茶树炭疽病病原菌的绿色荧光蛋白基因标记及其侵染研究  

Study on Green Fluorescent Protein Gene Markers and Infection of Pathogens of Tea Anthracnose

作　　者：李志伟 谭玉梅 任锡毅[2,3] 刘永翔 黄永会[2,3] 刘作易[4] Li Zhiwei;Tan Yumei;Ren Xiyi;Liu Yongxiang;Huang Yonghui;Liu Zuoyi(College of Agriculture College,Guizhou University,Guiyang,550025;Institute of Biotechnology,Guizhou Academy of Agricultural Sciences,Guiyang,550006;Guizhou Key Laboratory of Agricultural Biotechnology,Guiyang,550006;Guizhou Academy of Agricultural Sciences,Guiyang,550006)

机构地区：[1]贵州大学农学院,贵阳550025 [2]贵州省农业科学院生物技术研究所,贵阳550006 [3]贵州省农业生物技术重点实验室,贵阳550006 [4]贵州省农业科学院,贵阳550006

出　　处：《基因组学与应用生物学》2020年第8期3510-3518,共9页Genomics and Applied Biology

基　　金：贵州省科技支撑计划(黔科合支撑[2017]2561);国家基金(31660021);农科院自主创新专项(黔农科学院自主创新科研专项字(2014)004)共同资助。

摘　　要：茶炭疽病是茶树上的叶部病害之一,在茶树上发生严重时会导致茶叶品质和质量的下降,造成一定的经济损失。本研究以根癌农杆菌GFP基因表达载体质粒EGFP-PSK2251和一株茶胶孢炭疽菌(GZCC20-0218)为材料,通过根癌农杆菌介导转化的方法,构建茶胶孢炭疽菌的遗传转化体系,筛选获得稳定遗传的转化子。利用荧光显微镜技术揭示转化子在叶片组织内定殖的特点。研究结果表明:根癌农杆菌与茶胶孢炭疽菌的分生孢子在26℃,0.2 mol/L乙酰丁香酮(AS)诱导下得到转化子。对转化子进行绿色荧光检测、分子验证及遗传稳定性测定后,从而获得稳定遗传的绿色荧光蛋白(GFP)标记菌株。同时,试验表明GFP标记菌株仍具有致病性。GFP标记菌株进行茶叶侵染试验时,7 d后茶叶发病率达到100%。发病叶片进行组织切片观察,发现胶孢炭疽菌的分生孢子可以在叶片的气孔和叶片组织内定殖。Tea anthracnose is one of the leaf diseases on tea trees.When the fungus seriously occurs on the tea tree,it will lead to a decline in tea quality,causing some economic damage.In this study,Agrobacterium tumefaciens GFP gene expression vector plasmid EGFP-PSK2251 and a strain of Colletotrichum gloeosporioides isolated from tea(GZCC20-0218)were used as materials.Transformation of GFP gene to Colletotrichum gloeosporioides strain was established via Agrobacterium tumefaciens mediated transformation method and the stable GFP-labeled transformants of Colletotrichum gloeosporioides were obtained.The colonizating characteristics of transformant in leaf tissue were revealed by use fluorescence microscopy technique.The results showed that the transformants were obtained under the common medium(IM)after Agrobacterium tumefaciens and the conidia of Colletotrichum gloeosporioides were induced by 0.2 mol/L acetosyringone(AS)at 26℃.The stable inherited strains with green fluorescent protein(GFP)were confirmed via the green fluorescence detection,molecular verification and genetic stability determination of the transformants.At the same time,GFP-labeled strains are still pathogenic.The live tea seedlings were infected by using a stable inherited GFP-tagged strain,the incidence rate reached to 100%after 7 days.Tissue section observation of the diseased leaves revealed that the conidia of Colletotrichum gloeosporioides could colonize the leaf stomata and tissues.

关 键 词：茶叶 胶孢炭疽菌 根癌农杆菌 GFP标记 定殖 

分 类 号：S435.711[农业科学—农业昆虫与害虫防治]