利用荧光蛋白标记研究稻瘟病菌有性世代的细胞结构  

Lighting the Cellular Structures of Sexual Generation in Magnaporthe oryzae with Fluorescent Proteins and Fluorescent Dyes

作　　者：郭晓宇 李玲[2,3] 董波[4] 王教瑜[2] 柴荣耀[2] 张震[2] 毛雪琴[2] 邱海萍[2] 郝中娜[2] 王艳丽[2] 孙国昌[2] Guo Xiaoyu;Li Ling;Dong Bo;Wang Jiaoyu;Chai Rongyao;Zhang Zhen;Mao Xueqin;Qiu Haiping;Hao Zhongna;Wang Yanli;Sun Guochang(College of Chemistry and Lore Sciences,Zhejiang Normal University,Jinhua 321004,China;State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control,Institute of Plant Protection and Microbiology,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;School of Agricultural and Food Sciences,Zhejiang Agriculture and Forest University,Lin＇an 311300,China;Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,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]省部共建国家重点实验室培育基地-浙江省植物有害生物防控重点实验室,浙江省农业科学院植物保护与微生物研究所,杭州310021 [3]浙江农林大学农业与食品学院,临安311300 [4]浙江省农业科学院病毒与生物技术研究所,杭州310021

出　　处：《中国细胞生物学学报》2018年第7期1138-1145,共8页Chinese Journal of Cell Biology

基　　金：国家自然科学基金(批准号:31470249);浙江省自然科学基金(批准号:LQ17C010001)资助的课题~~

摘　　要：稻瘟病是水稻最重要的病害之一,同时稻瘟病菌(Magnaporthe oryzae)是植物病原真菌研究的模式生物。稻瘟病菌是异宗配合的子囊菌,但其有性世代的细胞学过程、形态结构、分子机制以及对病菌变异的贡献研究都较少,也缺乏必要的研究手段。该文利用荧光蛋白标记结合荧光染色的方法对稻瘟病菌有性世代结构进行了标记和显微观察,以期为稻瘟病菌有性生殖过程和机制研究提供方法与借鉴。将绿色荧光蛋白GFP和红色荧光蛋白m Cherry分别导入两个相对交配型的稻瘟病菌菌株Guy11(MAT1-2)和70-15(MAT1-1),各转化子及野生型按交配型组合对峙培养,观察有性世代结构中的荧光表达情况。结果表明,组蛋白H3启动子、核糖体蛋白RP27启动子和疏水蛋白MPG1启动子均可在稻瘟病菌有性孢子形成过程中高丰度表达。进一步利用这三种启动子和两种荧光蛋白对稻瘟病菌有性孢子的细胞器(细胞核、过氧化物酶体)进行标记和观察,结果表明,融合组蛋白H2B的m Cherry及融合核定位信号(NLS)的GFP均能有效标记子囊孢子细胞的细胞核,荧光集中而明亮;带有过氧化物酶体定位信号1(PTS1)的GFP可以有效地标记子囊孢子中的过氧化物酶体,每个细胞中均有数量不等的过氧化物酶体,表明子囊孢子中需要过氧化物酶体参与生化代谢。该文还利用脂肪染色剂尼罗红、BODIPY和细胞壁染色剂卡氏白结合荧光蛋白标记对子囊和子囊孢子进行组合染色。结果显示,尼罗红、BODIPY、卡氏白染色剂互不干扰,可以与不同颜色的荧光蛋白相互组合,从而更加清晰地标记子囊和子囊孢子的结构、细胞器和储藏物质。Magnaporthe oryzae causes rice blast disease, the most devastating disease on rice, and also is a model organism for the study on plant pathogenic fungi. M. oryzae is a heterogeneous fungus, but its sexual generation rarely occurs in nature. The studies are thus limited on cytological process, morphological structure and molecular mechanism of sexual generation, and on the contribution of sexual reproduction to the pathogenicity variation of the fungus. The methods for such studies are still required. In the present work, the sexual structures of M. oryzae were investigated microscopically by using fluorescent protein labeling combined with fluorescent staining. The green fluorescent protein（GFP） and the red fluorescent protein（m Cherry） were firstly introduced respectively into the M. oryzae strains with opposite mating types, Guy11（MAT1-2） and 70-15（MAT1-1）. The transformants, together with the wild types were cross-cultured on OMA medium basing their mating types to induce the sexual generation, which were then detected fluorescent-microscopically. The results showed that the promoters of histone H3, ribosomal protein RP27 and hydrophobin MPG1 could be highly expressed in ascospores of M. oryzae. Subsequently, we labeled and detected the nuclei and peroxisomes in asci and ascospores of M. oryzae via the three promoters and the two fluorescent proteins. The m Cherry fused with H2 B and the GFP fused with a nuclear localization signal（NLS） can be well distributed on the nuclei in ascospores. The GFP fused with peroxisomal localization signal 1（GFP-PTS1） can effectively label the peroxisomes in the cells of ascospores. The presence of peroxisomes in sexual structures may suggest that the metabolism in peroxisomes is involved in the sexual reproduction of the fungus. We also used fluorescent dyes Nile red, BODIPY and Calcofluor white to stain the asci and ascospores of the fungus. The data indicated that these fluorescent dyes did not interfere with the fluorescent proteins and brighten cle

关 键 词：稻瘟病菌 有性世代 荧光蛋白 BODIPY 尼罗红 卡氏白 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]