CN116926079A

CN116926079A - Application of transcription factor WRKY7 in regulation of rice seed size and yield

Info

Publication number: CN116926079A
Application number: CN202210359441.5A
Authority: CN
Inventors: 陈旭君; 蒋西子; 郭泽建; 彭友良
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2022-04-07
Filing date: 2022-04-07
Publication date: 2023-10-24

Abstract

The application discloses application of a transcription factor WRKY7 in regulating and controlling the size and yield of rice seeds. The transcription factor WRKY7 disclosed by the application is derived from rice, and the WRKY7 is as follows A1), A2) or A3): a1 A protein whose amino acid sequence is sequence 2; a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 2 in the sequence table and has the same function; a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that after the WRKY7 coding gene is knocked out, the length, thickness and thousand seed weight of the seeds are extremely obviously increased, and the WRKY7 and the coding gene thereof can regulate and control the size and weight of rice seeds, so that the WRKY7 coding gene has important significance for crop breeding.

Description

Application of transcription factor WRKY7 in regulation of rice seed size and yield

Technical Field

The application relates to application of a transcription factor WRKY7 in regulating and controlling the size and yield of rice seeds in the field of plant genetic engineering.

Background

Rice (Oryza sativa L.) is an important grain crop, and the yield of rice is determined by the effective tillering number, the spike number and thousand grain weight (depending on the length, width and thickness of each seed), and can be effectively increased by increasing the thousand grain weight of rice. Several genes affecting rice seed size have been reported in rice, such as GW2, MKK4, etc. The WRKY transcription factor is involved in a plurality of life processes such as plant growth, disease resistance, stress resistance and the like. The genes can be effectively utilized in crop disease-resistant breeding improvement through gene editing technology.

Disclosure of Invention

The application aims to solve the technical problem of how to increase the size and weight of rice seeds.

To solve the above technical problems, the present application provides, first, any one of the following applications of proteins or substances regulating the content or activity of the proteins:

d1 Regulating the size of plant seeds;

d2 Preparing a product for regulating and controlling the size of plant seeds;

d3 Regulating and controlling the weight of plant seeds;

d4 Preparing a product for regulating and controlling the weight of plant seeds;

d5 Cultivating a seed size-altering plant;

d6 Preparing a seed size-altering plant product;

d7 Cultivating the seed weight-altering plant;

d8 Preparing a cultivated seed weight altering plant product;

the protein is derived from rice and is named as OsWRKY7, and OsWRKY7 is as follows A1), A2) or A3):

a1 A protein whose amino acid sequence is sequence 2;

a2 A protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequence shown in the sequence 2 in the sequence table and has the same function;

a3 A fusion protein obtained by ligating a tag to the N-terminal or/and the C-terminal of A1) or A2).

In order to facilitate purification of the protein of A1), a tag as shown in the following table may be attached to the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in the sequence 2 in the sequence table.

Table: tag sequence

Label (Label)	Residues	Sequence(s)
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The OsWRKY7 protein in A2) has 75% or more identity with the amino acid sequence of the protein shown in the sequence 2 and has the same function. The identity of 75% or more is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

The OsWRKY7 protein in the A2) can be synthesized artificially or can be obtained by synthesizing the encoding gene and then biologically expressing.

The gene encoding the OsWRKY7 protein in A2) above can be obtained by deleting one or more amino acid residues from the DNA sequence shown in the sequence 1, and/or performing one or more base pair missense mutations, and/or ligating the coding sequences of the tags shown in the above table to the 5 'and/or 3' ends thereof. Wherein, the DNA molecule shown in the sequence 1 codes for OsWRKY7 protein shown in the sequence 2.

In the above application, the substance may be any one of the following B1) to B9):

b1 A nucleic acid molecule encoding OsWRKY 7;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b3 A recombinant vector comprising the nucleic acid molecule of B1) or a recombinant vector comprising the expression cassette of B2);

b4 A recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3);

b5 A transgenic plant cell line comprising the nucleic acid molecule of B1) or a transgenic plant cell line comprising the expression cassette of B2);

b6 A transgenic plant tissue comprising the nucleic acid molecule of B1) or a transgenic plant tissue comprising the expression cassette of B2);

b7 A transgenic plant organ comprising the nucleic acid molecule of B1) or a transgenic plant organ comprising the expression cassette of B2);

b8 A nucleic acid molecule that reduces OsWRKY7 content or activity, or a nucleic acid molecule that knocks out an OsWRKY7 encoding gene;

b9 An expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule of B8).

