CN118222623A - Application of Rice AGO12 Gene and Its Encoded Protein in Controlling Rice Grain Size and Yield - Google Patents

Abstract

The invention discloses application of rice AGO12 gene and its coding protein in controlling rice grain size and yield, and experiments show that over-expressing OsAGO12 gene in rice can increase rice grain length and width and raise rice grain size and thousand grain weight, thus increasing rice yield. The OsAGO12 protein can increase the grain size of plants and improve the plant yield by regulating the size of the exocaryopsis cells. The OsAGO12 protein has great application value in cultivating large-grain high-yield plant varieties.

Description

Application of rice AGO12 gene and its encoded protein in controlling rice grain size and yield

Technical Field

The invention relates to the fields of biotechnology and gene technology, and in particular to the application of rice AGO12 gene and its encoded protein in controlling rice grain size and yield.

Background technique

With the continuous growth of the global population, the issue of food security has become increasingly prominent and has become a focus of common concern in society. With the rapid development of the economy and the acceleration of urbanization, agricultural land is facing unprecedented pressure. The expansion of industrial and residential land has led to a continuous reduction in the area of traditional agricultural land, which poses a direct challenge to agricultural production. At the same time, with the improvement of residents' living standards, the demand for diversified agricultural products has increased, and the planting area of non-food crops has expanded accordingly, which has affected the planting area and yield of food crops to a certain extent, and thus posed a potential threat to national food security.

Among many food crops, rice occupies a pivotal position. Its yield is directly related to national food security and people's living standards. Therefore, increasing rice yield, especially improving the agronomic traits of rice through scientific means, plays a vital role in ensuring national food security.

Rice grain size is one of the key agronomic traits that determines yield. Grain size not only directly affects the yield per unit area, but is also closely related to the processing quality and appearance quality of rice, which in turn affects the market value of rice and consumer acceptance. Therefore, in-depth research on the regulatory mechanism of rice grain development and the discovery of genes related to grain size can not only improve rice yield and quality, but also provide a theoretical basis and technical support for rice molecular breeding.

In addition, with the continuous advancement of biotechnology, especially the development of gene editing technology, researchers are able to improve the genetic characteristics of rice more accurately. Through genetic engineering, new rice varieties that are more adaptable to environmental changes, have strong disease resistance and high yield can be cultivated, thereby achieving higher food output on limited land resources.

In summary, facing the dual pressures of population growth and tight land resources, strengthening rice genetic research, optimizing rice agronomic traits, and improving rice yield and quality have great practical significance and far-reaching strategic impact for ensuring my country's food security and promoting sustainable agricultural development.

Summary of the invention

In view of this, the object of the present invention is to propose a method for using the rice AGO12 gene and its encoded protein in controlling rice grain size and yield, which is reliable in implementation, flexible in application, and capable of increasing plant grain size and yield.

In order to achieve the above technical objectives, the technical solution adopted by the present invention is:

The use of OsAGO12 protein or OsAGO12 gene encoding the protein or a vector comprising the gene, wherein the use is any one of the following M1)-M6):

M1) Regulate plant yield;

M2) regulating plant seed growth and/or seed size;

M3) regulates plant spike development and branching;

M4) regulates the size of the outer glume cells of plant grains;

M5) Application in cultivating transgenic plants with enlarged grains and/or increased yield;

M6) Plant breeding;

Wherein, the OsAGO12 protein is one of the following:

a1) a protein with an amino acid sequence as shown in SEQ ID NO: 2;

a2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

a3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2;

a4) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having the same function.

In order to facilitate the purification of the OsAGO12 protein with the amino acid sequence in a1), the tag sequence shown in Table 1 can be connected to the amino terminus of the protein shown in SEQ ID NO:2.

Table 1

Label Residues sequence FLAG 8 DYKDDDDK

In a3), the substitution and/or deletion and/or addition of one or several amino acid residues is the substitution and/or deletion and/or addition of no more than 10 amino acid residues; at the same time, the protein in a3) can be artificially synthesized, or its encoding gene can be synthesized first and then obtained by biological expression.

In addition, in a3), the coding gene of the protein can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID NO: 1, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 to its 5′ end and/or 3′ end.

The homology in a4) includes amino acid sequences having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence shown in SEQ ID NO: 2 of the present embodiment.

