利用荧光蛋白标记西瓜枯萎病菌的过氧化物酶体及细胞核  

Fluorescent Labeling the Peroxisome and Nucleus in Fusarium oxysporum f.sp.niveum

作　　者：施笑笑 王教瑜[2] 肖琛闻[3] 王艳丽[2] 李大勇[4] 柴荣耀[2] 孙国昌[2] SHI Xiaoxiao;WANG Jiaoyu;XIAO Chenwen;WANG Yanli;LI Dayong;CHAI Rongyao;SUN Guochang(College of Chemistry and Life Sciences,Zhejiang Normal University,Jinhua 321004,China;State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products,Institute of Plant Protection and Microbiology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;College of Agriculture and Biotechnology,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江师范大学化学与生命科学学院,金华321004 [2]农产品质量安全危害因子与风险防控国家重点实验室,浙江省农业科学院植物保护与微生物研究所,杭州310021 [3]浙江省农业科学院畜牧兽医研究所,杭州310021 [4]浙江大学农业与生物技术学院,杭州310058

出　　处：《中国细胞生物学学报》2020年第2期195-203,共9页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31470249);浙江省重点研发计划(批准号:2019C02010)资助的课题。

摘　　要：西瓜枯萎病是一种世界范围的西瓜毁灭性病害,其病原菌为尖孢镰刀菌西瓜专化型(Fusarium oxysporum f.sp.niveum,FON)。研究病原菌生长发育和侵染的机制是解决病害的根本途径。利用荧光蛋白对细胞或细胞器进行标记,是病原菌研究中的重要方法。该研究利用绿色荧光蛋白和红色荧光蛋白对FON的细胞核和过氧化物酶体进行了荧光标记。通过农杆菌介导转化(Agrobacterium tumefaciens-mediated transformation,AtMT),该文将3种不同的荧光定位载体分别导入FON,获得了细胞核红色荧光标记的转化子(潮霉素抗性,含mCherry-H2B融合蛋白),以及过氧化物酶体绿色(潮霉素抗性,含GFP-PTS1融合蛋白)和红色(潮霉素抗性,含DsRED-PTS1融合蛋白)荧光标记的转化子各1种。在标记细胞核的菌株中,菌丝、孢子都可见明亮、圆形的红色荧光点,荧光点与DAPI染色标记的细胞核区域完全重合。在过氧化物酶体标记的菌株中,菌丝、孢子中可见明亮的红色或绿色荧光成小点状分布,符合过氧化物酶体的分布特征,而且在脂类物质诱导的条件下,荧光点的数量明显增加。此外,该文还利用细胞壁荧光染色剂卡氏白对3种荧光蛋白标记菌株进行染色。结果显示,卡氏白染色产生的蓝色荧光与红、绿荧光蛋白的荧光在FON中互不干扰。转化子继代培养和初步分析表明,其表型与野生型无差异,菌株继代后荧光表达稳定、定位明显。该结果为进一步研究FON细胞器动态、生长发育与致病分子机制提供了方法和工具。FON(Fusarium oxysporum f.sp.niveum)causes watermelon Fusarium wilt,a destructive disease on watermelon worldwide.Research on the development and pathogenesis of FON lays the foundation for the control of the disease.Fluorescent labeling of the organelles and cell structures using fluorescent proteins is an important strategy in the investigations on fungal development and pathogenesis.In the present work,the nuclei and peroxisomes of FON were labeled with green or red fluorescent proteins.Via AtMT(Agrobacterium tumefaciens-mediated transformation),we generated three types of FON transformants which carried nuclei labeled with mCherry,peroxisomes labeled with GFP or peroxisomes labeled with DsRED,respectively.In the strains with mCherry labeled nuclei,the bright red fluorescence in round dots were detected in hyphae and conidia,overlaying well with the fluorescence formed by DAPI staining.In the strains with GFP or DsRED labeled peroxisomes,green or red small fluorescent dots were present in hyphal and conidial cells,corresponding with the distribution of peroxisomes in fungal cells.Further,the numbers of the fluorescent dots was increased significantly on lipids.Calcofluor white staining was also performed on the three transformants.Under confocal microscopy,the blue fluorescence of Calcofluor white cooperated well with the fluorescence of green or red fluorescent proteins,producing ideal multi-fluorescent images.In addition,the fluorescent proteins could be stably expressed and well distributed during the subcultivation of the transformants.The growth and phenotypes of the transformants were unaltered compared with the wild type strain.We provided a useful tool for the study on the organelle dynamics,development and pathogenesis of FON.

关 键 词：西瓜枯萎病菌 GFP MCHERRY DSRED 荧光标记 

分 类 号：S436.5[农业科学—农业昆虫与害虫防治]