Rice grain type protein Os03g0122600 as well as coding gene and application thereof

Abstract

The invention discloses a rice grain type protein Os03g0122600 as well as a coding gene and application thereof. The rice grain type protein Os03g0122600 has an amino acid sequence as shown in SEQ ID NO.2, or is a functional equivalent variant formed by substitution, deletion or addition of one or more amino acid residues in the sequence as shown in SEQ ID NO.2. The invention also discloses a preparation method of the rice grain type protein Os03g0122600. The coding gene has a nucleotide sequence as shown in SEQ ID NO.1, or a nucleotide sequence which is hybridized with SEQ ID NO.1 and codes the same functional protein. The Os03g0122600 gene is knocked out through a CRISPR / Cas9 gene editing technology, it is found that compared with a wild type, the rice grain length is remarkably increased, the grain length-width ratio and the thousand grain weight are increased, and a new strategy is provided for molecular breeding of high-yield and high-quality rice varieties.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and in particular relates to rice grain-shaped protein Os03g0122600 and its encoding gene and application. Background Art

[0002] As one of the world's most important food crops, rice plays an irreplaceable role in ensuring food security. With the continued growth of the global population and the gradual reduction of arable land, increasing rice yields has become a critical issue in the agricultural sector. Rice grain length is a key factor influencing rice yield and quality. Generally speaking, appropriately increasing grain length can increase rice's thousand-grain weight, thereby increasing yield per unit area. Furthermore, in terms of rice quality, longer rice grains are often more attractive to consumers and have a higher market value.

[0003] The rapid advancement of molecular biology techniques, including the completion of whole-genome sequencing of rice and the continuous improvement of genetic linkage maps, has provided a solid foundation for the study of genes associated with rice grain length. These findings have enabled researchers to locate QTLs (quantitative trait loci) for rice grain length and subsequently clone and analyze the functions of key genes. However, the genetic mechanism of rice grain length is extremely complex, regulated by multiple loci and significantly influenced by environmental factors. While some genes associated with rice grain length have been identified, our understanding of the entire genetic regulatory network remains limited, and the genetic control mechanism of grain length remains incompletely understood. Numerous potential key genes in rice grain length regulation remain to be discovered and studied. Identifying more genes regulating rice grain length and developing effective editing targets will have important theoretical and practical implications for advancing rice molecular breeding and developing new high-yield, high-quality rice varieties.

[0004] In recent years, breakthroughs have been made in genome editing technologies, such as CRISPR / Cas9, which enables precise editing of plant genomes. This technology uses site-specific nucleases to create double-strand breaks at specific locations in the genome, activating the cell's own homologous recombination and non-homologous end-joining repair mechanisms to achieve site-specific modification of target genes. In rice research, using genome editing technology to identify targets that can effectively improve rice grain shape, particularly grain length, has become a key to gene-editing breeding. Summary of the Invention

[0005] The purpose of the present invention is to provide a rice grain-shaped protein Os03g0122600 and its encoding gene and application, and to illustrate its application method for improving rice grain length through gene editing technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions: Application of rice grain shape protein Os03g0122600 in regulating rice grain traits, wherein the amino acid sequence of the rice grain shape protein Os03g0122600 is as shown in SEQ ID NO.2; or a functionally equivalent variant formed by substitution, deletion or addition of one or more amino acid residues of this sequence.

[0007] The nucleotide sequence of the coding gene of the rice grain shape protein Os03g0122600 is as shown in SEQ ID NO.1; or a nucleotide sequence generated by base addition, substitution or deletion of the nucleotide shown in SEQ ID NO.1 and encoding the rice grain shape protein Os03g0122600.

[0008] Furthermore, the regulation of rice grain traits includes the length and 1000-grain weight of rice grains.

[0009] Application of rice grain shape protein Os03g0122600 in cultivating long-grain rice varieties, wherein the amino acid sequence of the rice grain shape protein Os03g0122600 is as shown in SEQ ID NO.2, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO.1.

[0010] A method for cultivating long-grain rice varieties, comprising the following steps: inhibiting the activity of the rice grain shape protein Os03g0122600 in rice, or inhibiting the expression of the gene encoding the rice grain shape protein Os03g0122600 in recipient rice, or knocking out the gene encoding the rice grain shape protein Os03g0122600 in the rice recipient to obtain transgenic rice; the amino acid sequence of the rice grain shape protein Os03g0122600 is as shown in SEQ ID NO.2, and the nucleotide sequence of its coding gene is as shown in SEQ ID NO.1.