In addition, the OsAGO12 gene is one of the following:

b1) a cDNA molecule having a nucleotide sequence as shown in SEQ ID NO: 1 in the sequence listing;

b2) a cDNA molecule or a genomic DNA molecule that has 75% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and encodes the OsAGO12 protein;

b3) a cDNA molecule or a genomic DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) and encodes the OsAGO12 protein.

In addition to the above, the present application also provides an application of a biomaterial comprising OsAGO12 protein, wherein the application is any one of the following M1)-M6):

M1) Regulate plant yield;

M2) regulating plant seed growth and/or seed size;

M3) regulates plant spike development and branching;

M4) regulates the size of the outer glume cells of plant grains;

M5) Application in cultivating transgenic plants with enlarged grains and/or increased yield;

M6) Plant breeding;

Wherein, the OsAGO12 protein is one of the following:

a1) a protein with an amino acid sequence as shown in SEQ ID NO: 2;

a2) a fusion protein obtained by connecting a tag to the N-terminus and/or C-terminus of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing;

a3) a protein having the same function obtained by replacing and/or deleting and/or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2;

a4) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing and having the same function.

As a possible implementation mode, further, the biomaterial described in this solution is any one of the following A1)-A8):

A1) a nucleic acid molecule encoding an OsAGO12 protein;

A2) an expression cassette containing the nucleic acid molecule described in A1);

A3) a recombinant vector containing the nucleic acid molecule described in A1);

A4) a recombinant vector containing the expression cassette described in A2);

A5) a recombinant microorganism containing the nucleic acid molecule described in A1);

A6) a recombinant microorganism containing the expression cassette described in A2);

A7) a recombinant microorganism containing the recombinant vector described in A3);

A8) A recombinant microorganism containing the recombinant vector described in A4).

As a preferred implementation option, preferably, the nucleic acid molecule described in scheme A1) is one of the following:

b1) a cDNA molecule having a nucleotide sequence as shown in SEQ ID NO: 1 in the sequence listing;

b2) a cDNA molecule or a genomic DNA molecule that has 75% or more identity with the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing and encodes the OsAGO12 protein;

b3) a cDNA molecule or a genomic DNA molecule that hybridizes with the nucleotide sequence defined in b1) or b2) and encodes the OsAGO12 protein.

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.

Based on the above, a person skilled in the art can easily mutate the nucleotide sequence encoding the OsAGO12 protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence encoding the OsAGO12 protein are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the OsAGO12 protein and have the same function.

The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence SEQ ID NO:2 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

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

The expression cassette (OsAGO12 gene expression cassette) containing a nucleic acid molecule encoding the OsAGO12 protein described in A2) above refers to a DNA capable of expressing the OsAGO12 protein in a host cell, and the DNA may include not only a promoter for initiating transcription of OsAGO12, but also a terminator for terminating transcription of OsAGO12. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters; tissue, organ and development-specific promoters and inducible promoters.

The recombinant vector containing the OsAGO12 gene expression cassette can be constructed using an existing expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pCambia2300, pCambia1300, pCambia1301, pCambia3301, pWM101, etc. The plant expression vector may also include the 3′ non-translated region of the exogenous gene, that is, a polyadenylic acid signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3′ end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the plant gene (such as the soybean storage protein gene) 3′ end transcribed non-translated region has similar functions. In the present invention, when constructing a plant expression vector, an enhancer sequence, such as a translation enhancer or a transcription enhancer, can be used to improve the expression efficiency of the gene. These enhancer sequences are usually located at the ATG start codon or its adjacent region, but must be consistent with the reading frame of the coding sequence to ensure the correct protein translation process. Translation control signals and start codons can be derived from nature, or can be obtained by synthetic methods, and they can also be derived from a part of the transcription start region or structural gene. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors involved in the present invention can contain a variety of marker genes. These marker genes can be enzyme genes (such as β-glucosidase gene GUS, luciferase gene, etc.) that can be expressed in plants and produce color changes or luminescent compounds, or genes that provide antibiotic resistance (such as nptII, bar, hph, dhfr and EPSPS genes), as well as marker genes for chemical resistance (such as herbicide resistance genes) and genes that provide specific metabolic capabilities (such as mannose-6-phosphate isomerase gene). Taking into account the biosafety and environmental impact of transgenic plants, the present invention also provides a method without the use of selective marker genes, that is, directly screening transformed plants through adverse screening. This method aims to reduce the ecological risks that may be caused by transgenic plants, while simplifying the screening and identification process of transgenic plants and improving the safety and controllability of transgenic technology. Through these innovative methods, the present invention aims to promote the efficiency and safety of plant genetic improvement and provide support for agricultural production and ecological environment protection. The above-mentioned vector can be a plasmid, a cosmid, a phage or a viral vector. In a specific embodiment of the present invention, the vector is pCambia2300-Actin. The recombinant vector is pCambia2300-Actin-OsAGO12. The pCambia2300-Actin-OsAGO12 is a double-stranded DNA molecule shown in nucleotides 1-3177 shown in SEQ ID NO:1 of the sequence list inserted between the Sal I and PstI restriction sites of the pCambia2300-Actin vector.

