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p C1300-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。
Pita2is a broad-spectrum rice blast resistance gene located in centromere region of chromosome 12.In the previous research,5 candidate genes were obtained from fine-mapping,but 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 specific target sequences with protospacer adjacent motif（PAM）sequences were selected from the exons of each candidate gene to construct the gateway vector SK-g RNA separately.Then the targeted g RNAs in the SK-g RNA vectors were separately digested by isocaudarner,and the two g RNAs were ligated into the binary expression vector p C1300-Cas9 using one step reaction.The results of PCR and sequencing showed that 5 multiple sites editing vectors for each candidate gene were successfully constructed.Our results lay the foundation for further study on the function of Pita2 candidate genes by genetic transformation and the relationship analysis between gene expression and resistance.
作者
王晓婉
邓森文
李开
王春台
徐鑫
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 43007)
出处
《中国农学通报》
2017年第19期40-45,共6页
Chinese Agricultural Science Bulletin
基金
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)
