Cloning, Expression And Enzymology Character Of Sesquiterpene Synthase Gene From Aquilaria Sinensis

Agarwood is a traditional Chinese rare medicinal herb, which is the black resinous portion from xylogen in the tree of Aquilaria sinensis(Lour.) Gilg. Aquilaria sinensis growth normal in the natural conditions, the xylem is white, soft and no flavor. the agarwood formation from the inside black resin When they are in the external condition such as wind, lightning strikes,the gnawing of ants or insects. The main medicinal composition in Agarwood is sesquiterpene, Modern Medicine confirmed that sesquiterpenes in agarwood showed obvious pHysiological activity to nervous system, Since the sesquiterpene synthase play a key role in the sesquiterpene biosynthetic pathway in Agargwood, the study on sesquiterpene synthase would be significant.In this study, The sesquiterpene synthase genes As-SesTPS and As-SesTPS1 were cloned from agarwood for the first time, and the expression level of the two genes were detected by qRT-PCR.Then As-SesTPS and As-SesTPS1 were expressed in E.coil,and the enzymatic property were analyzed by GC-MS. The main results obtained are as follows:A sesquiterpene synthase gene denoted As-SesTPS1 were obtained from total RNA of Aquilaria sinensis based on previous transcriptome sequencing data, the sequence contained an ORF of 1671 bp, encoding 556 amino acids and had conserved motifs of RRx8 W and DDxxD. According to bioinformatics analysis results, we found that As-SesTPS1 shared high similarity with other sesquiterpene synthase. We predicted that this gene is a new sesquiterpene synthase and it encodes new sesquiterpene synthase with different structure.Using Histone as reference gene, Analysis The expression levels of As-Ses TPS and As-Ses TPS1 in different positions of Aquilaria sinensis by qRT-PCR technique. It was confirmed that the gene was expressed in agarwood with high level and rarely no expressed in wood sample.The gene As-SesTPS and As-SesTPS1 Play a key role in the formation of agarwood.The two genes were inserted into Prokaryotic expression vector pET28 a, and recombiant plasmid pET-28a-As-SesTPS-BL and pET-28a-As-SesTPS1-BL were succefully expressed in E.coli. According to the results of SDS-PAGE、Western-blot and amino acid sequencing, the protein As-SesTPS and As-SesTPS1 were expressed in the form of inclusion bodies;The optimal induction condition is 1 mM IPTG, 37 ℃,6 h. The protein sesquiterpene synthase encoding by As-SesTPS and As-Ses TPS1 with enzymatic activity were obtained after purification and refolding process.The sesquiterpene synthases As-SesTPS catalyzed the substrate FPP, and three sesquiterpene product were obtained, they are Nerolidol, β-Bisabolene and Humulene. Nerolidol is the major product, which is also the characterized component of agarwood. And the optimal enzymatic reaction condition in different temperature,p H and time were obtained.This study not only provide dates in the similarity and characteristic difference between sesquiterpene synthases in Agarwood for future lucubrate, but also lay the foundation of improve the content of sesquiterpenes in Aquilaria sinensis and research of metabolism of Agarwood in gene level. The study on sesquiterpene synthases of A.sinensis will lay a foundation for the further investigation on sesquiterpene biosynthesis pathway and molecular mechanisms in the formaiton of agarwood.