The microorganisms can be yeast, bacteria, algae or fungi, such as Agrobacterium. In a specific embodiment of the present invention, the Agrobacterium EHA105 and the recombinant bacteria are Agrobacterium EHA105 containing the pCambia2300-Actin-OsAGO12.

As a preferred implementation option, preferably, in the above M1) of this scheme, the regulating plant yield is regulating the thousand-grain weight of plant grains, specifically increasing the thousand-grain weight of plant grains.

M2) The regulating of plant seed size is regulating plant seed length and/or seed width;

The regulation of the size of the outer husk cells of plant grains in M4) is to regulate the length and/or cell width of the outer husk cells of plant grains.

In this scheme, the purpose of plant breeding is to cultivate large-grain and high-yield plant varieties.

As a preferred implementation option, preferably, the plant described in this scheme is a monocotyledonous plant or a dicotyledonous plant;

Alternatively, the plant is rice.

Based on the above, the present solution also provides a method for cultivating transgenic plants with enlarged grains and/or increased yield, which comprises: obtaining a target plant having an OsAGO12 protein or an OsAGO12 gene encoding the protein, and increasing the expression amount and/or activity of the OsAGO12 protein in the target plant to obtain a transgenic plant;

Wherein, the seed size and/or yield of the transgenic plant are higher than those of the target plant.

As a preferred implementation option, preferably, the grain size and/or yield of the transgenic plant in this scheme is higher than that of the target plant, which is embodied in any one of the following N1)-N6):

N1) the transgenic plant ear grows larger than the target plant;

N2) the number of primary branches of the transgenic plant ear is greater than that of the target plant;

N3) The number of secondary branches on the ear of the transgenic plant is greater than that of the target plant.

N4) The width of the seeds of the transgenic plant is greater than that of the target plant;

N5) The length of the seeds of the transgenic plant is greater than that of the target plant;

N6) The thousand-grain weight of the transgenic plant grains is greater than that of the target plant.

As a preferred implementation option, preferably, the method of increasing the expression amount and/or activity of the OsAGO12 protein in the target plant in this scheme is: overexpressing the OsAGO12 protein in the target plant;

Wherein, the overexpression method is: introducing the OsAGO12 gene encoding the OsAGO12 protein into the target plant;

The OsAGO12 gene is a DNA molecule with a nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

In this scheme, the "introduction of the coding gene of the OsAGO12 protein into the target plant" is achieved by introducing a recombinant expression vector containing the coding gene of the OsAGO12 protein into the target plant. The recombinant expression vector is a plasmid that can express the OsAGO12 protein obtained by inserting the coding gene of the OsAGO12 protein into a plant expression vector.

In a specific embodiment of the present invention, the recombinant expression vector may be: a recombinant plasmid pCambia2300-Actin-OsAGO12 obtained by inserting a double-stranded DNA molecule shown in nucleotides 1-3177 of sequence 1 in the sequence listing into the multiple cloning site of the pCambia2300-Actin vector (e.g., between the Xba I and BamH I restriction sites).

In the above method, the transgenic plant is understood to include not only the first generation transgenic plant obtained by transforming the OsAGO12 gene into the target plant, but also its progeny. For transgenic plants, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. The transgenic plant includes seeds, callus, whole plants and cells.

In any of the above applications or methods, the plant is a monocot or a dicot. The monocot may be a Gramineae plant; the Gramineae plant may be a Rice plant. The Rice plant may be Rice, such as Rice 11.

The present invention scheme has found through experiments that overexpression of the OsAGO12 gene in rice can increase the length and width of rice grains, improve rice grain size and 1000-grain weight, and thus increase rice yield. OsAGO12 protein can increase plant grain size and improve plant yield by regulating the size of grain outer glume cells. OsAGO12 protein has great application value in cultivating large-grain and high-yield plant varieties.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

Figure 1 shows the Western identification of OsAGO12 transgenic rice.

