Use of protein OsCDKB2;1 or a substance regulating its expression in regulating rice yield

Abstract

The present invention discloses the application of the protein OsCDKB2;1 or substances regulating its expression in regulating rice yield. The present invention belongs to the field of biotechnology. The protein provided by the present invention is the OsCDKB2;1 protein, which may be as follows: A1) a protein with an amino acid sequence of SEQ ID No.1; A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues to the protein of A1), having more than 80% identity with the protein of A1) and having the function of regulating plant stress resistance; A3) a fusion protein obtained by connecting a protein tag to one end of A1) or A2). Using the CRISPR / Cas9-mediated gene editing technology to perform site-directed mutation or knockout of specific targets on the rice grain development-related coding gene OsCDKB2;1 provides new materials for the breeding of rice varieties with growth performance and has a positive effect on accelerating the improvement of rice varieties.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of a protein OsCDKB2;1 or a substance for regulating the expression of the protein OsCDKB2;1 in regulating rice yield. Background Art

[0002] In recent years, extreme temperatures and water shortages caused by climate change have become a global problem that needs to be solved urgently. With the continuous growth of population and the reduction of available fresh water, the imbalance between food supply and demand has been further aggravated. Rice is one of the most important food crops in my country and occupies an irreplaceable and important position in the development of the national economy. Extreme temperatures affect the changes in global extreme climate, which threatens crop yields. Data surveys show that increased salt concentrations lead to significant reductions in rice yields. The coordinated improvement of crop yield and stress resistance is an effective response strategy to address the frequent occurrence of extreme weather such as population growth and global warming. Exploring key genes that coordinately regulate rice yield and salt tolerance is the key to accelerating rice improvement. Summary of the invention

[0003] The main technical problem to be solved by the present invention is how to improve the growth performance of rice.

[0004] In order to solve the above technical problems, the present invention provides the use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in regulating plant growth performance.

[0005] The application of the protein or the gene expression regulating substance or the substance regulating the activity or content of the protein provided by the present invention in regulating plant growth performance can be any of the following:

[0006] P1) Use of the protein or the substance regulating the expression of the gene or the substance regulating the activity or content of the protein in regulating the growth performance of plants,

[0007] P2) Use of the protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in cultivating plants with improved growth performance,

[0008] P3) Application of the protein or the substance regulating the expression of the gene or the substance regulating the activity or content of the protein in germplasm resources for improving plant growth performance.

[0009] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene, wherein the gene encodes the protein OsCDKB2;1.

[0010] The protein OsCDKB2;1 can be the following protein A1), A2) or A3):

[0011] A1) A protein with an amino acid sequence shown in SEQ ID No. 1;

[0012] A2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein of A1), having more than 80% identity with the protein shown in A1) and having the function of regulating plant stress resistance;

[0013] A3) A fusion protein obtained by connecting a protein tag to one end of A1) or A2).

[0014] In the above text, the substance that regulates gene expression can be a substance that performs at least one of the following 6 regulations: 1) Regulation carried out at the gene transcription level; 2) Regulation carried out after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) Regulation of the RNA transport of the gene (that is, regulation of the transport of the gene's mRNA from the nucleus to the cytoplasm); 4) Regulation of the translation of the gene; 5) Regulation of the degradation of the gene's mRNA; 6) Post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0015] In this article, the regulation can be up-regulation or enhancement or increase, or can also be down-regulation or inhibition or decrease.

[0016] In this article, the regulation of plant growth performance can be up-regulation or enhancement or increase of plant growth performance, or can also be down-regulation or inhibition or decrease of plant growth performance.

[0017] In this article, the plant growth performance can include plant grain type such as grain width, or / and 1000-grain weight.

[0018] In this article, the regulation of the expression of the coding gene of the protein can be inhibition or decrease or down-regulation of the expression of the coding gene. Inhibition or decrease or down-regulation of the expression of the coding gene can be achieved by gene knockout or gene silencing.

[0019] The so-called gene knockout refers to the phenomenon that a specific target gene is inactivated by gene editing technology. Gene knockout inactivates a specific target gene through changes in the DNA sequence.

