Application of gene os03g0112700 in regulating rice grain width
Patent Information
- Application Number
- CN202510850587.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-24
- Publication Date
- 2026-11-03
- Estimated Expiration
- 2045-06-24
AI Technical Summary
Current technologies lack sufficient understanding of the genetic regulatory network of rice grain width, making it difficult to discover more regulatory genes and identify efficient editing targets, thus affecting rice molecular breeding and the cultivation of high-quality varieties.
The Os03g0112700 gene was knocked out using CRISPR/Cas9 gene editing technology. Specific sgRNAs were designed to target and edit rice plants. By constructing a targeting vector and performing genetic transformation, gene knockout and expression inhibition were achieved, reducing rice grain width and increasing the length-to-width ratio.
It significantly reduces the width of rice grains by 5% to 6.4% and increases the length-to-width ratio, providing an efficient rice breeding strategy and improving rice quality.
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Figure CN120796292B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and more specifically to the application of gene Os03g0112700 in regulating rice grain width. Background Technology
[0002] Rice, as a core crop for maintaining global food supply, occupies an irreplaceable position in strengthening food security. With the improvement of people's living standards and the continuous pursuit of a quality life, high-quality rice is attracting increasing attention from consumers, making the improvement of rice quality a major challenge that urgently needs to be overcome in the process of agricultural development. Rice grain width is one of the core factors affecting rice quality. Generally, appropriately reducing grain width helps to improve the length-to-width ratio of rice. In terms of rice quality, longer grains often better meet consumers' aesthetic preferences and have higher market value.
[0003] With the rapid development of molecular biology techniques, the successful completion of whole-genome sequencing of rice, and the continuous optimization of genetic linkage maps, a solid scientific foundation has been laid for in-depth research into rice grain width-related genes. These achievements have enabled researchers to conduct QTL mapping of rice grain shape, and subsequently clone and functionally analyze key genes. Furthermore, the recent leaps in CRISPR / Cas9 gene editing technology have provided technical support for gene function research. By targeting specific gene fragments or sites, it is possible to study which genes regulate grain width, and this also provides convenient conditions for breeding high length-to-width ratio rice.
[0004] However, the genetic regulation system of rice grain width is extremely complex, involving not only the combined effects of multiple gene loci but also being highly susceptible to strong interference from environmental factors. Currently, although some genes associated with rice grain width have been identified, our understanding of the entire genetic regulatory network remains insufficient. Many potential key genes await discovery. Uncovering more genes regulating grain width and identifying highly efficient editing targets is of significant theoretical and practical value for advancing rice molecular breeding and cultivating high-quality new rice varieties.
[0005] Therefore, how to further develop new rice grain width-related regulatory genes is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the present invention provides the application of gene Os03g0112700 in regulating rice grain width. The present invention knocks out the Os03g0112700 gene using CRISPR / Cas9 gene editing technology and finds that, compared with the wild type, the rice grain width of the gene knockout mutant plants is significantly reduced, and the length-to-width ratio is significantly increased, providing a new strategy for molecular breeding of high-quality rice.
[0007] The primary objective of this application is to provide the application of gene Os03g0112700 in regulating rice grain width, characterized in that the nucleotide sequence of gene Os03g0112700 is any one of the following:
[0008] 1) As shown in SEQ ID NO.3;
[0009] 2) Or a nucleotide sequence that, after substitution, deletion and / or addition of one or more bases, still encodes the same functional protein as the sequence shown in SEQ ID NO.3.
[0010] As a preferred technical solution, the amino acid sequence encoded by the gene Os03g0112700 is any one of the following:
[0011] 1) As shown in SEQ ID NO.4;
[0012] 2) Or an amino acid sequence that still has the same enzyme function after the amino acid sequence shown in SEQ ID NO.4 has been replaced, deleted and / or added to one or more amino acids.