Figure 2 shows the identification of OsAGO12 CRISPR/Cas9 transgenic rice.

Figures 3A-3D are photographs of the ears of transgenic strains and the statistical results of ear length and branch number. Specifically, Figure 3A is a photograph of rice ears of different materials; Figure 3B is a statistical analysis of the length of rice ears of different materials; Figure 3C is a statistical analysis of the number of primary branches of rice ears of different materials; Figure 3D is a statistical analysis of the number of secondary branches of rice ears of different materials.

Figures 4A-4E are grain photos and grain size statistics of transgenic lines; specifically, Figure 4A is a photo of the width of rice grains of different materials. Figure 4B is a statistical analysis of the width of rice grains of different materials; Figure 4C is a photo of the length of rice grains of different materials; Figure 4D is a statistical analysis of the length of rice grains of different materials; Figure 4E is a statistical analysis of the thousand-grain weight of rice grains of different materials.

Figures 5A-5C are photos of the outer husk cells of the grains of the transgenic lines and statistical analysis of the length and width of the outer husk cells; specifically, Figure 5A is an electron microscope photo of the outer husk cells of the grains of different materials. Figure 5B is a statistical analysis of the length of the outer husk cells of the grains of different materials; Figure 5C is a statistical analysis of the width of the outer husk cells of the grains of different materials.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings and examples. It is particularly noted that the following examples are only used to illustrate the present invention, but are not intended to limit the scope of the present invention. Similarly, the following examples are only partial embodiments of the present invention rather than all embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were repeated three times, and the results were averaged.

The full name of the pCambia2300-Actin vector in the following examples is pCambia2300-Actin-FLAG plant overexpression vector, which was constructed by the laboratory itself.

The Agrobacterium EHA105 in the following examples is a product of Bomade Biotechnology Co., Ltd., with a product catalog number of BC303.

The rice Zhonghua 11 in the following embodiments is recorded in the document "Cloning of the rice glutenin Gt1 gene of "Zhonghua 11" and construction of the wax gene promoter-guided Gt1 gene expression vector", Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204), which can be obtained by the public from the applicant. The biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

The protein shown in SEQ ID NO: 2 in the sequence list is named as OsAGO12 protein. In rice cDNA, the coding region of OsAGO12 protein is shown in SEQ ID NO: 1 in the sequence list, and it is named as OsAGO12 gene.

Example 1. Obtaining OsAGO12 protein and its encoding gene

Primers were designed according to the sequence SEQ ID NO: 2, and the primer sequences were:

AGO12-F: 5'-ATGTCTTCGCGCGGCGG-3', SEQ ID NO: 3

AGO12-R: 5’-TCAGCAGTAGAACATGAACCGCT-3’; SEQ ID NO: 4.

According to the instructions, total RNA from Oryza sativa L. japonica cv. Zhonghua 11 rice (Oryza sativa L. japonica cv. Zhonghua 11, Xu Yu et al. "Cloning of the rice glutenin Gt1 gene and construction of the wax gene promoter-guided Gt1 gene expression vector of Zhonghua 11", Journal of Shanghai Normal University (Natural Science Edition), Vol. 39, No. 2, April 2010, p. 204) was extracted with TRIzol Reagent from Invitrogen, and reverse transcription was performed using the company's SuperScript II reverse transcriptase to obtain cDNA. The primer used for reverse transcription was a 16-nucleotide Oligod (T) primer.

The cDNA obtained by reverse transcription was used as a template and the above-mentioned gene-specific primers AGO12-F and AGO12-R were used for PCR (Polymerase Chain Reaction) reaction to obtain a 3177 bp PCR product having the nucleotide sequence shown in SEQ ID NO: 2 in the sequence table.

After the PCR product was recovered, it was connected with the vector pEASY-Blunt Zero Cloning Kit (Quanshijin Company, product catalog number: CB501-01) and transformed into Escherichia coli strain DH5α to obtain transformants. The plasmid extracted from the transformant was sent for sequencing. The plasmid was obtained by inserting the gene OsAGO12 shown in the sequence SEQ ID NO: 2 in the sequence table into the vector pEASY-Blunt. The plasmid was named pCambia2300-Actin-AGO12, which is a recombinant vector.