[0011] Furthermore, the grain length and grain weight of the transgenic rice are increased compared with those of the recipient rice.

[0012] Furthermore, the sgRNA target sequence for gene knockout is 5'-ATCGGCG ATCGAAGATGGTGCGG-3'.

[0013] The present invention also provides primer sequences for detecting the knockout of the above gene, and the primers consist of 600-F and 600-R; namely: 600-F: 5’-CGGCAACAAGACAGTACAGCA-3’; 600-R: 5’-CATTCATCCAACTTTTCACCCA-3’.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention first reveals the function of the Os03g0122600 gene in regulating grain length, filling the gap in the research on the morphological development of grains for this gene. By optimizing the design of sgRNA targets and the transformation system, the editing efficiency of this technology reaches over 65%, providing reliable technical support for the rapid breeding of high-yield long-grain rice varieties. By targeting and knocking out this gene through the CRISPR / Cas9 system, the inhibitory effect on grain length can be significantly relieved, increasing the grain length of mutant rice by 6.5% - 6.8%. Compared with traditional cross-breeding, the efficient gene editing technology adopted in the present invention can accurately locate the target gene, avoid pleiotropic effects, and the traits of the edited mutants are stably inherited.

[0015] The present invention provides a precise gene editing strategy for cultivating long-grain rice varieties. By targeting and regulating the rice grain shape-related gene Os03g0122600, long-grain rice varieties can be effectively obtained; the effect of the grain shape regulatory gene of the present invention is significant. Compared with the wild type, it can increase the grain length and grain weight of rice, thereby significantly increasing the rice yield, and has important application value in the utilization of rice heterosis and variety improvement. Brief Description of the Drawings

[0016] Figure 1 For the sequence analysis of the rice Os03g0122600 gene and the design of gene editing targets; among them, the black boxes represent exons, the black lines represent introns, the white boxes represent untranslated regions, and the Cas9 recognition sequence is located at +6bp of the second exon.

[0017] Figure 2 For the acquisition of Os03g0122600 mutants; where WT is the wild-type sequence, the Arabic numerals represent the number of bases deleted or added (a "-" before the number indicates base deletion, and a "+" before the number indicates base addition), and the lowercase letters are added bases.

[0018] Figure 3 For the investigation of the grain shape of different genotypes of the Os03g0122600 gene; where WT is the wild-type control plant, and cas9-1 and cas9-2 are mutant plants.

[0019] Figure 4 For the grain shape analysis of the Os03g0122600 gene mutant and its wild type; where WT is the wild-type control plant, and cas9-1 and cas9-2 are mutant plants. Detailed Embodiments

[0020] The following examples are used to further illustrate the present invention, but do not constitute a limitation to the present invention.

[0021] During the implementation process, if the experimental method is not specifically specified, the conventional experimental methods in the art are adopted.

[0022] Unless otherwise specified, the experimental materials used were purchased from common suppliers on the market.

[0023] Example 1: Cloning of Rice Os03g0122600 Gene Based on the Os03g0122600 gene information stored in the Rice Data Center, full-length primer sequences (F: 5'-ATGGTGCGGGGGAAGACG-3'; R: 5'-TCATATTGCCCTCCGGAAT-3') were designed to amplify the Os03g0122600 genomic sequence using genomic DNA from Xiushui 134 as a template.

[0024] Example 2: Sequence Analysis of Rice Os03g0122600 Gene and Design of Gene Editing Targets The rice Os03g0122600 gene is shown in SEQ ID NO. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID NO. 2. The present invention experimented with variety Xiushui 134, and designed a Cas9 recognition site at 6 bp of the first exon of the Os03g0122600 gene, as shown in FIG. Figure 1 The target sequences for this gene recognition are: (sgRNA) ATCGGCGATCGAAGATGGTGCGG. This target was selected based on a comprehensive consideration of factors including gene function, editing efficiency, and off-target risk, aiming to achieve precise editing of the gene.

