| 稻瘟病抗性基因Pita~2候选基因多位点编辑载体的构建 |
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| 引用本文： | 王晓婉,邓森文,李 开,王春台,徐 鑫.稻瘟病抗性基因Pita~2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. |
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| 作者姓名： | 王晓婉 邓森文 李 开 王春台 徐 鑫 |
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| 作者单位： | 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,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,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,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,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 |
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| 基金项目： | 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)。 |
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| 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。
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| 关 键 词： | 稻瘟病抗性基因 Pita2 CRISPR/Cas9系统 多位点编辑 |
| 收稿时间： | 2017/2/28 0:00:00 |
| 修稿时间： | 2017/5/29 0:00:00 |
Multiple Sites Editing Vector Construction of Rice Blast Resistance Gene Pita2 Candidate Genes |
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| 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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| Authors: | Wang Xiaowan Deng Senwen Li Kai Wang Chuntai and Xu Xin |
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| Institution: | 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,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,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,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,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 |
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| 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. |
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| Keywords: | Rice blast resistance gene Pita2 CRISPR/Cas9 system Multiple sites editing |
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