In the above applications, the nucleic acid molecule of B1) may be B11) or B12) or B13) or B14) as follows:

b11 A cDNA molecule or a DNA molecule of which the coding sequence is a sequence 1 in a sequence table;

b12 A DNA molecule shown in a sequence 1 in a sequence table;

b13 A cDNA molecule or a DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and encoding OsWRKY 7;

b14 A cDNA molecule or DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b 11) or b 12) or b 13) and which encodes OsWRKY 7;

b8 The nucleic acid molecule is a nucleic acid molecule for knocking out an OsWRKY7 encoding gene by using a CRISPR-Cas9 system.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the OsWRKY7 protein of the present application can be easily mutated by one of ordinary skill in the art using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the OsWRKY7 protein isolated by the present application are derived from the nucleotide sequence of the present application and are equivalent to the sequence of the present application as long as they encode the OsWRKY7 protein and function as the OsWRKY7 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of a protein consisting of the amino acid sequence shown in the coding sequence 2 of the present application. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ And 1mM EDTHybridization in a mixed solution of A, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M NaPO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS; the method can also be as follows: hybridization and washing the membrane 2 times at 68℃in a solution of 2 XSSC, 0.1% SDS for 5min each time, and hybridization and washing the membrane 2 times at 68℃in a solution of 0.5 XSSC, 0.1% SDS for 15min each time; the method can also be as follows: hybridization and washing of membranes were performed at 65℃in 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution.

The 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

In the above application, the expression cassette (OsWRKY 7 gene expression cassette) described in B2) containing a nucleic acid molecule encoding an OsWRKY7 protein refers to a DNA capable of expressing the OsWRKY7 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of the OsWRKY7 gene but also a terminator for terminating transcription of the OsWRKY7 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters from tomato, leucine aminopeptidase ("LAP", chao et al (1999) Plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No.)5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean beta-cone (Beachy et al (1985) EMBO J. 4:3047-3053)). They may be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I) ⁹⁸⁵ ) Nature 313:810; rosenberg et al (1987) Gene,56:125; guerineau et al (1991) mol. Gen. Genet,262:141; proudroot (1991) Cell,64:671; sanfacon et al Genes Dev.,5:141; mogen et al (1990) Plant Cell,2:1261; munroe et al (1990) Gene,91:151; ballad et al (1989) Nucleic Acids Res.17:7891; joshi et al (1987) Nucleic Acid Res., 15:9627).

The existing expression vector can be used for constructing a recombinant vector containing the OsWRKY7 gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Co.), etc. The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). When the gene of the present application is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector. The plasmid may be in particular a pOs-sgRNA vector and/or a pH-Ubi-cas9-7 vector.

B9 The recombinant vector can be a recombinant vector which can reduce the OsWRKY7 content and is prepared by using a CRISPR-Cas9 system. The recombinant vector may express an sgRNA targeting the nucleic acid molecule of B1). The target sequence of the sgRNA can be 46 th-65 th positions of the sequence 1 in the sequence table. The recombinant vector can be pCS3-OsWRKY7, and the pCS3-OsWRKY7 is prepared according to the following steps: inserting a DNA molecule shown in the 46 th-65 th position of the 5' end of the sequence 1 in the sequence table between two Bsa I recognition sites of a pOs-sgRNA vector, and carrying out LR reaction on the obtained recombinant vector and pH-Ubi-cas9-7 to obtain a recombinant vector with the correct sequence, namely the pCS3-OsWRKY7. The pCS3-OsWRKY7 can transcribe sgRNA of a DNA molecule shown in 46 th-65 th positions of the targeting sequence 1 and express Cas9 protein.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacterium may be Agrobacterium, such as Agrobacterium tumefaciens EHA105.

In the above applications, none of the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs include propagation material.

In the above application, the substance that regulates the protein content or activity may be a substance that reduces the protein content or activity, the regulating the plant seed size may be increasing the plant seed size, the regulating the plant seed weight may be increasing the plant seed weight, the cultivating seed size changing plant may be cultivating seed size increasing plant, and the cultivating seed weight changing plant may be cultivating seed weight increasing plant.

In the above applications, the seed size may be embodied in the length, width, and/or thickness of the seed.

In the above application, the plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) a gramineous plant;

m3) rice.

