蛇足石杉内生真菌Shiraia sp.Slf14中Ⅲ型聚酮合酶的表达、纯化及生物信息学分析

The Prokaryotic Expression,Purification and Bioinformatics of Type Ⅲ Polyketide Synthase from Shiraia sp. Slf14,Which Is an Endophytic Fungus of Huperzia serrata

竹红菌素是我国特有的一类苝醌类光敏色素,在开发新型抗肿瘤抗病毒药物、光敏活性农药及新型光电转换材料等方面应用前景广阔.聚酮合酶(Polyketide synthase,PKS)是合成竹红菌素的一种关键酶,通过全基因组测序及分析发现在Shiraia sp.Slf14中存在一个III型聚酮合酶基因.利用RT-PCR技术,以其总RNA为模板,扩增得到目的片段,并成功构建表达载体p ET-22b(+)-PKSIII,采用Ni-NTA亲和层析法纯化目的蛋白,在大肠杆菌BL21(DE3)中成功表达出了目的蛋白,SDS-PAGE结果显示表达重组产物分子质量约为43 k Da,与理论值一致,为III型聚酮合酶的催化活性研究提供理论依据.

The hypocrellins are a unique class of perylenequinones characterized by a pentacyclic conjugated chromophore giving rise to photoactivity. One type III PKS gene was obtained from Shiraia sp. Slf14,which is the key synthase in the product process of the hypocrellin. The total RNA as the template to amplify the type III PKS fragment was used and then was cloned into p MDTM19-T vector. After identify,type III PKS gene was ligated into p ET-22b（ ＋） to obtain recombinant expressing vector p ET-22b（ ＋）-PKSIII. The expression vector into Escherichia coli BL21（ DE3） and cultured positive colonies of E. coli in liquid LB medium were introduced. Then the objective protein were obtained and isolated for purification using a Ni-NTA affinity chromatography. It can lay the foundation for the catalytic activity of type III polyketide synthase.