Analysis And Systematic Mining Of Genes Involved In The Biosynthetic Pathway Of Triterpenoids In Sanghuangporus Baumii

Sanghuangporus baumii is a kind of precious medicinal fungus that has been widely used as traditional herbal medicine in China for many years.Extracts from this mushroom have been demonstrated to be effective on a diversity of diseases,including anti-cancer,antioxidant,anti-diabetes,antiinflammatory,and so on.Wild resources of S.baumii have become scarce at present and the artificial cultivation of S.baumii is at the stage of small-scale production.Therefore,it is difficult to make the most of the medicinal value of S.baumii.The triterpenoids,one of the main bioactive components found in S.baumii.However,difficulties in the extraction and isolation process hinder their utilization as drug.Fortunately,the identification of key enzymes involved in the biosynthetic pathway of triterpenoids could help reveal the mechanism of triterpenoid biosynthesis at a molecular level.However,to the best of our knowledge,the triterpenoid biosynthesis pathway in S.baumii is still undetermined.In this work,based on our transcriptome data,key enzyme genes involved in the biosynthetic pathway of triterpenoids in S.baumii were digged and analyzed.In addition,the cloning and expression patterns of candidate genes were investigated.These results provide insights into the genetic background of S.baumii,which will be useful in future to reveal its medicinal mechanism of triterpenoid biosynthesis pathway.The main results obtained were as follows:1.Mining of candidate unigenes relating to the triterpenoid biosynthesis pathwayIn the S.baumii transciptome,94 unigenes(yield of 1.08%)were annotated to be involed in 3 terpene-relating pathways.In addition to gene annotation,homology-based BLAST and phylogenetic analysis with well characterized genes in the biosynthesis of triterpenoids from other species were also performed,and 19 candidate unigenes were obtained.These candidates includes 12 involved in the MVA pathway,and 7 CYP450s were found for the downstream pathway.These candidate unigenes lay resource foundation of triterpenoid biosynthesis for S.baumii.2.Analysis of expression characteristics of candidate genes involved in the triterpenoid biosynthesis pathway during different developmental stages of S.baumiiQuantitative real-time PCR technology was performed to measure transcript levels of several genes in the triterpenoid pathway including HMGR,HMGS,FPPS,SQS,SE and LS during different developmental stages of S.baumii.The results showed that the transcript levels of these genes were in dynamic change.Overall,the transcript levels of most genes in the mycelium stage were higher than the fruiting body stage.For instance,the highest transcript levels of HMGS and HMGR were at 14d and were 2.33 and 1.56-fold higher than the 5d,respectively.Moreover,the highest transcript levels of FPPS and SE were at 11d and were 2.20 and 3.55-fold higher than the 5d,respectively.3.Methyl jasmonate induces triterpenoid biosynthesis in S.baumiiIn this work,methyl jasmonate(MeJA)as an exogenous elicitor was used to investigate the feasibility of enhancing triterpenoid biosynthesis in S.baumii.Results revealed that the appropriate addition concentration of MeJA was determined to be 150 μM,and the triterpenoid yield was 12.61 mg/g dry weight(DW),which was 4.05-fold higher than the untreated control.Moreover,quantitative real-time PCR was performed to measure transcript levels of several genes in the triterpenoid pathway including HMGR,HMGS,FPPS,SQS,SE and LS.The results demonstrated that MeJA significantly induced expression of these genes.This is the first time to assess the novel use of MeJA to elicit triterpenoid biosynthesis in S.baumii,and the results indicated that MeJA was indeed a potent inducer of triterpenoid biosynthesis.4.Cloning and bioinformatics analysis of HMGS and SEFull-length cDNA of HMGS and SE from S.baumii was cloned by RACE.The sequencing results showed that the full-length nucleotide sequence of HMGS was 1 930 bp,containing a complete open reading frame of 1 458 bp which encoded a polypeptide of 485 amino acids.Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52.75 KDa,and theoretical isoelectric point was 5.60.The encoded protein comprisesed 7 373 atoms and its molecular formula was C2340H3668N626O717S22.This protein was a hydrophilic protein,without transmembrane and signal peptide sequence.In addition,the sequencing results showed that the full-length nucleotide sequence of SE was 1856bp,containing a complete open reading frame of 1 452 bp which encoded a polypeptide of 483 amino acids.Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 53.36 KDa,and theoretical isoelectric point was 8.41.The encoded protein comprisesed 7 567 atoms and its molecular formula was C2422H3804N658O665S18 This protein was a hydrophobic protein,with transmembrane and without signal peptide sequence.At last,the secondary and tertiary structures of HMGS and SE were predicted.The constructed phylogenetic tree showed that HMGS and SE from S.baumii were most similar to HMGS and SE from Fomitiporia mediterranea,respectively.5.Construction of prokaryotic expression and overexpression vectors of SEIn order to observe the expression of SE in E.coli,the entire protein-coding cDNA of SE was cloned into the expression vector pET-32a.Then the pET-32a-SE construct was transformed into E.coli BL21(DE3)cells.After the induction of IPTG,through SDS-PAGE analysis a significant protein band was found in the vicinity of the relative molecular weight of approximately 55 KDa which was consistent with molecular weight of the predicted protein.Additionally,we designed primers according to the gpd promoter sequence of Lentinula edodes in Genbank,and obtained the gpd promoter fragment by PCR.Subsequently,the plant binary expression vector pCAMBIA 1301 was selected as the basic vector,and then the 35S promoter replaced with L.edodes gpd promoter through the methods such as enzyme digestion and connection.Finally,the coding region of SE was cloned to the downstream of the gpd promoter.The results revealed that the overexpression vector pCAMBIA 1301-gpd-gpd-SE was constructed successfully.