The application also provides any one of the following methods:

x1) a method of growing a seed size-increasing plant comprising: reducing the content or activity of OsWRKY7 in the receptor plant, or knocking out the OsWRKY7 coding gene, or reducing the expression of the OsWRKY7 coding gene to obtain a target plant with increased seed size;

x2) a method of increasing plant seed size comprising: reducing the content or activity of OsWRKY7 in the receptor plant, or knocking out the OsWRKY7 coding gene, or reducing the expression of the OsWRKY7 coding gene to obtain a target plant with increased seed size, thereby realizing the increase of the seed size of the plant;

x3) a method of growing a seed weight increasing plant comprising: reducing the content or activity of OsWRKY7 in the receptor plant, or knocking out the OsWRKY7 coding gene, or reducing the expression of the OsWRKY7 coding gene to obtain the target plant with increased seed weight;

x4) a method of increasing plant seed weight comprising: reducing the content or activity of OsWRKY7 in the receptor plant, or knocking out the OsWRKY7 coding gene, or reducing the expression of the OsWRKY7 coding gene to obtain the target plant with increased seed weight, thereby realizing the increase of the seed weight of the plant.

In the method, the knockout of the OsWRKY7 coding gene can be completed by using a CRISPR-Cas9 system, and the knockout can be performed by using the recombinant vector of B9).

In the method, the knockout of the OsWRKY7 coding gene can be realized by deleting the T at the 62 th position of the sequence 1 in the sequence table or inserting a nucleotide A between the 62 th and 63 th positions of the sequence 1 in the genome DNA.

The recombinant vector may be introduced into plant cells by conventional biotechnological methods using Ti plasmids, plant viral vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach, 1998,Method for Plant Molecular Biology VIII,Academy Press,New York,pp.411-463;Geiserson and Corey,1998,Plant Molecular Biology (2 nd Edition)).

The plant of interest is understood to include not only the first generation plants in which the OsWRKY7 protein or its coding gene has been altered, but also their progeny. For the plant of interest, the gene may be propagated in that species, or may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The plants of interest include seeds, calli, whole plants and cells.

In the above method, the seed size may be embodied in the length, width and/or thickness of the seed.

The plant may be M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) a gramineous plant;

m3) rice.

OsWRKY7 or the substances for regulating the content or the activity of OsWRKY7 also belong to the protection scope of the application.

Experiments prove that the OsWRKY7 can regulate and control the size and the weight of plant seeds, and the length, the thickness and the thousand seed weight of the seeds are remarkably increased after the OsWRKY7 coding gene is knocked out, so that the OsWRKY7 and the coding gene thereof can regulate and control the size and the weight of rice seeds, and have important significance for crop breeding.

Drawings

FIG. 1 shows the sequencing results of homozygous mutants KO-W7-2 and KO-W7-4 fragments of interest and the protein sequences encoded by the mutants. WT is wild rice middle flower 17.

FIG. 2 shows KO-W7#2 and KO-W7#4 as width (A), length (B), thickness (C), thousand kernel weight (D), and appearance (E) of seeds.

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the application in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

The pOs-sgRNA and pH-Ubi-Cas9-7 vectors in the examples described below are described in the literature (Miao J, guo D, zhang J, huang Q, qin G, zhang X, wan J, gu H, qu LJ (2013), targeted mutagenesis in rice using CRISPR-Cas system. Cell Res 23:1233-1236), and are available to the public from the applicant, and the biomaterials are used only for repeated experiments related to the application and are not useful for other applications.

Example 1 obtaining transgenic plants

1. Construction of knockout vectors

The DNA molecule shown in the 46 th-65 th position of the 5' end of the sequence 1 in the sequence table is designed as a target sequence, the target sequence is inserted between two Bsa I recognition sites of a pOs-sgRNA vector, the obtained recombinant vector and pH-Ubi-Cas9-7 are subjected to LR reaction, and the obtained recombinant vector with the correct sequence is the knockout vector pCS3-OsWRKY7 (which is verified by sequencing), and the pCS3-OsWRKY7 can transcribe sgRNA of the DNA molecule shown in the 46 th-65 th position of the target sequence 1 and can express Cas9 protein.

2. The vector pCS3-OsWRKY7 was introduced into Agrobacterium tumefaciens EHA105 (Clontech Co.) to obtain recombinant EHA105/pCS3-OsWRKY7.

