指状青霉cyt b_5和cyt b_5r基因克隆及与cyp51A的共表达

Cloning of Gene cyt b_5 and cyt b_5r and Their Co-expression with cyp51A in Penicillium digitatum

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.