木薯MeLOB36基因的CRISPR/Cas9基因编辑载体构建及验证  

Construction and Verification of CRISPR/Cas9 Gene Editing Vector for Cassava MeLOB36 Gene

作　　者：阳慧 张彤 王亚杰 陆小花 刘姣[2] 李瑞梅[2] 郭建春[2] 耿梦婷[1] 姚远[2] Yang Hui;Zhang Tong;Wang Yajie;Lu Xiaohua;Liu Jiao;Li Ruimei;Guo Jianchun;Geng Mengting;Yao Yuan(College of Tropical Crops,Hainan University,Haikou,570228;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences,Haikou,571101;College of Life Sciences,Hainan University,Haikou,570228)

机构地区：[1]海南大学热带作物学院,海口570228 [2]中国热带农业科学院热带生物技术研究所,海口571101 [3]海南大学生命科学学院,海口570228

出　　处：《分子植物育种》2023年第11期3612-3618,共7页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2019YFD1001105);海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC297);现代农业产业技术体系建设专项(CARS-11-HNGJC)共同资助。

摘　　要：LBD基因家族是指含有侧生器官边界(lateral organ boundaries, LOB)结构域的一种植物特异性转录因子家族,在植物的生长发育和代谢调控等方面起着至关重要的作用。木薯MeLOB36基因是LBD基因家族成员,前期研究发现该基因在木薯根尖、体细胞胚、脆性胚性愈伤组织表达量较高,推测其参与了木薯植株的形态建成。为了鉴定MeLOB36基因的功能,本研究利用在线软件CRISPR-P v2.0在保守区域设计靶标MeLOB36基因的sg RNA,构建MeLOB36基因的编辑载体pCAMBIA1301-Cas9-MeLOB36-sg RNA。将重组载体转化LBA4404根癌农杆菌后侵染木薯脆性胚性愈伤组织,通过PCR扩增MeLOB36基因编辑靶点及潜在脱靶位点区段序列,进行Sanger测序。结果表明,MeLOB36基因被成功编辑,且未脱靶。本研究有助于进一步获得MeLOB36基因的突变体,从而鉴定MeLOB36转录因子对木薯植株形态建成的影响。The LBD gene family with a conserved lateral organ boundary(LOB)domain is a plant-specific tran-scription factor family,which play a crucial role in plant growth,development and metabolic regulation.The Me-LOB36 gene of cassava is a member of LBD gene family,previous studies exhibited that the gene highly expressed in cassava root tips,somatic embryos,and fragile embryogenic callus,thus it was speculated that MeLOB36 gene was related to the morphogenesis of cassava plants.In order to identify the function of MeLOB36 gene,the online software CRISPR-P v2.0 was used to design sgRNA sequence in a conserved region,and the gene editing vector pCAMBIA1301-Cas9-MeLOB36-sgRNA was constructed.To infect the fragile embryogenic callus of cassava through transforming the recombinant vector into A grobacterium LBA4404,and then the segments of the MeLOB36 gene editing target and potential off-target locations were amplified by PCR.The Sanger sequencing results showed that the MeLOB36 gene was successfully edited and no off-target phenomenon occurred.This study is helpful to further obtain the mutants of MeLOB36 gene,and identify the effect of MeLOB36 transcription factor on the morphogenesis of cassava plants.

关 键 词：木薯 转录因子 MeLOB36基因 基因编辑 

分 类 号：S533[农业科学—作物学]