靶向加工番茄elF4E1的CRISPR/Cas9载体有效性验证  

Verification of the Effectiveness of CRISPR/Cas9 Vectors Targeting to Processing Tomato eIF4E1

作　　者：付紫梅 袁伦 邵冬南 郝小军[3] 崔百明[1] 向本春[3] FU Zi-mei;YUAN Lun;SHAO Dong-nan;HAO Xiao-jun;CUI Bai-ming;XIANG Ben-chun(College of Life Sciences,Shihezi University/Key Laboratory of Agriculture Biotechnology of Shihezi University,Shihezi Xinjiang 832003,China;Western Biotechnology Corporation Lit.,Chongqing,400039,China;Key Laboratory for Oasis Agricultural Pest Management and Plant Resource Utilization at Universities of Xinjiang Uygur Autonomous Region,College of Agronomy,Shihezi University,Shihezi Xinjiang 832003,China)

机构地区：[1]石河子大学生命科学学院/石河子大学农业生物技术重点实验室,新疆石河子832003 [2]威斯腾生物医药科技有限责任公司,重庆400039 [3]新疆绿洲农业病虫害治理与植保资源利用自治区普通高校重点实验室/石河子大学农学院,新疆石河子832003

出　　处：《新疆农业科学》2018年第2期230-237,共8页Xinjiang Agricultural Sciences

基　　金：国家自然科学基金"一种新病毒对新疆加工番茄的影响"(0205/KZ0059)~~

摘　　要：【目的】验证基于CRISPR/Cas9系统构建的靶向编辑加工番茄(Solanum lycopersicum)e IF4E1基因载体的有效性,为CRISPR/Cas9系统在培育PVY抗性植株中的应用提供技术支持。【方法】构建靶向编辑番茄真核翻译起始因子el F4E1基因的CRISPR/Cas9系统表达载体,用农杆菌渗透法瞬时转化番茄植株,PCR扩增已转化植株靶位点周围DNA序列后用HaeⅢ进行酶切,回收未切开的条带与p GEM-T载体连接后进行单克隆测序。【结果】对测得的9个克隆序列进行比对分析,在PAM(protospacer adjacent motifs)上游的6~8 bp的碱基处均发生突变,并且都为单碱基的替换,导致多肽链中单个氨基酸的替换。【结论】利用CRISPR/Cas9基因组编辑系统构建的载体能够特异性地靶向加工番茄e IF4E1基因,为利用CRISPR/Cas9系统敲除e IF4E1基因,获得抗PVY病毒的番茄育种材料奠定了基础。【Objective】To verify the effectiveness of eIF4E1 gene vector constructed based on CRISPR/Cas9 system for targeted genome editing processing tomato(Solanum lycopersicum)and to provide technical support for the application of CRISPR/Cas9 system in the cultivation of PVY resistant plants.【Method】Constructing a CRISPR/Cas9 system expression vector targeting to the elF4E1 gene of tomato eukaryotic translation initiation factor,the tomato plants were transiently transformed by Agrobacterium tumefaciens infiltration method.The DNA sequences around the target sites of the transformed plants were amplified by PCR and digested with Hae III,the bands that had not been successfully digested were recovered and ligated with pGEM-T vector for monoclonal sequencing.【Result】An alignment analysis of the 9 cloned sequences revealed that mutations occurred at 6-8 bp upstream of the PAM(protospacer adjacent motifs),and they were all single-base substitutions,resulting in the replacement of a single amino acid in the polypeptide chain.【Conclusion】In this study,the vector constructed by CRISPR/Cas9 genome editing system can specifically target the tomato eIF4E1 gene,which has laid the foundation for the subsequent use of the CRISPR/Cas9 system to knock out the eIF4E1 gene and obtain the tomato breeding materials against PVY virus.

关 键 词：加工番茄 PVY elF4E1 CRISPR/Cas9 瞬时转化 

分 类 号：S641.2[农业科学—蔬菜学] S188[农业科学—园艺学]