不同寄主专化性甘薯长喙壳菌分泌蛋白质组学分析及其致病蛋白鉴定

Ceratocystis fimbriata Ellis & Halsted attacks an exceptionally wide range of economically important plants, causing wilt-type diseases, cankers, and rot of storage roots. It's showed strong host specialization in reciprocal inoculation experiments on mostly hosts. But the isolates from diseased pomegranate and taro produced representative diseased symptoms on both crops in China, which is firstly founded that monocotyledonous family Araceae (Colocasia esculenta) isolates of C. fimbriata can infect dicotyledonous host, pomegranate (Punica granatum) in the world. Additionally, we firstly confirmed that dicotyledonous host pomegranate isolates of C. fimbriata were strongly pathogenetic to monocotyledonous C. esculenta. In other words, it is not showed host specialization in reciprocal inoculation experiments on pomegranate and taro hosts. It is a new host-specialized genotypes different with other hosts isolates of C. fimbriata. But it's pathogenic mechanism has not been reported. This project aimed to analyze the expression profiles of secretory proteins produced by the pomegranate and taro isolates of C. fimbriata and C. fimbriata f.sp. platani strains by using SDS-PAGE, column chromatography, quantitative mass spectrometry and amino acid sequence database retrieval methods. In addition, different secretory proteins were identified through homology comparison by using peptide mass fingerprinting and protein databases. Further, some key proteins which related to pathogenicity will be selected to clone their genes, and primers were designed and reverse transcription polymerase chain reaction amplification was performed using total RNA extracted from these strains. The amplification products were expressed using the yeast expression system pYES2.1/V5-His-TOPO after cloning, and the pathogenic functions of the expression products were verified after identification and purification. These researches would help to build the theoretical bases for persistent control of C. fimbriata.

甘薯长喙壳菌是重要的植物病原真菌，具有明显寄主专化性。本项目前期研究发现，该种菌在单子叶植物（芋头黑腐病菌) 与双子叶植物 (石榴枯萎病菌) 间存在相互侵染现象，即不存在寄主专化性，或可认为该菌是区别于其它寄主专化性菌的新'专化型'，但其致病机理尚未明确。本项目拟主要采用SDS-PAGE及柱层析、MALDI-TOF-MS 技术和氨基酸序列数据库检索等方法，对石榴枯萎病菌与芋头黑腐病菌主要致病分泌蛋白类型进行鉴定分析；然后，通过生物信息学方法获得差异表达蛋白的编码基因，筛选出致病相关基因；进行RT-PCR 扩增，并利用酵母表达系统进行表达，表达产物经鉴定纯化后进行致病功能验证。通过本研究，找出与甘薯长喙壳菌毒力相关的蛋白或基因，明确该病原菌致病分子机制，为探索不同寄主菌株间如何突破寄主专化性关系及相互侵染的机理提供科学依据。

根据项目计划书目标要求本项目对榴枯萎病菌与芋头黑腐病菌主要致病分泌蛋白进行了分离、鉴定及功能验证分析，探讨了其互相侵染的致病机制。在项目实施过程中建立并优化甘薯长喙壳菌分泌蛋白的提取方法，在 PD 液体培养基，pH 6.5，25-30℃，黑暗振荡条件下培养 20d 以上病原菌产毒量和生长量最大；提取到的分泌蛋白毒素在浓度为 50μg·ml-1分泌蛋白毒素针刺接种到寄主植物叶片后，接种部位叶片可很快出现坏死斑 (2-3d)，分泌蛋白毒素在叶片上形成坏死斑的面积和浓度对数呈显著正相关性；进一步分离和纯化得到了分子量分别约在 10 kDa，15 kDa和95 kDa 左右的分泌蛋白质，这些蛋白质在这两种寄主 C. fimbriata 中的分泌量较大，并证实 15 kDa 分泌蛋白质为致病关键蛋白。石榴枯萎病菌株与芋头黑腐菌所产生的分泌蛋白质组分在大小和序列上高度相似，在致病性、遗传分化及互作酶活性等研究中也证实两菌株之间具有高度的一致性；在氨基酸测序和质谱分析的基础上，获得了能稳定表达目的蛋白质基因的阳性克隆。在 2.0μg/μl 的浓度接种到致伤的石榴植株和芋头植株叶片上，72h 时可观察接种叶片的表型变化 (致病斑点)，证实其具有致病活性。