[0025] Example 3: Targeting vector construction and rice genetic transformation This study used the CRISPR / Cas9 gene editing technology system to carry out related experiments. The vector used in the experiment was an artificially constructed recombinant plasmid pCXUN-Cas9-gRNA. The backbone vector pCXUN-Cas9 of this circular vector was purchased from Beijing Weishanglide Biotechnology Co., Ltd. Among them, the Cas9 protein coding sequence is 4200 base pairs in length and can encode 1400 amino acids. Its expression is driven by the Ubiquitin promoter, while the expression of gRNA is regulated by the U6 promoter. When constructing the targeting vector, a professional primer synthesis company first synthesized the gRNA fragment according to the designed sequence, and then used DNA ligase to accurately connect it to the downstream of the U6 promoter, thereby successfully constructing the recombinant plasmid pCXUN-Cas9-gRNA.

[0026] In the present invention, the vector pCXUN-Cas9-gRNA is transformed into the receptor material Xiushui 134, and genetic transformation is achieved by Agrobacterium-mediated method, and the resistant callus is screened, differentiated and rooted. The specific steps are as follows: 1. Select plump and mature japonica rice seeds, remove the hulls, and conduct strict disinfection treatment. After disinfection, inoculate the seeds into an induction medium (MS medium supplemented with 3 mg·L -1 of 2,4-D, with the pH adjusted to 5.6). Culture them in the dark at 26°C for 10 days, then carefully excise the radicles and continue to culture for 8 days. After the formation of embryogenic callus, transfer it to fresh induction medium and conduct subculture every 20 days. After two subcultures, select callus with firm texture, light yellow color, and a diameter of about 2 - 3 mm for subsequent Agrobacterium transformation experiments.

[0027] 2. Take a small amount from the stored recombinant Agrobacterium liquid, evenly streak and inoculate it onto an LB solid medium (pH 5.8) containing 50 mg·L -1 kanamycin and 50 mg·L -1 rifampicin, and culture it in the dark at 28°C for 48 hours to activate Agrobacterium. Then, pick a single colony from the activated plate for secondary streaking and continue to culture in the dark at 28°C for two days. After culturing, wash and resuspend Agrobacterium using AAM medium (pH 5.2) containing 150 μM·L -1 acetosyringone, adjust the cell concentration of the bacterial liquid to an OD600nm value between 1.2 - 1.8, and then let it stand for 1.5 hours to prepare the Agrobacterium suspension for transformation.

[0028] 3. Completely immerse the embryogenic callus obtained in step 1 in the Agrobacterium suspension prepared in step 2, let it stand for 30 minutes to allow Agrobacterium to fully contact with the callus. Then, take out the callus and place it on sterile filter paper to drain the excess bacterial liquid, and then inoculate it into a co-culture medium (NB medium supplemented with 2 mg·L -1 of 2,4-D and 100 μM·L -1 acetosyringone, pH 5.8), and co-culture it in the dark at 25°C for 3 days.

[0029] 4. Pick the callus after co-culture in step 3 into a wide-mouth culture bottle, rinse it 5 times with sterile water, shake it several times each time until no filamentous bacteria are seen in the water. For the last time, let it stand in sterile water containing 250 mg·L -1 carbenicillin for 1 h, then place it on sterile filter paper to dry the callus, and inoculate it into a selection medium (NB medium + 2,4-D 2 mg·L -1 + carbenicillin 250 mg·L -1 + hygromycin 50 mg·L -1, in the dark at 28°C in a medium with pH 5.8), and subcultured every two weeks. After about three weeks, tumor-like bright yellow resistant calli can be seen growing from the browning and shriveled calli.

[0030] 5. Transfer the resistant calli obtained in step 4 to a differentiation medium (NB medium + 2,4-D 2 mg·L -1 + KT 10 mg·L -1 + NAA 0.4 mg·L -1 , pH 5.8). After 2 weeks, the calli start to turn green, and after 3 weeks, young shoots can grow, followed by the development of young roots. When the seedlings grow to a certain stage, transfer them to a rooting medium (1 / 2 MS medium, pH 5.6). After the seedlings take root and mature, carefully wash the medium attached to the roots and transplant them to the experimental base for subsequent growth observation and research.

