WO2014205616A1

WO2014205616A1 - Uses of uch320 protein and coding gene thereof in adjusting and controlling growth and development of plant

Info

Publication number: WO2014205616A1
Application number: PCT/CN2013/001674
Authority: WO
Inventors: 王东辉; 白书农; 许智宏
Original assignee: 北京大学
Priority date: 2013-06-27
Filing date: 2013-12-31
Publication date: 2014-12-31
Also published as: CN103320468A; CN103320468B

Abstract

Provided are a UCH320 protein and a coding gene thereof. Also provided are uses of the UCH320 protein and the coding gene thereof in regulating growth and development of plants, particularly, uses in regulating plant traits related to seed production.

Description

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.

Background prison

Since the 1970s, the use of heterosis has made a significant contribution to rice production in China. Faced with the new major demand for high-yield, high-quality, high-efficiency, safe and ecological in China's economic development, it is a serious challenge for contemporary scientists to further explore the application potential of heterosis. .

The formation of 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.

At present, 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. According to current international rules, 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.

For a long time, people have been using conventional breeding methods to breed hybrid crops. These methods have long cycle times and are slow to meet the urgent needs of production development. 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.

Invention disclosure

It is an object of the present invention to provide a UCH320 protein and its encoding gene for use in regulating plant growth and development.

The UCH320 protein is as follows (a) or (b):

(a) a protein consisting of the amino groups and columns shown in SEQ ID NO: 1 in the Sequence Listing;

(b) Substituting and/or the deletion and/or addition of the amino acid sequence of the protein defined by (a) with one or several amino acid residues, and a protein associated with the regulation of plant growth and development.

The growth and development of the plant can be embodied in at least one of the following 1) -6):

1) seed seed setting rate;

2) seed cages;

3) seed volume;

4) seed length;

5) seed grain width;

6) Number of single ear seeds.

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. The shorter the grain length, and/or the narrower the seed granule width, and/or the fewer the number of single ear seeds; the UCH320 protein is highly expressed in the carp; the plant has a high seed setting rate, and/ Or the weight of the seed, and/or the larger the seed volume, and/or the longer the seed length, and/or The wider the seed granule width, and/or the more the number of single ear seeds.

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):

I) increased seed set rate or breakage;

II) increase or decrease in seed weight;

III) increase or decrease in seed volume;

IV) The growth or shortening of seed granules;

V) the seed grain width is broadened or narrowed;

VI) Increase in the number of single ear seeds or d.

In practical applications, when the selected plant species are increased in seed set rate, and/or seed weight increase, and/or seed volume increase, and/or seed grain length increase, and/or seed grain width broadening, and When the plant variety with an increased number of single ear seeds is used, 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. In the case of a plant variety having a reduced number of seeds, the plant having a lower expression level of UCH320 protein is required to be hybridized as a parent.

It is still another object of the present invention to provide a method of growing a transgenic plant.

The method for cultivating a transgenic plant provided by the present invention may specifically be as follows (A) or (B):

(A) A method of cultivating a transgenic plant having increased seed yield, comprising the steps of:

a) introducing a gene encoding the UCH320 protein into a plant of interest to obtain a transgenic plant expressing the gene encoding;

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);

(B) A method of cultivating a transgenic plant having reduced seed yield, comprising the steps of:

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).

In the above (A), the seed yield increase may be embodied by at least one of the following bl) - b6): bl) an increase in seed seed setting rate;

B2) increased seed weight;

B3) the seed volume is increased;

B4) seed grain growth;

B5) seed grain width broadening;

B6) the number of single ear seeds increased;

In the above (B), 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;

D2) seed weight reduction;

D3) seed volume reduction;

D4) seed length reduction; D5) the seed grain width is narrowed;

D6) The number of single ear seeds is reduced.

In the above application or method, the gene encoding the UCH320 protein (t/CH?20 gene) may be the DNA molecule according to any one of (1) to (4) below:

(1) 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;

(2) a DNA molecule as shown in SEQ ID NO: 2 in the Sequence Listing;

(3) a DNA molecule which hybridizes under the strict bovine to a DNA molecule defined by (1) or (2) and which encodes a protein consisting of the amino acid sequence shown by SEQ ID NO:1 in the Sequence Listing;

(4) A DNA molecule having a protein having 90% or more homology with any one of (1) to (3) and encoding a protein consisting of the amino acid sequence shown by SEQ ID NO: 1 in the Sequence Listing.