[0013] Another object of this application is to provide: a biomaterial, said biomaterial being any of the following:
[0014] 1) Expression cassettes containing the above gene Os03g0112700;
[0015] 2) A targeting vector containing the above-mentioned gene Os03g0112700, or a targeting vector containing the expression cassette described in 1);
[0016] 3) A transgenic cell line containing the above-mentioned gene Os03g0112700, or a transgenic cell line containing the expression cassette described in 1), or a transgenic cell line containing the targeting vector described in 2);
[0017] 4) Recombinant bacteria containing the above-mentioned gene Os03g0112700, or recombinant bacteria containing the expression cassette described in 1), or recombinant bacteria containing the targeting vector described in 2);
[0018] 5) A transgenic plant containing the above-mentioned gene Os03g0112700, or a transgenic plant containing the expression cassette described in 1), or a transgenic plant containing the targeting vector described in 2), or a transgenic plant containing the transgenic cell line described in 3).
[0019] As a preferred technical solution, 2) the targeting vector is pCXUN-Cas9-gRNA.
[0020] Another object of this application is to provide: the application of the above-mentioned biomaterials, wherein the application is any one of the following:
[0021] 1) Application in reducing rice grain width;
[0022] 2) Application in gene Os03g0112700 knockout;
[0023] 3) Application in improving the length-to-width ratio of rice;
[0024] 4) Application in the breeding of rice varieties with high length-to-width ratio.
[0025] Another object of this application is to provide a method for reducing the width of rice grains and / or increasing the length-to-width ratio of rice, comprising the following steps: inhibiting the expression of gene Os03g0112700 in rice plants or knocking out gene Os03g0112700 in rice plants or inhibiting the activity of protein encoded by gene Os03g0112700 in rice plants.
[0026] As a preferred technical solution, the nucleotide sequence of the gene Os03g0112700 is any one of the following:
[0027] 1) As shown in SEQ ID NO.3;
[0028] 2) Or a nucleotide sequence that encodes the same functional protein after substitution, deletion and / or addition of one or more bases as shown in SEQ ID NO.3;
[0029] The amino acid sequence of the protein encoded by the gene Os03g0112700 is any one of the following:
[0030] 1) As shown in SEQ ID NO.4;
[0031] 2) Or an amino acid sequence that still has the same enzyme function after the amino acid sequence shown in SEQ ID NO.4 has been replaced, deleted and / or added to one or more amino acids.
[0032] Another object of this application is to provide: an sgRNA targeting the gene Os03g0112700, the sequence of which is shown in SEQ ID NO.5.
[0033] Another object of this application is to provide: an amplification primer pair for detecting gene Os03g0112700 knockout, said amplification primer pair being 700-F and 700-R, the specific primer sequences being shown in SEQ ID NO.8 and SEQ ID NO.9.
[0034] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects:
[0035] 1) This application reveals for the first time the function of the Os03g0112700 gene in regulating rice grain width, further enriching the gene pool related to rice grain width. This invention uses the CRISPR / Cas9 system to target and knock out this gene. Compared to the wild type, the mutant rice grain width is reduced by 5%–6.4%, significantly decreasing the grain width and thus increasing the length-to-width ratio of the rice.
[0036] 2) This invention achieves a targeted editing efficiency of over 66% by optimizing the sgRNA target design and transformation system, providing reliable technical support for the rapid breeding of long-grain rice varieties. Attached Figure Description
[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0038] Figure 1 The purpose is to analyze the sequence of the rice Os03g0112700 gene and design sgRNA gene editing targets; where black boxes represent exons, black lines represent introns and untranslated regions, and the Cas9 recognition sequence is located in the first exon.
[0039] Figure 2 The gene is: Os03g0112700 mutant; where WT is wild type, and ehd4-1, ehd4-2, ehd4-3 and ehd4-4 are four mutant types; Arabic numerals indicate the number of deleted or added bases (a "-" before the number indicates a base deletion, and a "+" before the number indicates a base addition), and lowercase letters indicate added bases.
[0040] Figure 3 The study investigated the grain type of the Os03g0112700 gene mutant and its wild type; WT was the wild type, and ehd4-1 and ehd4-2 were mutants.
