to absorb the callus.
  • the callus was placed on a screening medium containing 25 mg/L Hygromycin for 14 days and then transferred to a screening medium containing 50 ml of hygromycin Hygromycin, followed by g ⁇ ! 2 Monday generation. Most of the callus was browned about 10 days after the screening, and then the milky white resistant callus was re-grown at the edge of the browned tissue. Choose to sell for 6-8 weeks.
  • the resistant callus of the milky yellow 3 ⁇ 4 dense was transferred to a differentiation medium containing 50 mg/L hygromycin and cultured for 3 days. Then, it was transferred to 16-20h/d, and the light intensity was 100-120Mmolm- 2s - 1 , and the seedlings were further differentiated after 3040 days.
  • the seedlings were transferred to a rooting medium and cultured for about two weeks. Select a seedling with a height of about 10 cm and roots. Wash the medium with warm water and transplant it into the soil. The surface of the water is not submerged. If it is fine, it needs to be shaded until the IJ seedling survives (subject to spit water).
  • RNAi transferred to step one is constructed.
  • the genomic DNA was extracted from the transgenic rice of the RNAi vector pCAM23A-[/CHJ20 and the control plants transformed into the pCAM23A empty vector, respectively.
  • PCR amplification was carried out with the bow 1 and the 1 antibody, and a band of about 460 bp was identified (with the UCH320 forward sequence and The plant of the GA20 intron sequence) was a positive plant transformed into the RNAi vector pCAM23A-[/CH?20.
  • PCR amplification was performed using primer 1 and the primer 2, and the plant with a size of about 200 bp (with the GA20 intron sequence) was identified as being transferred into pCAM23A. Positive plants of the vector.
  • Primer 1 5 '-ACTAGTAGATCTGATGGA-3 ';
  • Primer 2 5'-GGATCCCCTATATAATTTAAG-3' (reverse complement of positions 461-481 of SEQ ID NO: 3).
  • RNAi expression vector pCAM23A-[/CHJ20 of the transgenic rice were as shown in Fig. 2, and the results of the identification of the control plants partially transferred into the pCAM23A empty vector are shown in Fig. 3. After PCR identification, 24 PCR-positive generations were finally transferred into the RNAi vector pCAM23 A-[/CH320 transgenic rice plants.
  • the 24 positive clones were transferred into the RNAi expression vector pCAM23A-[/CHJ20 transgenic rice transgenic plants, the untransgenic wild type rice variety Zhonghua No.11, and the transferable pCAM23A empty vector obtained in Example 1
  • the control plants were experimental materials. The seeds of each experimental material were sown in a petri dish for germination (80-100 capsules of each experimental material), and the seedlings after germination were transplanted in pots, and then transferred to a large field in the suburbs of Beijing for growth. After the rice ears of each experimental material plant are harvested, the following aspects are analyzed and identified:
  • Example 1 24 of the positive clones of Example 1 were transformed into the seeds of the transgenic rice plants of the RNAi expression vector pCAM23A-[/CH?20, compared with the non-transgenic wild-type rice variety Zhonghua No.11.
  • the seed setting rate and seed weight were significantly reduced (P ⁇ 0.05), and even the fertile seeds were much longer.
  • the seed setting rate and seed weight were basically the same as those of the untransgenic wild type rice variety Zhonghua No.11, and there was no statistical difference.
  • Table 1 The seed setting rate of the tt3 ⁇ 4 spikes of each experimental material and the statistical analysis of the 100-seed weight of seeds
  • the 24 "20R-" in the table are 24 transgenic rice plants transgenic into the RNAi expression vector pCAM23A-[/CH?20; the wt indicates the untransformed wild type rice variety. Zhonghua No. 11.
  • total number of grains in the table is equal to the "real grain number (with rice grain)” plus the number of empty grains (no rice empty shell) "the solidity rate” is equal to the “solid grain number” divided by the total number of grains ".
