Rice OsFb34 gene and its encoded protein and application
Through genome-wide correlation analysis, the OsFb34 gene was localized and the gene was edited using CRISPR/cas technology, which solved the problem of improving the grain shape of rice grains and achieved effective regulation of grain length, grain width and aspect ratio of rice grains.
Patent Information
- Application Number
- CN202411478179.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-22
- Publication Date
- 2025-03-04
- Estimated Expiration
- 2044-10-22
AI Technical Summary
The prior art is difficult to effectively locate and regulate genes related to rice grain shape, making it difficult to achieve grain shape improvement.
The OsFb34 gene on rice chromosome 7 was localized through genome-wide association analysis, and the knockout vector of the OsFb34 gene was constructed. The OsFb34 gene was knocked out or edited in rice through CRISPR/cas technology, thereby regulating the grain length, grain width and aspect ratio of rice grains.
Transgenic rice plants with reduced grain length, increased grain width and decreased length/width ratio were successfully obtained, which proved that the OsFb34 gene deletion can significantly affect the shape characteristics of rice grains.
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Figure CN119177240B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a rice OsFb34 gene and its encoded protein and application. Background Art
[0002] Rice is one of the most important food crops in my country. Grain shape is an important agronomic trait in rice research, which is closely related to rice yield, taste quality, etc. Rice grain shape refers to the shape characteristics of rice grains, which is usually expressed by grain length, grain width, or length / width ratio. It is a typical quantitative trait controlled by multiple genes. Genome-wide association analysis is an important means to study complex quantitative traits of crops. Although genes related to G protein signaling, ubiquitin-proteasome pathway, hormone signaling, and MAPK signaling pathway have been identified to regulate rice grain shape, there are few studies using genome-wide association analysis to locate rice grain shape regulatory sites and identify functional genes. Based on grain shape phenotypic data from many years and multiple environments, it is of great theoretical and practical significance to explore new genes that regulate rice grain shape and use modern biotechnology to transform and regulate rice grain shape.
[0003] OsFb34 protein has four transmembrane domains and no typical known functional domains. At the same time, its sequence is relatively conserved during plant evolution, but its biological functions in the model plant Arabidopsis thaliana and other species have not been reported. Summary of the invention
[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a rice OsFb34 gene. Another technical problem to be solved by the present invention is to provide a protein encoded by the rice OsFb34 gene. Another technical problem to be solved by the present invention is to provide an application of the rice OsFb34 gene for regulating the shape of rice grains.
[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] A rice OsFb34 gene, the nucleotide sequence of which is shown as SEQ ID NO.1.
[0007] The amino acid sequence of the protein encoded by the rice OsFb34 gene is shown in SEQ ID NO.2.
[0008] A vector, recombinant bacteria or host cell containing rice OsFb34 gene.
[0009] Application of rice OsFb34 gene in regulating rice grain shape.
[0010] The application of rice OsFb34 gene in regulating rice grain shape is as follows:
[0011] 1) Construction of a knockout vector for the rice OsFb34 gene;
[0012] 2) Transforming the constructed knockout vector of rice OsFb34 gene into rice;
[0013] 3) Cultivate, screen and obtain transgenic rice plants with reduced grain length, increased grain width and reduced length / width ratio.
[0014] The method for constructing the rice OsFb34 gene knockout vector is as follows: designing oligo primers according to the gRNA target sequence that inhibits the expression of the OsFb34 gene, configuring a reaction system to synthesize an oligo dimer; and constructing the oligo dimer into a CRISPR / cas vector to obtain the rice OsFb34 gene knockout vector.
[0015] The gRNA target sequence for inhibiting the expression of the OsFb34 gene is: 5'-TGCGTACCACACCCGGTACAGGG-3'.
[0016] Application of rice OsFb34 gene knockout vector in regulating rice grain shape.
[0017] The invention discloses an application of a mutant of rice OsFb34 gene in regulating rice grain shape, wherein the CDS nucleotide sequence of the OsFb34 gene is shown in SEQ ID NO.1; the mutant is mutated at any position on the sequence shown in SEQ ID NO.2, and the mutation can cause the termination of protein expression of the OsFb34 gene.
[0018] The mutations include point mutations, deletion mutations, and insertion mutations.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] 1) The rice OsFb34 gene disclosed in the present invention has a CDS nucleotide sequence as shown in SEQ ID NO.1, and an amino acid sequence of the encoded protein as shown in SEQ ID NO.2. The present invention constructs a knockout vector of the rice OsFb34 gene; transforms the constructed knockout vector into rice; and cultivates, screens, and obtains transgenic rice plants with reduced rice grain length, increased grain width, and reduced length / width ratio. The results show that knocking out the OsFb34 gene significantly reduces the grain length, grain width, and length / width ratio of the transgenic rice.
