球孢白僵菌锌指蛋白基因Bbazf在卵孢素合成中的功能

Some secondary metabolites produced by entomopathogenic fungi can inhibit the host immune reaction or be toxic to insect, and play important roles in the fungal pathogenesis. Therefore, it's theoretically and practically significant to study the biosynthesis pathway and regulation mechanism of these secondary metabolites. Oosporein, produced by Beauveria species, has been shown to be toxic to insect and can inhibit the growth of fungi, bacteria, virus and cancer cells. Thus, it has great potential in the fields of pest management, medicine and so on. However, at present, only the structural and chemical properties of oosporein have been identified, and nothing is known about the genes and regulation mechanism about oosporein biosynthesis. In our previous work, a collection of B. bassiana random T-DNA insertion mutants was screened and one mutant was isolated that produced abundance oosporein. The random insertion site was mapped to a gene that displayed homology to a transcription factor (zinc finger protein) and was designated as Bbazf. This gene was disrupted by homologous recombinant and confirmed to negatively regulate oosporein production. In the present study, we will try to isolate the genes which were regulated by Bbazf using transcriptomic and biological information techniques or the genes interacted with it by CHIP and yeast two-hybrid methods. After finished this study, we expect to elucidate the function of Bbazf in the process of oosporein synthesis and strain virulence, or to obtain the genes related to oosporein biosynthesis.

杀虫真菌产生的一些次生代谢物具有抑制宿主免疫反应或毒杀昆虫的功能。因此，从基因水平上研究这些次生代谢物的生物合成及调控机制具有重要的理论和实践意义。白僵菌属产生的卵孢素具有抗虫、抑菌和抑制肿瘤生长的活性，具有广阔的应用前景，但目前还不清楚其生物合成基因及分子调控机理。课题组在前期研究中，经突变体库筛选和基因tagging证实，球孢白僵菌中一个具锌指结构的转录因子Bbazf负调控卵孢素的生物合成。敲除该基因，卵孢素合成显著提高，菌株毒力增强。在此基础上，本申请拟对Bbazf敲除子和野生型菌株进行转录组测序比较，获得该基因调控的下游基因；利用酵母双杂交和CHIP手段，分析与Bbazf相互作用的蛋白或基因组序列，进而分析这些基因在卵孢素合成过程中的作用及对菌株毒力的影响。项目的完成，有望获得卵孢素合成相关基因，并阐明其调控机制，为卵孢素在病虫害的生物防治和医药领域的应用奠定基础。

球孢白僵菌是一类重要的昆虫病原微生物，为了能吸收宿主营养，进化出了能致病昆虫宿主及与其它微生物竞争的能力。在真菌致病昆虫宿主的过程中能产生一种红色的次生代谢物卵孢素。但目前对于卵孢素的合成调节网络及生物学功能一直都不清楚。我们通过筛选卵孢素合成突变体，获得了一个调节卵孢素合成的转录因子Bbazf (后根据其调节次生代谢物合成的功能，命名为Bbsmr1，secondary metabolite regulator 1)。在球孢白僵菌中删除Bbsmr1，导致了卵孢素合成基因簇的表达上调及卵孢素的组成性产生。OpS3是卵孢素合成基因簇中的一个转录因子。在Bbsmr1突变体中进一步删除OpS3导致了卵孢素合成的消失，表明Bbsmr1负调节了OpS3的表达。OpS1是卵孢素聚酮合成酶基因，将其启动子与GFP融合，获得OpS1：：GFP。结合RT-PCR的表达分析显示，OpS1主要在真菌致死宿主后的24-48h内表达。细菌数分析显示，在真菌致死宿主后，细菌数急剧上升，但随着卵孢素的产生，细菌数下降了90%左右。体外分析证实了卵孢素对僵虫细菌抑制作用。上述结果表明，在球孢白僵菌致死昆虫后，卵孢素作为一种抑菌物质，可以抑制僵虫体内细菌的增殖，使真菌可以最大限度的获得营养物质。