Cloning And Analysis Of A Casbene Synthase Gene From Euphorbia Fischeriana Steud.

There are a large number of tepenoids in Euphorbiaceae plant, including monoterpene,sesquiterpene, diterpene, triterpene and so on. Most of these natural compounds are of interestdue to toxicity and/or potential therapeutic activity. Prostratin, belonged to phorbol esters, istigliane type diterpenoids. It was isolated in the1990s by the U.S. National Cancer Instituteand was proved to have good resistance to HIV. In recent years, the biosynthetic pathway oftigliane type diterpene has aroused the interest of scientists. The casbene synthase is consideredas a putative key enzyme in the pathway of tigliane biosynthesis. Under the catalyzing ofcasbene synthase, GGPP is cyclized to the intermediate cembrane, and then cembrane istransformed to casbene and turned into tigliane finally.Scientists had isolated prostratin from Euphorbia fischeriana Steud.. In this study, withEuphorbia fischeriana root as plant material, we cloned a casbene-synthase gene named EfCS,using RACE-PCR method. The sequence bioinformatics analysis was carried out with13otherspecies casbene synthase gene sequences accessed in Genbank. We constructed a prokaryoticexpression vector and expressed it in Escherichia coli. Fusion protein was purified and theenzymatic reaction was carried out. The final products of the enzymatic reaction was detectedby GC-MS. In summary, the main contents are as follows:1. Casbene synthase gene, named as EfCS (GenBank accession number JN862821), wascloned from the root of Euphorbia fischeriana Steud. through RACE-PCR. The fragment was1969bp in length and contained an open reading frame coding a polypeptide of602aminoacids.2. The bioinformatics methods were employed to analyze and predict the composition ofnucleic acid and amino acid sequences, leader peptides, signal peptide, teans-membranetopological structure, hydrophobicity or hydrophilicity, the secondary and tertiary structure andthe function domains of14Casbene synthases proteins whose whole gene sequences wereregistered in the GenBank. 3. Casbene synthase gene was successfully subcloned into the prokaryotic expressionvector pET32a, we got the recombinant plasmid pET32a-EfCS containing the target gene.4. The recombinant plasmid pET32a was transformed into E.coli BL21(DE3) competentcells, and the fusion protein was successfully induced by IPTG. The total protein of bacteriacultured with and without IPTG were extracted respectively and the total protein of bacteriainduced by IPTG was purified by Ni-Agarose His tag. Finally, the SDS-PAGE electrophoresiswas carried out to test these proteins.5. Concentrated putative EfCS protein was added to the solution containing substrate, andthe reaction product was extracted with hexane. We found that the enzymatic reaction productswere at17.86min with GC-MS analysis. By analyzing the mass spectra of enzymatic product,we found that the characteristic fragment ions and relative abundance of product are as follows:149,99.9%;121,65.7%;93,48.2%;107,34.7%;105,26.2%;79,25.8%;122,21.7%;69,21.5%;136,19.0%;67,17.8%.The molecular ion peak and the relative abundance are272and7.6%. The chemical formula is C20H32. The gene cloned in this study may be casbenesynthase or neocembrene synthase gene.