Application of rice OsSAP18 gene in regulation and control of rice grain length and thousand grain weight

Abstract

The invention discloses application of a rice OsSAP18 gene in regulation and control of rice grain length and thousand grain weight. According to the invention, a rice tissue gene expression database is utilized to screen high-level expression genes in seeds, and the expression mode of the OsSAP18 gene in the rice seeds indicates that the OsSAP18 gene may participate in the rice seed development process; a homozygous mutant of the ossap18 is constructed and obtained through a gene editing technology, and planting tests of the homozygous mutant of the ossap18 and a control plant ZH11 find that the ossap18 functional deletion mutant obtained through the method shows phenotypes that the grain length is increased and the thousand grain weight is increased.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology breeding, and in particular relates to an application of a rice OsSAP18 gene in regulating rice grain length and 1000-grain weight. Background Art

[0002] Rice is one of the most important food crops in my country, and its yield and quality play an extremely important role in ensuring my country's food security.

[0003] Rice yield is primarily determined by three factors: the number of panicles per mu, the number of grains per panicle, and grain weight. Grain weight has high heritability and a stable phenotype, making early selection for grain weight the most effective during high-yield breeding. Rice grain weight is typically determined by grain length, width, and thickness. To date, researchers have cloned several genes involved in regulating rice grain weight, but the vast majority exhibit multiple effects, limiting the availability of genetic resources directly applicable to rice breeding. Therefore, identifying new genes that regulate rice grain weight and utilizing modern biotechnology to modify and regulate rice grain shape for breeding improvement are of great theoretical and practical significance. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide an application of the rice OsSAP18 gene in regulating rice grain length and 1000-grain weight.

[0005] The present invention is achieved by applying the rice OsSAP18 gene in regulating rice grain length and 1000-grain weight, wherein the CDS nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.1.

[0006] Preferably, the application is to increase rice grain length and 1000-grain weight by inhibiting the expression of the OsSAP18 gene.

[0007] Preferably, the inhibiting of OsSAP18 gene expression is to inhibit OsSAP18 gene expression by constructing a knockout vector of the rice OsSAP18 gene or constructing a mutant of the rice OsSAP18 gene.

[0008] Preferably, the mutant of the rice OsSAP18 gene is mutated at any position in the sequence shown in SEQ ID NO. 2, and the mutation can cause the termination of protein expression of the OsSAP18 gene.

[0009] Preferably, the mutation includes point mutation, deletion mutation, and insertion mutation.

[0010] Preferably, in the knockout vector, the gRNA target sequence for knocking out the OsSAP18 gene is:

[0011] 5'-AGGGCCGCCGATGCAGTACCGGG-3'.

[0012] The present invention further discloses a method for increasing rice grain length and thousand-grain weight, the method comprising the following steps:

[0013] (1) Construction of a knockout vector for the rice OsSAP18 gene;

[0014] (2) Transforming the constructed rice OsSAP18 gene knockout vector into rice;

[0015] (3) Screen and obtain gene-edited rice lines with increased rice grain length and 1000-grain weight.

[0016] Preferably, in step (1), the method for constructing the knockout vector of the rice OsSAP18 gene is as follows: designing oligo primers according to the gRNA target sequence that inhibits the expression of the OsSAP18 gene, configuring a reaction system to synthesize oligo dimers; and constructing the dimer into a CRISPR / cas vector to obtain the knockout vector of the rice OsSAP18 gene.

[0017] Preferably, the gRNA target sequence is:

[0018] 5'-AGGGCCGCCGATGCAGTACCGGG-3'.

[0019] Preferably, the oligo primer is:

[0020] oligo primer F: 5'-TGTGTGGGGGCCGCCGATGCAGTACC-3';

[0021] oligo primer R: 5'-AAACGGTACTGCATCGGCGGCCCTCA-3'.

