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。

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.