  • the inventors of the present invention further transferred to the non-transgenic wild-type rice variety Zhonghua No. 11 (denoted as WT) in Table 1, and 24 positive expressions of Example 1 into the RNAi expression vector pCAM23A-[/CHJ20 transgene.
  • the 100-grain weight and seed setting rate of the rice plant (recorded as 320R) were statistically analyzed. The results are shown in Table 2.
  • a indicates P ⁇ 0.0001 (t test) compared with 100-weight for WT group
  • b indicates P ⁇ 0.0001 (t test) compared with WT group.
  • Example 3 Acquisition and identification of t/CH transgenic plants
  • the t/CH?20 gene involved in the present example is derived from rice (Oryza.saUva., the cDNA sequence thereof is shown in SEQ ID NO: 2 in the sequence listing, and the sequence 2 is composed of 974 nucleotides, of which 102-791
  • the coding sequence (ORF); the sequence 2 encodes the protein shown in SEQ ID NO: 1 in the sequence listing (UCH320 protein), and the sequence 1 consists of 229 amino acid residues.
  • PCR amplification was carried out using the sequence 2 in the sequence listing as a template, and using the primers 23A320-F and 23A320-R.
  • the PCR product was digested with restriction endonucleases Xba I and Sal I, and the fragment of interest was recovered and ligated with a large fragment of the pCAM23A vector backbone digested with restriction endonucleases Xba I and Sd I to obtain a recombinant plasmid.
  • the recombinant plasmid which was inserted into the DNA fragment shown in positions 102-791 of SEQ ID NO: 2 in the sequence listing between the restriction sites Xba I and Sal I of the pCAM23A vector was designated as pCAM23A-320.
  • the promoter for transcription of the DNA fragment shown in positions 102 to 788 of SEQ ID NO: 2 in the sequence listing is the Actin promoter.
  • the transforming receptor is the rice variety, and the specific operation is the same as in the first embodiment.
  • transgenic vaccines having hygromycin resistance that is, rice plants transformed into the recombinant expression vector PCAM23A-320 and pCAM23A empty vector constructed in the first step, were obtained.
  • the genomic DNA was extracted from the transgenic rice transformed with the recombinant expression vector pCAM23A-J20 and the control plants transferred to the pCAM23A empty vector, respectively.
  • PCR amplification was carried out with the products 23A320-F and 23A320-R, and the plant with the size of about 706 bp was identified as the recombinant expression vector pCAM23A- _? 20 positive plants. Since the endogenous t/CH?20 gene of rice contains multiple introns, using the plant genome as a template to amplify only the transgenic plants can obtain a fragment of about 706 bp, and the wild type plants do not.
  • PCR amplification was performed using the stalk 1 and the stalk 2 (see Example 1), and the plant having a size of about 200 bp (GA20 intron sequence) was identified. This is a positive plant that is transferred to the pCAM23A empty vector.
  • Step (1) Identification of 6 positive generations of transgenic rice transformed into recombinant expression vector pCAM23A-320, 320-6, 320-10, 320-34, 320-35 320-39 and 320-40, transferred to pCAM23A empty vector
  • R1 5 '-TTCGAAAGAGCCATGACGTT-3 ' (reverse complement of 762-781 of sequence 2); FC: 5 '- GCTGTCTTCCCCAGCATTGT-3 ';
  • the QBI experiment was performed using ABI Prism 7000 fluorescence quantitative PCR system and ower SYBR Green I mixing kit. Real-time fluorescence quantitative PCR reaction j3 ⁇ 4 95 °C pre-denaturation 30s; 95 °C 5s, 60 °C 34s, 40 cycles. Power (2, -AACt) was used to calculate the template ratio between different samples to represent the relative expression of different genes.
  • step (1) compared with the non-transgenic rice variety Zhonghua 11 (WT), step (1) identifies six positive strains! ⁇
  • the amount of 3 ⁇ 4 ⁇ 4 of UCH320 in the transgenic rice plants 320-6, 320-10, 320-34, 320-35 320-39 and 320-40 of the recombinant vector pCAM23A-J20 was significantly increased.
