紫色红曲菌色素和桔霉素合成关键基因的克隆及表达调控

The resources of Monascus in China were researched. Seven Monascus species were identified from samples collected around the country. A strain Mp-41 with the highest yield of pigment (5340.4 U/g) was screened out from 688 Monascus strains. This strain was used as T-DNA receptor and more than 800 inserting transformants were gained by Agrobacterium tumefaciens-mediated transformation (ATMT). Then screened pigment and citrinin yield-related mutants as sources of genes, pigment and citrinin yield-related genes would be cloned by TAIL-PCR (Thermal asymmetric interlaced PCR) and SON-PCR (single oligonucleotide nested PCR) and sequenced. Similarity of cloned genes are compared with related fungi such as Aspergillus nidulans, Aspergillus fumigatus, Aspergillus oryzae, 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.The objects of this research are to obtain 2-3 pigment and citrinin yield-related genes, analyze their functions and temporal and spatial expressions. Two genetic engineering Monasucus strains with highter pigment lower citrinin will be constructed. The pigment and citrinin yield of Monascus could be controlled in future by manipulating genes.

收集、分离和纯化我国红曲菌菌种资源，鉴定出7个红曲菌菌种；经筛选获高产红曲色素的紫色红曲菌Mp-41专利菌株1株（色价5340.4 U/g）。以Mp-41菌株为受体通过农杆菌介导的转化获一定量的T-DNA标签库。筛选红曲色素产量、桔霉素含量相关的突变体，采用TAIL-PCR、SON-PCR等方法克隆色素、桔霉合成关键基因，测序后通过信息生物学比对分析相关真菌（构巢曲霉、烟曲霉、米曲霉等）和突变体序列的同源性，预测其保守序列和可能的功能。构建基因缺失载体和基因恢复载体，用农杆菌介导法转化Mp-41菌株，实现关键基因阻断和过表达，并验证基因的功能。本项目研究目标是克隆2～3个新的红曲色素和桔霉素合成关键基因，掌握其功能和时空表达动态，构建低桔霉素含量高产色素的红曲菌基因工程菌株2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。

红曲菌（Monascus）是一种重要的微生物资源，在生长过程中能产生红曲色素、γ-氨基丁酸（γ-aminobutyric acid）、莫纳可林（Monacolins）等多种生理活性物质。调查、收集我国红曲霉菌种资源，筛选获得高产红曲色素、MonacolinK、淀粉酶等优质菌株7株，保藏于中国典型培养物保藏中心，丰富了国家菌种资源。以实验室筛选的紫色红曲菌（Monascus purpureus）Mp-41专利菌株为实验材料，建立了根癌农杆菌介导紫色红曲菌Mp-41菌株的高效转化体系；成功构建了含有5000多个Mp-41菌株转化子的T-DNA插入突变体库。利用紫外可见扫描和分光光度法测定发酵液中红曲色素色价，薄层层析法（TLC）和高效液相色谱法（HPLC）测定发酵液中桔霉素含量，筛选获得红曲色素产量、桔霉素含量提升和下降的正、负突变体各20株，作为TAIL-PCR实验材料。采用TAIL-PCR、RACE、降落PCR等技术克隆获得了红曲色素合成关键基因（MpigE），桔霉素合成关键基因（pksCT），谷氨酸脱羧酶（GAD）基因和γ-氨基丁酸转氨酶（GABA-T）基因等。对MpigE基因、pksCT基因、GAD基因 和GABA-T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）的表达情况；利用qRT-PCR技术，检测了不同培养时间MpigE基因、GAD基因和GABA-T的表达情况；掌握其功能和时空表达动态；对GAD基因进行了大肠杆菌原核表达分析。利用基因敲除和根癌农杆菌介导的红曲菌转化技术，对MpigE基因进行了敲除；分析MpigE基因缺失突变株的形态、生长速率、红曲色素、桔霉素含量等的变化，验证了MpigE基因功能。构建了高产色素低产桔霉素含量的基因工程菌株2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。. 项目实施期发表研究论文16篇，其中SCI收录论文4篇，申请国家发明专利8件，其中4件已授权；培养硕士生6名，在读研究生3名。参加国内学术交流会议6次，参加国际学术会议1次，国际学术会议上做了题为“红曲菌的资源、代谢产物与基因”的英文大会报告1次，承办国内学术会议“2016年浙江省微生物学会学术年会”1次。