绿僵菌微循环产孢调控途径研究

The conidia of filamentous fungi could be formed on extended hyphae (normal conidiation）or be occurred directly after conidia germination (microcycle conidiation). The regulatory pathways of normal conidiation were elucidated in many filamentous fungi, the central pathway of normal conidiation is conservative though there are some differences in the regulatory pathways among filamentous fungi. Microcycle conidiation is a universal mechanism of filamentous fungus to adapt adverse conditions, but there are very few studies on the regulatory mechanism of microcycle conidiation including the model organisms, thus no regulatory pathways of microcycle conidiation have been elucidated. In our previous studies, a medium for microcycle conidiation of Metarhizium acridum was established and advantages of microcycle conidiation were confirmed, 221 genes were up-regulated compared with normal conidiation, and two genes were identified to be regulatory gene by RNAi. In this project, the regulatory pathway of Metarhizium microcycle conidiation will be studied systematically. Firstly, all the 221 up-regulated genes will be knocked out. Secondly, the conidiation properties of all the knockout transformants will be investigated to identify the regulatory genes for microcycle conidiation. Thirdly, the gene expression profiles of the knockout transformants of the regulatory genes will be analyzed individually by Solexa sequencing to investigate the transcriptions of all the identified regulatory genes, thus would elucidate the regulatory pathway of Metarhizium microcycle conidiation. At last, the knockout transformants of the regulatory genes will be cultured in the medium for normal conidiation and the medium for microcycle conidiation to study the effects of the regulatory genes on virulence and the tolerance to heat and ultra radiation.

丝状真菌的孢子由菌丝分化的产孢细胞形成（正常产孢）或由不经过菌丝阶段直接形成（微循环产孢）。许多丝状真菌正常产孢调控途径都已经阐明，不同丝状真菌的调控中心途径十分保守。微循环产孢是丝状真菌中广泛存在的一种适应不良环境的机制，但是其调控机制研究很少，至今仍未弄清微循环产孢调控途径。申请人建立了绿僵菌的微循环产孢培养基，发现其优良的产孢特性，采用消减杂交技术筛选出221个微循环产孢上调表达的基因，已采用RNAi鉴定出2个调控基因。本项目将系统地研究绿僵菌微循环产孢调控途径：通过设计和构建221个微循环产孢上调表达基因的敲除载体，获得基因敲除转化子，观察产孢特性变化，鉴定出微循环产孢调控基因；采用Solexa测序法获得微循环调控基因敲除转化子的基因表达谱，分析其它微循环产孢调控基因的表达情况，从而确定出微循环产孢调控途径；并分析微循环调控基因敲除后对毒力、耐热和抗紫外等重要性状的影响。

昆虫病原真菌以气生分生孢子侵染寄主，在固态培养基上产生分生孢子是与杀虫真菌生产直接相关的重要性状。与其他丝状真菌一样，绿僵菌通常进行正常产孢，在不良条件下进行微循环产孢。微循环产孢具有产孢快、产孢多、孢子均一等优点，但是难以规模化生产杀虫真菌孢子粉。本项目采用数字表达谱等方法分析了不同营养元素（C、N、P）调控绿僵菌微循环产孢的差异基因，结果显示：137个基因参与正常产孢过程,436个基因参与微循环产孢过程，83个基因参与碳源对产孢模式转换机制的调控过程，216个基因参与氮源对产孢模式转换机制的调控过程，168个基因参与磷源对产孢模式转换的机制的调控过程。选取微循环产孢相关基因，构建了敲除转化子，通过在不同培养基(1/4SDAY、SYA)上进行产孢分析发现6个微循环产孢调控基因：3个基因（BrlA、Macpp、Mmc）在微循环产孢培养基（SYA）上调控微循环产孢，敲除基因后绿僵菌不能在SYA上微循环产孢；3个基因（MAH1、PP5、Ctf）在完全培养基（1/4SDAY）上调控微循环产孢，敲除基因后绿僵菌1/4SDAYY上微循环产孢，而不是正常产孢。通过构建回复转化子的产孢分析，进一步验证了6个微循环产孢调控基因的功能。采用定量RT-PCR等方法，分析了在SYA上参与微循环产孢调控的3个基因之间的调控关系。此外，还分析了4个微循环产孢调控基因的（Macpp、MAH1、PP5、Ctf）在绿僵菌生长、毒力和孢子抗逆特性（热、紫外线等）的作用；分析了在1/4SDAY上产孢时期特异上调表达的2个基因（ENA ATPase、乙醇脱氢酶1）的功能，揭示了ENA ATPase在孢子耐热和耐紫外线照射的机理; 乙醇脱氢酶1在低氧条件下调控绿僵菌生长、产孢,分析了乙醇脱氢酶1调控的分子机制,在正常氧浓度下不影响生长、产孢、抗逆、毒力等重要性状。. 丝状真菌的微循环产孢途径至今尚未不清楚.本项目首次发现了6个微循环产孢调控基因，并初步弄清了在营养受限的微循环产孢培养基（SYA）上调控微循环产孢的3个基因之间的调控关系；发现ENA ATPase与孢子抗逆境特性相关，但是不影响生长、产孢、毒力等重要性状；乙醇脱氢酶1通过调控在低氧条件下的乙醇代谢而影响绿僵菌产孢。本项目研究结果为进一步阐明绿僵菌等丝状真菌的微循环产孢调控机制奠定了基础，对于弄清微循环产孢与正常产孢之间的调控网络都具有