[0020] The gene silencing refers to the phenomenon that a gene is not expressed or is expressed at a low level without damaging the original DNA. Gene silencing, on the premise of not changing the DNA sequence, causes the gene not to be expressed or to be expressed at a low level. Gene silencing can occur at two levels. One is transcriptional gene silencing caused by DNA methylation, heterochromatinization, and position effect, etc. The other is post-transcriptional gene silencing, that is, at the post-transcriptional level of the gene, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, quelling, RNA interference (RNAi), and translational inhibition mediated by microRNA (miRNA), etc.

[0021] Among the above-mentioned proteins, the protein is derived from rice (Oryza sativa).

[0022] Among the above-mentioned proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology, so as to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein-tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0023] Among the above-mentioned proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined by using homology search sites on the Internet, such as the BLAST web page of the NCBI homepage website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0024] Among the above-mentioned proteins, the identity of more than 80% can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0025] Among the above-mentioned biological materials, B1) the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0026] In the above applications, the substance for regulating gene expression and the substance for regulating the activity or content of the protein may be biological materials related to the protein, and the biological materials may be any one of the following:

[0027] B1) A nucleic acid molecule encoding the protein;

[0028] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0029] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0030] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0031] B5) A transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2);

[0032] B6) A transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

[0033] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2);

[0034] C1) A nucleic acid molecule that inhibits or reduces or silences the expression of the encoding gene of the protein;

[0035] C2) The encoding gene expressing the nucleic acid molecule described in C1);

[0036] C3) An expression cassette containing the encoding gene described in C2);

[0037] C4) A recombinant vector containing the encoding gene described in C2), or a recombinant vector containing the expression cassette described in C3);

[0038] C5) A recombinant microorganism containing the encoding gene described in C2), or a recombinant microorganism containing the expression cassette described in C3), or a recombinant microorganism containing the recombinant vector described in C4);

[0039] C6) A transgenic plant cell line containing the encoding gene described in C2), or a transgenic plant cell line containing the expression cassette described in C3), or a transgenic plant cell line containing the recombinant vector described in C4);

[0040] C7) A transgenic plant tissue containing the encoding gene described in C2), or a transgenic plant tissue containing the expression cassette described in C3), or a transgenic plant tissue containing the recombinant vector described in C4);

[0041] C8) A transgenic plant organ containing the coding gene described in C2), or a transgenic plant organ containing the expression cassette described in C3), or a transgenic plant organ containing the recombinant vector described in C4).

[0042] B1) In the said nucleic acid molecule, those skilled in the art can easily mutate the nucleotide sequence of the protein OsCDKB2;1 of the present invention by using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides with 75% or more identity to the nucleotide sequence of the protein OsCDKB2;1 isolated from the present invention, as long as they encode the protein OsCDKB2;1 and have the function of the protein OsCDKB2;1, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0043] Among the above biological materials, the nucleic acid molecule described in B1) can be the coding gene of the said protein.

[0044] The nucleic acid molecule described in B1) can be a DNA molecule of any of the following (a1), (a2), or (a3):

[0045] (a1) A DNA molecule whose coding region is as shown in Sequence 2 in the Sequence Listing;

[0046] (a2) A DNA molecule that hybridizes with the defined DNA molecule under stringent conditions and encodes the said protein;

[0047] (a3) A DNA molecule derived from rice and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the defined DNA molecule and encoding the said protein.

[0048] Specifically, the nucleic acid molecule described in B1) can be a DNA molecule whose coding sequence of the coding strand is as shown in Sequence 2.

[0049] In this article, the said vector is well-known to those skilled in the art and includes, but is not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), Ti plasmids, or viral vectors. Specifically, it can be vector pFGC5941 and / or pCAMBIA1300 and / or vector pEASY-Blunt simple;

[0050] Among the above-mentioned biomaterials, the expression cassettes described in B2) and C3) refer to DNAs capable of expressing the said gene in a host cell, which may include not only a promoter for initiating gene transcription but also a terminator for terminating gene transcription. Further, the said expression cassette may also include enhancer sequences. 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. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); the tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); the heat shock promoter (U.S. Patent 5,187,267); the tetracycline-inducible promoter (U.S. Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 2007 1 0099169.7)), the promoters specific for seed storage proteins (for example, the promoters of phaseolin, napin, oleosin and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can 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: the Agrobacterium tumefaciens nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the terminators of nopaline and octopine synthases (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (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).

[0051] For the convenience of identifying and screening transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes encoding enzymes or luminescent compounds that can produce color changes and can be expressed in plants (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical reagent-resistant marker genes (such as herbicide-resistant genes).