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.

Wherein, the 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.

In the above method (A), 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. ■ When the recombinant gene is constructed into a recombinant expression vector, 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. a ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter 9 and the like, which can be conjugated to other plant promoters alone or in combination with other plant promoters; in addition, when the recombinant expression vector is constructed using the gene of the present invention, 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. In order to facilitate the identification and screening of transgenic plant cells or plants, 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. Or 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.

In an embodiment of the invention, the promoter that initiates transcription of the t/CH?2 gene in the recombinant expression vector is an Actin promoter.

More specifically, 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

In the above method (B), the gene encoding the UCH320 protein is inhibited in the plant of interest, A method of reducing the expression of the t/CH?203 in the plant of interest can be a cocoa.

In the present invention, 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:

SEQ forward - X - SEQ reverse ( I )

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;

Is the spacer sequence between SEQ _fi ^ of the SEQ _E ^ ^, in the sequence, the family; f3⁄4X is not complementary to the SEQ forward and the SEQ.

In the present invention, 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. Wherein, 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 (positions 276-474 for the GA20 intron nucleotide sequence) corresponds to X in the above formula (1), and 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).

Further, 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 promoter ^Actin promoter. Specifically, 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.

In the above method (A) and method (B) for cultivating a transgenic plant, 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.

In each of the above applications or methods, the plant may be a monocot or a dicot. In the present invention, the plant is specifically a monocotyledonous rice, such as the rice variety Zhonghua 11.

In the present invention, 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/稻, 2006, 21(2): 64-68").

A DNA fragment represented by the following formula (I) is also within the scope of the present invention:

SEQ forward - X - SEQ reverse ( I )

The SEQ is the nucleotides 14 to 268 of SEQ ID NO: 3 in the sequence listing;

The sequence of SEQ is inversely complementary to the sequence forward of the SEQ;

Is a spacer sequence between the SEQ _fi ^ of the SEQ _E ^ ^, in the sequence, the family; f3⁄4X is not complementary to the SEQ forward and 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. In one embodiment of the present invention, 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 recombinant plasmid obtained after the interference sequence of the [/03⁄4203⁄4 (SEQ ID NO: 3); more specifically, the interfering expression vector is prepared according to the method comprising the steps of: using the sequence of the ^el and the double restriction enzyme sequence 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.

DRAWINGS

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. Among them, 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. Among them, 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. Among them, 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. Among them, 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. Among them, 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. Among them, 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. Among them, 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. Among them, in A and B, 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. Among them, 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. 11 which is not transgenic; the upper two lines are positively transferred to the recombinant expression vector pCAM23A. -_?20 of transgenic rice plants planted 320-39 and 320-40. B is the grain length statistical result; C is the grain thickness statistical result; D is the grain width statistical result.

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. Among them, 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 best invented by Xiong

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" . In the pUCCRNAi interference vector, the recognition sites for the restriction enzymes Spe I and Bgl 位于 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".

Agrobacterium EHA105: Beijing Quanjin Biological Engineering Co., Ltd.

The medium involved in the process of obtaining transgenic plants in the following examples is as follows:

1. Rice callus induction and subculture medium (indica rice) NB minimal medium

Reagent lx mother liquor

KC1 2.95g/L 29.5 g/L (10x)

AAM large amount of NaH ₂ P0 ₄ 0.15g / L l.5g / L (10x)

MgS0 ₄ -7H ₂ 0 0.25g/L 2.5 g/L (10x)

CaCl ₂ -2H ₂ 0 0.15mg/L l.5g/L (10x)

MnS0 ₄ 4H ₂ 0 lmgL 1000 mg/L (lOOOx)

H3BO3 0.3 mg/L 300 mg/L (lOOOOx)

ZnS0 ₄ 7H ₂ 0 0.2 mg/L 200 mg/L (lOOOOx)

AAM trace Na ₂ Mo0 ₄ -2H ₂ 0 0.025 g / L 25 mg / L (lOOOOx)

CuS0 ₄ 5H ₂ 0 0.0025 g/L 2.5 mg/L (lOOOOx)

CoCl ₂ -6H ₂ 0 0.0025 g/L 2.5 mg/L (lOOOx)

KI 0.075 g/L 15 mg/L (lOOOOx) 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.

