Method for directionally knocking out OsRopGEF11 gene to reduce rice plant height based on CRISPR / Cas9 technology
Knocking out the OsRopGEF11 gene through CRISPR/Cas9 technology solved the problem of limited dwarf gene resources in rice breeding, and achieved a significant reduction in rice plant height and an increase in yield.
Patent Information
- Application Number
- CN202411829815.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-12
- Publication Date
- 2025-04-18
AI Technical Summary
The prior art is used in rice breeding with limited resources for dwarfing genes, which is difficult to effectively reduce rice plant height and affect yield.
The OsRopGEF11 gene knockout recombinant vector was constructed by CRISPR/Cas9 technology, and the embryonic callus of Japanese Qing rice was transformed by Agrobacterium mediating method, and the T0 generation OsRopGEF11 gene knockout homozygous plants were obtained to reduce the rice plant height.
Through OsRopGEF11 gene knockout, the rice plant height decreased by 25.3%, significantly improving rice yield.
Smart Images
Figure CN119842779A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular biology and relates to a method for reducing rice plant height, and specifically relates to a method for targeted knockout based on CRISPR / Cas9 technology. OsRopGEF11 Genetic methods to reduce rice plant height. Background Art
[0002] Rice is one of the main food crops in the world, and plant height is one of the important agronomic traits that affect its yield (Yu Dejin, Shui Yang, Guo Faping, et al. QTL analysis of rice plant height under different nitrogen application conditions [J / OL]. Hybrid Rice, 2024, (11): 1-7.). Too high rice plant height will cause a certain degree of lodging, resulting in a reduction in rice yield. Therefore, appropriately reducing rice plant height will help increase rice yield. In the history of rice breeding, the application of the semi-dwarf gene sd1 has promoted the selection and breeding of semi-dwarf rice varieties, greatly increasing rice yield (Ma Yuyin, Wang Ruping, Li Lei, et al. Research progress on genetics and breeding of rice plant height [J]. Henan Agricultural Science, 2008, (11): 12-17.). Existing studies believe that rice plant height is a quantitative trait regulated by multiple genes (Yu Dejin, Shui Yang, Guo Faping, et al. QTL analysis of rice plant height under different nitrogen application conditions [J / OL]. Hybrid Rice, 2024, (11): 1-7.). Although there are many studies on genes regulating rice plant height, the dwarfing gene resources used for rice breeding are limited. Therefore, in-depth research on the functions of rice genes is needed to further develop new semi-dwarf rice varieties.
[0003] In recent years, CRISPR / Cas9 gene editing technology has been widely used in genome editing of various organisms including rice. For example, the br1 gene knocked out in corn using the CRISPR / Cas9 system can significantly reduce plant height (MuQ, Wei J, Longest HK, et al. A MYB transcription factor underlying plantheight in sorghum qHT7.1 and maize Brachytic 1 loci. Plant J. Publishedonline November 1, 2024.). Zhang et al. knocked out the ZmCOLD1 gene and found that the ZmCOLD1 gene regulates plant height by affecting the cell division process (Zhang X, Zhang Z, Peng H, et al. GPCR-like proteinZmCOLD1 regulate plant height in an ABA manner. Int J Mol Sci. 2024;25(21):11755.). Studies on the rice OsPH9 gene found that knocking out the gene led to a decrease in rice plant height (Du, X, Wang, Han, et al. Fine mapping of qPH9, a major quantitative trait locus, responsible for plant height in foxtail millet [Setaria italica (L.) P.Beauv.]. Mol Breeding 41, 77 (2021).). Bing et al. found that knocking out the OsNADP-ME2 gene inhibited the expression of GA synthesis and signal transduction genes, thereby significantly reducing rice plant height (Li, B, Zhou, X, Yao, W. et al. NADP-malic Enzyme OsNADP-ME2 Modulates Plant Height Involvingin Gibberellin Signaling in Rice. Rice 17, 52 (2024).). These studies have demonstrated that CRISPR / Cas9 technology has important application value in dwarf rice breeding. Summary of the invention
[0004] The technical problem solved by the present invention is to provide a targeted knockout based on CRISPR / Cas9 technology. OsRopGEF11A method for genetically reducing rice plant height constructed using CRISPR / Cas9 technology OsRopGEF11 Gene knockout recombinant vector, and then OsRopGEF11 The gene knockout recombinant vector was transformed into embryonic callus of the recipient material Nipponbare rice (abbreviated as WT) by Agrobacterium-mediated method. After screening and molecular identification, T0 generation knockout homozygous plants were obtained. The rice plant height was counted at the maturity stage, confirming that the rice plant height was significantly reduced.
[0005] The present invention adopts the following technical solution to solve the above technical problems: targeted knockout based on CRISPR / Cas9 technology OsRopGEF11 The method of reducing rice plant height by gene is as follows: constructing by CRISPR / Cas9 technology OsRopGEF11 Gene knockout recombinant vector, and then OsRopGEF11 The knockout recombinant vector was transformed into embryonic callus of Nipponbare rice by Agrobacterium-mediated method, and the T0 generation was obtained after screening and molecular identification. OsRopGEF11 Knockout rice.
