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

doi:10.6048/j.issn.1001-4330.2019.02.004

Abstract

Abstract: 【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.

Key words: cotton; CRISPR/Cas9; gene editing; GhPAO3

摘要： 【目的】研究棉花GhPAO3基因的功能,通过CRISPR/Cas9技术构建棉花GhPAO3基因的基因编辑载体。【方法】筛选合适的两个PAM位点设计引物,在引物中添加接头,重叠延伸PCR,将两个靶标片段连接在一起。通过限制性核酸内切酶BsaⅠ对pRGEB32-7载体进行单酶切,使用一步法连接重叠延伸产物与线性化的pRGEB32-7载体。【结果】使用电击转化法将构建好的棉花GhPAO3基因的基因编辑载体转入农杆菌LBA4404感受态,通过卡那霉素筛选阳性单克隆菌株。【结论】条带大小与预期一致,棉花GhPAO3基因的基因编辑载体构建成功。

关键词: 棉花, CRISPR/Cas9, 基因编辑, GhPAO3

CLC Number:

S562

ZHU Xue-feng, TIAN Wen-gang, XIONG Xian-peng, XUE Fei, ZHANG Li, ZHU Hua-guo. Construction of CRISPR/Cas9 Expression Vector of GhPAO3 Gene in Cotton[J]. Xinjiang Agricultural Sciences, 2019, 56(2): 226-232.

朱雪峰, 田文刚, 熊显鹏, 薛飞, 张莉, 朱华国. 棉花GhPAO3基因的CRISPR/Cas9表达载体的构建[J]. 新疆农业科学, 2019, 56(2): 226-232.

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