鹿儿岛被毛孢分生孢子胞外黏液蛋白提取与鉴定  

Extraction and identification of extracellular mucilage proteins from the conidia of Hirsutella satumaensis

作　　者：段林 邹晓[1] 瞿娇娇 DUAN Lin;ZOU Xiao;QU Jiaojiao(Institute of Fungal Resources,College of Life Sciences,Guizhou University,Guiyang,Guizhou 550025,China;College of Tea Sciences,Guizhou University,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州大学生命科学学院真菌资源研究所,贵州贵阳550025 [2]贵州大学茶学院,贵州贵阳550025

出　　处：《微生物学通报》2021年第12期4836-4843,共8页Microbiology China

基　　金：国家自然科学基金(31860037,32060038);贵州省科技计划项目(黔科合基础[2021]080,黔科合基础[2020]1Z009,黔科合支撑[2019]2405号);贵州大学引进人才基金([2019]10)。

摘　　要：【背景】分生孢子表层黏液在虫生真菌与昆虫的互作中扮演着重要角色。鹿儿岛被毛孢是寄主专一、孢子表面具黏液层的虫生真菌,但其胞外黏液成分尚不清楚。【目的】优化鹿儿岛被毛孢分生孢子黏液蛋白的提取方法,对黏液蛋白组分进行质谱鉴定,探究分生孢子胞外黏液的蛋白成分。【方法】用低浓度还原剂二硫苏糖醇(Dithiothreitol,DTT)对胞外黏液进行温和洗脱并优化提取条件,聚丙烯酰胺凝胶电泳(Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis,SDS-PAGE)检测蛋白提取浓度,用碘化丙啶(Propidium Iodide,PI)染色法检测孢子细胞膜选择通透性,通过肽段酶解、LC-MS/MS数据采集及蛋白质比对分析鉴定蛋白质。【结果】鹿儿岛被毛孢分生孢子黏液蛋白的最佳提取条件:孢子浓度为5×107 cells/mL,DTT浓度为2 mmol/L,4℃提取24 h;黏液蛋白提取后不影响孢子细胞膜通透性。提取的黏液蛋白共鉴定到蛋白质77个,蛋白丰度排前10的蛋白多为分泌性小分子蛋白,包括未注释的蛋白、几丁质酶、脂酶或是与昆虫体壁降解相关的酶类。【结论】鹿儿岛被毛孢胞外黏液中富含与寄主识别、穿透相关的蛋白质,与被毛孢的黏附、识别功能密切相关。[Background]The surface mucilage layer of conidia plays an important role in the interaction between insects and fungi.Hirsutella satumaensis is a host-specific entomopathogenic fungus,and its conidia are covered with a mucilage layer.However,the composition of the mucilage remains unclear.[Objective]The extraction method of mucilage proteins from the conidia of H.satumaensis was optimized,and the extracted proteins were identified by mass spectrometry.[Methods]The extracellular mucilage was eluted with a low concentration of dithiothreitol(DTT) and the extraction conditions were optimized.We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) to determine the extracted proteins concentrations and propidium iodide(PI) to detect the permeability of conidial membrane.Finally,the proteins from extracellular mucilage were identified by peptide enzymatic hydrolysis,LC-MS/MS,and protein comparative analysis.[Results]The optimized extraction condition was as follows:extraction of the conidia at a concentration of 5×107 cells/mL with 2 mmol/L DTT at 4 ℃ for 24 h,and the cell membrane integrity was not affected after extraction.Seventy-seven proteins were identified from the extracellular mucilage of H.satumaensis conidia,most of which were small molecular secretory proteins,including unannotated proteins,chitinase,lipase,and other insect cuticle-degrading enzymes.[Conclusion]The extracellular mucilage of H.satumaensis has rich proteins involved in host recognition and penetration,which is associated with adhesion and recognition of the host insect.

关 键 词：鹿儿岛被毛孢 胞外黏液蛋白 温和洗脱法 附着性 

分 类 号：Q936[生物学—微生物学]