[0006] Furthermore, the OsRopGEF11 The specific construction process of the gene knockout recombinant vector is as follows: design two pairs of primers as target sites: The primers used for Target1 were F1: 5'-AAACTGGCTTCCACAAACACGGCTA-3'; R1: 5'-GGCATAGCCGTGTTTGTGGAAGCCA-3'; The primers used for Target2 were F2: 5'-GGCATGTGTCGTCGTTCTCCGACAA-3'; R2: 5'-AAACTTGTCGGAGAACGACGACACA-3'; Two gRNAs were prepared by primer annealing and the pC1300-Ubi-Cas9 vector was used Kpn I and Bam HI was used for restriction digestion, and the two gRNAs were used Kpn I and Bgl II was used to digest and recover the fragments, and the fragments were connected to the pC1300-Ubi-Cas9 vector to construct OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 .
[0007] Furthermore, the OsRopGEF11 The specific construction process of gene knockout recombinant vector is as follows: OsRopGEF11 The sequences were submitted to NCBI for retrieval. OsRopGEF11The CDS sequence of the gene selected two candidate sequences with a PAM site of "NGG" and a length of 21 bp. After Blastn alignment, the sequence with high specificity was selected as the editing target, and the two pairs of primer sequences were added Aar I restriction site, based on which primers F1 / R1 and F2 / R2 were designed to prepare two gRNAs. The reaction system for preparing gRNA from F1 / R1 was: 5µL of F1 / R1 and 40µL of H2O; the reaction conditions were: denaturation at 95℃ for 5min, annealing at 55℃ for 5min. F2 / R2 prepared gRNA fragments according to the above reaction system to obtain two gRNA fragments. The two gRNA fragments and pC1300- Ubi-Cas9 were used separately. Aar I digested and then connected. The reaction system was: 5µL of each gRNA fragment after digestion, 3µL of vector after digestion, 1µL of T4 DNA ligase, 2µL of T4 Buffer and 9µL of H2O. After mixing, react at 37℃ for 2h. The ligation product was transformed into E. coli competent cells, and positive clones were screened and confirmed by identification and sequencing. OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 Build successful.
[0008] Furthermore, the OsRopGEF11 The specific preparation process of gene knockout rice is as follows: freeze-thaw method is used to OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 Agrobacterium tumefaciens EHA 105 competent cells, and the Agrobacterium transformant solution was used to infect the Nipponbare rice callus to obtain knockout regenerated plants. Hi-TOM high-throughput sequencing was used for second-round PCR detection to screen OsRopGEF11 The sequencing results of the knockout rice were analyzed using SnapGene software and compared with the expected gene sequence. The sequencing results screened out a homozygous mutant, one of which lacked a base C in the target sequence and the second target sequence lacked 16 bases. It was named strain C11-1. OsRopGEF11 Knockout rice.
[0009] Compared with the prior art, the present invention has the following advantages: compared with Nipponbare rice, OsRopGEF11 The plant height of gene knockout rice decreased by 25.3%, reaching an extremely significant level. BRIEF DESCRIPTION OF THE DRAWINGS
[0010] Figure 1 Schematic diagram of the Cas9-OsRopGEF11 gene knockout recombinant vector.
[0011] Figure 2 for OsRopGEF11 Identification of knockout rice C11-1.
[0012] Figure 3 This is a comparative analysis of the plant height of WT and C11-1 rice.
[0013] Figure 4 This is a statistical analysis chart of plant height of WT and C11-1 rice. DETAILED DESCRIPTION
[0014] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention. Example
[0015] 1. Rice OsRopGEF11 Construction of gene knockout vector Will OsRopGEF11 The sequences were submitted to NCBI for retrieval. OsRopGEF11 The CDS sequence of the gene selected two candidate sequences with a PAM site of "NGG" and a length of 21 bp. After Blastn alignment, the sequence with high specificity was selected as the editing target, and the two pairs of primer sequences were added Aar I restriction site, based on which primers F1 / R1 and F2 / R2 were designed to prepare two gRNAs. The reaction system for preparing gRNA from F1 / R1 was: 5µL of F1 / R1 and 40µL of H2O; the reaction conditions were: denaturation at 95℃ for 5min, annealing at 55℃ for 5min. F2 / R2 prepared gRNA fragments according to the above reaction system to obtain two gRNA fragments. The two gRNA fragments and pC1300- Ubi-Cas9 were used separately. Aar I digested and then connected. The reaction system was: 5µL of each gRNA fragment after digestion, 3µL of vector after digestion, 1µL of T4 DNA ligase, 2µL of T4 Buffer and 9µL of H2O. After mixing, react at 37℃ for 2h. The ligation product was transformed into E. coli competent cells, and positive clones were screened and confirmed by identification and sequencing. OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 Build successful.