Glutamine 876mg/L 8.76g/L (lOOx)

AAM amino aspartic acid 226 mg L 2.26g/L (lOOx) acid arginine 174 mg/L 1.74g/L (lOOx)

Glycine 7.5 mg/L 750mg^ (lOOx)

AAM Vitamins Thiamine Hydrochloride VB1 1 mg L 0.1g/L (lOOx)

Pyridoxine hydrochloride VB ⁶ 0.5mg / L 50 mg / L (lOOx) MS MS niacin 0.5mg / L 50 mg / 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.

Hydrolyzed casein 500 mg/L

Glucose 36g/L

Sucrose 68.5g/L

Plant gel 2.6 g/L solid is added, liquid is not added

AS (acetosyringone, acetyl clove ΙΟΟμΜ medium is added to about 55 °C)

H 5.2

±h+立类+立类

• 6, six mustard

4, anti-[raw screening medium 1L, poured in a disposable flat stomach about 40-50 water

; 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.

Plant Gel 3.5-2.6 g/L

pH 5.8,

6. Rice rooting medium (indica rice)

The preparation method of the hormone mother liquor involved in each of the above media:

(1) 0.5mgml 2,4-D mother liquor: weigh 100mg 2,4-D, placed in a small beaker; add a small amount of absolute ethanol to completely dissolve; 2,4-D alcohol solution slowly Add to the water on the magnetic stirrer, if precipitation occurs, need to be reconfigured; dilute to 200ml with water, store at 4 °C.

(2) 0.5mgml α-ΝΑΑ mother liquor preparation: Weigh lOOmgNAA in a small beaker; dissolve NAA with 1M KOH solution; dilute to 200ml with water, and store at 4°C.

(3) 0.5mgml 6-BA mother liquor preparation method: Weigh 100mg 6-BA in a small beaker; add a small amount of concentrated hydrochloric acid, use glass 5 to grind the shape, then add a small amount of concentrated hydrochloric acid to make it completely dissolved; Dilute with water and dilute to 200 ml and store at 4 °C.

(4) Preparation of lOOmM acetosyringone (As): Weigh 196.2mg As, dissolve directly in 5ml DMSO, dilute to 10ml, dispense into sterile tubules, and store at -20 °C.

(5) 5mgml 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.

^Example 1, t/CH?20 gene RNAi 7j 3⁄4 plant acquisition and identification

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.

I. RNAi 3⁄43⁄4 vector construction of pCAM23A-[/CH?20

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)

Using the sequence 2 in the sequence listing as a template, 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 ΒωηΗ 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. In the RNAi vector pCAM23A-[/CHJ20, the promoter for transcription of the DNA fragment shown in SEQ ID NO:3 in the Sequence Listing is the Actin promoter. Among them, the sequence 3 consists of 748 nucleotides. Wherein, 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.

2. UCH320 gene Acquisition and identification of RNAi rice plants

1. UCH320 gene acquisition of RNAi rice plants

(1) Preparation of rice transformed receptors

A. Induction culture of rice immature embryo callus

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.

B. Induction culture of rice mature embryo callus

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.

(2) Transformation and cultivation of Agrobacterium

A. Extraction of purified plasmid

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.

B. Take the electric shock cup and soak it in 3⁄4K ethanol and let it dry.

C. Agrobacterium EHA105 shock preparation

I. 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.

l. Collect 1 ml of bacterial solution in a 1.5 ml centrifuge tube, centrifuge at 8000 rpm for 30 s at 4 °C. ΙΠ. To the residual liquid, precipitate with 200μ1 (1 (1Η ₂ Ο fill, 4 ° C, 8000ipm, centrifuge for 30s).

IV. Repeat the steps ΠΙ three times.

V. Residual liquid, the precipitate is floated with ddH ₂ 0, which is the competent state of Agrobacterium EHA105 for electric shock. Add 200 μl of sterilized glycerin and mix at -80 °C until use.

D. Electric shock

I. Take the plasmid (the RNAi expression vector pCAM23A-[/CH?20 or _P CAM23A empty vector constructed in step 1) to 200μ1 ΕΗΑ105 competent state, mix it gently, then transfer it to the electric shock cup and place it on ice.

II. Prepare the electric shock device (BioRad), the voltage is 2.5V, hold the shock button by hand until the electric shock is completed.

