张彤 王亚杰 郭文雅 李瑞梅 刘姣 郭建春 胡新文 姚远 耿梦婷

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（Xanthomonas axonopodis 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。

关键词：木薯;细菌性枯萎病;MeSWEET10a;CRISPR/Cas9

中图分类号：S533      文献标识码：A

Abstract： 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 MeSWEET10a 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 MeSWEET10a promoter and enhance the resistance of cassava to bacterial blight.

Keywords： cassava; bacterial blight; MeSWEET10a; CRISPR/Cas9

DOI： 10.3969/j.issn.1000-2561.2021.11.010

木薯是熱带地区重要的粮食作物，而病害是木薯产量提高的重要制约因素之一[1-2]。木薯细菌性枯萎病（cassava bacterial blight， CBB）是我国木薯面临的主要病害，该病是由地毯草黄单胞菌木薯萎蔫致病变种（Xanthomonas axonopodis pv. Manihotis， Xam）引起的细菌性病害[3]。我国木薯主栽品种均不抗Xam病原菌的侵染，CBB曾导致我国木薯大面积减产。因此，培育抗CBB木薯新品种有益于我国木薯产业持续健康发展。

Xam病原菌侵染木薯时，可通过Ⅲ型分泌系统将转录激活因子样效应物蛋白（transcription activator-like effectors，TAL）分泌到宿主细胞中[4]，TAL效应蛋白的可变重复区（repeat variable diresidues，RVD）识别宿主感病相关基因启动子上的效应蛋白结合元件（effector-binding element，EBE），并激活下游感病基因表达，以促进Xam病原菌的侵染[5]。……