桦褐孔菌菌核发育关键酶基因调控序列的克隆分析及gpd启动子的克隆

The very rare and valuable medicinal fungi, Inonotus obliquus (Fr.) Pilát, of which the sclerotia are often taken as medicine, only distribute in several northern countries,and now in danger of extinction while the large-scale cultivation of the sclerotium has not succeeded due to the low biological efficiency.The breeding of good strains is the precondition of large-scale cultivation of Inonotus obliquus sclerotia, however the study on the breeding of Inonotus obliquus is still blank.In the previous study, we found that Inonotus obliquus sclerotia formation based on laccase activityies and the production had positive correlations with cellulose enzymes and protease activities. This project will ascertain the functions of the enzymes coding genes based on cloning, meanwhile, the upstream regulatory sequences will be cloned and analysed, furthermore, the gpd promoter of Inonotus obliquus will be cloned and analysed, and the activities will be identified. These will lay the foundations to explore the molecular mechanisms and expression regulation of sclerotium formation and genetic improvement of Inonotus obliquus.

桦褐孔菌Inonotus obliquus（Fr.）Pilát是十分珍稀而又名贵的药用真菌，仅分布在北半球的少数国家，以其菌核入药，野生资源濒临灭绝，菌核人工栽培由于生物学效率低尚未实现规模化栽培。优良菌株的选育是实现桦褐孔菌菌核人工规模化栽培的前提，而桦褐孔菌育种工作的研究目前尚属空白。前期研究我们发现，漆酶活性大小决定桦褐孔菌菌核形成与否，菌核产量与纤维素酶及蛋白酶活性成正相关。本项目在对桦褐孔菌菌核发育关键酶基因克隆的基础上对其进行功能验证，同时对上游调控序列进行克隆和分析，并对桦褐孔菌的gpd启动子进行克隆分析和活性验证，为进一步揭示桦褐孔菌菌核形成的分子机制及调控表达和优良菌株的选育提供依据。

桦褐孔菌Inonotus obliquus（Fr.）Pilát是一种十分珍稀而又名贵的药用真菌。本项目采用RACE技术克隆得到桦褐孔菌菌核形成关键酶基因内切葡聚糖酶基因、β-葡萄糖苷酶基因和酸性蛋白酶基因，并进行相关的生物信息学分析；利用荧光定量PCR技术研究关键酶基因在菌核形成期间的表达量变化情况；构建纤维素酶和酸性蛋白酶原核表达载体，获得纯化的蛋白；克隆得到gpd基因和启动子，并进行生物信息学分析。结果表明：内切葡聚糖酶基因序列全长为1966 bp，cDNA序列的全长为1233 bp，编码410个氨基酸，该基因包含14个外显子和13个内含子；β-葡萄糖苷酶基因包含14个外显子和13个内含子，DNA序列全长为3382 bp，cDNA序列的全长2583 bp，编码860个氨基酸；酸性蛋白酶基因cDNA序列全长为990 bp，编码330个氨基酸；测定桦褐孔菌菌核发育期间内切葡聚糖基因表达量一直升高，而β-葡萄糖苷酶基因的表达量先上升后下降；克隆获得gpd基因和启动子序列，为建立高效的外源基因表达系统奠定了基础。