Cloning and bioinformatics analysis of cytochrome P450 reductase gene in Poria cocos / 中草药

ABSTRACT

Objective: To clone cytochrome P450 reductase (PcCPR) gene from Poria cocos and to characterize with bioinformatics methods. Methods: According to annotated transcriptome of P. cocos, the PcCPR gene was cloned through RACE, and the genomic DNA sequence was further obtained through PCR. The characteristics of the encoded protein were analyzed using bioinformatics, the 3D structure of the protein was modeled with I-TASSER server, and phylogenetic tree of CPR was carried out with MEGA. Results: The 2 514 bp full-length cDNA sequence (GenBank Accession No. KP768251) and the 5 292 bp genomic DNA sequence (GenBank Accession No. KP896487) of PcCPR was obtained, which contained four exons and three introns. PcCPR encoded a protein with 732 amino acids. The protein was predicted to be an unstable hydrophilic protein with calculated molecular weight of 81 147 and isoelectric point 5.39. PcCPR does not have a signal peptide but has a transmembrane segment (aa residues 7 to 22), and it is anchored to endoplasmic reticulum. There are two flavin binding domains and many predicted FAD and NADP binding sites, these sites are adjacent respectively at 3D structure level. The homologous analysis indicates that PcCPR has a higher similarity with CPR from basidiomycetes than CPR from ascomycetes. Conclusion: The PcCPR was successfully cloned, which will provide a foundation for researches on PcCPR and PcCPR associated metabolic.