稻瘟病抗性基因Pita2候选基因多位点编辑载体的构建

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

中国农学通报 ›› 2017, Vol. 33 ›› Issue (19): 40-45.doi: 10.11924/j.issn.1000-6850.casb17020123

所属专题：生物技术；水稻；农业生态

稻瘟病抗性基因Pita2候选基因多位点编辑载体的构建

王晓婉,邓森文,李开,王春台,徐鑫

(中南民族大学生命科学学院/武陵山区特色资源植物种质保护与利用湖北省重点实验室/生物技术国家民委重点实验室,武汉 430074）

收稿日期:2017-02-28 修回日期:2017-05-29 接受日期:2017-03-22 出版日期:2017-07-11 发布日期:2017-07-11
通讯作者: 徐鑫
基金资助:
国家自然科学基金面上项目“水稻稻瘟病真性抗性基因Pita-2 的克隆及功能研究”(30971563)；中央高校专项基金“稻瘟病抗性基因Pi63 进化机制及应用研究”(CZZ16002)。

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

Wang Xiaowan, Deng Senwen, Li Kai, Wang Chuntai, Xu Xin

(Hubei Provincial Key Laboratory for Protection and Application of Special Plants in Wuling Area of China/Key Lab for Biotechnology of State Ethnic Affairs Commission/ College of Life Science,South-Central University for Nationalities, Wuhan 430074)

Received:2017-02-28 Revised:2017-05-29 Accepted:2017-03-22 Online:2017-07-11 Published:2017-07-11

摘要/Abstract

摘要： Pita2是定位于水稻第12 号染色体着丝粒区域的稻瘟病广谱抗性基因，前期研究中通过精细定位得到了5 个候选基因，其功能及作用原理还不清楚。本研究利用CRISPR/Cas9 系统构建Pita2候选基因的多位点编辑载体。首先在候选基因外显子区域找到2 个含有PAM序列的特异性靶位点序列，并构建候选基因入门载体SK-gRNA，然后利用同尾酶将SK-gRNA重组载体上的目的片段gRNA酶切，采用一步法将2 个gRNA连接到双元表达载体pC1300-Cas9。质粒PCR鉴定以及测序的结果表明，以上5 个多位点编辑载体构建成功。后期通过遗传转化并鉴定候选基因表达量与抗病性之间的关系，为水稻稻瘟病抗性基因Pita2功能研究奠定基础。

关键词: 简析, 简析, 祁隽藻, 清代, 农书, 马首农言

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.

中图分类号:

S511

王晓婉,邓森文,李开,王春台,徐鑫. 稻瘟病抗性基因Pita2候选基因多位点编辑载体的构建[J]. 中国农学通报, 2017, 33(19): 40-45.

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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稻瘟病抗性基因Pita2候选基因多位点编辑载体的构建

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

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