摘要
为探究指状青霉细胞色素b5(Cyt b5)与细胞色素b5还原酶(Cyt b5r)在细胞色素P450 CYP51A电子传递方面的功用,研究了指状青霉CYP51A与Cyt b5-Cyt b5r共表达机制;并检测了其对于cyp51A基因表达水平的影响。通过转录组分析筛选并PCR克隆获得了cyt b5与cyt b5r基因,分别命名为HS-Pdcyt b5和HS-Pdcyt b5r。以多基因串联克隆载体p PICZαA为骨架构建了指状青霉共表达质粒ppbr A(p PIC-Pdcyp51A-cyt b5-cyt b5r);电转化法将重组质粒ppbr A导入毕赤酵母X-33中。q RT-PCR分析结果显示,CYP51A与Cyt b5-Cyt b5r共表达后,其基因表达水平升高54%-97%,并维持较长时间(48-72 h)。表明Cyt b5-Cyt b5r系统可将电子高效转移给CYP51A,从而增强cyp51A基因的转录表达。从指状青霉中克隆表达HS-Pd Cyt b5和HS-Pd Cyt b5r蛋白,并通过共表达的方式研究cyp51A基因的功能尚为首次报道。
In order to investigate the role of Cytochrome bs ( Cyt b5 ) and Cytochrome b5 reductase ( Cyt bsr ) in the electron transport of eytoehromeP450CYP51AinPenicilliumdigitatum, the co-expression mechanism of CYP51A and Cyt bs-Cyt b:inP, digitatum was studied, and its effects on the expression of gene cyp51A was detected. By analyzing and screening transcriptome as well as PCR cloning, gene cyt b5 and cyt b: were acquired and designated as HS-Pdcyt bs and HS-Pdcyt b:. Further, a co-expressed plasmid vector ppbrA ( pPIC-Pdcyp51A-cyt bs- cyt b: ) was constructed successfully using multiple-gene series cloning vector pPICZctA. This recombinant plasmid ppbrA was transformed into Pichia pastoris X-33 by electroporation. Analysis by qRT-PCR revealed that after CYP51A was co-expressed with Cyt b5-Cyt b5r, the expression level of cyp51A increased 54%-97% and it remained in a long period ( 48-72 h ) . This indicated that the Cyt b5-Cyt b5r complex was capable of transferring electrons to CYP51A, which thus enhanced the transcript expression level of cyp51A. This is the first report regarding cloning and expressing HS-PdCyt b5 and HS-PdCyt b5r proteins from P. digitatum, and explore the function of P. digitatum' s gene cyp51A by coexpressing system.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第9期179-188,共10页
Biotechnology Bulletin
基金
国家自然科学基金项目(31371893
31071653)