[0041] Figure 4 The analysis included the grain size and thousand-grain weight of the Os03g0112700 gene mutant and its wild type; WT was the wild type, and ehd4-1 and ehd4-2 were mutants. Detailed Implementation
[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] Example 1
[0044] Cloning of the rice Os03g0112700 gene
[0045] Based on the Os03g0112700 gene information collected by the Rice Data Center, the full-length primer amplification sequence was designed as follows:
[0046] F:5'-ATGCTGAGTCTCTCGGAAAGAGCAC-3', SEQ ID NO.1;
[0047] R:5'-TCAGGGGTGCAGGTAGTTACAAG-3', SEQ ID NO.2;
[0048] Using commercially available Xiushui 134 rice as raw material, RNA was extracted and then reverse transcribed into cDNA. The extracted genomic cDNA was used as a template and amplified using the primers described above to clone the rice Os03g0112700 gene. The specific amplification conditions are as follows:
[0049] Amplification system: Total volume 25 μL, 5 μL FLU-ARMS2×PCR Mix, 2 μL cDNA, 1.5 μL Primer mix, 16.5 μL ddH2O;
[0050] Amplification program: 94℃ pre-denaturation for 4 minutes, 38 cycles (95℃ denaturation for 30 seconds; 58℃ annealing for 45 seconds; 72℃ extension for 30 seconds), followed by an extension at 72℃ for 7 minutes outside of the cycles.
[0051] After PCR amplification, the gene Os03g0112700 was cloned, and its nucleotide sequence is as follows:
[0052] Os03g0112700 nucleotide sequence:
[0053]
[0054] Amino acid sequence corresponding to Os03g0112700:
[0055] MPQTSSSAHSNVESHHDHYPSWSQVQHDPKNSLQAGSGKDLNSRYDIAQKLPSQRITRDVPSPVFAWSPSESRTASSQHEGSCLSSTTNLCTHDELHSSIASAKAKSFAPATPVEDRGSSSPSGMLSLSERAPICSPQSAPSASASDTCKMEENMNQQKTLEADISNTSVNQSPQSKILPESSPDNQDAEHEYRSPPPISESKELSPQSRTTPGSSPDNQDTEREYPSPPPISGSKEISPQSRTILESSPDNQDNGHEYPSPPPIPESIELSPHSKALPESSPDNQDIEPECPSPPQIPESKELSRQSKILPESSPGNQDIEPECPSPPQIPESKELSQQSKILPESSPDNHDIKCEYSSPTPIPESKELSLQSKILPESSSDNQDIKCEDPSPTPISKSKEVSPQSKILSESYLDNQDVERECPSSILITESKELAVDLPGSISLAPEKTASTDVGENSSLAFIFPKSTLAGDDALKSVFDMAKAHLECEDSKVKEELYVESTVVIRDDMVVNPASGVESIDMSENLLESLMEQSCGTFYMDGTTALEGFLSGSTKEEPQCSSPIALSTCSSPIALSPWGEHGYYQGDSVGSSLWGVQDDDPIGNIWPLSSQAPALQYSSAGSTAHFIDEATVTHGNNGVVLSSTPGEEVGLPNSGVCTDWGLVEQVNPETNDASVSMIDKNSGLVDSQPSANDGSDVGTARNTNHNTNLSLNHETAVPLSRSSGEASRKHGFITDLNVATSEEALGNTKNWNPYAGNANRGSQRNHHRDRYSQISESWLLSSNYSRSRSDGFGTGGSSRSTPRGQTQRGICKFHENGYCRKGASCNYLHP*, SEQ ID NO. 4.
[0056] Example 2
[0057] Construction of targeting vector and genetic transformation of rice
[0058] (1) Design of sgRNA of rice Os03g0112700 gene
[0059] This invention employs the CRISPR / Cas9 gene editing technology system to conduct related experiments, designing a Cas9 recognition site at 6 bp in the first exon of the Os03g0112700 gene. Figure 1 A specific sgRNA was designed based on this site, and its nucleotide sequence is as follows:
[0060] sgRNA nucleotide sequence:
[0061] 5'-CCTAGCCCGCCTCCAATATCTGG-3', SEQ ID NO.5;
[0062] (2) Construction of recombinant plasmid pCXUN-Cas9-gRNA
[0063] The pCXUN-Cas9 vector (purchased from Beijing Weishang Lide Biotechnology Co., Ltd.) was used as the backbone vector for constructing the recombinant plasmid pCXUN-Cas9-gRNA. The full-length Cas9 protein coding sequence is 4200 base pairs, encoding 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, the corresponding reverse strand fragment was synthesized according to the sgRNA positive strand in step (1). 1 μL of the positive and reverse strands were mixed with 8 μM Anneal Buffer (TE + 50 mM NaCl) and annealed at 95℃ for 10 min, then cooled to 20℃ at 0.1℃ / s to fully anneal and form double-stranded DNA. Subsequently, DNA ligase was used to accurately ligate it downstream of the U6 promoter of the linearized pCXUN-Cas9 vector by Bsa I enzyme digestion. Sequencing confirmed that the sgRNA was correctly inserted and without mutation, thus successfully constructing the recombinant plasmid pCXUN-Cas9-gRNA.
