茄子U6启动子克隆及CRISPR/Cas9介导的基因编辑体系建立  

Cloning of U6 Promoters and Establishment of CRISPR/Cas9 Mediated Gene Editing System in Eggplant

作　　者：王丹[1,2] 王谧[1,2] 刘军 周晓慧[2] 刘松瑜[2] 杨艳 庄勇 WANG Dan;WANG Mi;LIU Jun;ZHOU Xiaohui;LIU Songyu;YANG Yan;ZHUANG Yong(College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement,Institute of Vegetable Crops,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]南京农业大学园艺学院,南京210095 [2]江苏省农业科学院蔬菜研究所,江苏省高效园艺作物遗传改良重点实验室,南京210014

出　　处：《园艺学报》2022年第4期791-800,共10页Acta Horticulturae Sinica

基　　金：国家自然科学基金项目(31701932)。

摘　　要：利用拟南芥(Arabidopsis thaliana)U6 RNA保守序列从茄子(Solanum melongena L.)基因组中鉴定到7个U6 RNA,并采用PCR技术成功克隆其启动子序列。GUS染色结果表明4个茄子U6启动子(SmU6-1P、SmU6-2P、SmU6-4P和SmU6-7P)具有转录活性。构建以SmU6-1P/AtU6-P驱动SmWRKY4 sgRNA的CRISPR/Cas9基因编辑载体,通过农杆菌介导侵染茄子子叶外植体得到T_(0)代植株。以T_(0)代植株的DNA为模板扩增WRKY4片段并对产物测序,测序结果显示以SmU6-1P构建的CRISPR/Cas9载体能靶向编辑SmWRKY4,编辑率为27.0%。在基于AtU6-P的T_(0)代植株中未检测到碱基突变,表明在茄子遗传转化中,茄子SmU6启动子编辑效率高于拟南芥AtU6启动子。Seven U6 RNAs were identified from the eggplant(Solanum melongena L.)genome based on the conserved U6 RNA sequence in Arabidopsis and the responding promoters were cloned by PCR amplification.GUS staining results showed that four SmU6 promoters(SmU6-1P,SmU6-2P,SmU6-4P,SmU6-7P)were capable of transcriptional activity.The CRISPR/Cas9 vectors with AtU6-P/SmU6-1P driving SmWRKY4 sgRNA were constructed.T_(0) plants were obtained by Agrobacterium-mediated transformation of eggplant cotyledons.The DNA of T_(0) plants were extracted to amplify the target fragments,which were subsequently sequenced to analyze whether SmWRKY4 gene were edited or not.As a result,the CRISPR/Cas9 vector containing SmU6-1P could edit SmWRKY4 with an editing rate of 27.0%.However,no mutation was detected in T_(0) plants based on AtU6-P.These results indicated that the editing efficiency of eggplant U6 promoter was better than that of Arabidopsis U6 promoter in eggplant genetic transformation.

关 键 词：茄子 U6启动子 CRISPR/Cas9 基因编辑 

分 类 号：S641.1[农业科学—蔬菜学]