[0022] The present invention overcomes the shortcomings of the prior art and provides a method for regulating rice grain length and 1000-grain weight using the rice OsSAP18 gene. To screen candidate genes for regulating rice grain weight, the present invention utilized the Rice Tissue Gene Expression Database (https: / / rapdb.dna.affrc.go.jp) to screen for genes highly expressed in seeds (embryo sac, embryo, and endosperm). The present invention identified that the OsSAP18 gene has the highest expression level in the rice embryo sac and a relatively high expression level in the seed embryo. The expression pattern of the OsSAP18 gene in rice seeds suggests that it may be involved in rice seed development. To further analyze whether OsSAP18 has a biological function in regulating rice grain weight, the present invention utilized gene editing technology to create and obtain the ossap18 loss-of-function mutants ossap18-1 and ossap18-2. Through the planting experiment of the homozygous mutant of ossap18 and the control plant ZH11, it was found that the grain length of ZH11 was about 7.44mm, while the grain length of the ossap18-1 mutant was 8.13mm, and the grain length of the ossap18-2 mutant was 8.06mm, and the grain length was significantly different from ZH11; the statistical results of grain width showed that the grain width of ZH11 was about 3.10mm, while the grain width of the ossap18-1 mutant was 3.06mm, and the grain width of the ossap18-2 mutant was 3.14mm, and the grain width difference was not significant compared with ZH11; the statistical results of thousand-grain weight showed that the thousand-grain weight of ZH11 was about 22.37g, while the thousand-grain weight of the ossap18-1 mutant was about 26.30g, and the thousand-grain weight of the ossap18-2 mutant was about 26.72g, and the thousand-grain weight differences were significant compared with ZH11.

[0023] Compared with the shortcomings and deficiencies of the prior art, the present invention has the following beneficial effects: compared with ZH11, the mutants of ossap18-1 and ossap18-2 obtained by the method of the present invention both exhibit phenotypes of increased grain length and increased 1000-grain weight. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the analysis of tissue expression pattern of OsSAP18 gene;

[0025] Figure 2 This is the sequencing result of the gene-edited mutant of OsSAP18;

[0026] Figure 3This is an analysis of rice grain shape of ossap18 gene-edited mutants; Figure A: grain length phenotype of ZH11, ossap18-1 and ossap18-2; scale bar, 5 mm; Figure B: grain width phenotype of ZH11, ossap18-1 and ossap18-2; scale bar, 5 mm; Figure C: statistical analysis of grain length, grain width and 1000-grain weight of ZH11, ossap18-1 and ossap18-2; **, P < 0.01. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0028] 1. OsSAP18 gene is expressed at high levels in rice seeds

[0029] To screen candidate genes regulating rice grain weight, the present invention used the Rice Tissue Gene Expression Database (https: / / rapdb.dna.affrc.go.jp) to screen genes with high expression levels in seeds (embryo sac, embryo, and endosperm). Among them, the present invention identified the OsSAP18 gene as having the highest expression level in the rice embryo sac and a relatively high expression level in the seed embryo ( Figure 1 The expression pattern of OsSAP18 gene in rice seeds suggests that it may be involved in the rice seed development process.

[0030] 2. Creation of ossap18 loss-of-function mutants using gene editing technology

[0031] To further analyze whether OsSAP18 has the biological function of regulating rice grain weight, the present invention used gene editing technology to create and obtain an ossap18 loss-of-function mutant.

[0032] 1. Selection of OsSAP18 target sites

[0033] OsSAP18 is located on rice chromosome 2 and contains 6 exons and 5 introns. The target site selected for the knockout vector is located in the first exon region ( Figure 2 The target sequence of the OsSAP18 gene is AGGGCCGCCGATGCAGTACCGGG.

[0034] 2. Construction of recombinant vector

[0035] (1) Synthesize primers based on the sgRNA target sequence:

[0036] F:5'-TGTGTGGGGGCCCGCCGATGCAGTACC-3';

[0037] R:5'-AAACGGTACTGCATCGGCGGCCCTCA-3'.

[0038] (2) Preparation of primer dimers:

[0039] The synthesized primers were dissolved in water and diluted to 10 μmol, and PCR was performed according to the following reaction system. The reaction conditions were as follows: heating at 95°C for 3 minutes using a PCR instrument, and then slowly decreasing the temperature to 20°C at a rate of approximately 0.2°C / second.

[0040] The system is as follows:

[0041]

[0042]

[0043] (3) Constructing primer dimers into CRISPR / cas vectors:

[0044] After mixing according to the following reaction system, react at 20°C for 1 hour, and construct the primer dimer into the CRISPR / cas vector BGK032 through homologous recombination.

[0045]

[0046] 3. The above reaction system was transformed into Escherichia coli competent cells according to the CaCl2 heat shock transformation method for identification to obtain positive recombinants.

[0047] 4. DNA level identification of transformed rice and mutant plants:

[0048] The plasmids of the positive recombinants constructed above were extracted and transformed into the EHA105 strain, and then into the rice variety Zhonghua 11 (ZH11) through Agrobacterium infection.

