基于CRISPR/Cas9系统的蒺藜苜蓿高效基因组编辑载体构建  

Construction of efficient genomic editing vector for Medicago truncatula by CRISPR/Cas9 system

作　　者：周嫦嫦 张海玲[2] 李佶恺[2] 尚晨[2] 朱瑞芬[2] 刘慧莹[2] 付春祥[3] 申忠宝[2] ZHOU Changchang;ZHANG Hailing;LI Jikai;SHANG Chen;ZHU Ruifen;LIU Huiying;FU Chunxiang;SHEN Zhongbao(College of Life Science and Technology,Harbin Nonnal University,Harbin 150080,China;Grass and Science Instilute of Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China;Qingdao Institute of Bioenergy and Bioprocess Technology,Chinese Academy of Sciences,Qingdao 266101,China)

机构地区：[1]哈尔滨师范大学生命科学与技术学院,哈尔滨150080 [2]黑龙江省农业科学院草业研究所,哈尔滨150086 [3]中国科学院青岛生物能源与过程研究所,山东青岛266101

出　　处：《黑龙江畜牧兽医》2021年第3期109-113,162,163,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金青年科学基金项目(31802124);黑龙江省博士后资助经费项目(LBH-Z18262);黑龙江省农业科学院院级科研项目(2018JJPY002)。

摘　　要：为了构建基于CRISPR/Cas9系统的蒺藜苜蓿高效基因组编辑载体,便于开展蒺藜苜蓿的基因组编辑研究,试验以蒺藜苜蓿基因组中的PDS基因为靶标,应用CRISPR-P软件设计2条特异sgRNA靶位点序列,以pYLCRISPR/Cas9-P35S为骨架载体,以适合双子叶植物的AtU3b作为sgRNA转录启动子,并引入可以显著提高基因编辑效率的tRNA保守序列,采用Golden Gate克隆法构建双靶点CRISPR/Cas9植物表达载体。结果表明:针对蒺藜苜蓿PDS基因设计的双靶点pYLCRISPR/Cas9植物表达载体的各个表达盒组装顺序完全正确,DNA碱基序列准确无误。说明pYLCRISPR/Cas9-AtU3b-tRNA-sgRNA基因编辑载体构建成功,可以为进一步开展农杆菌介导的蒺藜苜蓿基因组编辑研究提供载体,为双子叶植物开展基因编辑研究提供新的载体构建策略。In order to construct an efficient genome editing vector of Medicago truncatula(M. truncatula) based on CRISPR/Cas9 system and facilitate the genome editing research of M.truncatula, PDS gene in the genome of M.truncatula was used as target, and two specific sgRNA target sequences were designed by software CRISPR-P in this work. pYLCRISPR/Cas9-P35 S was used as the skeleton vector, AtU3 b suitable for dicotyledonous plants was used as the sgRNA transcription promoter, and tRNA conserved sequence that could significantly improve the efficiency of genome editing was introduced. Then double target CRISPR/Cas9 plant expression vector was constructed by the Golden Gate cloning technology. The results showed that the assembly sequence of each expression box of the dual target pYLCRISPR/Cas9 plant expression vector designed for PDS gene of M.truncatula was completely correct and DNA base sequence was correct. The results indicated that the successful construction of pYLCRISPR/Cas9-AtU3 b-tRNA-sgRNA gene editing vector could provide a vector for further research on Agrobacterium mediated genome editing of M.truncatula, and a new vector construction strategy for gene editing research of dicots.

关 键 词：CRISPR/Cas9系统 蒺藜苜蓿 基因组编辑 载体构建 靶位点 

分 类 号：S854[农业科学—临床兽医学] Q78[农业科学—兽医学]