  • transgenic plants transgenic plants expressing the recombinant expression vector pCAM23A-J20 positively identified in Example 3, wild-type rice cultivar Zhonghua No. 11 which was not transgenic, and control plants transferred to pCAM23A empty vector obtained in Example 3
  • the seeds of each experimental material were sown in a petri dish for germination (80-100 seeds were sown for each experimental material), and the seedlings after germination were transplanted in pots, and then transferred to the Datian of the suburbs of Beijing for growth. After the rice ears of each experimental material plant are harvested, the following aspects are analyzed and identified:
  • the seed weight and seed volume (seed) were basically the same as those of the wild-type rice variety Huahua No. 11 which was not genetically modified, and there was no statistical difference.
  • ZH-11 is the untransgenic wild-type rice variety Zhonghua No. 11 as a control.
  • the number of plants per system was 20 or more.
  • the inventors of the present invention further directed to the non-transgenic wild-type rice cultivar Zhonghua No. 11 (denoted as WT) in Table 3, and six transgenic rice plants positively expressed in Example 3 into the recombinant expression vector pCAM23A-320.
  • the statistical analysis of the difference between the 1000-grain weight of the difficult is shown in Table 4.
  • a 3 ⁇ 4 ⁇ is P ⁇ 0.0001 (t test) compared to the 1000-grain weight of the WT group.
  • Number of seeds per panicle The ratio of the total number of grains on the plant to the number of rice ears on the plant.
  • Table 4 Statistical analysis of single ear number in UCH320 transgenic rice plants During the development of rice, the invention down-regulates the expression level of UCH320 protein in rice by RNA interference technology, which can cause rice seeds to exhibit the following phenotype: the seed setting rate is lower than that of wild type rice seeds, even if fertile The seeds are also much longer and the weight of the seeds is also significantly reduced.
  • up-regulation of UCH320 protein expression in rice by gene overexpression technology can lead to rice seed showing the following phenotype: seed weight increase, volume increase, and number of single ear seeds compared to wild type rice seeds increase.
  • the invention lays a foundation for finding a simpler idea and method for producing high-yield traits.

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Abstract

La présente invention concerne une protéine UCH320 et un gène la codant. L'invention concerne également des utilisations de la protéine UCH320 et le gène la codant dans la régulation de croissance et le développement de plantes, en particulier, dans la régulation de caractères de plantes liés à la production de graines.
PCT/CN2013/001674 2013-06-27 2013-12-31 Utilisations de protéine uch320 et gène codant cette protéine par ajustement et contrôle de la croissance et du développement de plantes WO2014205616A1 (fr)

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JP2005185101A (ja) * 2002-05-30 2005-07-14 National Institute Of Agrobiological Sciences 植物の全長ｃＤＮＡおよびその利用
CN101466259A (zh) * 2005-05-10 2009-06-24 孟山都技术有限公司 用于植物改良的基因及其用途
CN102372767A (zh) * 2010-08-19 2012-03-14 北京大学 植物性状相关蛋白及其编码基因和应用
CN103003432A (zh) * 2010-07-19 2013-03-27 巴斯夫植物科学有限公司 具有增强的产量相关性状的植物和用于产生该植物的方法
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CN101466259A (zh) * 2005-05-10 2009-06-24 孟山都技术有限公司 用于植物改良的基因及其用途
CN103003432A (zh) * 2010-07-19 2013-03-27 巴斯夫植物科学有限公司 具有增强的产量相关性状的植物和用于产生该植物的方法
CN102372767A (zh) * 2010-08-19 2012-03-14 北京大学 植物性状相关蛋白及其编码基因和应用
CN103320468A (zh) * 2013-06-27 2013-09-25 北京大学 Uch320蛋白及其编码基因在调控植物生长发育中的应用

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