Example 2: Obtaining OsAGO12 overexpressing rice

1. Construction of expression vector

Using pEASY-Blunt-AGO12 as a template, PCR amplification was performed with primers AGO12-5'Sal I (sequence: 5'-aggggatcctctagagtcgac ATGTCTTCGCGCGGCGG-3' (SEQ ID NO: 5)) and AGO12-3'Pst I (sequence: 5'-taaagcagggcatgcctgcag TCAGCAGTAGAACATGAACCGCT-3' (SEQ ID NO: 6)) to obtain a PCR product of 3177 bp.

The above PCR product was purified and recovered, and connected with the pCambia2300-Actin-Flag plasmid vector backbone digested with Sal I and Pst I by seamless cloning (refer to Novezan, product catalog number: C112-01) to obtain the recombinant plasmid pCambia2300-Actin-FLAGAGO12. After sequencing, the recombinant plasmid pCambia2300-Actin-FLAGAGO12 was a vector obtained by inserting OsAGO12 between the Sal I and Pst I restriction sites of the pCambia2300-Actin-Flag plasmid.

2. Obtaining transgenic rice overexpressing OsAGO12

1) Callus induction culture

The seeds of Zhonghua 11 rice (hereinafter also referred to as wild-type rice) are shelled, first soaked in 70% ethanol for 10 minutes, and then soaked in 0.1% mercuric chloride for 30 minutes; surface sterilization is performed. The solution on the surface of the seeds is washed off with a large amount of sterile water, and the moisture on the surface of the seeds is absorbed with sterile filter paper. The seeds are placed on a mature embryo callus induction medium plate, the edge of the plate is sealed with Parafilm film, and cultured in a 26°C incubator away from light. After about 15 days, the grown callus tissue is carefully removed and transferred to the mature embryo subculture medium, and the culture is continued under the same conditions. Subculture is required every two weeks. When used for transformation, it is necessary to select granular callus tissue that has been subcultured for about 5 days and is light yellow.

2) Cultivation of Agrobacterium

pCambia2300-Actin-FLAGAGO12 was electroporated into Agrobacterium EHA105 to obtain the recombinant bacteria EHA105/pCambia2300-Actin-FLAGAGO12.

Streak EHA105/pCambia2300-Actin-FLAGAGO12 on an LB plate containing antibiotics (50 mg/L Kanamycin, 50 mg/L Rifampicin) and culture at 28°C for 2 days. Pick a single colony and inoculate it into liquid LB medium, culture it at 28°C with shaking until OD600 is about 0.5, add acetosyringone to a final concentration of 100 mM, and obtain an Agrobacterium suspension for transforming rice callus.

3) Co-culture of rice callus and Agrobacterium

Place the subcultured callus in a sterilized conical flask and pour in the Agrobacterium suspension to submerge the callus. Leave at room temperature for 20 minutes and gently shake from time to time to allow the callus to fully contact the bacterial solution. Gently remove the callus with sterile tweezers, place it on a sterile filter paper to absorb the excess bacterial solution, and transfer it to a co-culture medium plate covered with a layer of sterile filter paper. Culture in the dark at 28°C for 3 days to obtain co-cultured callus.

4) Screening and differentiation of resistant callus

The co-cultivated callus was washed with an appropriate amount of sterile water to remove the residual Agrobacterium on the surface, placed on a screening medium, and cultured in the dark at 26°C for screening. After two weeks, it was transferred to a new screening medium and continued to be screened for two weeks. Select the callus in good condition after two rounds of screening, transfer it to a differentiation medium plate, culture it in the dark for 3 days, and then transfer it to a light incubator (15hr/day) for light culture. Differentiated seedlings can be seen after one month. When the differentiated seedlings grow to about 2cm, transfer them to a rooting medium in a conical flask and continue to culture for about two weeks. Select seedlings with good growth and well-developed root systems, wash the culture medium on the roots with tap water, and transplant them into the soil. Collect the seeds to obtain T1 generation FLAGAGO12 rice seeds, and sow them to obtain T1 generation FLAGAGO12 rice.

The rice seeds of the T1 generation were preliminarily screened by G418 (the pCambia2300 vector carries the G418 resistance screening gene), and the germinated seeds indicated that the vector was transferred into the rice. The germinated seeds were planted in the soil, and after growing for 2 weeks, 0.1 g of leaves were taken and ground into powder with liquid nitrogen.

