Study On The Biosynthetic Pathway Of Hypocrellin A Based On Transcriptome Sequencing

Shiraia bambusicola Henn.belongs to the phylum Ascomycota.As a traditional Chinese medicine,it was used for the treatment of rheumatic arthritis,sciatica,pertussis,tracheitis and so on.Its major active metabolic product is hypocrellin.Hypocrellin A(HA)is the main component in hypocrellin,and it has been found that HA has antiviral and antitumor activities.HA showed great potential in clinical photodynamic therapy.Although HA has exhibited a variety of pharmacological functions and has been used widely,its productivity is still lower.Therefore,it will be hot and in the research frontier to identify HA biosynthetic pathway and improve the HA production with genetic technology.In this paper,we firstly irradiated the wild strain S4201-W that produce HA by a 800 mj/cm2 dose of ultraviolet radiation treatment And we found the sample S4201-D1 that did not produce HA.Then,S4201-D1 was inoculated and determined by high-performance liquid chromatography(HPLC).And it showed the heridity stability.To identify putative genes that are involved in the biosynthesis of hypocrellin A and determine the pathway,we performed transcriptome sequencing for Shiraia bambusicola S4201-W and the mutant S4201-D1.Then,we obtained 38,056,034 and 39,086,896 clean reads from S4201-W and S4201-D1,respectively.Afterwards,all of the high-quality reads were assembled into 17,923 unigenes with an average length of 1,287.45 bp and an N50 of 1,970 bp.The functional annotation of these unigenes were performed using different protein databases,including the KOG,GO,KEGG,NR and SwissProt databases.In all,7,225 unigenes were clustered into 25 functional categories in KOG.9,647 unigenes were assigned to their main GO categories,including 52 sub-categories.A total of 3,581 unigenes were annotated and assigned to 323 different pathways.Furthermore,14,243 and 9,011 unigenes were respectively annotated to another two databases.Based on the negative binomial distribution test,716 unigenes were found to be upregulated,and 188 genes were downregulated in S4201-D1,compared with S4201-W.We have found seven unigenes involved in the biosynthesis of hypocrellin A and proposed a putative hypocrellin A biosynthetic pathway.These 7 unigenes encoding multicopper oxidase,fasciclin,polyketide synthase,o-methyltransferase/FAD-dependent monooxygenase,o-methyltransferase,hydroxylase and FAD/FMN-dependent oxidoreductase respectively.By exploring the HA biosynthetic pathway and its main enzyme genes,this study will provide a theoretical basis for future genes functional studies and valuable resources for molecular mechanism about its metabolic pathways.