Application of OsCRKS2 gene in controlling drought resistance of rice

A technology of drought resistance and rice, applied in the fields of application, genetic engineering, plant genetic improvement, etc.

Pending Publication Date: 2022-04-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The OsCRKS2 gene involved in the present invention belongs to the rice CRKs family, and no rice CRKs related to abiotic stress response has been reported

Method used

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  • Application of OsCRKS2 gene in controlling drought resistance of rice
  • Application of OsCRKS2 gene in controlling drought resistance of rice
  • Application of OsCRKS2 gene in controlling drought resistance of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Isolation and Cloning of OsCRKS2 Gene

[0026]Primers OsCRKS2-FL-F: 5'-ATGCAGATGCTGATCGTTTC-3', OsCRKS2-FL-R: 5'-CTACCTTGCCAATGTGATACT-3' were designed. Using the cDNA of rice variety Nipponbare leaves as a template, primers OsCRKS2-FL-F and OsCRKS2-FL-R were used to amplify the CDS sequence encoded by the OsCRKS2 gene. The amplified sequence is shown in SEQ ID NO: 1, and the encoded amino acid sequence is SEQ ID NO: 1 Shown in ID NO.2.

[0027] The PCR reaction conditions were: pre-denaturation at 95°C for 3 minutes; 33 cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min; extension at 72°C for 5 min.

[0028] The amplified PCR product was connected into the pGEM-T Easy vector by TA cloning method, positive clones were screened and confirmed by sequencing, the CDS sequence of OsCRKS2 was obtained, the sequence is shown in SEQ ID NO.1, and the clone was named pGEM OsCRKS2 plasmid .

Embodiment 2

[0030] Construction of Overexpression Vector of OsCRKS2 Gene

[0031] The positive clone pGEM-OsCRKS2 plasmid obtained in Example 1 was amplified with primers OsCRKS2-OE-F: 5'-tacgaacgatagccggtacc ATGCAGATGCTGATCGTTTC-3' and OsCRKS2-OE-R: 5'-ttgcggactctagaggatcc CTACCTTGCCAATGTGATACT-3' to amplify the gene containing OsCRKS2 DNA fragments of complete coding segments;

[0032] The PCR reaction conditions were: pre-denaturation at 94°C for 3 minutes; 30 cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min; extension at 72°C for 5 min. The obtained PCR product was connected into the pU1301 vector digested with restriction endonucleases KpnI and BamHI by the method of Gibson Assembly, the vector was sequenced and confirmed, and finally an OsCRKS2 gene overexpression vector that could be used for genetic transformation was obtained.

Embodiment 3

[0034] Construction of oscrks2 CRISPR mutants

[0035] The gene sequence of OsCRKS2 gene was obtained from rice gene database Rice Data (http: / / www.ricedata.cn / gene / ). Pick a target site according to CRISPR-P v2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ). The vector construction of CRISPR mutant strains can refer to relevant literature (He Yubing et al. Programmed self-elimination of the CRISPR / Cas9 construct greatly accelerates the isolation of edited and transgene-free rice plants. Mol. Plant. 2018, 05.005.).

[0036] The target sites selected in the CRISPR-P v2.0 website are as follows:

[0037] Target site: TCCTAATGCTTCTCCTCTCT.

[0038] The constructed CRISPR vector OsCRKS2-CRISPR was transferred into the rice variety "Zhonghua 11" (a conventional rice variety from the Institute of Rice Science, Chinese Academy of Agricultural Sciences) through the method of Agrobacterium-mediated genetic transformation of rice. Infection, co-cultivation, selection of hygromycin-resistant ...

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Abstract

The invention belongs to the technical field of plant genetic engineering. The invention particularly relates to application of the OsCRKS2 gene in controlling the drought resistance of rice. According to the invention, the OsCRKS2 gene capable of improving the drought tolerance of rice is obtained through separation, cloning and functional verification. The nucleotide sequence of the OsCRKS2 gene is as shown in SEQ ID NO: 1, and the sequence of protein coded by the gene is as shown in SEQ ID NO: 2. The rice drought response control gene OsCRKS2 is cloned, CRISPR mutant phenotype identification is carried out on candidate genes, drought stress phenotype identification in the seedling stage and the adult-plant stage shows that when a gene segment is deleted, the drought stress resistance of rice is reduced, and the function and the application way of the gene are proved.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to the application of the OsCRKS2 gene in controlling the drought resistance of rice, and the CDS sequence of the gene is shown in SEQ ID NO.1. Background technique [0002] Plants will be affected by many environmental factors during the growth process. Drought, cold damage and high temperature will lead to large-scale crop yield reduction, which is the bottleneck of agricultural development in many areas. Cultivating stress-tolerant crop varieties has always been one of the main goals of agricultural science and technology research. In order to resist or adapt to these unfavorable factors, the plant body senses the changes in the extracellular environment and transmits them into the cells through various channels, which will induce the expression of some response genes, and produce some genes that can protect the cells from drought, high salt, low temp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12A01H5/00A01H6/46
CPCC12N9/12C12N15/8273
Inventor 熊立仲叶田田
Owner HUAZHONG AGRI UNIV
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