ΙΠ. After standing for 2 minutes at room temperature, add YEP liquid culture solution, let stand at 28 ° C for 1 h, then incubate at 28 ° C, 200 rpm.

2h.

IV. Centrifuge at 8000ipm for 30s, collect the bacterial solution, suspend the pellet with ddH ₂ 0, and coat the gelatin-containing 50 mg^ and streptomycin-containing Sm50mg/L YEB solids in a glass rod for 3 hours, and incubate at 28 °C for 48 hours. . The scraped lawn is resuspended in YEB liquid medium and cultured at 28 ° C until the late log growth period; then 0.5 ml is transferred to 100 ml of the same YEB liquid medium for 2-3 h to OD600 of 0.5 or so. The cultured recombinant Agrobacterium 4000g was centrifuged 10 and the pellet was suspended in 100 ml of AAM liquid medium into a recombinant Agrobacterium suspension.

(3) Co-cultivation of rice callus and Agrobacterium

The passage of the state» obtained in step 1 to a certain time of rice callus (subculture 4-5 days, light yellow color, granular) was placed in a 100 ml sterile triangular flask, and then added to the appropriate amount of 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.

(4) Screening of resistant callus

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.

(5) Differentiation of resistant callus

From the resistant callus grown after two- or three rounds of screening, 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.

(6) Rooting, strong seedlings and transplanting

When the buds of the resistant callus differentiated to about 24 cm, 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).

Through the above operation, the two transgenic seedlings with the hygromycin resistance are most earned, that is, the RNAi transferred to step one is constructed. Vector CAM23A-[/CHJ20 and pCAM23A empty vector rice plants (Ti generation).

2. Identification of UCH320 gene RNAi rice plants

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. For the transgenic rice transformed into the RNAi expression vector pCAM23A-[/CH?20, 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. For the control plants transferred to the pCAM23A empty vector, 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).

The results obtained in the partial step 1 transformation into the 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.

2. UCH320 gene RNAi 7j 3⁄4 plant function identification

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:

1. Analysis of rice phenotype

The results showed that 24 of the wild-type rice cultivar Zhonghua No.11, which was not transgenic, were transferred to the RNAi vector pCAM23A-[/CHJ20 transgenic rice plants with dysplasia. Phenotype (Figure 4). For the control plants obtained in Example 1 and transferred to pCAM23A empty vector, the rice blast type was basically the same as that of the non-transgenic wild type rice cultivar Zhonghua No.11, and there was no statistical difference.

2. Statistical analysis of seed set rate and seed weight

The results showed that 24 of the wild-type rice cultivar Zhonghua No. 11 which was not transgenic, 24 of the positive expression of the Ti-transformed RNAi vector pCAM23A-[/CHJ20 transgenic rice plants had many cognac grains ( Figure 5) . The seed setting rate of the grain on the rice ears of each experimental material and the statistical analysis of the seed weight per 100 seeds were obtained. The detailed results are shown in Table 1 and Figure 6 and Figure 7. Among them, Figure 6 shows the change rate of rice seed weight for each experimental material, calculated according to the following formula with reference to the knot line of Table 1: Rice seed weight change ratio = 100 parts of the sample weight of the sample - 100 pieces of the wild type sample Solid grain weight) / wild type sample per 100 grain solid weight. From the above results, it can be seen that 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. For the control plants obtained in Example 1 and transferred to the pCAM23A empty vector, 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

Note: 1. 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.

2. The "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.

Table 2 Differences in 100-grain weight and seed setting rate between WT and 320R

Seed setting rate 0.9910 0.0433 0.2196 ^b 0.153

Note: 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

I. Construction of recombinant vector pCAM23A-320

According to the sequence 2 in the sequence table, the following sequence is designed:

23A320-F: 5,-TC TCT AGA ATG GGG AAG CGG TGG ATC-3 ' (underlined enzyme cleavage site

The recognition sequence of Xto l, followed by positions 102-119 of sequence 2);

23A320-R: 5,-CC GTC GAC TTA CAC AAC TTT CGA AAG AGC-3' (underlined is the recognition sequence of the restriction site Sail, followed by the reverse complement of sequence 771-791 of sequence 2 ).

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. In the recombinant expression vector _P CAM23A-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.

