食线虫真菌Pochonia chlamydosporia菌株退化的分子机制

As the strain using for producing commercial nematicidal agent of "Xian Chong Bi Ke", degeneration of Pochonia chlamydosporia ZK7 strain negatively influences the quality and application of the agent.In previous study, we have obtained several degenerated strains of ZK7 with degeneration characteristics respectively on the pathogenicity, extracellular enzymes, conidial production and mycelial growth. By using serial methods including iTRAQ/mTRAQ - HPLC - LC-MS/MS, this project will be performed to characterize the differential proteomics of these degeneration strains comparing with the original. Based on obtained results on the differential proteins, including protein species, protein species with differential expression, functions, metabolism pathway of differential proteins,and gene knockout of vital proteins, this study attempts to elucidate the molecular mechanism of fungal degeneration under the condition of sub-cultured on artificial media.Results of study will expand our knowledges to understand the common issue of fungal degeneration from the proteomics perspective.

厚垣孢普可尼亚霉（Pochonia chlamydosporia）ZK7菌株是商品化线虫生防菌剂"线虫必克"的生产菌株，ZK7菌株的退化问题严重影响该生物农药的产品质量和产业化应用。本项目利用前期工作中获得的ZK7致病力退化、胞外酶退化、产孢量退化和菌丝生长速度退化菌株，与原始菌株组成关联差异样本，采用蛋白组学研究中的iTRAQ/mTRAQ - HPLC - LC-MS/MS系列研究技术方法，分析各差异样本中种类差异和表达量差异的蛋白种类；通过对差异蛋白的分类、功能注释及其代谢通路分析，以及关键蛋白基因敲除的功能验证，从蛋白质层面揭示生防真菌多次继代培养后菌株退化的分子机制。研究结果有助于从蛋白质组学的视角拓展目前对真菌菌株退化这一共性科学问题的认知。

利用食线虫真菌厚垣孢普可尼亚霉(Pochonia chlamydosporia)YMF1.1301菌株研制的线虫生防制剂“线虫必克”是国内首个利用微生物研发并产业化应用的线虫生防制剂。在推广应用中，P. chlamydosporia YMF1.1301菌株的退化影响了防治效果，进而影响到该生物农药的推广应用。了解其退化机制为选择生防菌株的适宜保藏方法和菌种改良有重要的科学意义和应用价值。本项目从蛋白质组学层面揭示该菌株的退化机制，获得了以下主要结果：. 1. 通过传代培养，从原始菌株Pochonia chlamydosporia YMF1.1301中筛选获得产孢量和丝氨酸蛋白酶浓度显著退化的菌株YMF1.613；YMF1.613的产孢量和丝氨酸蛋白酶浓度分别为1.75×106个/皿，49.97 μg/mL，而YMF1.1301的产孢量和丝氨酸蛋白酶浓度分别为3.19×107个/皿和232.83 μg/mL。. 2. 运用从头预测法对P. chlamydosporia进行了编码基因预测，预测了12,781个基因，并利用GO、COG等注释方法注释了其中的8,409个基因。. 3. 运用差异蛋白质组学方法分析原始菌株与退化菌株的蛋白质组差异，退化菌株YMF1.613相对于原始菌株YMF1.1301的下调蛋白共19个，涉及到11个功能分类；上调蛋白共426个，涉及到18个功能分类。. 4. P. chlamydosporia YMF1.1301可能的退化机制是：（a）调控细胞生长、分化的信号转导机制中的信号分子（Rho GTP等）出现了下调，导致生长速度变慢；（b）调控细胞生长、发育的关键酶出现下调（Cytokinin oxidase等），导致生长速度变慢；（c）调控细胞衰老、凋亡、自噬途径的相关蛋白（Transmembrane 9 superfamily member 1、Histidinol-phosphate aminotransferase、Prohibitin、Mitochondrial prohibitin complex protein 1等）上调，导致细胞生长受抑制；（d）调控细胞壁合成相关蛋白下调（Chitobiase、GlmS等），导致产孢量、生长速度受抑制。