稻瘟病新抗性基因Pi39候选基因CRISPR/Cas9敲除载体的构建

doi:10.11924/j.issn.1000-6850.casb15100071

中国农学通报 ›› 2016, Vol. 32 ›› Issue (6): 91-95.doi: 10.11924/j.issn.1000-6850.casb15100071

所属专题：生物技术；水稻；农业生态

稻瘟病新抗性基因Pi39候选基因CRISPR/Cas9敲除载体的构建

刘早利,陈亚红,王春台,刘新琼

(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:2015-10-19 修回日期:2016-01-25 接受日期:2015-11-18 出版日期:2016-03-07 发布日期:2016-03-07
通讯作者: 刘新琼
基金资助:
转基因专项“稻瘟病抗性基因的克隆及其应用”(2014ZX0800904B)。

Knock-out Vector Construction of Novel Blast Resistance Gene Pi39 Candidate Gene by CRISPR/Cas9 System

Liu Zaoli, Chen Yahong, Wang Chuntai, Liu Xinqiong

(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:2015-10-19 Revised:2016-01-25 Accepted:2015-11-18 Online:2016-03-07 Published:2016-03-07

摘要/Abstract

摘要： Pi39是位于水稻第12染色体上的稻瘟病新抗性基因，为了明确其功能，本研究利用CRISPR/Cas9系统构建候选基因的敲除载体。通过对候选基因序列分析，将合成的靶位点序列插入入门载体pENTR-sgRNA，再与表达载体Cas9发生重组。本研究首先在候选基因的第一个外显子区域找到了2个带有PAM序列的靶位点，将2个目的小片段依次克隆到pENTR-sgRNA载体上。通过菌落PCR检测及测序，结果表明目的小片段插入位置正确。将入门载体与表达载体进行重组反应后，阳性克隆经菌落PCR和质粒DNA酶切鉴定并测序，结果表明重组载体构建成功。本研究利用CRISPR/Cas9系统构建敲除载体操作简单，耗时短，为进一步开展Pi39候选基因的功能研究奠定了基础。

关键词: 速生杨木胶合板, 速生杨木胶合板, 胶合强度, 载药量, 阻燃性能

Abstract: Pi39 is a novel rice blast resistance gene located on rice chromosome 12. To clarify the resistant functions of Pi39, the knock-out vector of the candidate gene was constructed by the CRISPR/Cas9 system. Based on the analysis of the candidate gene, the two targeted site sequences were synthesized and inserted into the entry vector pENTR-sgRNA that was recombined into the expression vector Cas9 later. Firstly, the two target sites with PAM sequence were selected in the candidate gene, and then the two small target fragments were cloned into pENTR-sgRNA vector in turn. The results of colony PCR and Sanger sequencing indicated that the little purpose fragments inserted correctly. After the recombination of the entry vector and the expression vector, the positive clones were verified by colony PCR and restriction enzyme digestion and further confirmed by Sanger sequencing. Those results showed that the recombinant vector was successfully constructed. The knock-out vector was built of easy manipulation and time-saving by CRISPR/Cas9 system, which laid a foundation for further research on the function of Pi39 candidate gene.

刘早利,陈亚红,王春台,刘新琼. 稻瘟病新抗性基因Pi39候选基因CRISPR/Cas9敲除载体的构建[J]. 中国农学通报, 2016, 32(6): 91-95.

Liu Zaoli,Chen Yahong,Wang Chuntai and Liu Xinqiong. Knock-out Vector Construction of Novel Blast Resistance Gene Pi39 Candidate Gene by CRISPR/Cas9 System[J]. Chinese Agricultural Science Bulletin, 2016, 32(6): 91-95.

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稻瘟病新抗性基因Pi39候选基因CRISPR/Cas9敲除载体的构建

Knock-out Vector Construction of Novel Blast Resistance Gene Pi39 Candidate Gene by CRISPR/Cas9 System

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