[0052] The present invention also provides a method for upregulating or enhancing or improving the growth performance of rice.

[0053] The method for upregulating or enhancing or improving the growth performance of rice provided by the present invention includes the following steps: downregulating or inhibiting or reducing the expression of the coding gene of the protein in rice, so as to improve the growth performance of rice.

[0054] In a specific embodiment, the method for upregulating or enhancing or improving the growth performance of rice may include the following steps:

[0055] A) Using the CRISPR / Cas9 system to perform gene editing on the coding gene OsCDKB2;1 of the OsCDKB2;1 protein in the recipient rice, and causing the OsCDKB2;1 gene to mutate, resulting in premature termination of the translated protein, to obtain the target rice;

[0056] B) Self-crossing the target rice to obtain homozygous rice, which is the target rice, and the growth performance of the target rice is better than that of the recipient rice.

[0057] The OsCDKB2;1 gene is a DNA molecule of the following (a1) or (a2) or (a3):

[0058] (a1) A DNA molecule in which the coding region of the OsCDKB2;1 gene is as shown in Sequence 2 in the Sequence Listing;

[0059] (a2) A DNA molecule that hybridizes with the defined DNA molecule under stringent conditions and encodes the protein OsCDKB2;1;

[0060] (a3) A DNA molecule derived from rice and having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology with the defined DNA molecule and encoding the protein.

[0061] The present invention also provides a method for cultivating rice with enhanced growth performance.

[0062] The method for cultivating rice with enhanced growth performance provided by the present invention comprises the following steps: down-regulating, inhibiting or reducing the expression of the coding gene of the protein in the rice to obtain the target rice, and the growth performance of the target rice is stronger than that of the rice.

[0063] In this article, the enhancement of the growth performance is mainly reflected in the increase in grain width and 1000-grain weight.

[0064] In the above method, the down-regulation, inhibition or reduction of the expression of the coding gene of the protein in the rice is to knockout the coding gene of the protein in the target rice.

[0065] In the above method, the knockout is achieved through the CRISPR / Cas9 system.

[0066] In the above method, the target for gene editing by the CRISPR / Cas9 system is the 2763-2782nd position of sequence 3 or the 211-230th position of the corresponding sequence 2.

[0067] In the above method, the knockout of the coding gene of the protein in the target rice may be to perform at least one of the following mutations on the coding gene of the protein of sequence 3 in the rice genome:

[0068] 1) Replace 5'-ACGGCGCTCAGGGAGGTGTC-3' in the coding gene of the protein in the rice genome DNA with 5'-ACGTCGCTCAGGGAGGTGTC-3', thereby knocking out the gene encoding OsCDKB2;1 protein;

[0069] 2) Replace 5'-ACGGCGCTCAGGGAGGTGTC-3' in the coding gene of the protein in the rice genome DNA with 5'-GCGGCGCTCAGGGAGGTGTC-3', thereby knocking out the gene encoding OsCDKB2;1 protein;

[0070] The present invention also provides a substance for improving the grain size of rice, which is a CRISPR / Cas9 system for gene editing the gene encoding OsCDKB2;1 protein; the target for gene editing by the CRISPR / Cas9 system is the 2763-2782nd position of sequence 3 or the 211-230th position of the corresponding sequence 2.

[0071] The present invention uses the CRISPR / Cas9-mediated gene editing technology to perform site-directed mutation or knockout of specific targets on the coding gene of the rice grain development-related protein OsCDKB2;1, providing new materials for the breeding of rice varieties with increased grain size, and playing a positive role in accelerating the improvement of rice varieties. Description of the Drawings

[0072] Figure 1 It is a schematic structural diagram of the starting plasmid SG2027.

[0073] Figure 2 It is the sequencing result of the mutation site and its surrounding nucleotides.

[0074] Figure 3 They are the phenotypes and statistical charts of key agronomic traits of rice. Among them, A is the grain width phenotype of Nip and oscdkb2;1 mutants; B is the statistical data of the grain width of Nip and oscdkb2;1 mutants; C is the statistical data of the 1000-grain weight of Nip and oscdkb2;1 mutants. Specific implementation manners

[0075] The present invention will be further described in detail below in combination with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided examples can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0076] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0077] Unless otherwise specified, the quantitative tests in the following examples are all set with three repeated experiments, and the results are averaged.