3. And (3) transforming the recombinant bacterium obtained in the step (2) into rice middle flower 17.

The specific method comprises the following steps:

3.1 Induction culture of Rice callus

Husking 17 seeds of mature rice, sterilizing with 70% ethanol for 1 min, sterilizing with 40% (v/v) sodium hypochlorite for 15min, washing with sterile water for 3 times, blow-drying on sterilized filter paper, uniformly sowing on induction medium NBi (N6 minimal medium added with 0.3g/L CEH (enzymatic hydrolysis casein), 0.5g/L glutamine, 0.5g/L L-proline, 2.0mg/L2,4-D,30g/L sucrose; pH 5.8), culturing at 28deg.C for about 2 weeks to give callus, and culturing the callus on new NBi medium for 4 days.

3.2 preparation of Agrobacterium suspension

Coating the recombinant Agrobacterium solution to be transferred on a solid YEP plate, culturing for 2-3 days at 28 ℃, flushing with sterile water to obtain a bacterial solution, adding the bacterial solution obtained by flushing into a co-culture solution (0.3 g/LCEH,2g/L inositol, 30g/L sucrose; pH 5.3) containing 100 mu M acetosyringone, and adjusting the suspension OD of the agrobacterium to be transferred ₆₀₀ To 0.3-0.5.

3.3 Co-culture of Rice callus and Agrobacterium

The yellow rice callus is selected and put into agrobacterium suspension for 15-20 min, and is gently shaken from time to time. The Agrobacterium suspension was poured off, the calli were blotted onto sterilized filter paper and subsequently transferred to co-culture medium NBco containing 100. Mu.M acetosyringone (N6 minimal medium supplemented with 0.3g/L CEH,0.5g/L glutamine, 0.5g/L proline, 2.0mg/L2,4-D,30g/L sucrose; pH 5.3) and incubated in the dark at 28℃for 2-3 days.

3.4 selection and differentiation of Rice resistant callus

The calli cultured for 2 days were washed 3 times with sterile water, then soaked in sterile water containing 400mg/L of timentin for 15 minutes, after pouring off the water, the calli were blotted onto sterilized filter paper and transferred to pre-screening medium NBps (N6 minimal medium with 0.3g/L CEH,0.5g/L glutamine, 0.5g/L proline, 2.0mg/L2,4-D,30g/L sucrose; pH 5.8) containing 400mg/L timentin, 40mg/L hygromycin B, after 1 week of culture, onto screening medium NBs (N6 minimal medium with 0.3g/L CEH,0.5g/L glutamine, 0.5g/L proline, 2.0mg/L2,4-D,30g/L sucrose; pH 5.8) containing 400mg/L timentin, and cultured for about 4 weeks at 28 ℃.

The newly grown pale yellow resistant calli were selected, transferred to regeneration medium NBr (N6 minimal medium supplemented with 0.3g/L CEH,0.5g/L glutamine, 0.5g/L proline, 30g/L sucrose; pH 5.8) containing 30mg/L hygromycin B, 3.0 mg/L6-BAP (6-benzylaminopurine) and 0.5mg/L NAA (naphthylacetic acid) and cultivated at 28℃with light until the tissues developed green and germinated.

3.5 rooting, transplanting and seed collection

When the buds grow out with the length of 2-3 cm, transferring the buds to rooting culture medium (3 g/L sucrose and 7g/L glucose are added to 1/2MS minimal medium) containing 40mg/L hygromycin B, and culturing at 28 ℃ under illumination; after the young seedlings grow out of developed root systems for about 2-3 weeks, the young seedlings are moved to a greenhouse for cultivation until the seeds are harvested, and the T0 generation transgenic rice seeds are obtained. The seed obtained by selfing the T0 generation transgenic plant and the plant grown from the seed are T1 generation. The seed obtained by selfing the T1 generation transgenic plant and the plant grown from the seed are T2 generation. The seed obtained by selfing the T2 generation transgenic plant and the plant grown from the seed are T3 generation.

4. Identification method of knocked-out plants

Identification of transgenic Rice knocked out OsWRKY7 Gene: genomic DNA of T0, T1, T2 and T3 generation transgenic rice is extracted respectively, and an identification primer jd-WRKY7F is utilized: 5'-AGACGTGCGTGTATGTGCTA-3' and jd-WRKY7R:5'-ACGATCGAGCTAACCAAGGG-3' the gene fragment containing the knockdown target sequence was amplified and the resulting PCR product was verified by sequencing. The homozygous mutants KO-W7-2 (KO-W7 # 2) and KO-W7-4 (KO-W7 # 4) were obtained.

