莱氏野村菌微菌核发育相关Rho/Nox基因家族的功能研究

ABSTRACT：Nomuraea rileyi is an economic sound entomopathogenic fungus which can infect several noctuids and other insects. However, the fungi’s large-scale production is limited due to its fastidious growth and stimulating sporulation conditions. Our pioneer researches confirmed that a special liquid fermentation conditions are capable of induction microsclerotia (MS) development of N. rileyi. The MS with thick wall and dominating structure can replace small conidia as the infectious agent for insect control. Our previous works indicated that MS development results from polar hyphal intertwining each other, and also company with reactive oxidative species (ROS) increasing in hyphal cells. But the molecular regulation mechanism of induction MS development still remain many unknown to be clarification. Based on our previous study about comparison transcriptomic analysis results of MS development in N. rileyi, we try to explore genomic functions of those up-regulation gene families such as Rho protein genes (RhoA, Rho B and Bem) and Nox protein genes (NoxA, Nox B and Nox R) when MS development of N. rileyi. We will use modern molecular biological methodologies of knock out, RNA interfered and supper-express for Rho and Nox genomic families, construct knock out, supper-express vectors and deleted target gene of N. rileyi respectively by genomic transformation via co-culture with Agrobacteria tumerfacens, phenotypic difference analysis of mutants and wild type; decipher of priorities regulation gene families during microsclerotium development, probe interaction of Nox / Rho gene families and regulation pass way net. This work will reveal the oxidative stress molecular mechanism of microsclerotia development, promoting MS large scale production of N. rileyi, and provide useful references for inducing microsclerotia in other entomopathogenic filamentous fungi.

摘要：莱氏野村菌是一种环境友好的虫生真菌，自然感染多种夜蛾科害虫。因产孢理化条件特殊、限制其量产和应用。前期研究表明壁厚、抗逆耐贮的微菌核能够替代分生孢子作为活性成分。并已证实微菌核诱导形成伴随着细胞内活性氧的升高，而活性氧来源及蛋白家族调控基因及其调控机理至今未见报道，存在诸多疑问。本项目凭借前期比较转录组学分析上调表达基因为基础，针对微菌核形成相关开展对野村微菌核发育过程中与活性氧产生及调控相关的关键基因家族功能预测分析。通过构建缺失靶基因的野村菌敲除和超表达载体; 以农杆菌共培养遗传转化方法，对Rho、Nox基因家族进行敲除/干扰、超表达，分析突变株与野生型的表型差异，解析与微菌核形成相关的关键调控基因及调控途径。研究对于阐明野村菌微菌核氧胁迫发育的分子机理、提高微菌核的发酵水平都具有重要意义，并可为其他丝状真菌微菌核生产提供借鉴。

真菌的活性氧代谢、极性生长在真菌生长、发育中起着重要作用，特别是我们前期的研究发现，适度的氧胁迫和菌丝的极性诱导是真菌微菌核形成的关键因子。项目执行期间，根据项目申请书拟定的 内容和研究目标开展工作，利用基因敲除和沉默敲除或沉默方法，结合突变体表达谱和转录组分析，对莱氏野村菌涉及极性生长相关以及活性氧代谢相关的20余个基因或转录因子及其相互之间的互作开展研究，分析基因在莱氏野村菌两型转化，生长发育，产孢，微菌核形成以及应对非生物环境胁迫的作用。发现了莱氏野村菌铁载体NrSidA介导的铁转运是满足莱氏野村菌铁需要和解铁毒的关键因子；在微菌核形成中，Hog信号通路与CW1信号通路有着密切的交互作用，pbs2基因在Hog信号途径中起着核心作用；莱氏野村菌的微菌核形成与两型转化有大量的活性氧代谢相关基因的参与，涉及多条调控信号通路，这些信号通路和基因之间有拮抗、互补、相交、冗余，形成一个复杂的调控网络。对这个复杂调控网络还有很多工作需要深入进行。. 我们的研究还显示，许多与氧代谢相关的基因，不仅影响着莱氏野村菌的两型转化，对胁迫的耐受性，产孢和微菌核形成，而且对菌株的毒力也有很大影响。这种影响可能通过破坏菌丝的侵入结构或降低水解酶活性而影响到菌株对寄主的侵染过程，也可能通过对菌株逃避寄主免疫能力和菌株在寄主体内增殖能力的抑制而降低对寄主的致病力。. 以上研究结果为真菌发育、虫生真菌致病机理、真菌功能基因挖掘，菌株特性、真菌培养条件尤其是微菌核培养优化提供了有力支撑，一些成果已经被用于生产实践。. 到目前为止，项目共发表研究论文10篇，其中第一标注4篇，第二标注6篇。申请发明专利2项，获授权专利1项，申请PCT检索1项，美国专利1项。通过项目完成，培养博士后1人，博士研究生3人，硕士研究生9人。