侧耳纤维二糖水解酶基因的克隆与表达研究

pDL1是能够转化细菌和真菌的双功能质粒。将pDL1与糙皮侧耳(Pleurotus ostreatus)的DNA片段连接,然后转化玉米黑粉菌(Ustilago maydis)的原生质体。通过新霉素选择平皿的筛选,转化率达到800转化子/μgDNA,建立了糙皮侧耳的基因文库。随机取15个转化子进行点渍法(Dotting blotting)检验,结果均呈阳性。此结果表明,抗性克隆不是源于玉米黑粉菌敏感细胞的回复突变。萨慎法(Southern blotting)检验显示,本研究在玉米黑粉菌细胞中克隆糙皮侧耳纤维二糖水解酶基因是成功的。基因表达试验表明,玉米黑粉菌克隆株具有糙皮侧耳纤维二糖水解酶的活性。

Studies on Cloning and Expression of Cellobiosehydrolase Gene of P. ostreatus

pDL1 is a kind of bifunctional plasmid which can transform bacteria and fungi. And pDLl transformed the protoplasts of Ustilago maydis after being ligated with DNA fragments of Pleurotus ostreatus. Through the screening of neomycin selective plates, the transformation ratio reached 800 transformants per microgram of DNA, and the gene library of Pleurotus ostreatus was established. 15 randomly selected transformants expressed positive by dotting blotting test. The result showed that the resistant clones were not originated from back mutation of sensitive cell of Ustilago maydis. And Southern blotting test revealed that studies were successful for cloning the cellobiosehydrolase gene of Pleurotus ostreatus in the cells of Ustilago maydis. While the gene expression test showed that the cloned strains of Ustilago maydis expressed the enzyme activity of cellobiosehydrolase of Pleurotus ostreatus.