Application of LOC_Os02g37970 gene in regulating rice grain width and length

Abstract

The application belongs to the technical field of plant genetic engineering, and discloses LOC_Os02g37970 Application of a gene in regulation of rice grain width and grain length LOC_Os02g37970 LOC_Os02g37970 LOC_Os02g37970 LOC_O Gene knockout mutant m1497 Phenotype identification shows that, compared with the wild type, the mutant m1497 Grain width and grain length are significantly increased. The application provides a new gene resource and breeding material for molecular breeding of high-yield rice, and is suitable for gene editing, molecular marker assisted breeding and genetic improvement of yield traits.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving LOC_Os02g37970 Application of genes in regulating rice grain width and length. Background Technology

[0002] Rice is the staple food for more than half of the world's population, and its yield and quality are directly related to food security and the development of the agricultural industry. In previous studies, our team constructed a genetic mapping population using wild rice introgression lines. Combined with multi-environment phenotypic identification and genome-wide association analysis, we detected a major-effect QTL site (tentatively named qPN2.2) controlling both grain width and grain length in the 22.86–23.05 Mb region of rice chromosome 2. However, the key candidate genes in this region have not been precisely cloned, and the molecular mechanism by which they regulate panicle grain width and grain length remains unclear. Therefore, cloning the key genes regulating grain width and grain length in this region, verifying their functions, and clarifying their application value are of significant theoretical and practical value for enriching the gene resources for high-yield molecular breeding of rice and elucidating the molecular mechanism of synergistic regulation of yield and resistance. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of existing technologies, such as the scarcity of high-yield regulatory gene resources in rice, the long cycle of traditional hybridization breeding, the low efficiency of target trait aggregation, susceptibility to environmental interference, and the lack of precise cloning of key candidate genes at the major QTL site qPN2.2 on rice chromosome 2, and the unclear molecular regulatory mechanism. This invention provides... LOC_Os02g37970 Application of genes in regulating rice grain width and length.

[0004] The first aspect of the present invention provides LOC_Os02g37970 Applications of gene knockout, wherein the application is any of the following: A1) Application in increasing the grain width and / or grain length of rice grains; A2) Application in the preparation of rice with increased grain width and / or grain length; The LOC_Os02g37970 The nucleotide sequence of the gene is shown in SEQ ID NO.1. LOC_ Os02g37970 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0005] The second aspect of this invention provides a method for preparing trait-improved rice, the method comprising: Methods for preparing rice with increased grain width and / or grain length: Utilizing gene knockout technology to reduce the... LOC_ Os02g37970The expression level of the gene was adjusted to obtain rice with increased grain width and length; preferably, the gene was knocked out using the CRISPR / Cas9 system, and the target site sequence is shown in SEQ ID NO.3.

[0006] Compared with the prior art, the present invention has the following beneficial effects: First time revealed LOC_Os02g37970 This gene negatively regulates both grain width and grain length in rice. Knocking out this gene significantly increases both grain width and grain length in rice. Attached Figure Description

[0007] Figure 1 It is knockout LOC_Os02g37970 The mode of gene mutation. A is... LOC_Os02g37970 Gene structure diagram, and mutants m1497 The single-base insertion site (at 321 bp). B represents wild-type ZH11 (WT) and the mutant. m1497 Sequence alignment showed that the mutant had an additional G (marked in red) inserted at the target site, consistent with the mutation site of A.

[0008] Figure 2 It is wild-type ZH11 (WT) and mutant m1497 Comparison of grain width and grain length.

[0009] Figure 3 mutant m1497 Statistical chart of particle width results for wild-type ZH11 (WT).

[0010] Figure 4 mutant m1497 Statistical chart of grain length results for wild-type ZH11 (WT). Detailed Implementation

[0011] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0012] SEQ ID NO.1: ATGGCCGGCCTGAGTGTCCTTCTTGAGACACACAAGAACGATCACCACCCCAACATGAGGCCTCCCCAAATCATCAGCAAGGCCACCCTCCATAGCCACCCAGAGACGATGTCCTCCTCCTCTCCGGCGACTGCGACGACGGCGACGATGAGCTCCTTCTTGCAGCGCTGCTTCCTCTGCCGCAGGGAGC TCGCCGACGGCAAGGACATCTACATGTACAGAGGGGACAGAGCGTTCTGCAGCGTGGATTGCCGGTGCAAGCAGATCTTCATGGACGAGGACGCCGCCGCCGCCGGCGGCAACTGCGCCGCGGTGCGCGCCGGCCGGCGGCGGGCGGCCGTACCGCGCGAGCAAACCGGCGCCGGTGGCTTCGCGTACTGA SEQ ID NO.2: MAGLSVLLETHKNDHHPNMRPPQIISKATLHSHPETMSSSSPATATTATMSSFLQRCFLCRRELADGKDIYMYRGDRAFCSVDCRCKQIFMDEDAAAAGGNCAAVRAGRRRAAVPREQTGAGGFAY* SEQ ID NO.3: CGGCGAGCTCCCTGCGGCAG SEQ ID NO.4: CTGCCGCAGGGAGCTCGCCG SEQ ID NO.5: AGCTTAGATGCATGCCATGGA SEQ ID NO.6: CGATCACCACCCCAACATGA Example 1: Construction of gene knockout vector and transformation in rice 1. Materials and Methods 1.1 Plant materials Zhonghua 11, a conventional japonica rice variety. 1.2 Strains and Plasmids The vector used in this experiment was the commercial vector pGEL031, purchased from Shanghai Newp Biotechnology Co., Ltd.; the competent E. coli DH5α cells used were purchased from Nanjing Novizan Biotechnology Co., Ltd.; and Agrobacterium EHA105 was preserved in our laboratory.