[0031] Example 4: Genotype Detection and Phenotype Investigation of the Os03g0122600 Gene Editor First, extract genomic DNA from the transformed plants using a conventional DNA extraction method. Using the extracted DNA as a template, perform a PCR amplification reaction with primers CZT-F (5'-GGGAGATCCAGCTAGAGGTC-3') and CZT-R (5'-GGAAGGAGGAAGACAAGG-3') to preliminarily identify the transgenic plants. To further determine the specific situation of gene editing, use specific primers 600-F (5’-CGGCAACAAGACAGTACAGCA-3’) and 600-R (5’-CATTCATCCAACTTTTCACCCA- 3’) for the target gene to perform PCR amplification on the genomic DNA of the above preliminarily identified transgenic plants, so as to obtain the Os03g0122600 gene fragment containing the target sequence. Send the amplified gene fragment to a professional sequencing company for sequencing analysis. Through careful comparison and analysis of the sequencing results, it is found that the Os03g0122600 gene has produced 4 different types of genotypes, covering various gene editing methods such as heterozygous mutation, biallelic modification, and homozygous mutation ( Figure 2 ). Conduct detailed phenotype observations on the mutant plants cas9-1 and cas9-2 ( Figure 3 ). The results show that compared with wild-type japonica rice, the rice grain length of these two mutants has increased significantly. Further weigh and count the dry weight of the seeds of the mutant lines, and the results are as Figure 4As shown, compared with wild-type seeds, the grain length of mutant rice cas9-1 and cas9-2 increased by 6.5% - 6.8%, and the 1000-grain weight of cas9-1 and cas9-2 rice seeds increased by 18.0% and 16.5% respectively. This result fully demonstrates that gene editing has a significant effect on increasing rice yield. In other yield-related traits, such as panicle length, primary branch number, secondary branch number, and grain number per panicle of rice, there were no significant differences between cas9-1 and cas9-2 mutant lines and the wild type. The present invention successfully obtained mutants of Os03g0122600 gene by using CRISPR / Cas9 gene editing technology. The operation process of this technology is relatively simple and the experimental cost is low. Most laboratories with basic molecular biology experimental conditions can carry out relevant research. By self-crossing and separating the offspring of the obtained ideal grain type rice variety, transgenic foreign fragments can be effectively removed, which is safer and more reliable than traditional transgenic breeding methods. Compared with conventional cross-breeding methods, this technology greatly shortens the breeding cycle and provides an efficient and feasible method for rice molecular breeding.

[0032] As described above, the above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all should be covered by the protection scope of the present invention.

Claims

1. Application of rice grain shape protein Os03g0122600 in regulating rice grain traits, characterized in that: The amino acid sequence of the rice grain shape protein Os03g0122600 is shown in SEQ ID NO.2; or a functionally equivalent variant formed by substitution, deletion or addition of one or more amino acid residues of this sequence.

2. The application according to claim 1, wherein: The nucleotide sequence of the coding gene of the rice grain shape protein Os03g0122600 is shown in SEQ ID NO.1; or a nucleotide sequence generated by addition, substitution or deletion of bases of the nucleotide shown in SEQ ID NO.1 and encoding the rice grain shape protein Os03g0122600.

3. The application according to claim 1, wherein: The regulation of rice grain traits includes the length and 1000-grain weight of rice grains.

4. Application of rice grain shape protein Os03g0122600 in cultivating long grain rice varieties, characterized in that: The amino acid sequence of the rice grain shape protein Os03g0122600 is shown in SEQ ID NO.2, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.

1.

5. A method for cultivating a long-grain rice variety, characterized in that: It includes the following steps: inhibiting the activity of the rice grain shape protein Os03g0122600 in rice, or inhibiting the expression of the gene encoding the rice grain shape protein Os03g0122600 in the recipient rice, or knocking out the gene encoding the rice grain shape protein Os03g0122600 in the rice recipient to obtain transgenic rice; The amino acid sequence of the rice grain shape protein Os03g0122600 is shown in SEQ ID NO.2, and the nucleotide sequence of its coding gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that: The grain length and grain weight of the transgenic rice are increased compared with those of the recipient rice.

7. The method according to claim 5, wherein: The sgRNA target sequence for gene knockout is 5'-ATCGGCG ATCGAAGATGGTGCGG-3'.

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