The Cloning, Expression And Location Analysis Of Squalene Synthase From Endophytic Fungi Penicillium Minioluteum Isolated From Eleutherococcus Senticosus

Objective To clone P. minioluteum (Penicillium minioluteum) squalene synthase,which is an Endophytic fungi of Eleutherococcus senticosus. And analyze the prokaryoticexpression and localization of. And we analysed the SS codon usage and its influence onexpression of the gene.Methods PmSS conservative squence was cloned. The unknown5’ sequence was cloneby5’ RACE method. After PCR amplification and cloning, recombinant plasmid ofPmSS gene was successfully constructed. Plasmid of PmSS gene by double enzymerecovery and was connected to the expression vector pET-30a(+), connected productpET-30a-PmSS was conversion of BL21(DE3), and the PmSS gene was induced toexpress by different temprture from20°C～37°C and different IPTG concentration from0.2mmol/L to1.2mmol/L. The expressed extraction product was analysed by SDS-PAGE, after isolated by protein extraction kit. PmSS gene cloning plasmid was doubleenzymed digestion and recovery was connected to the fluorescence expression vectorpEGFP-N1and pDsRed2-N1. Plasmid was converted into P. minioluteum, to determinethe localization of SS protein. Using codon W, CUSP analysis of47SS genes fromdifferent species of and SPSS16software was used for statistical analysis andcorrespondence analysis. And compare with phylogenetic tree constructed by MEGA5.0neighbor-joining connecting method.Results PmSS gene was cloned scussfuly. The full length was1881bp, conteining aORF of1413bp. The predicted protein contein the conservative sequence and activeamino acid. PmSS gene prokaryotic expressed in BL21(DE3) host, the expressionconditions were optimized, temperature and IPTG concentration was analysised. PmSSexpressed. PmSS expressed better in30℃and IPTG concentration of0.6mmol/L. Inthe P. minioluteum SS were was distributed as punctiform or facet, the same whit theexpected results. The GC content of squalene synthase gene codon1to3base (GC1,GC2and GC3) were51.33%,34.65%and54.37%, respectively. The content of GCcontent in three different sites showed a significant correlation (P<0.01), correspondenceanalysis results show that, the first axis showed30.71%differences, the correlationbetween the mean and the effectiveness of GC3codon number and GC3, GC1and GC2 were significant (P<0.01).26synonymous codons were the optimum codons which wereall ended by C or G. Compared cluster analysis dendrogram of RSCU for SS genes withthe evolutionary tree build based on SS gene sequences through MEGA5.0software，both fit traditional viewpoint.Conclusion SS of Eleutherococcus senticosus Endophytic fungi P. minioluteum wassuccessfully expressed in prokaryotic, and PmSS localized in the endoplasmic reticulum.PmSS gene codon preference end in G/C, using the model of selection and mutation,mutation has greater influence on codon preference, to lay the foundation for improvingthe mechanism of Eleutherococcus senticosus saponins content and further study of P.minioluteum.Figure18; Table5; Reference117...