Construction of CRISPR/Cas9 Expression Vector of GhPAO3 Gene in Cotton

【Objective】 In order to further study the function of the cotton GhPAO3 gene, the gene editing vector for the cotton GhPAO3 gene was constructed by CRISPR/Cas9 technique. 【Method】 Primers were designed by screening the appropriate two PAM sites, and a linker was added to the primers, and the two target fragments were ligated together by overlap extension PCR. The pRGEB32-7 vector was subjected to single enzyme digestion by restriction endonuclease Bsa I, and the overlap extension product and the linearized pRGEB32-7 vector were ligated using a one-step method. 【Results】 The gene editing vector of the constructed cotton GhPAO3 gene was transferred into Agrobacterium LBA4404 competent state by electroporation transformation method and the positive monoclonal strain was screened by kanamycin. 【Conclusion】 The result of PCR showed that the band size was consistent with expectation, which indicated that the gene editing vector of cotton GhPAO3 gene had been constructed successfully.