2. Acquisition and identification of t/CH?20 transgenic rice 3⁄4 strain

1. Acquisition of UCH320 transgenic rice plants

(1) Preparation of rice transformed receptors

The transforming receptor is the rice variety, and the specific operation is the same as in the first embodiment.

(2) Transformation and cultivation of Agrobacterium

The specific operation was the same as in Example 1. Finally, two 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.

2. Identification of t/CH?20 transgenic rice plants

(1) PCR identification

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. For the transgenic rice transformed into the recombinant expression vector pCAM23A-J20, 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. For the control plants transferred to the pCAM23A empty vector, 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.

The results of the identification of the transgenic rice transformed into the recombinant expression vector pCAM23A-J20 obtained in part step 1 are shown in FIG. Transgenic rice transformed into recombinant expression vector pCAM23A-J20, which was positive by 3⁄4 PCR Six plants were randomly selected from the plants, which were recorded as 320-6, 320-10, 320-34, 320-35 320-39 and 320-40, respectively. (2) Real-time quantitative PCR detection

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 The control plants, as well as the non-transgenic rice variety Zhonghua 11, extracted total RNA from the leaves, reverse-transcribed cDNA, and used the cDNA as a template to quantify the cDNA of the gene UCH320 with the F1 and R1. PCR amplification with actinl as an internal reference and primers for FC and RC.

F1 : 5 '-CTGGCGACACTGATGCTAAT-3 ' (positions 565-584 of sequence 2);

R1 : 5 '-TTCGAAAGAGCCATGACGTT-3 ' (reverse complement of 762-781 of sequence 2); FC: 5 '- GCTGTCTTCCCCAGCATTGT-3 ';

RC: 5'- GCTCGATGGGGTACTTGAGG-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.

The experiment was repeated three times and the results were averaged.

The results are shown in Fig. 9. It can be seen that 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.

^Example 4, t/CH?20 transgenic 7j 3⁄4 plant function identification

6 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 For experimental materials. 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:

1. Analysis of rice phenotype

The results showed that 6 rice plants of the transgenic rice plants positive for the recombinant expression vector PCAM23A-320 which were positive in Example 3 were larger and fuller than the non-transgenic wild type rice variety Zhonghua No.11 (Fig. 10). ). For the control plants obtained by transferring the pCAM23A empty vector obtained in Example 3, the rice phenotype was basically the same as that of the non-transgenic wild rice variety Zhonghua No.11, and there was no statistical difference.

2. Statistical analysis of seed weight S3⁄4 seed volume

For the statistical analysis of the seed volume (grain length, thickness and width) of the seed on the shed of each experimental material, see Figure 11. It can be seen that compared with the non-transgenic wild-type rice variety Zhonghua No.11, 6 of the transgenic rice plants transformed into the recombinant expression vector pCAM23A-J20 with positive expression of Example 3 have larger seed volume, mainly in the grain. Longer length (P<0.05), wider grain width (PO.05).

Statistical analysis was carried out on the 1000-grain weight of the seeds on the shed ear of each experimental material. The detailed results are shown in Figure 3 and Figure 12. It can be seen that compared with the non-transgenic wild-type rice variety Zhonghua No.11, the seed cages of the transgenic rice plants transformed into the recombinant expression vector PCAM23A-320 by the 6 positive samples were significantly decreased (P<0.05). ).

For the control plants obtained in Example 3, which were transferred to the pCAM23A empty vector, the seed weight and seed volume (seed) The length, thickness and width of the grain 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.

Note: In the table, 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 (recorded as 320R) is shown in Table 4.

Table 4 Difference analysis of 1000-grain weight of WT and 320R

Note: a 3⁄4 ^ is P < 0.0001 (t test) compared to the 1000-grain weight of the WT group.

3. Statistical analysis of the number of seeds per ear

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.

The results showed that the average number of seeds per plant of transgenic rice plants transformed with the recombinant expression vector pCAM23A-_?20 was more than that of the non-transgenic wild-type rice variety Zhonghua No.11. Large CPO.05), the detailed results are shown in Table 4 and Figure 13. For the control plants obtained by transferring the pCAM23A empty vector obtained in Example 3, the number of single ear seeds was basically the same as that of the non-transgenic wild type rice variety Zhonghua No. 11, and there was no statistical difference.

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. On the other hand, 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.

