基于农杆菌介导的茄子枯萎病菌遗传转化体系建立及GFP标记菌株的构建  

Establishing of the Transformation System of Fusarium oxysporum f.sp.melongenae by Agrobacterium tumefaciens-mediated Transformation(ATMT)and Constructing of GFP Labeled Strain

作　　者：严亚琴 胡天华[1] 王五宏[1] 胡海娇[1] 魏庆镇 汪精磊 包崇来[1] YAN Yan-Qin;HU Tian-Hua;WANG Wu-Hong;HU Hai-Jiao;WEI Qing-Zhen;WANG Jing-Lei;BAO Chong-Lai(Institute of Vegetable,Zhejiang Academy of Agricultural Science,Hangzhou 310000,China)

机构地区：[1]浙江省农业科学院蔬菜所研究所,杭州310000

出　　处：《农业生物技术学报》2023年第9期1980-1988,共9页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金(LQ22C150007);浙江省“十四五”农业新品种选育重大科技专项(2021C02065-1-3)。

摘　　要：由尖孢镰刀菌茄子专化型(Fusarium oxysporum f. sp. melongenae)引起的茄子(Solanum melongena)枯萎病严重制约茄子的生产,其致病机制尚不明确。本研究以茄子枯萎病菌的分生孢子为转化受体,利用携带GFP表达载体pCAMsgfp的根癌农杆菌(Agrobacterium tumefaciens)介导转化茄子枯萎病菌,建立并优化了农杆菌介导的茄子枯萎病菌遗传转化体系。结果表明,当农杆菌光密度(optical density)值为0.5时,在200 μmol/L乙酰丁香酮诱导条件下,与浓度为1×10^(6)个/mL的病原菌分生孢子悬浮液等体积混合后,在共培养温度为25℃~28℃条件下共培养48 h,茄子枯萎病菌的转化效率最高可达319±10.21个转化子/106个孢子。GFP的特异性引物PCR鉴定结果以及荧光观察表明GFP基因能够正常表达。本研究建立并优化了根癌农杆菌介导的茄子枯萎病菌的遗传转化体系,并获得了遗传稳定的GFP标记菌株,为病原菌与茄子互作的机制研究提供技术支撑。Fusarium wilt caused by Fusarium oxysporum f.sp.melongenae seriously restricts the production of eggplant(Solanum melongena).However,its pathogenic mechanism is still unclear.In this study,a transformation system of F.oxysporum f.sp.melongenae was established and optimized,in which GFP expression vector pCAMsgfp was transformed into conidia using,ATMT(Agrobacterium tumefaciensmediated transformation)method.The results showed that a satisfactory transformation with an efficiency of 319±10.21 transformants per 1×10^(6) spores could be achieved in a system of 1×10^(6) spores per milliliter of F.oxysporum f.sp.melongenae spore suspension which were co-cultured with Agrobacterium tumefaciens suspension at a concentration of OD6000.5 under the co-culture medium containing 200μmol/mL acetosyringone at 25~28℃ for 48 h.PCR identification of the GFP gene and fluorescence observation showed that GFP gene could be expressed normally.In this study,the genetic transformation system of F.oxysporum f.sp.melongenae mediated by ATMT was established and optimized,and the GFP-labeled strain was successfully obtained,which could provide powerful technic for further explore the infection process and the pathogenic mechanism of F.oxysporum f.sp.melongenae.

关 键 词：茄子枯萎病 尖孢镰刀菌茄子专化型 农杆菌介导的遗传转化 GFP 

分 类 号：S182[农业科学—农业基础科学]