Cloning And Function Of Cytochrome P450 Gene PcCYPs In Poria Cocos

The dried sclerotium of Poria cocos(Schw.)Wolf is a commonly used fungal medicinal material,pachymic acid is one of the main active ingredients of P.cocos and has multiple functions.t The cultivation of P.cocos is based on pine wood,the increasing demands for P.cocos has led to the destruction of forest resources.Therefore,it is urgent to seek new method to produce pachymic acid.The development of synthetic biology technology provides a possible way to achieve sustainable development of P.cocos industry.Cytochrome P450 is widely present in living organisms and is involved in metabolism from energy metabolism to secondary metabolites and developmental regulation of the organism.At present,some catalytic enzyme genes that may be involved in the synthesis of pachymic acid have been identified based on the Poria cocos genome and transcriptome,but many cytochrome P450 oxidase genes and their functions have not been identified.which is the hotspot and difficulty in research pachymic acid synthetic biology technology.In this study,PcCYP1 and PcCYP3 genes were cloned based on the differentially expressed cytochrome P450 enzyme superfamily gene genes found in the transcriptome library of P.cocos sclerotium and mycelium.The possible functions of the two genes were studied through bioinformatics analysis,overexpression technology,RNAi technology,and eukaryotic expression technology,which laid the foundation for the in-depth study of the biological functions of PcCYP1 and PcCYP3.The main research results obtained are as follows:1.The CDS sequences of cytochrome P450 gene PcCYP1,PcCYP3 and the alternative splicing body PcCYP2 of PcCYP1 gene were cloned from P.cocos,and analyzed by bioinformatics.The CDS lengths of PcCYP1,PcCYP2,and PcCYP3 are1590 bp,1542bp,and 1581 bp,which encode 529,526,and 513 amino acids,respectively,and all have multiple phosphorylation sites.The 1-17 th amino acid sequence of PcCYP1 and PcCYP2 are signal peptides,and the subcellular localization in mitochondria is a secreted unstable hydrophilic protein.PcCYP3 is predicted to have transmembrane regions at the N-term by transmembrane region prediction,subcellular is located on plasma membrane,and it is a stable hydrophilic membrane protein.PcCYP1,PcCYP2 and PcCYP3 are closest to CYP502 of Coprinopsis cinerea,and all contain the CYP450 conserved domains Fxx Gx Rx Cx G,Exx R,PER,AGx DTT.Quantitative PCR results showed that the expression of alternative splicing variant PcCYP2 differs significantly indifferent periods,so it is speculated that PcCYP2 may have a regulatory effect on the growth and development of P.cocos.2.Successfully constructed the overexpression vector p Blue Script SK-PcCYPs,and transformed into P.cocos strain GIM5.219 by the PEG-mediated protoplast transformation method,3 PcCYP1 overexpression strains,3 PcCYP2 overexpression strains,and 3 PcCYP3 overexpression strains were obtained respectively by plate resistance screening and hygromycin resistance gene hph amplification detection.Based on the analysis of PcCYP1,PcCYP2,PcCYP3 expression levels,colony growth morphology,triterpene product content,and mycelium viability of the overexpressing strains,it can be known that overexpression of PcCYP1,PcCYP2,and PcCYP3 can significantly increase the growth rate of mycelium,the content of pachymic acid,and mycelial activity,but the content of total triterpenes was not different from the control.Therefore,it is inferred that the gene overexpression of PcCYPs can promote the synthesis of pachymic acid,but its function needs further verification.3.Silent expression vector p Blue Script SK-PCIT-PcCYPs was successfully constructed,and 1 silent transformant of PcCYP1 and 6silent transformants of PcCYP3 were obtained.Among them,the mycelial growth rate and the content of pachymic acid were significantly reduced in transformants with significantly reduced expression of the target gene.Therefore,it was further verified that PcCYP1 and PcCYP3 may have a regulating effect on the growth of mycelium and the synthesis of pachymic acid.4.The secretory eukaryotic expression vector p PICZ?-PcCYPs and the intracellular expression vector p GAPZC-PcCYPs were constructed and transformed into Pichia X33 strain for heterologous expression.After SDS-PAGE analysis,PcCYP1,PcCYP2,and PcCYP3 initially achieved secretory expression in Pichia pastoris,but the expression level was low,which laid the foundation for the next step of enzymatic reactions in vitro.The above research has initially revealed that PcCYP1,PcCYP2,and PcCYP3 are involved in the growth and development of P.cocos and the synthesis of pachymic acid,which lays the foundation for further revealing its function through in vitro catalytic function analysis,and provides an idea for studying other CYP450 genes with unknown functions.