鹿儿岛被毛孢的侵染方式及其胞外黏液功能研究

Some species of the teleomorph Ophiocordyceps of Hirsutella Pat., also known as important pathogenic fungus of arthropod and nematode in the nature, are rare Chinese traditional medicine. There are the infection ways and thick hydrophobic mucous layer outside their spores,Whether or not the mucous layer has special functions during the infection process or life history is not completely known. The study, which Hirsutella satumaensis and its host Bombyx mori will be used as materals, include three parts. Part 1: find the reasons of infections through the experiments of physical contact after surface inoculation and feeding spores to host, make clear whether or not it caused by surface invasion or by alimentary infection and tracking the characteristic DNA fragments for the hypothesis of Vertical infection. Part 2: according to the results of Part1, using the moderate way to elute the mucous layer meanwhile keep the spores alive, then according to the variation of indexes of the vitality, the toxicity, the environment adaptability, find the functions of the mucous layer during the process of infections. Part 3: collect lots of spores and elute their mucous layers, then by using the biochemical analysis technology, analysis the constitutive property and preliminary estimate their functions,focus on the lectin. The study will provide references in deepen understanding the ralatively host-specificity, the invasion mechannism of the slow growing fungus and the artificial cultivation of some important species of cordyceps sinensis.

被毛孢（Hirsutella Pat.）是自然界节肢动物和线虫的重要病原真菌，其有性型虫草的一些种类是我国名贵中药。该菌侵染寄主方式和其胞外黏液层的功能，目前尚未完全了解。本项目拟采用鹿儿岛被毛孢(Hirsutella satumaensis)和其寄主家蚕(Bombyx mori)作为实验材料，研究以下内容: [1]用孢子经口饲喂和体壁接触的接种方法来明确被毛孢侵染方式是体表入侵还是经消化道感染？用跟踪特征性DNA片段方法验证其经虫卵垂直传播的可能性。[2]利用温和的洗脱方法去除黏液层并保证细胞存活，通过观察孢子去除黏液后孢子活力、毒力和环境适应力等指标变化，确定黏液层在侵染过程中的作用。[3]大量收集孢子分离其黏液，利用非标记定量技术(Label-free)，研究其黏液中蛋白组成，重点关注与识别功能相关的凝集素类蛋白。研究对于加深理解虫生真菌侵染传播机制和重要虫草的发生机理有重要意义。

被毛孢（Hirsutella Pat.）是自然界节肢动物和线虫的重要病原真菌，寄主范围包括昆虫、蜘蛛、螨类和线虫等类群，其有性型虫草的一些种类是我国名贵中药。该属真菌在世界上除南极洲外均有分布，但分布相对集中在亚热带地区，在我国大部地区均有分布。该菌侵染寄主方式和其孢外黏液层的功能目前尚未完全了解。本项目采用鹿儿岛被毛孢Hirsutella satumaensis和其寄主家蚕Bombyx mori为实验材料，1）研究被毛孢侵染方式是从体表入侵还是经消化道感染？是否具有经虫卵垂直传播的可能性。2）探索黏液层与被毛孢孢子的环境适应力和毒力的相关性。3）分析孢外黏液中蛋白组成与功能。结果发现，体表侵染实验中，H. satumaensis可对家蚕生理产生影响，但致死率极低；后采用大蜡螟做试虫，高浓度、低剂量孢子注射是较好的侵染方式。采用特异性引物定性检测H. satumaensis在家蚕基因和蚕卵基因中的存在情况，未能检测到H. satumaensis基因；孢子外粘液主体为疏水性物质，能够增加孢子耐受高渗透压、紫外线、极端温度等胁迫条件，去除粘液后孢子的黏附率显著降低40%左右。粘液与昆虫的致病率密切相关。首次发现被毛孢外粘液分泌受昆虫激素影响，并且发现鹿儿岛被毛孢中有家蚕同源基因。通过非标记定量技术(Label-free)和飞行质谱技术在粘液中发现一个分子量为21 KDa并具有凝集活性的蛋白。为了进一步研究相关基因的功能，成功构建了鹿儿岛被毛孢的遗传转化体系。本项目研究意义在于，明确了被毛孢的侵染方式和孢外粘液的生态学功能，同时发现粘液分泌受昆虫激素调控，这对于理解虫生真菌与寄主的相互作用机制有重要意义，也为重要虫草的人工栽培提供理论支持。