[0064] Enzyme digestion steps: 5 μL plasmid (approximately 1 μg) + 10 μL 10* buffer + 1 μL Bsa I enzyme + 84 μL water, 37°C for 2-3 hours.
[0065] (3) Rice genetic transformation
[0066] The recombinant plasmid pCXUN-Cas9-gRNA constructed in step (3) was transformed into the recipient material Xiushui 134 using Agrobacterium-mediated transformation, and resistant callus was screened, differentiated, and rooted. The specific steps are as follows:
[0067] A: Callus culture
[0068] Select plump and mature Xiushui 134 japonica rice seeds, remove the outer shell and disinfect them, then inoculate the seeds into induction medium (MS medium + 3 mg / L 2,4-D, pH 5.6) and culture for 10 days. Remove the radicle and continue to culture for 8 days to form embryogenic callus.
[0069] Embryogenic callus was transferred to a new induction medium and subcultured every 20 days. After two subcultures, embryogenic callus with firm texture, light yellow color and diameter of 2-3 mm was selected for subsequent Agrobacterium transformation.
[0070] B: Preparation of Agrobacterium suspension
[0071] Recombinant Agrobacterium bacterial suspension containing the recombinant plasmid pCXUN-Cas9-gRNA was streaked evenly onto LB solid medium (pH 5.8) containing 50 mg / L kanamycin and 50 mg / L rifampin, and incubated in the dark at 28°C for 48 h to activate Agrobacterium. Then, single colonies were picked from the activated plates for secondary transfection and streaked, and incubated in the dark at 28°C for 2 days. After incubation, the Agrobacterium was washed and resuspended in AAM medium (pH 5.2) containing 150 μM / L acetylsuccinone, and the bacterial concentration was adjusted to OD. 600nm The pH value was set between 1.2 and 1.8, and then the mixture was allowed to stand for 1.5 hours to prepare an Agrobacterium suspension.
[0072] C: Transfection
[0073] The embryogenic callus was completely immersed in Agrobacterium suspension and allowed to stand for 30 min. The embryogenic callus was then removed, the bacterial suspension was drained, and the callus was inoculated into co-culture medium (NB medium + 2 mg / L 2,4-D + 100 μM / L acetylsylgenone, pH 5.8) and co-cultured in the dark at 25℃ for 3 days.
[0074] D: Resistant callus culture
[0075] After co-culture, embryogenic callus tissue was collected, washed, and placed in sterile water containing 250 mg / L carbenicillin. The mixture was left to stand for 1 hour, then removed and air-dried. It was then inoculated into selection medium (NB medium + 2,4-D 2 mg / L + carbenicillin 250 mg / L + hygromycin 50 mg / L, pH 5.8) and cultured in the dark at 28°C. The tissue was subcultured every two weeks. After three weeks, nodular bright yellow resistant callus tissue was observed growing from the browned and shriveled callus tissue.
[0076] E: Differentiation and rooting culture
[0077] The resistant callus was transferred to differentiation medium (NB medium + 2,4-D 2 mg / L + KT 10 mg / L + NAA 0.4 mg / L, pH 5.8) for culture. After 2 weeks, the callus began to turn green, and after 3 weeks, the seedlings sprouted, followed by the development of the young roots. When the seedlings reached a certain stage of growth, they were transferred to rooting medium (1 / 2 MS medium, pH 5.6). After the seedlings had developed roots and matured, the medium adhering to the roots was carefully washed off, and the seedlings were transplanted to the experimental base for subsequent genotyping and phenotypic evaluation.