[0021] 2) The present invention finally obtained a gene-edited homozygous plant with a site-directed mutation at the target site of the rice OsFb34 gene, which was characterized by the insertion of one base A and the deletion of four bases CGGT, respectively, and was named osfb34-1 and osfb34-2. Both homozygous mutation forms caused a protein coding frameshift, resulting in a loss of protein function.
[0022] 3) Compared with the control group, the mutants of osfb34-1 and osfb34-2 showed a phenotype of reduced grain length and increased grain width, which led to a decrease in the length / width ratio. The results showed that the loss of the OsFb34 gene would lead to a decrease in rice grain length and an increase in grain width, which in turn led to a decrease in the length / width ratio. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 This is the QTL map for stable grain shape in rice, qGS7-1;
[0024] Figure 2 This is a diagram of sequencing results of the gene-edited mutant of OsFb34;
[0025] Figure 3 These are rice grain shape analysis diagrams of osfb34 gene-edited mutants (A is the grain length phenotype diagram of ZH11, osfb34-1 and osfb34-2, with a scale bar of 3.5 mm; B is the grain width phenotype diagram of ZH11, osfb34-1 and osfb34-2, with a scale bar of 3.0 mm; C is the statistical analysis diagram of grain length, grain width and length / width ratio of ZH11, osfb34-1 and osfb34-2, ** indicates P<0.01). DETAILED DESCRIPTION
[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.
[0027] Example 1
[0028] 1. Genome-wide association analysis locates the key locus of rice grain width, qGS7-1
[0029] A genome-wide association analysis was performed using 276 rice accessions and the corresponding 497K single nucleotide polymorphism markers from the 3,000 Rice Genomes project (3K-RG) to explore potential candidate genes regulating rice grain shape. Based on the phenotypic data of grain length, grain width and length / width ratio of the rice population in 2019 and 2020, the FaST-LMM model was used for association analysis, and a new stable grain shape quantitative trait QTL was identified on chromosome 7, named qGS7-1 ( Figure 1 ). Transcriptome data were used to analyze the tissue expression patterns of upstream and downstream genes in this interval (~200kb) (http: / / bar.utoronto.ca / / efp_rice / cgi-bin / efpWeb.cgi), and LOC_Os07g27810 (OsFb34) was screened to be highly expressed in grains. Therefore, OsFb34 was selected as a candidate gene for grain width phenotype analysis in rice.
[0030] 2. Target gene cloning
[0031] Total RNA from rice seeds grown for 3 days was extracted using an RNA extraction kit (Tiangen, DP441), followed by reverse transcription (Invitrogen, K1622) to obtain cDNA. Using cDNA as a template, primers were synthesized and PCR amplification was performed. The primer sequences are as follows:
[0032] OsFb34-cDNA-F: 5'-ATGGCGTCCATCATCGTG-3';
[0033] OsFb34-cDNA-R: 5'-TTAGCTGTAAGTGCCCATGCCA-3',
[0034] The CDS sequence of the OsFb34 gene finally cloned is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.
[0035] 3. Selection of OsFb34 gene target site
[0036] OsFb34 is located on rice chromosome 7 and contains 3 exons and 2 introns. The target site selected for the knockout vector is located in the 3rd exon region ( Figure 2 ). The gRNA target sequence of OsFb34 gene is: 5'-TGCGTACCACACCCGGTACAGGG-3'.
[0037] 3. Construction of OsFb34 gene editing knockout vector and acquisition of gene editing mutants
[0038] ①Synthesize oligos according to the gRNA target sequence of the OsFb34 gene:
[0039] Oligo-F: 5'-TGTGTGGTGCGTACCACACCCGGTACA-3';
[0040] Oligo-R: 5'-AAACTGTACCGGGTGTGGTACGCACA-3'.
[0041] ② Preparation of oligo dimer:
[0042] The synthesized primers were dissolved in water to 10 μmol, mixed according to the reaction system (Buffer Aneal 18 μL, F Primer 1 μL, R Primer 1 μL), heated at 95°C for 3 minutes using a PCR instrument, and then the temperature was slowly lowered to 20°C at about 0.2°C / sec.