[0078] The SG2027 vector in the following examples has been described in Zhang Q, Liang Z, Cui X, et al. N6-methyladenine DNA methylation in Japonica and Indica rice genomes and its association with gene expression, plant development, and stress responses. Molecular plant, 2018, 11(12): 1492 - 1508. The public can obtain this biological material from the applicant. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0079] EXCEL software is used for statistical analysis of data, and SPASS software is used for multiple difference comparison of data.

[0080] Example 1. Creation of OsCDKB2;1 gene knockout rice

[0081] 1. Obtaining of the rice OSCDKB2;1 gene

[0082] Extract the leaf DNA of the rice variety Nipponbare. Using this DNA as a template, with primers OSCDKB2;1-F: 5’-ATGGCGGCGCTCCACCACCA-3’; OSCDKB2;1-R: 5’-GTAGAGCTCCTTGTTCACGTCGT-3’, perform PCR amplification using Max Super-Fidelity DNA Polymerase (product number: P505-d1, Vazyme) to obtain the amplification product, which is the coding region (CDS) of the OSCDKB2;1 gene.

[0083] The coding sequence of the OSCDKB2;1 gene in the rice variety Nipponbare is SEQ ID No.2, encoding the OSCDKB2;1 protein with the amino acid sequence of SEQ ID No.1. In the genomic DNA of rice Nipponbare, the nucleotide sequence of the genomic gene encoding the OSCDKB2;1 protein is SEQ ID No.3. The 2553-2963rd positions of SEQ ID No.3 are the first exon, the 3113-3286th positions are the second exon, the 3399-3492nd positions are the third exon, the 3582-3651st positions are the fourth exon, the 3758-3896th positions are the fifth exon, and the 4059-4151st positions are the sixth exon.

[0084] 2. Construction of the recombinant plasmid SG2027-OsCDKB2;1 for knocking out the OsCDKB2;1 gene

[0085] The recombinant plasmid SG2027-OsCDKB2;1 contains a sequence with the nucleotide sequence of sequence 4 in the sequence listing. Sequence 4 is the gene sequence obtained by Sanger sequencing, including the sgRNA sequence, the coding region sequence of OsCDKB2;1, and the SG2027 vector sequences on both sides of the inserted sequence. SG2027-OsCDKB2;1 expresses an sgRNA targeting the OsCDKB2;1 gene, and the target sequence is: 5’-ACGGCGCTCAGGGAGGTGTC-3’ (SEQ ID No.5). The target site of this sgRNA is located in the first exon of the OsCDKB2;1 gene, and the nucleotide sequence of the target site of this sgRNA is at positions 2763-2782 of SEQ ID No.3 (corresponding to positions 211-230 of sequence 2). SG2027-OsCDKB2;1 contains an sgRNA gene expression cassette with the nucleotide sequence at positions 59-7247 of sequence 4. The sgRNA gene is located at nucleotides 519-538 of sequence 4. The nucleotides at positions 59-268 are the promoter for initiating the transcription of the sgRNA gene, and the nucleotides at positions 7052-7247 are the terminator for terminating the transcription of the sgRNA gene.

[0086] SG2027-OsCDKB2;1 contains a Cas9 protein gene expression cassette with the nucleotide sequence at positions 3801-8072 of sequence 4. The nucleotides at positions 3801-8072 in sequence 4 encode the Cas9 protein. The nucleotides at positions 725-2725 are the promoter for initiating the transcription of the Cas9 protein gene, and the nucleotides at positions 7052-7247 are the terminator for terminating the transcription of the Cas9 protein gene.

[0087] 3. Obtaining and identification of OsCDKB2;1 gene knockout rice

[0088] The recombinant plasmid SG2027-OsCDKB2;1 obtained in step 2 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium. Using the Agrobacterium infection method, the recombinant Agrobacterium was used to genetically transform the embryonic callus of rice Nipponbare, and then resistant callus was screened (resistance screening was carried out using 100 mg / L hygromycin), and then differentiation and regeneration culture were carried out, and then rooting culture was carried out to obtain regenerated plants.

[0089] The specific steps are as follows:

[0090] (1) Take out the mature seeds of rice Nipponbare, remove the husks, and pick the seeds that are plump, smooth, and free of sterile spots for disinfection.

