Cloning And Expression Analysis Of Sesquiterpene Synthase Gene From Inonotus Baumii

Inonotus baumii is a kind of precious medicinal fungus which has very important medicinal value,and it is one of the fungal which have best anticancer effect in the field of biological anti-cancer.Sesquiterpene is one of the medicinal ingredients from Inonotus baumii,and it has important pharmacological activities.In this study,with Inonotus baumii as the research object and based on transcriptome data,a full-length gene of sesquiterpene synthase gene PLS1 was cloned by RACE technique,and the bioinformatic analysis of its full-length sequence was performed;By constructing the prokaryotic expression vector,the target protein was successfully induced to express,which verified the function of PLS1 preliminarily;Influence of different culture time and different concentrations of methyl jasmonate(MeJA)on expression levels of sesquiterpene synthase gene from Inonotus baumii was studied by using qRT-PCR.The main results obtained were as follows:1.Cloning and bioinformatics analysis of PLS1Full-length PLS1 from Inonotus baumii was cloned by RACE.The sequencing results showed that the full-length nucleotide sequence of PLS1 was 1 448bp,containing a complete open reading frame of 1 008bp which encoded a polypeptide of 547 amino acids.Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 37.95Kda,and theoretical isoelectric point was 4.79;The encoded protein comprisesed 5281 atoms and its molecular formula was C1687H2618N444O512S20;This protein was a hydrophilic protein,without transmembrane and signal peptide sequence;At last,the secondary and tertiary structures of this protein were predicted.2.Prokaryotic expression of PLS1The suitable restriction sites BamH ? and Hind ? was introduced into ORF ends of PLS1 by PCR.Constructed the recombinant plasmid pMD18-T-PLS1,and transformed it into E.coli DH5a competent cells.Positive clone was obtained by Blue-white screening,after that,expanded culture positive clones bacteria solution and extracted the recombinant plasmid pMD18-T-PLS1.After double digestion by BamH ? and Hind ?,purification,constructed the recombinant plasmid pET-32a-PLS1,then pET-32a-PLS1 was transformed into expression strain E.coli BL21(DE3)competent cells,and positive clones were screened finally.After the induction of IPTG,through SDS-PAGE analysis a significant protein band found was found in the vicinity of the relative molecular weight of approximately 37 kDa which was consistent with molecular weight of the predicted protein.3.The influence of culture time and MeJA on the expression levels of sesquiterpene synthase genesInfluence of different culture time and different concentrations of MeJA on expression levels of 5 sesquiterpene synthase genes in sesquiterpene metabolic pathway from Inonotus baumii was studied by using qRT-PCR.The results showed that expression of sesquiterpene synthase genes changed with culture time and concentrations of MeJA.With the extension of culture time,The relative expression of sesquiterpene synthase genes gradually increased,subject to 1th day was essentially flat with the control group,the expression of sesquiterpene synthase genes during other days was higher than control group and reached the maximum on 4th day or 5th day;MeJA induced by different concentrations,Unigene1790?Unigene5544?Unigene6151 and Unigene27582,the relative expressions of those 4 sesquiterpene synthase genes had different degrees of increase,besides,only PLS1 was reduced.Therefore,expression of sesquiterpene synthase genes was upregulated when MeJA induction on the whole.