[0078] Example 3
[0079] Genotyping and phenotypic analysis of the Os03g0112700 gene-edited variant
[0080] Using the plants obtained in Example 2 as raw materials, genomic DNA was extracted. The extracted DNA was then used as a template for PCR primer amplification to preliminarily identify the transgenic plants. The specific process is as follows:
[0081] The PCR amplification primer sequences include CZT-F and CZT-R, and the specific sequences are as follows:
[0082] CZT-F: 5'-GGGAGATCCAGCTAGAGGTC-3', SEQ ID NO.6;
[0083] CZT-R: 5'-GGAAGGAGGAAGACAAGG-3', SEQ ID NO.7;
[0084] PCR amplification reaction program: 25 μL;
[0085] PCR amplification reaction system: 94℃ pre-denaturation for 4 minutes, 38 cycles (95℃ denaturation for 30 seconds; 58℃ annealing for 45 seconds; 72℃ extension for 30 seconds), followed by an extension at 72℃ for 7 minutes outside of the cycles.
[0086] Preliminary identification indicates that all strains are positive for transgenic genes.
[0087] To further determine the specifics of the gene editing, DNA was extracted from the preliminarily identified transgenic plants. Using the extracted DNA as a template, specific amplification primer sequences were designed targeting the target gene, and PCR amplification was performed to obtain the Os03g0112700 gene fragment containing the target sequence. The amplified gene fragment was then sent to a professional sequencing company for sequencing analysis.
[0088] The primer sequences for target gene-specific amplification are as follows:
[0089] 700-F: 5'-TCCTTCTGCGTCTGCTTC-3', SEQ ID NO.8;
[0090] 700-R: 5'-ATCGGAGTTGGACTGGAATA-3', SEQ ID NO.9;
[0091] PCR amplification reaction program: Total volume 25 μL, 5 μL FLU-ARMS2×PCRMix, 2 μL cDNA, 1.5 μL Primermix, 16.5 μL ddH2O;
[0092] PCR amplification reaction system: 94℃ pre-denaturation for 4 minutes, 38 cycles (95℃ denaturation for 30 seconds; 58℃ annealing for 45 seconds; 72℃ extension for 30 seconds), followed by an extension at 72℃ for 7 minutes outside of the cycles.
[0093] Through careful comparison and analysis of the sequencing results, it was found that the Os03g0112700 gene produced four different types of genotypes, covering various gene editing methods such as heterozygous mutation, biallelic modification, and homozygous mutation. Figure 2 ).
[0094] Detailed phenotypic observations were conducted on the mutant plants cas9-1 and cas9-2, and the experimental results are as follows: Figure 3 As shown.
[0095] Results analysis: The results showed that, compared with wild-type japonica rice, the grain width of both cas9-1 and cas9-2 mutants was significantly reduced.
[0096] Further statistical analysis was conducted on the length-to-width ratio of the seeds of the mutant lines, and the experimental results are as follows: Figure 4 As shown.
[0097] Results analysis: Compared with wild-type seeds, the grain width of mutant rice cas9-1 and cas9-2 was reduced by 6.0% to 7.2%, and the length-to-width ratio of cas9-1 and cas9-2 rice seeds was increased by 10.2% and 11.2%, respectively. This result fully demonstrates that gene editing has a significant effect on improving the length-to-width ratio of rice.
[0098] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0099] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. The application of gene knockout Os03g0112700 in reducing rice grain width, characterized in that, The amino acid sequence encoded by the gene Os03g0112700 is shown in SEQ ID NO.
4.
2. The application of the knockout gene Os03g0112700 according to claim 1 in reducing rice grain width, characterized in that, The nucleotide sequence of the gene Os03g0112700 is shown in SEQ ID NO.
3.
3. An application of a biomaterial, wherein the biomaterial is any one of the following: 1) A knockout vector for the gene Os03g0112700; 2) Recombinant bacteria containing the knockout vector described in 1); The application is any one of the following: 1) Application in reducing rice grain width; 2) Application in improving the length-to-width ratio of rice grains; 3) Application in the breeding of high length-to-width ratio grain rice varieties; The amino acid sequence encoded by the gene Os03g0112700 is shown in SEQ ID NO.
4.
4. A method for reducing rice grain width and / or increasing the length-to-width ratio of rice grains, characterized in that, The method includes the following steps: inhibiting the expression of gene Os03g0112700 in rice plants or knocking out gene Os03g0112700 in rice plants or inhibiting the activity of the protein encoded by gene Os03g0112700 in rice plants; the nucleotide sequence of gene Os03g0112700 is shown in SEQ ID NO. 3; the amino acid sequence encoded by gene Os03g0112700 is shown in SEQ ID NO. 4.