[0043] ③Construct oligo dimer into CRISPR / cas vector
[0044] According to the reaction system shown (H2O 6μL, CRISPR / Cas Vector 2μL, Oligo dimer 1μL, EnzymeMix 1μL), mix well and react at 20°C for 1 hour. The oligo dimers were constructed into the CRISPR / cas vector BGK032 by homologous recombination.
[0045] ④ Obtaining gene-edited mutants
[0046] The ligated fragments were transformed into competent Escherichia coli cells using the CaCl2 heat shock transformation method and then identified. After obtaining positive recombinants, the plasmids were extracted and then transformed into Agrobacterium tumefaciens GV3101 strains. The target gene was transferred into the rice variety Zhonghua 11 using the Agrobacterium infection method.
[0047] The T0 transgenic lines were obtained by hygromycin resistance screening. The DNA of the resistant plants was extracted, and the genomic DNA was amplified by PCR using specific primers containing the target site. The PCR products were sequenced for the first generation to obtain transgenic homozygous plants with site-directed mutations in the target site of the rice OsFb34 gene.
[0048] The T0 transgenic lines were obtained by hygromycin resistance screening. The DNA of the resistant plants was extracted, and the genomic DNA was amplified by PCR using specific primers containing the target site. The PCR products were sequenced for the first generation to obtain gene-edited homozygous plants with site-directed mutations in the target site of the rice OsFb34 gene, which were characterized by the insertion of one base A and the deletion of four bases CGGT, respectively. They were named osfb34-1 and osfb34-2. The sequencing results of two of the mutants are shown in Figure 2 Since the number of bases inserted and deleted in osfb34-1 and osfb34-2 is not a multiple of 3 bases, both will cause protein frameshift mutations, which are gene-edited mutants of OsFb34. Both homozygous mutations cause protein coding frameshifts, resulting in protein function loss.
[0049] The primer sequences used to amplify the OsFb34 gene target site are as follows:
[0050] OsFb34-F: 5'-GACCAAAGTACATAGGGTAAATGG-3';
[0051] OsFb34-R: 5'-GCAAGCTCCTTTTAGCTGTAAG-3'.
[0052] 4. Functional identification of OsFb34 gene regulating rice grain shape
[0053] In 2022 and 2023, the homozygous mutant plants of osfb34 mentioned above were planted simultaneously with the control plant ZH11 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 length / width ratio were carried out on ZH11, osfb34-1 and osfb34-2, and the number of statistical lines was ≧20.
[0054] The results are as follows Figure 3 As shown in the figure, the grain length of ZH11 is about 7.21mm and the grain width is 2.84mm; the grain length of osfb34-1 mutant is 6.71mm and the grain width is 3.06mm, which are significantly different from ZH11; the grain length of osfb34-2 mutant is 6.73mm and the grain width is 3.05mm, which are significantly different from ZH11. Both osfb34-1 and osfb34-2 mutants show a phenotype of reduced grain length and increased grain width, which leads to a decrease in the length / width ratio.
[0055] In summary, the deletion of the OsFb34 gene will lead to a decrease in rice grain length and an increase in grain width, which will in turn lead to a decrease in the length / width ratio.
[0056] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.
Claims
1. Rice OfB34 Application of gene in regulating rice grain shape, the rice OfB34 The CDS nucleotide sequence of the gene is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that: Here are the steps: 1) Construction of rice OfB34 Gene knockout vector; 2) The constructed rice OfB34 The gene knockout vector was transformed into rice; 3) Cultivate, screen and obtain transgenic rice plants with reduced grain length, increased grain width and reduced length / width ratio.
3. The use according to claim 2, characterized in that: The rice OfB34 The method for constructing a gene knockout vector is as follows: OfB34 Design oligo primers for the gRNA target sequence of gene expression and configure the reaction system to synthesize oligo dimers; The oligo dimer was constructed into the CRISPR / cas vector to obtain rice OfB34 Gene knockout vectors.
4. The use according to claim 3, characterized in that: The inhibition OfB34 The gRNA target sequence for gene expression was: 5'-TGCGTACCACACCCGGTACAGGG-3'.
5. The rice according to claim 3 OfB34 Application of gene knockout vector in regulating rice grain shape.
6. Rice OfB34 Application of a mutant of a gene in regulating rice grain shape, the OfB34 The CDS nucleotide sequence of the gene is shown in SEQ ID NO. 1; the mutant is a mutation at any position in the sequence shown in SEQ ID NO. 2, and the mutation can cause OfB34 The protein expression of the gene is terminated.
7. The use according to claim 6, characterized in that: The mutations include point mutations, deletion mutations, and insertion mutations.