MabrlA-Mawet途径在绿僵菌产孢方式转换中的调控机制

The conidia of filamentous fungi could be produced by the normal conidiation or microcirculation conidiation，which are mutually interchangeable. However, there are very few studies on the regulatory mechanism of conidiation pattern shift in the filamentous fungi, thus no regulatory pathways of conidiation pattern shift have been elucidated. The mechanisms underlying the conidiation pattern shift of Metarhizium, one of the model organism for researching the conidiation pattern shift of filamentous fungi, will be studied in this study. In our previous study, knockout transformants of 8 regulatory genes in MabrlA-MawetA pathway of the normal conidiation in M. acridum have been constructed, and screening the knockout transformants of four regulatory genes has identified two regulatory genes of conidiation pattern shift, MabrlA and Mawet. To elucidates the mechanisms of MabrlA-MawetA pathway of the normal conidiation on the conidiation pattern shift of M. acridum, we will further screen the knockout transformants of another four regulatory genes for the genes regulate conidiation pattern shift. Then, the gene expression profiles of these knockout transformants of the regulatory genes in conidiation pattern shift will be analyzed individually in microcycle medium and normal condiation medium by Solexa sequencing to investigate the regulatory networks of all the identified regulatory genes of conidiation pattern shift, thus would sort out new genes potentially related to the conidiation pattern shift. Thirdly, the new candidate genes will be knocked out to identify their function in regulation of conidiation pattern shift. Fourthly, the expression of all the regulatory genes of conidiation pattern shift will be further analyzed by RT-PCR for clarifying the pathway regulating the conidiation pattern shift. Lastly, the pathway regulating the conidiation pattern shift will be compared with the MabrlA-MawetA pathway to elucidate the the regulatory mechanism of the conidiation pattern shift. This study could deepen our understanding of the conidiation regulatory mechanism in the filamentous fungi.

丝状真菌产孢存在正常产孢和微循环产孢两种方式，两者可以发生转换，但其转换调控机制研究很少，调控途径尚不清楚。绿僵菌是研究丝状真菌产孢方式转换的模式真菌之一。前期研究中，申请人敲除了绿僵菌正常产孢调控途径（MabrlA-MawetA途径）中8个基因，对其中4个基因的研究发现MabrlA、Mawet参与产孢方式转换的调控。为了阐明该途径在产孢方式转换中的调控机制，本项目将进一步筛选途径中产孢方式转换调控基因；在正常产孢和微循环产孢培养基上，测定这些基因敲除转化子的基因表达谱，筛选差异基因；通过基因敲除方法，确定与产孢方式转换相关的新基因；采用定量RT-PCR方法，分析产孢方式转换相关基因的调控关系，弄清与MabrlA-MawetA途径相关的产孢方式转换调控途径，从而阐明MabrlA-MawetA途径调控产孢方式转换机制。本研究将加深对丝状真菌产孢调控机制的理解，为提高孢子生产效率提供理论依据。

丝状真菌产孢存在正常产孢和微循环产孢两种方式，两者可以发生转换，但其转换调控机制研究很少，调控途径尚不清楚。绿僵菌是研究丝状真菌产孢方式转换的模式真菌之一。本研究采用基因敲除的方法，确定绿僵菌正常产孢调控途径（MabrlA-MawetA途径）中MabrlA参与产孢方式转换的调控。为了阐明该MabrlA在产孢方式转换中的调控机制，本研究分析MabrlA敲除后在正常产孢和微循环产孢培养基上与野生型（WT）的差异表达基因，其中上调表达538个基因，下调表达478个基因。Mac01240（磷酸转移酶，MaPTS1）、MAC_02010、MAC_02940（C6转录因子MaC6）、Mac05756（锌指蛋白Macc1）、MAC06509、MAC06816（转录因子MaFadA）表达差异在10倍以上。通过基因敲除技术，确定MaPTS1、MaC6、Macc1和MaFadA基因影响产孢。另外，在两种培养基上发现编码疏水蛋白（XP_007810716.1）的共同差异基因，在正常产孢过程上调表达，而在微循环产孢过程中下调表达，推迟这个基因可能参与产孢转换过程。研究中采用Chip-seq技术进一步探索MabrlA参与产孢方式转换的调控机制，构建了ΔMabrlA 菌株的C端带有HA标签的回复菌株，通过Anti－HA抗体检测到回复转化子中表达MabrlA-HA蛋白，目前利用Chip-seq试剂盒纯化的MabrlA-HA蛋白样品正在进行测序，接下来工作分析在产孢转化子MabrlA直接调控的下游基因。本研究将加深对丝状真菌产孢调控机制的理解，为提高孢子生产效率提供理论依据。