200 μl of protein extraction buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 8% β-mercaptoethanol, 20% glycerol) was added to the leaf powder of the transgenic rice strain with overexpression of OsAGO12, incubated on ice for 10 min, boiled at 100°C for 10 min, centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant was taken for SDS-PAGE. After transfer to the membrane, Western detection was performed. SDS-PAGE and Western Blot were performed according to the known methods and product instructions. The antibody used was anti-FLAG-HRP (sigma), and the endogenous Actin protein of rice was detected with the antibody anti-Actin as an internal reference. As shown in Figure 1, the band at 120 KDa was positive, indicating that the gene was transferred and the protein was expressed. Two strains #4 and #19 were selected for subsequent disease resistance analysis experiments (Zhonghua 11 in Figure 1 is wild-type rice, used as a negative control).

Example 3. Obtaining OsAGO12 CRISPR/Cas9 rice

1. Construction of expression vector

1) According to the website http://skl.scau.edu.cn/. Based on the PAM site with the terminal sequence of NGG, a 20 bp specific target sequence targeting the N-terminus of the OsAGO12 coding region was selected as follows: GCGGCGCGTCGACCCGTAGG;

2) Design primers according to the target DNA sequence and add BsaI restriction sites at both ends of the primers. The primer sequences are:

U-F: CTCCGTTTTACCTGTGGAATCG, SEQ ID NO: 7;

AGO12-gRT1: 5′-GCGGCGCGTCGACCCGTAGGgttttagagctagaaat-3′, SEQ ID NO: 8;

gRNA-R: CGGAGGAAAATTCCATCCAC, SEQ ID NO: 9;

AGO12-OsU3T1: 5′-CCTACGGGTCGACGCGCCGCgccacggatcatctgc-3′, SEQ ID NO: 10;

B1':TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG, SEQ ID NO: 11;

BL:AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC, SEQ ID NO: 12;

3) Using the intermediate vector as a template, three pairs of primers, U-F/AGO12-OsU3T1, AGO12-gRT1/gRNA-R, and B1’/BL, were used for three rounds of PCR amplification to obtain an expression cassette with a linker;

4) After amplification, the final product is cut and purified, and the final vector pYLCRISPR/Cas9, restriction endonuclease and T4 ligase are added to perform cutting and ligation according to a certain system;

5) Transform the Escherichia coli strain DH5α, apply kanamycin-resistant culture medium to obtain transformants, extract the plasmid of the transformants and send them for sequencing. The positive transformants are the final recombinant vector, named pYLCRISPR/Cas9-AGO12:sgRNA.

2. Obtaining OsAGO12 CRISPR/Cas9 Rice

1) Callus culture and transformation

See Example 2, 2, 1)-4)

2) Identification of OsAGO12 CRISPR/Cas9 rice

Leaf powder of the transgenic rice lines of OsAGO12 CRISPR/Cas9 was used to extract genomic DNA, and the specific method was referred to the High-Efficiency Plant Genomic DNA Extraction Kit (Tiangen Biochemical Technology Co., Ltd., Cat. No.: DP350). Afterwards, 0.5 g of genomic DNA was used as a template, and primers AGO12-C-F: 5'-TCGTCGGCGAGCGGCAAG-3' (SEQ ID NO: 13) and primer AGO12-C-R: 5'-TTGTCCGCGACTTGCACCAG-3' (SEQ ID NO: 14) were used for PCR reaction. The PCR products were directly sent for sequencing, and then sequence alignment was performed. As shown in Figure 2, the comparison of DNA sequencing results showed that the two positive strains were named ago12#1 and ago12#2, among which ago12#1 inserted an "A" between the 332nd and 333rd bases in the OsAGO12 coding region, and ago12#2 deleted 5bp between the 327th and 333rd bases in the OsAGO12 coding region, both of which caused the premature termination of the OsAGO12 amino acid sequence, resulting in the loss of OsAGO12 protein.

Example 4. Analysis of panicle length, branching and grain size of transgenic OsAGO12 rice

According to the production specifications, the following test materials: T ₃ generation transgenic rice with OsAGO12 gene OE-4 line, T ₃ generation transgenic rice with OsAGO12 gene OE-19 line, T ₃ generation transgenic rice with OsAGO12 knockout rice ago12#1 line, T ₃ generation transgenic rice with OsAGO12 knockout rice ago12#2 line and rice Zhonghua 11 plant (WT) were planted in the experimental field. The grains were harvested after the plants were strong and the grains were mature. The panicle length, branching, grain width, length and 1000-grain weight of plants with different test materials were compared.

