水稻锌指蛋白基因CRISPR/Cas9突变体的构建及突变分析  

CRISPR/Cas9 mutants construction of rice zinc finger protein gene and mutation analysis

作　　者：易勇 郑瑞 杨波[2] 栾维江[2] 郭嗣斌[3] 沙爱华 YI Yong;ZHENG Rui;YANG Bo;LUANWei-jiang;GUO Si-bin;SHAAi-hua(College of Agriculture,Yangtze University/Hubei Collaborative Innovation Center for Industrialization of Major Grain Crops,Jingzhou,Hubei 434025,China;College of Life Science,Tianjin Normal University,Tianjin 300387,China;Rice Research Institute,Guangxi Academy of Agricultural Sciences/Guangxi Key Laboratory of Rice Genetics and Breeding,Nanning 530007,China)

机构地区：[1]长江大学农学院/主要粮食作物产业化湖北省协同创新中心,湖北荆州434025 [2]天津师范大学生命科学学院,天津300387 [3]广西农业科学院水稻研究所/广西水稻遗传育种重点实验室,南宁530007

出　　处：《南方农业学报》2020年第11期2607-2613,共7页Journal of Southern Agriculture

基　　金：国家重点研发计划项目(2018YFD0301301-5-2);广西自然科学基金项目(2017GXNSFDA198039);广西水稻遗传育种重点实验室开放基金项目(160-380-16-1)。

摘　　要：【目的】通过CRISPR/Cas9基因编辑技术对水稻锌指蛋白基因(OsC3H54)进行基因编辑,筛选鉴定出其突变体植株,为深入研究OsC3H54的生物学功能提供良好材料,也为水稻锌指蛋白研究提供参考依据。【方法】通过E-CRISP在Os C3H54基因的外显子上设计靶点序列,将靶点序列连接至OsU6SK载体上,再与Cas9一起连接到pCAMBIA1300双元载体上,获得CRISPR/Cas9重组双元载体,通过农杆菌介导将其转入日本晴水稻愈伤组织,利用潮霉素进行抗性筛选,获得突变体植株,并分析其靶点位置的碱基及编码氨基酸突变情况。【结果】在OsC3H54基因第2个外显子上找到2个符合靶点设计要求的靶点,分别为TG1:5'-CCGCCGCGGCTGCCTTTGGATAC-3'和TG2:5'-CCTTCCC CAATGGCGGGGGTGGC-3'。将OsU6SK载体和靶点序列正确连接的重组载体与Cas9一起连接至pCAMBIA1300双载体上,成功获得CRISPR/Cas9重组双元载体(pCAMBIA1300-Cas9-TG1和pCAMBIA1300-Cas9-TG2)。通过农杆菌介导转入日本晴水稻,经潮霉素抗性筛选获得TG1靶点株系和TG2靶点株系,共16株CRISPR/Cas9突变体植株。CRISPR/Cas9突变体植株在靶点序列的突变位点位置附近出现套峰,表明2个株系的植株均发生碱基突变,其中Y1、Y2、Y3和Y4突变体植株均为单碱基插入突变,最终导致编码的氨基酸发生移码突变,蛋白翻译提前终止。【结论】水稻OsC3H54基因CRISPR/Cas9突变体植株的获得为进一步研究水稻锌指蛋白生物学功能提供了良好材料。【Objective】The rice zinc finger protein gene(OsC3H54)was genetically edited based on CRISPR/Cas9 gene editing technology,and the mutant plants were screened and identified,which would provide materials for in-depth study of the biological functions of OsC3H54 as well as reference for the research of rice zinc finger protein.【Method】The target sequence was designed on the exon of rice OsC3H54 gene through E-CRISP,and the target sequence was ligated to the OsU6 SK vector to obtain the target link vector.Then the target link vector together with Cas9 were integrated to pCAMBIA1300 to obtain the CRISPR/Cas9 recombination binary vector,which was then transformed into Nipponbare rice callus with Agrobacterium-mediated method.The positive transformed plants were screened out using hygromycin,and the mutated bases at the target site and encoded amino acids were analyzed.【Result】Two targets were found on the second exon of OsC3H54,which met the target design requirements,named TG1:5’-CCGCCGCGGCTGCCTTTGG ATAC-3’and TG2:5’-CCTTCCCCAATGGCGGGGGTGGC-3’.The OsU6 SK vector and recombinant vector with correct target sequence and the empty Cas9 vector were ligated to the pCAMBIA1300 binary vector,and the CRISPR/Cas9 recombinant binary vector(p CAMBIA1300-Cas9-TG1 and p CAMBIA1300-Cas9-TG2)was successfully obtained.The CRISPR/Cas9 recombinant vectors were transferred to Nipponbare based on Agrobacterium-mediated method with hygromycin resistance for transforms screening.A total of 16 CRISPR/Cas9 mutant lines of TG1 and TG2 target were obtained.A set of peaks near the mutation site of the target sequence in the CRISPR/Cas9 mutant lines were detected,indicating that base mutations happened in two transformed lines.Among them,the Y1,Y2,Y3 and Y4 mutant lines were all singlebase insertions mutations that led to premature termination of amino acid traanslation with frameshift mutations.【Conclusion】The obtained CRISPR/Cas9 mutant plant of OsC3H54 provides good materials for biological functions studying of rice zi

关 键 词：水稻 锌指蛋白 C3H54 CRISPR/Cas9 基因编辑 突变体植株 突变分析 

分 类 号：S511.035.3[农业科学—作物学]