Claims

Rights request

1. The use of a protein or a gene encoding the same for regulating growth and development of a plant, wherein the protein is as follows: (a) or (b): (a) a protein consisting of an amino group represented by the sequence 1 in the sequence listing;

2. The use according to claim 1, wherein: said plant growth and development is embodied in at least one of the following 1) -6):

1) seed seed setting rate;

2) seed SS;

3) seed volume;

4) seed length;

5) seed grain width;

6) Number of single ear seeds.

3. The use of a protein or a gene encoding the same for plant varieties having improved seed yield or disruption, the protein being as follows (a) or (b):

4. The use according to claim 3, wherein: said increase or decrease in said seed yield is as follows:

At least one of -VI):

I) increased seed set rate or 氐;

II) increase or decrease in seed weight;

III) increase or decrease in seed volume;

IV) The growth or shortening of seed granules;

V) the seed grain width is broadened or narrowed;

VI) increased number of single ear seeds or

5. The method of cultivating genetically modified plants is as follows (A) or (B):

a) introducing a gene encoding the protein according to claim 1 into a plant of interest to obtain a transgenic plant expressing a leg-encoding gene;

c) inhibiting expression of the gene encoding the protein of claim 1 in the plant of interest to obtain a transgenic plant;

d) obtaining transgenic plants in which the seed yield is broken compared to the plant of interest obtained from the transgenic plants obtained in step c).

6. The method according to claim 5, wherein: in said (A), said seed yield improving body Now at least one of the following bl ) -b6):

Bl) increased seed set rate;

B2) increased seed weight;

B3) the seed volume is increased;

B4) seed grain growth;

B5) seed grain width broadening;

B6) the number of single ear seeds increased;

In the above (B), the seed yield reduction is embodied in at least one of the following dl) - d6):

Dl ) seed seed set rate is reduced;

D2) seed weight reduction;

D3) seed volume reduction;

D4) seed length reduction;

D5) the seed grain width is narrowed;

D6) The number of single ear seeds is reduced.

The application or method according to any one of claims 1 to 6, wherein the gene encoding the protein is a DNA molecule according to any one of (1) to (4) below:

(1) The coding sequence is a DNA molecule of sequence 2 in the sequence 2 from the 5' end of the 5' end nucleoside;

(2) a DNA molecule as shown in SEQ ID NO: 2 in the Sequence Listing;

(3) a DNA molecule which hybridizes under stringent conditions to a DNA molecule defined by (1) or (2) and which encodes a protein consisting of the amino acid sequence shown by SEQ ID NO:1 in the Sequence Listing;

(4) A DNA molecule having 90% or more homology with any of the defined DNA molecules of (1) to (3) and encoding a protein consisting of the amino acid sequence shown by SEQ ID NO:1 in the Sequence Listing.

The method according to any one of claims 5 to 7, wherein the method comprises: inhibiting expression of the gene encoding the protein in the plant of interest, wherein the formula 1(1) is as shown in the following formula (I) Transfer of DNA fragments into the household; realized in M-target plants:

SEQ forward - X - SEQ reverse (I)

The SEQ is the nucleotides 14 to 268 of SEQ ID NO: 3 in the sequence listing;

The X is a spacer sequence between the SEQ and the SEQ, and in the sequence, the X is not complementary to the SEQ forward and the SEQ reverse.

A method according to claim 8, wherein the nucleotide sequence of the DNA fragment represented by the formula (I) is from positions 14 to 736 of the sequence 3 in the sequence listing.

The method according to any one of claims 5 to 9, wherein: the DNA fragment represented by the formula (I) is transferred into the plant of interest in the form of an RNA interference expression vector; The promoter for initiating transcription of the DNA fragment represented by the formula (I) on the RNA interference expression vector is the Actin promoter.

11. Use according to any of claims 1-10; or an application or method of M, characterized in that the plant is a monocotyledonous plant or a dicotyledonous plant.

12. A DNA fragment as shown in the following formula (I):

SEQ forward - X - SEQ reverse (I) The SEQ forward is the nucleotides 14 to 268 of SEQ ID NO: 3 in the sequence listing;

The DNA fragment according to claim 12, wherein the nucleoside alcohol of the leg DNA fragment is ranked from positions 14 to 736 of the sequence 3 in the sequence listing.

14. A recombinant vector, recombinant plasmid, expression cassette or transgene containing the DNA fragment of claim 12 or 13

15. The resulting transgenic plant is grown using the method of any of claims 5-11.