冬虫夏草菌逃避蝙蝠蛾免疫相关基因功能研究

The Chinese caterpillar fungus Ophiocordyceps sinensis, regarded as the "Himalayan Viagra", is highly valued for its medicinal benefits but becomes deterioration due to over-exploitation. The fungus infects the ghost moth and finally produces the fruiting body in the cadaver of the larva. The molecular mechanism of the pathogenesis is still unclear. As the fungus needs several months of colonization in haemolymph before killing the larva, the ghost moth challenge by O. sinensis is a valuable model for studying interactions between entomopathogentic fungi and insect hosts. Because the genetic background of O. sinensis is complex, the rich phenotypic changes are much more important for identification of gene function. Thus, we established a genetic transformation system for O. sinensis strain KD1223 using Agrobacterium mediated transformation with the random insertion binary vector pEX4. Base on the constructed random insertion mutant library for KD1223 strain, in this study, we will combine the culture characteristics on the solid and liquid PPDA medium and the ability of colonization in haemolymph of the ghost moth larvae to screen both enhanced and defective mutants on invasion of host immune defenses. Then, we will isolate both sides of the flanking sequences of the T-DNA insertion sites of these mutants by hiTAIL PCR and FPNI PCR and obtain the genes involved in evasion of host immune defenses by blastn and blastx in NCBI database. We will also compare the relative expression levels of the candidate genes involved in evasion of host immune defenses in the mutants and wildtype strain. Finally, we will design and construct the knockout vector based on the reconstructed p821 vector, and the knockout vector will then be transformed into O. sinensis mediated by A. tumefaciens. The target gene knockout mutant will be screened by hygromycin and identified by southern blot. The gene complementary system is essential for functional identification of target genes. The promotor sequence and the ORF sequence of the target gene will be cloned into the complementary vector, and this vector will also be transformed into O. sinensis knockout mutant mediated by A. tumefaciens. The complementary mutant will be screened by chlorimuron and identified by southern blot. The physiological and virulence changes and the expression level of genes involved in host immune defenses will be compared among the wild type, knockout and complementary strains. By screening and identifying the genes involved in evasion of host immune system, we aim to reveal the molecular mechanism of O. sinensis evading its host immune defenses. These results will enrich the adaptive evolution theories of interaction between entomopathogentic fungi and insect hosts and will support the artifical cultivation of O. sinensis.

冬虫夏草是我国传统的名贵中药材，是冬虫夏草菌侵染蝙蝠蛾幼虫形成的僵虫和子座复合体，目前对其致病发生机制尚不清楚。本项目针对冬虫夏草菌在蝙蝠蛾幼虫血腔定殖数月才致死寄主这一现象，围绕昆虫病原真菌如何逃避昆虫免疫这一科学问题，以冬虫夏草菌株KD1223为研究对象，在业已构建KD1223菌株遗传转化体系的基础上，比较分析突变体和野生菌株的培养特征及其在蝙蝠蛾幼虫血淋巴中的定殖能力，筛选于蝙蝠蛾幼虫血淋巴中生长增强型和缺陷型突变体；通过hiTAIL和FPNI等PCR方法克隆突变体T-DNA插入失活基因，利用实时荧光定量PCR方法分析这些基因的表达水平，获得冬虫夏草菌逃避蝙蝠蛾免疫相关基因；最后，利用基因敲除和回补的方法验证基因的功能。研究结果有助于揭示冬虫夏草菌逃避蝙蝠蛾幼虫免疫的分子机制，丰富病原真菌与寄主昆虫互作适应性进化理论，为冬虫夏草人工培养和可持续利用提供理论和实践依据。

病原真菌定殖寄主血腔是其致死寄主的一个非常重要的阶段，病原真菌能否成功逃避寄主免疫系统的识别，干扰或抑制寄主的免疫系统，决定了真菌能否成功致死昆虫。冬虫夏草菌定殖宿主蝠蛾幼虫血腔长达数月甚至一年以上，是非常独特的研究宿主昆虫与病原真菌免疫互作的模型。研究冬虫夏草菌逃避蝙蝠蛾幼虫免疫的分子机制可以为提高真菌杀虫速度提供理论依据，同时为冬虫夏草人工培养和可持续利用提供技术支撑。本研究通过突变体库筛选，获得72株固体和液体培养表型与WT有明显差异的突变体以及12株芽生孢子生长增强型和3株芽生孢子生长缺陷型菌株；通过 hiTAIL 和 FPNI 等 PCR 方法分离得到8个与气生菌丝形成、子实体生长表型相关基因以及3个定殖血腔逃避宿主免疫相关基因；利用同源重组基因敲除方法验证了这3个基因的功能，取得了如下主要结果。其一，发现Chp在分生孢子起始期和成熟期的表达上调，OCS_06094在分生孢子起始期和萌发期表达上调，Hbp在各个发育时期的表达均下调，参与调控冬虫夏草菌分生孢子的产量，Chp和Hbp同时影响芽生孢子的产量；真菌进入血腔后，Chp在芽生孢子增殖期表达下调，OCS_06094和Hbp在芽生孢子增殖期表达上调，影响芽生孢子在血腔的定殖速度及其对宿主的致病力。其二，发现小金蝠蛾幼虫在芽生孢子未形成菌丝体前可以携菌化蛹羽化为成虫，也有发现在蛹期转化形成菌丝体的蛹不能羽化为成虫而长出子实体。其三，冬虫夏草菌致死宿主的过程非常复杂，发现其致死宿主过程与宿主的生理状态密切相关，宿主昆虫肠道和血腔微生物、昆虫激素等因素均对冬虫夏草菌注射芽生孢子感染小金蝠蛾幼虫的僵化率有影响。