[0049] Through hygromycin resistance screening, T0 generation transgenic lines were obtained. DNA of resistant plants was extracted, and genomic DNA was amplified by PCR using specific primers containing the target site. The PCR products were sequenced for the first generation, and gene-edited homozygous plants with site-directed mutations in the target site of the rice OsURM1 gene were obtained, showing the insertion of one base "T" and the deletion of two bases "GT", respectively. They were named ossap18-1 and ossap18-2. The sequencing results of two of the mutants are shown in Figure 2 Since the number of bases inserted and deleted in ossap18-1 and ossap18-2 is not a multiple of 3 bases, both will cause frameshift mutations. Therefore, the present invention obtains a gene-edited mutant of OsSAP18.

[0050] The primer sequences used to amplify the OsSAP18 gene target site are as follows:

[0051] OsSAP18-F:5'-TCGCATCCCCTTCTGAAGTG-3';

[0052] OsSAP18-R:5'-TGCTCTCCTCAGTCAACCACACT-3'.

[0053] Functional identification of ossap18 in regulating rice grain weight

[0054] In 2023 and 2024, the homozygous mutant of ossap18 and the control plant ZH11 were planted simultaneously in the experimental field of the Biotechnology Park of Huaiyin Normal University (119°01′E / 33°36′N), and the phenotypic observations and data statistics of rice grain length, grain width and 1000-grain weight of ZH11, ossap18-1 and ossap18-2 were carried out. The number of statistical lines was ≥20.

[0055] The statistical results of grain length showed that the grain length of ZH11 was about 7.44 mm, while the grain length of ossap18-1 mutant was 8.13 mm and that of ossap18-2 mutant was 8.06 mm, which were significantly different from those of ZH11. The statistical results of grain width showed that the grain width of ZH11 was about 3.10 mm, while the grain width of ossap18-1 mutant was 3.06 mm and that of ossap18-2 mutant was 3.14 mm, which were not significantly different from those of ZH11. The statistical results of thousand-grain weight showed that the thousand-grain weight of ZH11 was about 22.37 g, while the thousand-grain weight of ossap18-1 mutant was about 26.30 g and that of ossap18-2 mutant was about 26.72 g, which were significantly different from those of ZH11. Figure 3 Therefore, compared with ZH11, both ossap18-1 and ossap18-2 mutants showed increased grain length and 1000-grain weight phenotypes.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Application of the rice OsSAP18 gene in regulating rice grain length and 1000-grain weight, wherein the CDS nucleotide sequence of the rice OsSAP18 gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The application is to increase rice grain length and thousand-grain weight by inhibiting the expression of OsSAP18 gene.

3. The use according to claim 2, characterized in that The method of inhibiting the expression of the OsSAP18 gene is to inhibit the expression of the OsSAP18 gene by constructing a knockout vector of the rice OsSAP18 gene or constructing a mutant of the rice OsSAP18 gene.

4. The use according to claim 3, characterized in that The mutant of the rice OsSAP18 gene is a mutation at any position in the sequence shown in SEQ ID NO. 2, and the mutation can cause the protein expression of the OsSAP18 gene to be terminated.

5. The use according to claim 4, characterized in that The mutations include point mutations, deletion mutations, and insertion mutations.

6. The use according to claim 3, characterized in that In the knockout vector, the gRNA target sequence for knocking out the OsSAP18 gene is: 5'-AGGGCCGCCGATGCAGTACCGGG-3'.

7. A method for increasing rice grain length and thousand-grain weight, characterized in that: The method comprises the following steps: (1) Construction of a knockout vector for the rice OsSAP18 gene; (2) Transforming the constructed rice OsSAP18 gene knockout vector into rice; (3) Screen and obtain gene-edited rice lines with increased rice grain length and 1000-grain weight.

8. The method according to claim 7, characterized in that In step (1), the method for constructing the knockout vector of the rice OsSAP18 gene is as follows: designing oligo primers according to the gRNA target sequence that inhibits the expression of the OsSAP18 gene, configuring a reaction system to synthesize oligo dimers; and constructing the dimers into a CRISPR / cas vector to obtain the knockout vector of the rice OsSAP18 gene.

9. The method according to claim 7, wherein The gRNA target sequence is: 5'-AGGGCCGCCGATGCAGTACCGGG-3'.

10. The method according to claim 8, wherein The oligo primers are: oligo primer F: 5'-TGTGTGGGGGCCGCCGATGCAGTACC-3'; oligo primer R: 5'-AAACGGTACTGCATCGGCGGCCCTCA-3'.

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