Cloning And Function Of Sterol O-acyltransferase Gene In Wolfiporia Cocos

Wolfiporia cocos is a kind of medicinal and edible fungi.With its dry sclerotia as medicine,it has the functions of invigorating the spleen and stomach,infiltrating water and dampness,tranquilizing the mind and tranquilizing the mind.It has a long history of medicinal use in China.It was first published in Shennong Herbal Classic,according to which it was called the top-grade of traditional Chinese medicine.Pachymic acid is one of the main quality indicators and active ingredients of Wolfiporia cocos.A large number of pharmacological studies have shown that pachymic acid has anti-tumor,anti-inflammatory,antioxidant effects,as well as hypoglycemic,sedative and other effects.At present,with the increasing demands for Wolfiporia cocos and the growing consumption of forest resources,the contradiction between Wolfiporia cocos production and forest protection has become increasingly acute.Wolfiporia cocos mycelium fermentation to produce pachymic acid and cultivation of Wolfiporia cocos as substitutes has become a new way for sustainable development of Wolfiporia cocos industry.The study on the biosynthetic pathway and metabolic regulation of pachymic acid provides a theoretical basis for the production of effective ingredients of Wolfiporia cocos or itself by using biotechnology and substitution cultivation techniques.Based on the qualitative analysis of triterpenoids in Wolfiporia cocos sclerotia by liquid chromatography-mass spectrometry,the possible biosynthesis pathway of pachymic acid was deduced according to its chemical structure formula.According to this approach,the related catalytic enzymes and coding genes were annotated in combination with transcriptome sequencing and reference to published Poria cocos genome database,KEGG,NCBI and other public databases.The annotated Wcsoat may catalyze the formation of the final product of tumoric acid,pachymaric acid.In this study,the full length of the gene was cloned on the basis of transcriptome sequencing,and the related functional analysis was carried out to understand the basic characteristics of the gene,and to analyze the possible function of the gene.The results provide a methodological basis for further analysis of the biosynthetic pathway of pachymic acid.The results are as follows:1.Total triterpenoids of Wolfiporia cocos were extracted and analyzed qualitatively.Eleven triterpenoids including pachymic acid,lanosterol,eburicoic acid,trametenolic acid and tumulosic acid were identified.The synthetic pathway of pachymic acid was deduced by chemical structural analysis,and the related catalytic enzymes and coding genes were annotated.Sterol O-acyltransferase catalyzed the formation of tumulosic acid to pachymic acid.2.The SOAT gene Wcsoat was cloned from Wolfiporia cocos and sequenced.A complete CDS coding region was obtained,which was 1839 bp in length and encoded 612 amino acids.There was no proteolytic enzyme in the amino acid sequence.The protein is an unstable hydrophobic protein and has no signal recognition function.The protein encoded by this gene is a membrane protein with seven transmembrane regions,belonging to the membrane-bound O-acyltransferase superfamily,about 68 kDa in size.3.The over-expression vector pBlueScript SK-Wcsoat was constructed and successfully transformed into Wolfiporia cocos strain GIM5.219 by PEG-mediated protoplast transformation.Three over-expression strains OE-1,OE-2 and OE-3 were obtained.The growth rate and mycelial growth pattern of the three over-expression strains changed significantly.The expression of Wcsoat of OE-1 strain was 5.44 times higher than that of wild type strain,and the mycelial growth rate was higher.Compared with other strains,the mycelial growth of OE-2 was obviously better than that of other strains.The mycelium grew densely and could be cultured by liquid fermentation without browning.The pachymaric acid content of over-expression strains was higher than that of wild type strains in the absence of MeJA.It is presumed that Wcsoat is involved in the synthesis of pachymaric acid,but its function needs further verification.4.The secretory eukaryotic expression vector pPICZαA-Wcsoat was constructed and transformed into Pichia pastoris X33 strain.Methanol induced the expression of the target protein and obtained the protein of the same size as expected,which laid a foundation for further enzymatic reaction in vitro.