[0091] (2) Inoculate the disinfected rice Nipponbare seeds onto the induction medium, and culture them in the dark at 28°C for about 14 days, and select the callus with good appearance and strong growth ability.

[0092] (3) Take the recombinant vector SG2027-OsCDKB2;1 constructed in step (2) above and introduce it into Agrobacterium tumefaciens EHA105 to obtain the recombinant bacterium EHA105 / SG2027-OsCDKB2;1.

[0093] (4) Take the recombinant bacterium EHA105 / SG2027-OsCDKB2;1 obtained in step (3) and resuspend the bacterial cells with an infection medium (MS liquid medium + 50 g / L sucrose + 50 μL / L Silwet L-77) to obtain an EHA105 / SG2027-OsCDKB2;1 bacterial suspension.

[0094] (5) Immerse the callus of Nipponbare in step (2) in the EHA105 / SG2027-OsCDKB2;1 bacterial suspension prepared in step (4) and infect for 20 min. After infection, pour out the bacterial suspension, take the callus, dry it with sterile filter paper, and then place it on a co-culture medium (MS basal medium) containing acetosyringone and glucose, and culture it in the dark at 28 °C for 50 - 55 h.

[0095] (6) After completing step (5), select the callus with no obvious Agrobacterium on the surface and transfer it to a bacteriostatic medium (MS basal medium) containing cefotaxime, and culture it in the dark at 28 °C for 3 - 4 days.

[0096] (7) Transfer the callus cultured above to a selection medium (MS basal medium) containing hygromycin and cefotaxime and culture it in the dark at 28 °C for 30 days, and subculture it every 10 days.

[0097] (8) After completing step (7), take the fresh hygromycin-resistant callus and inoculate it into a pre-regeneration medium (MS basal medium), culture it in the dark at 28 °C for 7 days, then place it in a light culture room (12 h light / 12 h dark) and continue to culture for 7 days, and then transfer it to a regeneration medium and continue light culture until regenerated plants grow out to obtain candidate OsCDKB2;1 gene knockout rice plants.

[0098] Denote the transgenic plants obtained using the recombinant vector SG2027-OsCDKB2;1 as OsCDKB2;1 transgenic plants.

[0099] The formulations of the induction medium and the differentiation medium are both MS medium.

[0100] 4. Identification of OsCDKB2;1 gene knockout rice

[0101] Test plants: Nipponbare Nip (control group) and the candidate OsCDKB2;1 gene knockout plants obtained in step 3.

[0102] Extract the genomic DNA from the leaves of the rice plants with the OsCDKB2;1 gene knockout to be tested. Using the genomic DNA as a template, perform PCR amplification with the primer pair consisting of primer OsCDKB2;1-F1 and primer OsCDKB2;1-R1.

[0103] OsCDKB2;1-F1: 5’-CGAGCTGCGGGCGATGGAC-3’;

[0104] OsCDKB2;1-R1: 5’-GCCGGATGGATACCTTGACGGT-3’.

[0105] Use the SG2027-OsCDKB2;1 plasmid as the positive control (V), and use the recipient variety Nipponbare as the negative control (Nip). Then sequence the obtained products.

[0106] After sequencing and identification, 2 homozygous edited plants (i.e., the mutations on the two homologous chromosomes are the same) were obtained, named OsCDKB2;1-1 and OsCDKB2;1-2 respectively.

[0107] Compared with the genomic DNA of Nip, in the two homologous chromosomes of the mutant plants with the mutation type cdkb2;1-1, the genes encoding the OsCDKB2;1 protein both underwent the following mutations: “5’-ACGGCGCTCAGGGAGGTGTC-3’” mutated to “5’-ACGTCGCTCAGGGAGGTGTC-3’”, resulting in amino acid mutations after the editing site. The sequencing results of this mutation site and its surrounding nucleotides are shown in Figure 2 .

[0108] Compared with the genomic DNA of Nip, in the two homologous chromosomes of the mutant plants with the mutation type cdkb2;1-1, the genes encoding the OsCDKB2;1 protein both underwent the following mutations: “5’-ACGGCGCTCAGGGAGGTGTC-3’” mutated to “5’-GCGGCGCTCAGGGAGGTGTC-3’”, resulting in amino acid mutations after the editing site. The sequencing results of this mutation site and its surrounding nucleotides are shown in Figure 2 .

