竹黄菌中一个聚酮合酶基因的初步研究

Preliminary Study on One Polyketide Synthase from Shiraia sp. SUPER- H168

目的:从竹黄菌Shiraia sp.SUPER-H168中扩增出一个聚酮合酶的编码基因g4 PKS,并扩增出其中一段酮基合成酶(KS)基因片段,构建表达载体p ColdⅡ-KS,在大肠杆菌BL21(DE3)中表达。方法:从竹黄菌Shiraia sp.SUPER-H168中提取基因组DNA和总RNA后,采用RT-PCR方法扩增获得目的片段g4 PKS,克隆至p MD18-T进行保存。以T载体为模板,利用引物KSfor/KSrev扩增KS基因片段,构建原核表达载体p ColdⅡ-KS,并将重组质粒转化至大肠杆菌BL21(DE3)中表达。结果:成功获得g4 PKS基因;成功构建表达载体p ColdⅡ-KS,并在大肠杆菌BL21(DE3)中成功表达出了目的蛋白。结论:成功构建出了表达载体p ColdⅡ-KS,表达出目的蛋白。

Objective： One fungal polyketide synthase gene named g4 PKS was obtained from Shiraia sp. SUPER- H168,and one ketosynthase（ PKS） domain fragment of g4 PKS was amplified. The expression vector p ColdⅡ- KS was constructed and expressed in E. coli BL21（ DE3）. Method： Genomic DNA and total RNA were extracted from Shiraia sp. SUPER- H168. The fragment g4 PKS was obtained by the method of RT- PCR,and cloned to vector p MD18- T. T vector as template,the PCR product of KS domain with the KSfor / KSrev- primer was amplified and expression vector p ColdⅡ- KS was constructed. Then recombinant plamid was transformed into E. coli BL21（ DE3） and expressed. Result： Successfully obtained g4 PKS gene and expression vector p ColdⅡ- KS with expressing the purpose protein. Conclusion： The expression vector p ColdⅡ- KS was successfully constructed and expressed.