KO-W7-2 mutant type is d1T (deletion of T at 62 th site of sequence 1 in sequence table, resulting in frame shift of 21 st amino acid of encoded protein, premature termination of protein translation); KO-W7-4, mutation type i1A (insertion of a nucleotide A between positions 62-63 of sequence 1, resulting in a frame shift from amino acid 22 of the encoded protein), sequencing and mutant encoded proteins are shown in FIG. 1.

Example 2 detection of seed size of transgenic plants

And (3) measuring plants: wild type rice middle flower 17 (ZH 17), T3 generation homozygous mutants KO-W7#2 and KO-W7#4 of example 1.

After the rice seeds are ripe, drying the seeds at 37 ℃ and threshing, counting the length, width, thickness and thousand grain weight of the seeds. 2020 2021 was repeated for two consecutive years on a transgenic test base in beijing, and the above data were analyzed with Excel 2016 and GraphPad Prism 8.

As shown in Table 1 and FIG. 2, the width, length, thickness and thousand seed weight of the mutant KO-W7#2 and KO-W7#4 seeds are extremely remarkably increased compared with those of the wild rice, which indicates that OsWRKY7 and the encoding gene thereof can regulate the size and weight of the rice seeds, and the size and weight of the rice seeds can be improved by knocking out the gene.

The CDS sequence of the OsWRKY7 gene in China 11 is a sequence 1 in a sequence table, and the OsWRKY7 protein is shown in a coding sequence 2.

TABLE 1

In table 1, it is shown that the difference reaches a very significant level, p < 0.001, compared to ZH 17.

The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

<110> Chinese university of agriculture

Application of <120> transcription factor WRKY7 in regulation of rice seed size and yield

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 741

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

atgtcgtcgc tgtacccgtc tctcctctcg ctgagcgaga gccccgccga gtaccggcag 60

gtcggcggtg gccgctacgc gggggaggac gtcgtcgacg acgacgacga catggctgcc 120

gtcgccgacg ctgtgtccag ctacctctcc ttcgacatgg acgacgtcga gtattacacg 180

ccggaggtgg gcttccactc aaagcagcac aacccgccgc cagttgctgc ggcgccactg 240

gaagccggag gaggcaggga gcaaagccgg cgggaagccg ccgtcaatct tggcaagatg 300

gacaggggac cggcgccggt gagcggcggc gcggcgactg gcggcgtgcc gaggagcaag 360

aacggcagca agatcgcgtt caagacgagg tcggaggtgg acgtgctgga cgacggctac 420

cggtggagga agtacggcaa gaagatggtc aagaacagcc ccaacccaag gaactactac 480

cggtgctcga gcgaggggtg ccgcgtgaag aagcgggtgg agcgcgcccg ggacgacgcg 540

cgcttcgtcg tcaccaccta cgacggcgtc cacaaccacc cggcgccgct gcacctcagg 600

ccgcagctgc cgccgcccgg cggctactcg atcgccgggg caccggccgt ggtggcgccg 660

cacggccgcc tcgggctgga ggaggccgag gtgatcgctc tcttcagggg caccaccgcc 720

acctcgctgc tgcttccttg a 741

<210> 2

<211> 246

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 2

Met Ser Ser Leu Tyr Pro Ser Leu Leu Ser Leu Ser Glu Ser Pro Ala

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Glu Tyr Arg Gln Val Gly Gly Gly Arg Tyr Ala Gly Glu Asp Val Val

20 25 30

Asp Asp Asp Asp Asp Met Ala Ala Val Ala Asp Ala Val Ser Ser Tyr

35 40 45

Leu Ser Phe Asp Met Asp Asp Val Glu Tyr Tyr Thr Pro Glu Val Gly

50 55 60

Phe His Ser Lys Gln His Asn Pro Pro Pro Val Ala Ala Ala Pro Leu

65 70 75 80

Glu Ala Gly Gly Gly Arg Glu Gln Ser Arg Arg Glu Ala Ala Val Asn

85 90 95

Leu Gly Lys Met Asp Arg Gly Pro Ala Pro Val Ser Gly Gly Ala Ala

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Thr Gly Gly Val Pro Arg Ser Lys Asn Gly Ser Lys Ile Ala Phe Lys

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Thr Arg Ser Glu Val Asp Val Leu Asp Asp Gly Tyr Arg Trp Arg Lys