The results are shown in Figures 3 (A-D) and 4 (A-E). Figure 3A is a photo of rice panicles of different materials; Figure 3B is a statistical analysis of the panicle length of rice of different materials; Figure 3C is a statistical analysis of the number of primary branches of rice panicles of different materials; Figure 3D is a statistical analysis of the number of secondary branches of rice panicles of different materials; the results show that the panicle length, the number of primary branches and the number of secondary branches of rice panicles of transgenic OsAGO12 gene rice are significantly greater than those of wild-type controls, while the panicle length, the number of primary branches and the number of secondary branches of rice panicles of ago12 gene knockout rice are significantly less than those of wild-type controls, indicating that overexpression of OsAGO12 gene plays an important role in improving the development and branching of rice panicles. Figure 4A is a photo of rice grain width of different materials; Figure 4B is a statistical analysis of rice grain width of different materials; Figure 4C is a photo of rice grain length of different materials; Figure 4D is a statistical analysis of rice grain length of different materials; Figure 4E is a statistical analysis of thousand-grain weight of rice grains of different materials.

The results showed that the grain width, length, width and 1000-grain weight of the rice with transgenic OsAGO12 gene were significantly greater than those of the wild-type control, while the grain width, length, width and 1000-grain weight of the rice with ago12 gene knockout were significantly smaller than those of the wild-type control, indicating that overexpression of the OsAGO12 gene plays an important role in increasing rice grain size and 1000-grain weight.

Example 5: Electron microscopic observation and analysis of hull cells of OsAGO12 transgenic rice grains

According to the production specifications, the following test materials: T ₃ generation transgenic rice with OsAGO12 gene OE-4 line, T ₃ generation transgenic rice with OsAGO12 gene OE-19 line, T ₃ generation transgenic rice with OsAGO12 knockout rice ago12#1 line, T ₃ generation transgenic rice with OsAGO12 knockout rice ago12#2 line and rice Zhonghua 11 plant (WT) were planted in the experimental field. The rice panicle grains were cross-linked, fixed and dehydrated with glutaraldehyde, and then 10 um thick samples were cut from the middle of the husk. The outer husk cells were analyzed by scanning electron microscopy. The length and width of the outer husk cells of the grains harvested from different test material plants were compared and statistical analysis was performed.

The results are shown in Figure 5 (A-C). Figure 5A is a photo of the outer hull cells of rice panicles of different materials; Figure 5B is a statistical analysis of the length of the outer hull cells of rice panicles of different materials; Figure 5C is a statistical analysis of the width of the outer hull cells of rice panicles of different materials; the results show that the length of the outer hull cells of rice grains transgenic for OsAGO12 gene is significantly greater than that of the wild-type control, while the width of the outer hull cells of rice grains knocked out for ago12 gene is significantly smaller than that of the wild-type control, indicating that the OsAGO12 gene plays an important role in increasing the size of the outer hull cells of rice grains. The OsAGO12 gene can promote the growth of the outer hull cells of rice grains, promote the development of rice seeds, and then increase the size and thousand-grain weight of rice seeds, thereby increasing the yield of rice.

The above descriptions are only some embodiments of the present invention, and are not intended to limit the protection scope of the present invention. Any equivalent device or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims (10)

1. Use of an OsAGO12 protein or an OsAGO12 gene encoding the protein or a vector comprising the gene, characterized in that the use is any one of the following M1) -M6):

   M1) regulating plant yield;

   m2) regulating plant grain growth and/or grain size;

   M3) regulating plant ear development and branching;

   M4) regulating plant grain exome cell size;

   m5) use in the cultivation of transgenic plants with increased grain size and/or yield;

   M6) plant breeding;

   wherein, the OsAGO12 protein is one of the following:

   a1 Protein with the amino acid sequence shown as SEQ ID NO. 2;

   a2 A fusion protein obtained by connecting a tag to the N end and/or the C end of an amino acid sequence shown in a sequence table SEQ ID NO. 2;

   a3 Protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence table SEQ ID NO. 2;

   a4 Protein which has 75% or more homology with the amino acid sequence shown in the sequence table SEQ ID NO. 2 and has the same function;

