Cloning of Gene cyt b5 and cyt b5r and Their Co-expression with cyp51A in Penicillium digitatum

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.024

Abstract

Abstract: In order to investigate the role of Cytochrome b₅（Cyt b₅）and Cytochrome b₅ reductase（Cyt b₅r）in the electron transport of cytochrome P450 CYP51A in Penicillium digitatum,the co-expression mechanism of CYP51A and Cyt b₅-Cyt b₅r in P. 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 b₅r were acquired and designated as HS-Pdcyt b₅ and HS-Pdcyt b₅r. Further,a co-expressed plasmid vector ppbrA（pPIC-Pdcyp51A-cyt b₅-cyt b₅r）was constructed successfully using multiple-gene series cloning vector pPICZαA. 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 co-expressing system.

Key words: Penicillium digitatum CYP51A, co-expression, cytochrome b5, cytochrome b5 reductase, Pichia pastoris X-33

QIN Ting-ting, GENG Hui, WANG Sheng-qiang, NIU Yu-hui, WU Zhi, LIU De-li. Cloning of Gene cyt b₅ and cyt b₅r and Their Co-expression with cyp51A in Penicillium digitatum[J]. Biotechnology Bulletin, 2016, 32(9): 179-188.

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Cloning of Gene cyt b₅ and cyt b₅r and Their Co-expression with cyp51A in Penicillium digitatum

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