棉花GhPAO3基因的CRISPR/Cas9表达载体的构建

doi:10.6048/j.issn.1001-4330.2019.02.004

新疆农业科学 ›› 2019, Vol. 56 ›› Issue (2): 226-232.DOI: 10.6048/j.issn.1001-4330.2019.02.004

棉花GhPAO3基因的CRISPR/Cas9表达载体的构建

朱雪峰, 田文刚, 熊显鹏, 薛飞, 张莉, 朱华国

石河子大学/新疆生产建设兵团绿洲生态农业重点实验室,新疆石河子832003

收稿日期:2018-10-16 出版日期:2019-02-20 发布日期:2019-05-22
通信作者: 朱华国(1979-),男,湖北黄冈人,副教授,博士,研究方向为棉花分子育种与植物生物技术利用,(E-mail)57530422@qq.com
作者简介:朱雪峰(1993-),男,新疆乌鲁木齐人,硕士研究生,研究方向为棉花分子育种,(E-mail)403713204@qq.com
基金资助:
国家自然科学基金(31660427);兵团现代农业科技成果转化计划(2015AC007)

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

ZHU Xue-feng, TIAN Wen-gang, XIONG Xian-peng, XUE Fei, ZHANG Li, ZHU Hua-guo

Key Laboratory of Oasis Eco-agriculture of Xinjiang Production and Construction Corps, College of Agronomy, Shihezi University, Shihezi Xinjiang 832003, China

Received:2018-10-16 Online:2019-02-20 Published:2019-05-22
Correspondence author: ZHU hua-guo(1979), Associate Professor, Doctoral Degree, Field:Cotton molecular breeding, Plant biotechnology utilization
Supported by:
the National Natural Science Foundation of China(31660427); Tackling Hard-Nut Problems in Modern Science and Technology and Research Results Transformation Project of XPCC (2015AC007)

摘要/Abstract

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

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

中图分类号:

S562

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.

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.

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棉花GhPAO3基因的CRISPR/Cas9表达载体的构建

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

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