130 135 140

Tyr Gly Lys Lys Met Val Lys Asn Ser Pro Asn Pro Arg Asn Tyr Tyr

145 150 155 160

Arg Cys Ser Ser Glu Gly Cys Arg Val Lys Lys Arg Val Glu Arg Ala

165 170 175

Arg Asp Asp Ala Arg Phe Val Val Thr Thr Tyr Asp Gly Val His Asn

180 185 190

His Pro Ala Pro Leu His Leu Arg Pro Gln Leu Pro Pro Pro Gly Gly

195 200 205

Tyr Ser Ile Ala Gly Ala Pro Ala Val Val Ala Pro His Gly Arg Leu

210 215 220

Gly Leu Glu Glu Ala Glu Val Ile Ala Leu Phe Arg Gly Thr Thr Ala

225 230 235 240

Thr Ser Leu Leu Leu Pro

245

Claims

1. Any of the following uses of a protein or a substance that modulates the content or activity of said protein:

d1 Regulating the size of plant seeds;

d2 Preparing a product for regulating and controlling the size of plant seeds;

d3 Regulating and controlling the weight of plant seeds;

d5 Cultivating a seed size-altering plant;

d6 Preparing a seed size-altering plant product;

d7 Cultivating the seed weight-altering plant;

d8 Preparing a cultivated seed weight altering plant product;

the protein is A1), A2) or A3) as follows:

a1 A protein whose amino acid sequence is sequence 2;

2. The use according to claim 1, characterized in that: the substance is any one of the following B1) to B9):

b1 A nucleic acid molecule encoding the protein of claim 1;

b2 An expression cassette comprising the nucleic acid molecule of B1);

b8 A nucleic acid molecule which reduces the protein content or activity of claim 1, or which knocks out a gene encoding the protein of claim 1;

3. The use according to claim 2, characterized in that: b1 The nucleic acid molecule is b 11) or b 12) or b 13) or b 14) as follows:

b12 A DNA molecule shown in a sequence 1 in a sequence table;

b13 A cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b 11) or b 12) and encoding the protein according to claim 1;

b14 A cDNA molecule or a DNA molecule which hybridizes under stringent conditions to the nucleotide sequence defined in b 11) or b 12) or b 13) and which codes for a protein according to claim 1;

b8 The nucleic acid molecule is a nucleic acid molecule that knocks out the gene encoding the protein of claim 1 using a CRISPR-Cas9 system.

4. A use according to any one of claims 1-3, characterized in that: the material for regulating the protein content or the activity is a material for reducing the protein content or the activity, the size of the plant seeds is regulated to be increased, the weight of the plant seeds is regulated to be increased, the size of the cultivated seeds is changed to be increased, and the weight of the cultivated seeds is changed to be increased.

5. Use according to any one of claims 1-4, characterized in that: the seed size is embodied in the length, width and/or thickness of the seed.

6. Use according to any one of claims 1-5, characterized in that: the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) a gramineous plant;

m3) rice.

7. The method comprises the following steps:

x1) a method of growing a seed size-increasing plant comprising: reducing the content or activity of the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1, or reducing the expression of the gene encoding the protein of claim 1, to obtain a plant of interest with increased seed size;

x2) a method of increasing plant seed size comprising: reducing the content or activity of the protein of claim 1 in a recipient plant, or knocking out the protein coding gene of claim 1, or reducing the expression of the protein coding gene of claim 1, to obtain a target plant with increased seed size, thereby realizing the increase of the seed size of the plant;

x3) a method of growing a seed weight increasing plant comprising: reducing the amount or activity of the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1, or reducing the expression of the gene encoding the protein of claim 1, to obtain a plant of interest with increased seed weight;

x4) a method of increasing plant seed weight comprising: reducing the content or activity of the protein of claim 1 in a recipient plant, or knocking out the protein-encoding gene of claim 1, or reducing the expression of the protein-encoding gene of claim 1, to obtain a target plant with increased seed weight, thereby achieving the increase in seed weight of the plant.

8. The method according to claim 7, wherein: knocking out the protein-encoding gene of claim 1 is accomplished using a CRISPR-Cas9 system.

9. The method according to claim 7 or 8, characterized in that: the seed size is embodied in the length, width and/or thickness of the seed;

and/or, the plant is M1) or M2) or M3):

m1) monocotyledonous or dicotyledonous plants;

m2) a gramineous plant;

m3) rice.

10. A protein as claimed in claims 1 to 3 or a substance regulating the content or activity of said protein.