   The OsAGO12 gene is one of the following:

   b1 A cDNA molecule having a nucleotide sequence shown in a sequence table SEQ ID NO. 1;

   b2 A cDNA molecule or a genomic DNA molecule which has 75% or more identity with the nucleotide sequence shown in SEQ ID No.1 of the sequence table and codes for the OsAGO12 protein;

   b3 A cDNA molecule or a genomic DNA molecule which hybridizes to the nucleotide sequence defined in b 1) or b 2) and encodes the OsAGO12 protein.
2. 2. Use of a biological material comprising an OsAGO12 protein, characterized in that said use is any one of the following M1) -M6):

   M1) regulating plant yield;

   m2) regulating plant grain growth and/or grain size;

   M3) regulating plant ear development and branching;

   M4) regulating plant grain exome cell size;

   m5) use in the cultivation of transgenic plants with increased grain size and/or yield;

   M6) plant breeding;

   wherein, the OsAGO12 protein is one of the following:

   a1 Protein with the amino acid sequence shown as SEQ ID NO. 2;

   a2 A fusion protein obtained by connecting a tag to the N end and/or the C end of an amino acid sequence shown in a sequence table SEQ ID NO. 2;

   a3 Protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence table SEQ ID NO. 2;

   a4 Protein which has 75% or more homology with the amino acid sequence shown in the sequence table SEQ ID NO. 2 and has the same function.
3. 3. The use according to claim 2, wherein the biological material is any one of the following A1) -A8):

   a1 A nucleic acid molecule encoding an OsAGO12 protein;

   a2 An expression cassette comprising A1) said nucleic acid molecule;

   A3 A) a recombinant vector comprising the nucleic acid molecule of A1);

   A4 A recombinant vector comprising the expression cassette of A2);

   a5 A) a recombinant microorganism comprising the nucleic acid molecule of A1);

   a6 A) a recombinant microorganism comprising the expression cassette of A2);

   A7 A) a recombinant microorganism comprising the recombinant vector of A3);

   A8 A recombinant microorganism comprising the recombinant vector of A4).
4. 4. The use according to claim 3, wherein the nucleic acid molecule of A1) is one of the following:

   b1 A cDNA molecule having a nucleotide sequence shown in a sequence table SEQ ID NO. 1;

   b2 A cDNA molecule or a genomic DNA molecule which has 75% or more identity with the nucleotide sequence shown in SEQ ID No.1 of the sequence table and codes for the OsAGO12 protein;

   b3 A cDNA molecule or a genomic DNA molecule which hybridizes to the nucleotide sequence defined in b 1) or b 2) and encodes the OsAGO12 protein.
5. 5. Use according to one of claims 2 to 4, wherein the modulated plant yield in M1) is modulated plant kernel thousand kernel weight;

   The size of the plant seeds is regulated and controlled in M2) to regulate and control the length and/or width of the plant seeds;

   M4) is to regulate the length and/or cell width of the plant exotic cells.
6. 6. The use according to any one of claims 2 to 4, wherein the plant is a monocotyledonous plant or a dicotyledonous plant;

   Or, the plant is rice.
7. 7. A method of growing a transgenic plant having increased grain size and/or yield comprising: obtaining a target plant with an OsAGO12 protein or an OsAGO12 gene encoding the protein, and increasing the expression level and/or activity of the OsAGO12 protein in the target plant to obtain a transgenic plant;

   wherein the grain size and/or yield of the transgenic plant is higher than that of the plant of interest.
8. 8. The method of claim 7, wherein the transgenic plant has a grain size and/or yield higher than that of the plant of interest as represented by any one of the following N1) -N6):

   N1) the spike length of the transgenic plant is larger than that of the target plant;

   n2) the primary branch number of the transgenic plant ear is greater than that of the plant of interest;

   N3) the secondary branch number of the transgenic plant ear is greater than that of the target plant.

   N4) the width of the transgenic plant grain is larger than that of the target plant;

   n5) the length of the transgenic plant grain is greater than that of the plant of interest;

   n6) thousand kernel weight of the transgenic plant kernel is greater than the plant of interest.
9. 9. The method of claim 7, wherein the method for increasing the expression level and/or activity of the OsAGO12 protein in the plant of interest comprises: overexpressing the OsAGO12 protein in the plant of interest;

   Wherein the over-expression method comprises the following steps: introducing the encoding gene OsAGO12 of the OsAGO12 protein into a target plant;

   the OsAGO12 gene is a DNA molecule with a nucleotide sequence shown in a sequence table SEQ ID NO. 1.
10. 10. The method according to any one of claims 7 to 9, wherein the plant of interest is a monocotyledonous plant or a dicotyledonous plant;

    Or, the target plant is rice.