木薯MeSWEET10a基因启动子EBE区编辑载体的构建及验证  

Construction and Verification of Editing Vector for EBE Region of MeSWEET10a Promoter in Cassava

作　　者：张彤 王亚杰 郭文雅 李瑞梅[2] 刘姣[2] 郭建春[2] 胡新文[1] 姚远[2] 耿梦婷[1] ZHANG Tong;WANG Yajie;GUO Wenya;LI Ruimei;LIU Jiao;GUO Jianchun;HU Xinwen;YAO Yuan;GENG Mengting(Hainan University,Haikou,Hainan 570228,China;Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences/Hainan Institute for Tropical Agricultural Resources,Haikou,Hainan 571101,China)

机构地区：[1]海南大学,海南海口570228 [2]中国热带农业科学院热带生物技术研究所/海南热带农业资源研究院,海南海口571101

出　　处：《热带作物学报》2021年第11期3120-3125,共6页Chinese Journal of Tropical Crops

基　　金：海南省自然科学基金项目(No.320RC492);中国热带农业科学院基本科研业务费专项资金(No.1630052020033);海南省研究生创新科研课题(No.Hys2019-07)。

摘　　要：木薯细菌性枯萎病(cassava bacterial blight,CBB)是由地毯草黄单胞木薯萎蔫致病变种(Xanthomon asaxonopodis pv. Manihotis, Xam)引起的木薯重要病害,影响木薯产量,我国主栽的木薯品种均不抗Xam。Xam分泌的TAL20效应蛋白可结合木薯MeSWEET10a基因启动子的EBE区,激活该基因表达,促进糖类运输至Xam侵染的部位,为病菌的繁殖提供碳水化合物。本研究利用在线软件CRISPR-P v2.0设计EBE区的基因编辑靶点,构建CRISPR/Cas9基因编辑质粒pCAMBIA1301-Cas9-EBE-sgRNA。将重组质粒转化LBA4404根癌农杆菌,利用农杆菌介导法侵染木薯脆性胚性愈伤组织。提取侵染和未侵染的木薯脆性胚性愈伤组织DNA,PCR扩增靶点EBE区及潜在的脱靶位点,并进行Sanger测序分析编辑效果。结果发现EBE区被成功编辑,且没有脱靶现象。本研究有助于进一步获得MeSWEET10a基因启动子EBE区域的木薯突变体,以增强木薯抗细菌性枯萎病的能力。Cassava bacterial blight(CBB) is an important cassava disease caused by Xanthomonas axonopodis pv.manihotis(Xam), which affects the yield of cassava. The main cassava varieties in China are not resistant to Xam. The TAL20 effector protein secreted by Xam, could bind to the EBE region of the cassava MeSWEET10 a promoter, activate the gene expression, promote the transport of sugars to the site of Xam infection, and provide carbohydrates for the reproduction of the pathogen. In this study, the gene editing target of EBE region was designed by using online software CRISPR-Pv2.0, and the CRISPR/Cas9 gene editing plasmid pCAMBIA1301-Cas9-EBE-sgRNA was constructed. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404, and the fragile embryogenic calli of cassava was infected by the A. tumefaciens-mediated method. DNA was extracted from the fragile embryogenic calli of infected and uninfected cassava. The target EBE region and potential miss sites were amplified by PCR, and the editing effect was analyzed by Sanger sequencing. It was found that the EBE region was edited successfully without off-target phenomenon. This study is helpful to further obtain cassava mutants in the EBE region of MeSWEET10 a promoter and enhance the resistance of cassava to bacterial blight.

关 键 词：木薯 细菌性枯萎病 MeSWEET10a CRISPR/Cas9 

分 类 号：S533[农业科学—作物学]