Multiple Sites Editing Vector Construction of Rice Blast Resistance Gene Pita2 Candidate Genes

doi:10.11924/j.issn.1000-6850.casb17020123

Abstract

Abstract: Pita2 is a broad-spectrum rice blast resistance gene located in centromere region of chromosome 12. In our previous research, 5 candidate genes were obtained from fine-mapping and their function and molecular mechanism were unknown. In this study, we constructed multiple sites editing vectors of those 5 candidate genes by high-efficiency and designated-editing CRISPR/Cas9 system. 2 protospacer adjacent motif (PAM) sequences were selected from the exons of each candidate genes to construct the gateway vector SK-gRNA separately. Then 2 targeted gRNAs on the SK-gRNA vectors were separately digested by isocaudarner and ligated into the binary expression vector pC1300-Cas9 using one step reaction. The results of PCR and Sanger sequencing from plasmid DNA showed that 5 multiple sites editing vectors for each candidate genes were successfully constructed. Our results lay the foundation for the further study on the function of Pita2 candidate genes by agrobacterium-mediated transformation and the analysis of the relationship between gene expression and resistance.

CLC Number:

S511

Wang Xiaowan,Deng Senwen,Li Kai,Wang Chuntai and Xu Xin. Multiple Sites Editing Vector Construction of Rice Blast Resistance Gene Pita2 Candidate Genes[J]. Chinese Agricultural Science Bulletin, 2017, 33(19): 40-45.

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