Functional Characterization Of Sesquiterpene Synthases From Aquilaria Sinensis

Aquilaria sinensis (Lour.) Gilg is the main species of Aquilaria which is one of the most precious and endangered medicinal plants in China. It possessed various biological activities such as sedative, analgesic and digestive, which is used to treat rheumatism, spasm of bronchus and respiration, abdominal cramp, diarrhea nausea, vomiting, anxiety and so on. The main fragrant compounds of agarwood are sesquiterpenes and phenylethy chromone derivatives, and a great variety of sesquiterpenes are contained in high-quality agarwood. The sesquiterpene synthases were considered as a key enzyme in the pathway of biosynthesis. Study the structure and the function of the sesquiterpene synthase gene, using genetic engineering to improve the content of sesquiterpene, will have important theoretical and practical value. Therefore, in this study, we isolated a sesquiterpene synthase gene, constructed an expression vector and expressed it in E.coli. The expression patten of target gene was analyzed with real-time PCR. The expressed protein of target gene was purified and analyzed the activity. We constructed the over-expression vector, obtained transgenic Arabidopsis, for the further research of target gene’s function.In this study, a pair of specific primers were designed according to the results of high-throughput sequencing, we cloned the sesquiterpene synthase from Aquilaria sinensis by RT-PCR. The sequencing results showed that the nucleotide sequence contained a complete open reading, frame, its full length was1644bp, encoding a polypeptide of547amino acids. Bioinformatics analysis of the amino acid sequence showed that it shared above95%identities to Aquilaria crassna.It confirmed that we had cloned the sesquiterpene synthase in A.sinensis, named ASS. We submitted it to NCBI GenBank database, the number is JQ712683. And we predicted the structure and biological properties of the protein using bioinformatics software.Only wounded trees can produce agarwood. In this study, calluses originating from the stems of the healthy A.sinensis plants were used to investigate the effect of wound-stress and MJ/Eth treatment. The results show that the short-term damage, the expression of sesquiterpene synthase gene was up-regulated significantly, to reach a certain level, the expression of ASS decreased rapidly.Sesquiterpene synthase gene was successfully cloned into the prokaryotic expression vector pET-28a, we got the recombinant plasmid pET-28a-ASS containing the target gene, and transformed into E.coli BL21(DE3) pLysS, the fusion protein was successfully induced by IPTG. The total protein of bacteria cultured with and without IPTG were extracted respectively and the total protein of bacteria induced by IPTG was purified by Ni-Agarose His Tag. Finally, the SDS-PAGE test the proteins. The proteins were analyzed using GC-MS, the sesquiterpene synthases catalyzed the substrate FPP, the results showed that ASS had activity, and the major product was δ-guaiene, it also produced other sesquiterpene, like β-elemene, a-guaiene. The gene we cloned in this study was a sesquiterpene synthases gene.In this study, we successfully constructed plant expression vector pBI121-ASS. The pBI121-ASS was transformed into Agrobacterium tumefaciens strain LBA4404. The plant expression vector with ASS gene were introduced into the Arabidopsis to obtain transgenic Arabidopsis by Agrobacterium-mediated transfrmation, It provided essential data for further study of ASS gene.