昆虫病原真菌蜡蚧轮枝菌对干旱胁迫的分子响应机制

Lecanicillium lecanii, a well-known entomopathogenic fungus, plays an important role in agricultural insect control. But the requirement for high humidity during the conidial germination and host infection had often led to the fluctuation of application effect in field. Therefore, it is the general belief that high environmental humidity is essential for the effective use of L. lecanii in insect control. In this proposal, the suppression subtractive hybridization (SSH) is used to construct the cDNA library consisting of L. lecanii genes differentially expressed under the drought stress. Several differentially expressed sequences are screened form the SSH library and their functions are identified by the bioinformatics analysis. Through the electronic cloning and rapid amplification of cDNA ends (RACE), the full-length cDNA sequences of these putative drought-responsive genes are obtained. The expression levels of putative drought-responsive genes under the drought stress are analyzed by the reverse transcriptase PCR (RT-PCR). The functions of these putative drought-responsive genes are confirmed by suppressing their expression using the method of antisense RNA-mediated gene silencing. Finally, we will elucidat the molecular mechanism of responding to drought stress in L. lecanii. This study will be expected to find out novel drought-responsive functional genes or their regulator genes and enrich the scientific content of responding to drought stress in biocontrol fungus. In practice, our results help to provide a firm theoretical foundation for the transformation of L. lecanii strains through molecular technology in order to increase the tolerance to drought-stress and solve the problem of bad application stability in field.

蜡蚧轮枝菌是一种重要的昆虫病原真菌，对多种农业害虫具有良好的防控效果，但是该生防真菌孢子的萌发与侵染高度依赖于高的环境湿度，致使其在田间的应用效果常不稳定，因此环境湿度就成为了该生防真菌在田间控制害虫的关键限制因子。本项目通过抑制性消减杂交技术构建蜡蚧轮枝菌干旱胁迫下的表达文库，筛选出差异表达序列,经生物信息学分析鉴定出该生防真菌应对干旱胁迫的相关基因；利用电子克隆、RACE等技术克隆出这些基因的cDNA全长，通过RT-PCR分析明确这些基因在干旱胁迫下的的表达情况；结合RNA干扰技术验证这些基因与该生防真菌耐旱性的关系，阐明蜡蚧轮枝菌响应干旱胁迫的分子机制。该研究结果有望发现新的干旱响应相关功能基因及其调节基因，丰富生防真菌对干旱胁迫响应机制的科学内容，为通过分子改造提高菌株耐旱性，解决该真菌杀虫剂田间应用稳定性差的问题提供理论依据，对促进真菌杀虫剂在绿色农业中发挥更大作用具有现实意义。

以虫生真菌建蜡蚧轮枝菌为研究对象，采用抑制性消减杂交技术和基因克隆与功能验证技术，分析该生防真菌应对干旱胁迫的分子机理。取得的主要研究结果有：（1）构建了蜡蚧轮枝菌干旱胁迫下的表达文库，筛选出蜡蚧轮枝菌干旱胁迫下差异表达的序列，RT-PCR结果显示，差异基因在蜡蚧轮枝菌干旱胁迫中增强表达；（2）克隆出干旱胁迫相关基因Llhog的cDNA全长，其与细胞分裂原活化蛋白激酶MAPK高度同源，并成功构建蜡蚧轮枝菌的Llhog基因的RNAi干扰突变体；（3）蜡蚧轮枝菌RNAi干扰突变体的生物学和旱耐特性性研究结果表明，Llhog基因沉默导致蜡蚧轮枝菌的菌丝生长减慢、产孢减少及其致病性降低，而且也导致蜡蚧轮枝菌的耐干旱能力降低。通过3年的研究，顺利完成了项目的研究任务，发表SCI论文4篇、中文核心论文2篇、已授权发明专利2项、培养硕士研究生1名。