红曲霉分生孢子形成相关基因的克隆、功能分析和表达调控

The Monascus in China was a kind of edible and medicinal microorganism resource. 685 Monascus strains were obtained from samples collected around the country. Five quality Monascus strains were screened. The patent strains Mp-41 and Mr-5 were used as T-DNA receptors and more than 5000 inserting transformants were gained by Agrobacterium tumefaciens-mediated transformation (ATMT). The transformant library will be amplified in this study further. Then screened conidiation-related mutants as sources of genes, conidiation-related genes will be cloned by TAIL-PCR (Thermal asymmetric interlaced PCR), SON-PCR (single oligonucleotide nested PCR), iPCR, RT-PCR, RACE and sequenced. Similarity of cloned genes are compared with related fungi such as Aspergillus nidulans, etc. The functions of cloned genes would be predicted based on the necleotide sequence with blast tool on line supplied by NCBI. Deletion mutants and restored mutants are constructed in order to proved the functions of genes. After construction of fusion expression vectors, target genes-GFP, the temporal and spatial expressions of genes are observed and analyzed using laser scanning confocal microscope and qRT-PCR. The objects of this research are to obtain 2～3 conidiation-related genes, analyze their functions and temporal and spatial expressions. The conidiation mechanism of Monascus could be clarified in future. Monascus gene resources could be excavated and utilized. The research about molecular mechanism of growing development in filamentous fungi could be promoted.

收集和分离红曲霉菌种资源685株，筛选获5株高产红曲霉色素等优质菌株；以紫色红曲霉Mp-41和红色红曲霉Mr-5专利菌株为出发菌株，建立了农杆菌介导高效转化红曲霉技术体系及克隆相关基因技术平台，构建了含5000多个转化子的T-DNA转化子库。本项目将继续扩增T-DNA转化子库，筛选分生孢子形成相关的突变体，采用TAIL-PCR、反向PCR、RT-PCR、RACE等基因克隆的技术，克隆分生孢子形成相关的基因，测序后通过信息生物学比对分析近缘真菌基因序列的同源性，预测其保守序列和可能的功能。构建基因敲除载体和基因恢复载体，用农杆菌介导等方法转化原始出发菌株，获敲除突变体和遗传恢复突变体，鉴定相关基因的功能。研究分生孢子形成相关基因的时空表达动态。本项目研究目标是克隆2～3个分生孢子形成相关的基因，掌握其功能和表达调控机制，挖掘红曲霉的功能基因，促进丝状真菌生长发育的分子机理研究。

丝状真菌与人类的生活和医药卫生密切相关，分生孢子的形成是丝状真菌无性繁殖的主要方式，对于降低生产成本，控制污染以及菌株保藏具有重要意义。红曲霉（Monascus）和曲霉（Aspergillus）是我国传统的食用和药用微生物，实验室收集筛选我国红曲霉和曲霉菌种资源，获得高产红曲色素、MonacolinK、Lovastatin等优质菌株6株，保藏于中国典型培养物保藏中心，丰富了国家菌种资源。利用实验室筛选的2株优质红曲霉和曲霉专利菌株为出发菌株，建立了农杆菌介导高效转化红曲霉和曲霉的技术体系和克隆相关基因的技术平台，已获得含5000多株和2800多株的红曲霉和曲霉转化子库；筛选获得了分生孢子形成缺失的突变体10株，作为TAIL-PCR实验材料。克隆获得了红曲霉分生孢子形成关键基因brlM、wetM，色素相关基因PigE和曲霉分生孢子形成关键基因brlA、flbA等。分别对brlM基因、wetM基因、PigE基因、brlA基因、flbA基因进行了序列和信息生物学分析。利用激光共聚焦显微镜分析了转化子绿色荧光蛋白（GFP）的表达；利用RT-PCR技术，检测了不同培养时间brlM基因、PigE基因、brlA基因和flbA基因的表达。构建了克隆基因的敲除载体，用农杆菌介导的转化技术，对brlM基因、PigE基因、brlA基因和flbA基因进行了敲除，获得了基因缺失突变株。分析了基因缺失突变株在形态、生长速率、红曲色素、桔霉素、MonacolinK、Lovastatin、棒曲霉素等的变化，鉴定了克隆基因的功能。基于第二代高通量测序技术对红曲霉Mp-41菌株的cDNA进行测序和功能注释。构建了高产红曲黄色素低产桔霉素的基因工程菌株1株，实现从基因水平上对红曲色素产量和桔霉素含量进行调控。并促进丝状真菌生长发育和代谢产物合成的分子机理研究。.项目实施期发表研究论文15篇，其中SCI收录论文4篇，申请国家发明专利7件，其中4件已授权；培养硕士生8名，在读研究生2名。参加国内学术会议4次、国际学术会议2次，做了题为“红曲霉的资源、代谢产物与基因”等英文大会报告2次、中文大会报告2次，主办国内学术会议“2016年浙江省微生物学会学术年会”1次。