根癌农杆菌介导的尖孢镰刀菌遗传转化体系构建  

Construction of agrobacterium tumefaciens-mediated genetic transformation system of tobacco Fusarium oxysporum

作　　者：李娟[1,2] 邱睿 张盈盈[1] 李小杰 李成军[1] 李淑君[1] LI Juan;QIU Rui;ZHANG Yingying;LI Xiaojie;LI Chengjun;LI Shujun(Key Laboratory for Green Preservation&Control of Tobacco Diseases and Pests in Huanghuai Growing Area,Tobacco Research Institute,Henan Academy of Agricultural Sciences,Xuchang 461000,China;Dingxi Plant Protection and Quarantine Station,Dingxi 743000,China)

机构地区：[1]河南省农业科学院烟草研究所,黄淮烟区烟草病虫害绿色防控重点实验室,河南省许昌市461000 [2]定西市植保植检站,甘肃省定西市743000

出　　处：《中国烟草学报》2022年第4期106-113,共8页Acta Tabacaria Sinica

基　　金：河南省农业科学院基础性科研工作项目“烟草尖孢镰刀菌致病相关基因的筛选与功能研究”(2021ZC25);河南省烟草公司科技项目“烟草根腐病害发生的微生物群落分析和生态风险预警研究”(2020410000270012);河南省农业科学院优秀青年科技基金计划项目“烟草茄病镰刀菌复合群系统发育与致病特性研究”(2022YQ09);河南省农业科学院科技创新团队“烟草主要病虫害绿色防控关键技术研究与应用”(2022TD26)。

摘　　要：【背景和目的】尖孢镰刀菌(Fusarium oxysporum)可引起烟草镰刀菌根腐病(Fusarium root rot of tobacco),为明确病原菌致病相关基因。【方法】以烟草强致病性尖孢镰刀菌菌株B-9-1的分生孢子为受体,利用携带潮霉素B(hygromycin B,hyg)质粒的根癌农杆菌(Agrobacterium tumefaciens)介导转化,获得能够稳定表达绿色荧光蛋白(green fluorescent protein,GFP)的尖孢镰刀菌转化菌株,构建烟草尖孢镰刀菌遗传转化体系。【结果】(1)尖孢镰刀菌的最优转化体系为:潮霉素B最适质量浓度50μg/mL,在25℃条件下,以1 cm宽的条形硝酸纤维膜为载体,将携带有pCAM-GFP-hyg质粒的根癌农杆菌摇菌培养至OD600=0.7,与震荡培养24 h获得的浓度为106个/mL的尖孢镰刀菌分生孢子悬浮液在含有200μmol/L乙酰丁香酮的共培养基上共培养48 h。(2)转化子继代培养、PCR检测和荧光显微观察结果表明外源GFP基因成功整合到烟草尖孢镰刀菌基因组中并可以进行稳定遗传表达。(3)Sourthern blot分析结果进一步证明T-DNA成功整合到尖孢镰刀菌基因组中,且60%以上为单拷贝插入。【结论】本研究成功构建了根癌农杆菌介导的烟草尖孢镰刀菌遗传转化体系,为烟草尖孢镰刀菌基因功能及病原菌的致病机制研究提供基础。[Methods]Tobacco root rot caused by Fusarium oxysporum is one of the main devastating diseases in tobacco(Nicotiana tabacum L.)production.Identifying the pathogenic genes of plant pathogen could provide new targets for disease accurate control.The objectives of this study are to construct a stable genetic transformation system of Fusarium oxysporum by using Agrobacterium tumefaciens carrying pCAM-GFP-hyg plasmid to transform the conidia of pathogenic Fusarium oxysporum(B-9-1),and obtain the transformant strains of B-9-1 with green fluorescent protein(GFP).[Results](1)The optimal transformation system of F.oxysporum was obtained under following condition:the optimal lethal concentration of hygromycin was 50μg/mL,co-culturing of the conidia suspension with a concentration of 106/mL(that obtained by shaking culture for 24 h)and Agrobacterium tumefaciens with an OD600 value of 0.7 on co-IM agar containing 200μmol/L Acetosyringone under 25℃for 48 h.(2)The T-DNA was successfully inserted into the genome of F.oxsporum,which was capable of conducting stable genetic expression by subculturing,molecular analysis and fluorescence microscopic verification.(3)The results of Sourthern blot analysis further demonstrated that T-DNA was successfully integrated into F.oxysporum genome,and more than 60%was single copy insertion.[Conclusion]Agrobacterium tumefaciens-mediated genetic transformation of tobacco F.oxysporum was successfully established,which laid foundation for the study of pathogenic mechanism and analysis of pathogenic genes of tobacco F.oxysporum.

关 键 词：烟草尖孢镰刀菌 遗传转化 转化子 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]