Cloning,Expression,and Functional Verification Of Lanosterol Synthase Gene Pclss From Poria Cocos

AS is known to all,Poria cocos(Schw.)Wolf)can be used as medicinal and edible.It can be compatible with a very large number of medicinal materials.An advanced fungi belonging to Polyporaceae.It’s main function elements are triterpenes and polysaccharides.It have the functions like as drive to dehumidify,Strengthen the resistance,Anti-Tumor,anti-inflammatory,spirit of the soul and other kinds of pharmacological and so on.In the process of producing cholesterol-type substances,lanosterol is the intermediates which catalyzed by the Lanosterol synthase(LSS).This is a key enzyme.In fungi and plants triterpene substances metabolic pathways,they have a common upstream material basis.There are no detailed reports on the biological function of lanosterol synthase in poria.Using a series of molecular biologically related methods to clone the genes of Lanosterol synthase(PcLSS)in the Poria cocos.The functional activity of the gene was verified by the heterogeneous complementary system of saccharomyces cerevisiae.The expression and purification conditions of this enzyme were studied by cold shock expression system.The expression level of genes in different days were also studied at the same time.The main results are show as follows:1 Successful cloning of PcLSS genes The PcLSS gene that CDS and gDNA area wre obtained by using molecular biology methods.They are 2187bp(GeneBanK No:KR336544),5408bp(GeneBanK No:KR336543),respectively.The promoter region contains transcriptional sequesters associated with transcriptional regulation,including acid,light,methyl jasmine,and ao on.PLSS is composed of 728 amino acids,the molecular weight is 83.4 kDa,it is an unstable hydrophilic protein.By analyzing the sequence of PcLSS proteins,found that the sequence of the protein was more similar with mycobacterium.The NJ evolution tree shows that the taxonomic status of the species is consistent with the clustering of proteins.2 The PcLSS gene can complement the function of Saccharomyces cerevisiae ERG7 gene The pyes2-pclss plasmid is obtained by using the traditional double-enzyme cutting reconnection method.Then convert it to ERG7 hybrid diploid Saccharomyces cerevisiae strain YHR072W.The defective haploid strain of the target gene was selected during the tetrad.The two types of defective strains are underlined growth in YPG and YPG + ergosterol medium.A phenotype analysis was carried out,the results show the strain which was conformity with the growth characteristics of the strain.The obtained PcLSS gene mediated the formation of ergo sterol in Saccharomyces cerevisiae,with the function of LSS gene.3 Protein expression and purification The recombinant plasmid of pColdl-pclss,which is required to construct the prokaryotic expression,is constructed using the method of double enzyme.Then convert it to the E.coli strain BL21(pG-KJE8)to expression by cold shock system(15 ℃).Optimize different conditions of proteins Express.The objective protein obtained under optimal expression conditions was purified by the pure column and then obtained PcLSS protein.4 The PcLSS gene has the highest expression level at 12 days of mycelium Using qRT-PCR method were compared the gene expression level under the condition of hypha growth to 6,9,12,15,18 days at 28 ℃,the relative expression of genes CYP as internal,the results showed that when hypha growth to 12 days,the highest amount of gene expression were reached.As mentioned above,The PcLSS gene was cloned by the method of molecular biology,and the function was verified by heterogeneous complementary methods through the defective haploid yeasts.The physicochemical properties and regulatory sites of PcLSS genes and proteins were analyzed by means of online gene analysis tools and means.The target protein PcLSS were obtained and purified by cold stimulus.Real-time fluorometric PCR results showed that the PcLSS gene was the highest relative expression level when the mycelium grow reach to 12 days.