APSES类型蛋白在莱氏绿僵菌微菌核发育中的功能研究

In preliminary research, the microsclerotia of Metarhizium rileyi, a well-known environmentally friendly dimorphic entomogenous fungus, successfully induced in liquid amended medium and exhibiting excellent facility fermentation, stress resistance and storage stability, could be used as a mycoinsecticide for insect control. Furthermore, we had investigated the molecular mechanism of microsclerotia formation and found that the polar hypha was the foundation for microsclerotia formation. However, little is known about the molecular mechanism of the cell differentiation during microsclerotia development. Nevertheless, we found that the genes of APSES type proteins that regulate morphogenesis, including two APSES family transcription factors (StuA and Swi6) and two APSES type DNA-binding domain proteins (KilA and Xbp) as transcription regulator, were up-regulated and down-regulated, respectively, during microsclerotia differentiation in the comparative transcriptomic analysis. In the present study, we will investigate the function of APSES type proteins during the morphogenesis, including microsclerotia formation, yeast-hypha switching, and conidial development. The functions of genes and proteins will be examined by analyzing the expression patterns and subcellular location. We will analysis the phenotype change through constructing disruption, complementation and overexpression strains. Furthermore, we will investigate and identify the functional genes as the downstream targets of the transcription factor and the function of transcription regulators during microsclerotia development. These researches will clarify the mechanism of the cell differentiation during M. rileyi microsclerotia formation. These results will complete the molecular mechanism of M. rileyi microsclerotia formation and promote the commercial scale-up and cost-effective of liquid fermentation of this entomogenous fungus. Furthermore, the results will also provide scientific parameter for other filamentous fungi to complete microsclerotia production.

液体诱导产生且作为活性成分的莱氏绿僵菌微菌核具有易生产、抗逆耐贮的优点，证实极性菌丝是其形成的前体，但其发育过程中调控细胞分化的机理尚未明确。前期工作发现调控菌体形态发生的APSES转录因子(StuA、Swi6)和调节蛋白基因(KilA、Xbp)在微菌核初始形成时分别上、下调表达。本项目拟开展莱氏绿僵菌形态发生过程中APSES类型蛋白的功能研究，分析菌体形态发生时基因表达谱变化和蛋白亚细胞定位。通过构建基因缺失/回复、超表达菌株，研究各突变体菌株在菌体形态发生时表型变化，解析微菌核发育过程中APSES转录因子调控的通路基因和调节蛋白如何参与调控。研究结果将阐述APSES类型蛋白在菌体形态发生时的生物学功能以及协同调控关系，不仅揭示微菌核发育过程中细胞分化的分子调控机理，进而完善微菌核发育分子机理，有助于进一步促进微菌核的产业化，而且对其他丝状真菌微菌核的形成也具有重要的参考价值。

液体诱导产生且作为活性成分的莱氏绿僵菌微菌核具有易生产、抗逆耐贮等优点。本项目通过分别构建APSES转录因子(MrStuA、MrSwi6、MrXbp)、HOG和CWI等保守信号通路核心基因成员、氧化胁迫应答的关键转录因子Mrap1等基因缺失/回复突变体，研究各突变体在菌体形态发生时(包括微菌核形成、酵母-菌丝型转换、分生孢子发育)的表型变化，解析了APSES转录因子及多条保守氧化胁迫应答转录因子的交叉调控通路基因，揭示了微菌核发育过程中关键转录因子交互调控微菌核发育的分子调控机理。研究结果揭示了细胞分化在微菌核发育过程中的功能，完善了丝状真菌微菌核分子发育机理，促进了莱氏野村菌及其他生防真菌的微菌核产业化进程。