[0109] Continue to cultivate the T1 generation rice mutant plants of the above-mentioned OsCDKB2;1 gene homozygous mutation types OsCDKB2;1-1 and OsCDKB2;1-2 strains, screen to obtain the T2 generation OsCDKB2;1 plants without transgenic elements, and conduct phenotypic identification.

[0110] The OsCDKB2;1-1 plants were self-crossed and the seeds were harvested. The seeds were cultivated into plants, which were the T1 generation plants. The T1 generation plants were self-crossed and the seeds were harvested, which were the T2 generation seeds. The OsCDKB2;1-1 plants and their self-crossed offspring were called the OsCDKB2;1-1 strain.

[0111] The OsCDKB2;1-2 plants were self-crossed and the seeds were harvested. The seeds were cultivated into plants, which were the T1 generation plants. The T1 generation plants were self-crossed and the seeds were harvested, which were the T2 generation seeds. The OsCDKB2;1-2 plants and their self-crossed offspring were called the OsCDKB2;1-2 strain.

[0112] Example 2. Comparison of rice production traits

[0113] The plants to be tested were: rice Nip, the mutant OsCDKB2;1-1, and the T3 generation homozygous lines of the OsCDKB2;1-2 strain.

[0114] The seeds of each plant to be tested were germinated and seedlings were raised in the greenhouse (starting from when the seeds showed white tips and cultivating for a total of 3 weeks), obtaining 3-week-old seedlings. The 3-week-old seedlings were transplanted to the fields in Langfang, Hebei and were normally cultivated and managed. The grain shape and 1000-grain weight data of the test lines were measured, and at least 30 individual plant data were statistically analyzed for each material.

[0115] The growth conditions of the plants to be tested are shown in Figure 3 , compared with Nip, the grain width ( Figure 3 A and B in Figure 3 ) and 1000-grain weight of OsCDKB2;1-1 and OsCDKB2;1-2 were significantly increased (

[0116] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Method for increasing the 1000-grain weight of rice, comprising the following steps: mutating the coding gene of the protein in the recipient rice to increase the 1000-grain weight of the rice, wherein the mutation is achieved through the CRISPR / Cas9 system, and the gene editing target of the CRISPR / Cas9 system is positions 2763-2782 of Sequence 3 or positions 211-230 of the corresponding Sequence 2; The protein is the protein of the following A1) or A2): A1) The protein with the amino acid sequence shown in SEQ ID No.1; A2) The fusion protein obtained by connecting a protein tag to one end of A1); The protein is derived from rice; The coding gene of the protein in the mutated recipient rice is one of the following mutations of the gene of the protein shown in Sequence 3 in the rice genome: 1) Replace 5'-ACGGCGCTCAGGGAGGTGTC-3' in the coding gene of the protein in the rice genome DNA with 5'-ACGTCGCTCAGGGAGGTGTC-3'; 2) Replace 5'-ACGGCGCTCAGGGAGGTGTC-3' in the coding gene of the protein in the rice genome DNA with 5'-GCGGCGCTCAGGGAGGTGTC-3'.

2. Method for cultivating rice with increased 1000-grain weight, comprising the following steps: mutating the coding gene of the protein in the recipient rice in the method of claim 1 to obtain the target rice, and the 1000-grain weight of the target rice is higher than that of the recipient rice; The mutation is achieved through the CRISPR / Cas9 system, and the gene editing target of the CRISPR / Cas9 system is positions 2763-2782 of Sequence 3 or positions 211-230 of the corresponding Sequence 2; The coding gene of the protein in the mutated recipient rice in the method of claim 1 is one of the following mutations of the gene of the protein shown in Sequence 3 in the rice genome: 1) Replace 5'-ACGGCGCTCAGGGAGGTGTC-3' in the coding gene of the protein in the rice genome DNA with 5'-ACGTCGCTCAGGGAGGTGTC-3'; 2) Replace 5'-ACGGCGCTCAGGGAGGTGTC-3' in the coding gene of the protein in the rice genome DNA with 5'-GCGGCGCTCAGGGAGGTGTC-3'.

3. A substance for increasing the 1000-grain weight of rice, which is the CRISPR / Cas9 system for gene editing the coding gene of the protein in the method of claim 1; the gene editing target of the CRISPR / Cas9 system corresponds to positions 2763-2782 of Sequence 3.

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