[0016] 2. OsRopGEF11 Genetic transformation and screening of gene knockout rice Freeze-thaw method OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 Agrobacterium tumefaciens EHA 105 competent cells, and the Agrobacterium transformant solution was used to infect the Nipponbare rice callus to obtain knockout regenerated plants. Hi-TOM high-throughput sequencing was used for second-round PCR detection to screen OsRopGEF11The sequencing results of the knockout rice were analyzed using SnapGene software and compared with the expected gene sequence. The sequencing results screened out a homozygous mutant, one of which lacked a base C in the target sequence and the second target sequence lacked 16 bases. It was named strain C11-1. OsRopGEF11 Knockout rice.
[0017] 3. Measurement of rice plant height during heading stage Waiting for the Japanese rice and OsRopGEF11 The knockout rice C11-1 strain grew to the heading stage, and the plant heights of WT and C11-1 were measured and photographed. The plant heights of WT and C11-1 strains grown to the heading stage were measured, and the measurement results showed that the plant height of C11-1 was reduced by 25.3% compared with WT, and reached an extremely significant level.
[0018] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.
Claims
1. Targeted knockout based on CRISPR / Cas9 technology OsRopGEF11 A method for reducing rice plant height by gene, characterized in that The specific process is: constructed through CRISPR / Cas9 technology OsRopGEF11 Gene knockout recombinant vector, and then OsRopGEF11 The knockout recombinant vector was transformed into embryonic callus of Nipponbare rice by Agrobacterium-mediated method, and the T0 generation was obtained after screening and molecular identification. OsRopGEF11 Knockout rice.
2. The CRISPR / Cas9-based targeted knockout according to claim 1 OsRopGEF11 A method for reducing rice plant height by gene, characterized in that Said OsRopGEF11 The specific construction process of the gene knockout recombinant vector is as follows: design two pairs of primers as target sites: The primers used for Target1 are F1: 5’-AAACTGGCTTCCACAAACACGGCTA-3’; R1: 5’-GGCATAGCCGTGTTTGTGGAAGCCA-3’; The primers used for Target2 are F2: 5’-GGCATGTGTCGTCGTTCTCCGACAA-3’; R2: 5’-AAACTTGTCGGAGAACGACGACACA-3’; Two gRNAs were prepared by primer annealing and the pC1300-Ubi-Cas9 vector was used Kpn I and Bam HI was used for restriction digestion, and the two gRNAs were used Kpn I and Bgl II was used to digest and recover the fragments, and the fragments were connected to the pC1300-Ubi-Cas9 vector to construct OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 .
3. The CRISPR / Cas9-based targeted knockout according to claim 1 OsRopGEF11 A method for reducing rice plant height by gene, characterized in that Said OsRopGEF11 The specific construction process of gene knockout recombinant vector is as follows: OsRopGEF11 The sequences were submitted to NCBI for retrieval. OsRopGEF11 The CDS sequence of the gene selected two candidate sequences with a PAM site of "NGG" and a length of 21bp. After Blastn alignment, the sequence with high specificity was selected as the editing target, and the two pairs of primer sequences were added Aar I restriction site, based on which primers F1 / R1 and F2 / R2 were designed to prepare two gRNAs. The reaction system for preparing gRNA from F1 / R1 was: 5µL of F1 / R1 and 40µL of H2O; the reaction conditions were: denaturation at 95℃ for 5min, annealing at 55℃ for 5min. F2 / R2 prepared gRNA fragments according to the above reaction system to obtain two gRNA fragments. The two gRNA fragments and pC1300-Ubi-Cas9 were used separately. Aar I digested and then connected. The reaction system was: 5µL of each gRNA fragment after digestion, 3µL of vector after digestion, 1µL of T4 DNA ligase, 2µL of T4 Buffer and 9µL of H2O. After mixing, react at 37℃ for 2h. The ligation product was transformed into E. coli competent cells, and positive clones were screened and confirmed by identification and sequencing. OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 Build successful.
4. The CRISPR / Cas9-based targeted knockout according to claim 1 OsRopGEF11 A method for reducing rice plant height by gene, characterized in that Said OsRopGEF11 The specific preparation process of gene knockout rice is as follows: freeze-thaw method is used to OsRopGEF11 Gene knockout recombinant vector Cas9- OsRopGEF11 Agrobacterium tumefaciens EHA 105 competent cells, and the Agrobacterium transformant solution was used to infect the Nipponbare rice callus to obtain knockout regenerated plants. Hi-TOM high-throughput sequencing was used for second-round PCR detection to screen OsRopGEF11 The sequencing results of the knockout rice were analyzed using SnapGene software and compared with the expected gene sequence. The sequencing results screened out a homozygous mutant, one of which lacked a base C in the target sequence and the second target sequence lacked 16 bases. It was named strain C11-1. OsRopGEF11 Knockout rice.