[0013] 1.3 Main Reagents The high-fidelity 2× Phanta Max Master Mix and 2× Rapid Taq Master Mix used in the experiment were purchased from Nanjing Novizan Biotechnology Co., Ltd.; T4 ligase, restriction enzyme and high-fidelity polymerase were purchased from NEB Company; kanamycin, carbenicillin and hygromycin were purchased from Beijing Solarbio Science & Technology Co., Ltd.; sodium chloride (NaCl) was purchased from Sinopharm Chemical Reagent Co., Ltd. Shanghai Test Brand.

[0014] 1.4 Identification of gene target sites The target gene, with its nucleotide sequence shown in SEQ ID NO. 1, was retrieved from the Rice Genome Database (RGAP, https: / / rice.uga.edu / ). LOC_Os02g37970 Sequence retrieval revealed that the gene encodes a protein containing the DUF581 domain. The gene is located on chromosome 2 (Chr2: 22,942,416–22,943,822), and the amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0015] Based on the design principles of sgRNA, LOC_Os02g37970 Target sequences selected from gene coding regions: CGGCGAGCTCCCTGCGGCAG AGG (The underlined part is the PAM sequence) serves as the CRISPR / Cas9 editing target site, with the corresponding sgRNA sequence being CTGCCGCAGGGAGCTCGCCG. The sgRNA sequence was ligated downstream of the promoter via PCR, and then the PCR fragment was seamlessly cloned and ligated into the enzyme-digested pGEL031 vector, successfully constructing a CRISPR / Cas9 single-target editing vector.

[0016] 1.5 Genetic transformation of rice (1) Seed sterilization and callus induction: Rice seeds were disinfected by soaking in alcohol for 30 seconds and 30% sodium hypochlorite solution for 15 minutes. After rinsing with sterile water 5 times, they were placed on induction medium for callus induction culture. After callus formation, the buds and seeds were removed by sterile tweezers, and well-developed callus tissues were selected and transferred to fresh medium for further amplification culture.

[0017] (2) Agrobacterium activation and infection: A small amount of Agrobacterium bacterial suspension was taken with a 200 μL pipette tip and streaked on a YEP solid plate. The plate was activated and cultured at 28°C in the dark for 2 days. Then, the Agrobacterium on the plate was rinsed with the infection solution to make a uniform bacterial suspension. The callus tissue was immersed in the bacterial suspension for 15 minutes, transferred to sterile filter paper to absorb excess liquid, and then co-cultured at 25°C in the dark for 3 days.

[0018] (3) Screening, differentiation and rooting: After co-culture, the callus tissue was thoroughly cleaned with sterile water containing carbenicillin, the surface moisture was dried in a clean bench, and the callus was transferred to the screening medium for positive callus screening. The positive callus particles were transferred to the differentiation medium to induce regeneration, and the resulting seedlings were transferred to rooting tubes for root strengthening culture. After the root system was developed, the seedlings were transplanted to the greenhouse.

[0019] 1.6 Identification of Mutant Materials Agrobacterium-mediated transformation of japonica rice Zhonghua 11 (ZH11) yielded T0 generation transgenic plants. Genomic DNA was extracted from seedling leaves, and PCR amplification of the target region was performed using specific primers (forward primer: AGCTTAGATGCATGCCATGGA; reverse primer: CGATCACCACCCCAACATGA). The products were then sequenced using Sanger sequencing and compared with wild-type reference sequences to identify the edited type. Gene knockout mutants were obtained. m1497 The mutation mode is (G insertion), which leads to premature termination of translation. Figure 1 ) Example 2: Determination of agronomic traits of knockout lines The rice seeds tested were mutants. m1497 T2 generation homozygous seeds and their wild-type ZH11. Three biological replicates were set up for each material; within each replicate, three groups of intact and plump seeds, 50 seeds per group, were randomly selected. The length and width of each seed (in mm) were measured. The average length and width of each group (50 seeds) were calculated as one data point. Therefore, a total of nine independent data points (3 biological replicates × 3 groups / replica) were obtained for each material for subsequent analysis. Figure 2 As can be seen from this, knockout LOC_Os02g37970 The gene can significantly increase the width and length of rice grains. Mutant m1497 The statistical data on the grain characteristics of wild-type ZH11 are shown in Table 1.

[0020] Table 1

[0021] Table 1 shows that knocking out rice... LOC_Os02g37970 After gene modification, the average grain width increased from 3.175 mm in the wild type to 3.499 mm. Figure 3The average grain length increased from 7.221 mm to 7.655 mm. Figure 4 ).

[0022] The present invention has been described in detail above. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0023] For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments are given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Knockout LOC_Os02g37970 The application of genes is characterized by, The application is any one of the following: A1) Application in increasing the grain width and / or grain length of rice grains; A2) Application in rice with increased grain width and / or grain length; The LOC_Os02g37970 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. LOC_Os02g37970 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

2. A method for preparing rice grains with increased grain width and / or grain length, characterized in that, The method described is to use gene knockout technology to knock out genes in rice. LOC_Os02g37970 Genes that result in rice with increased grain width and / or grain length, the aforementioned LOC_Os02g37970 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. LOC_Os02g37970 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The method according to claim 2, characterized in that, The use of gene knockout technology to knock out the gene in rice LOC_ Os02g37970 Genes are processed using the CRISPR / Cas9 system. LOC_Os02g37970 This is achieved through targeted gene knockout, with the target site of the CRISPR / Cas9 system shown in SEQ ID NO.

3.

4. A type of rice with increased grain width and / or grain length obtained by the method according to any one of claims 2 to 3.

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