Cloning Of The Sesquiterpene Synthase Gene In Aquilaria Sinensis And The Function Analysis Of Its Promoters

Agarwood is an expensive resinous heartwood derived from Aquilaria plants that is widely used in traditional medicines, incense and perfume. Studies have shown that sesquiterpenes derivatives are the main compounds in agarwood. Thus, understanding the biosynthesis and regulation of sesquiterpenes in Aquilaria spp. is critically important in determining the mechanism of agarwood formation. Sesquiterpene synthases are enzymes used in the final step to form sesquiterpenes. Currently, many studies on the functional identification and regulation of these enzymes have been reported, but biosynthesis and regulation of sesquiterpenes in Aquilaria sinensis are still unknown. In this work, two genes encoding sesquiterpene synthases (designated as AsSGS and AsSXS) were isolated from A. sinensis. The function of their promoters in A. sinensis was also investigated. The present study perhaps contributes towards an understanding of the biosynthesis of sesquiterpenes in A. sinensis.AsSGS was2342bp in length. There were7exons and6introns in AsSGS. AsSGS contained a1644bp open reading frame encoding547amino acids. The deduced AsSGS protein was predicted to possess the conserved RRx8W and DDxxD domain. The amino acid sequence of AsSGS was aligned with those of the other plant sesquiterpene synthase proteins that exhibit high sequence identities. AsSGS is a member of the guaiene synthase family. AsSXS was2156bp in length.There were6exons and5introns in AsSXS, AsSXS contained a1668bp open reading frame encoding555amino acids, The deduced AsSXS protein was predicted to possess the conserved RRx8W and DDxxD domain. The amino acid sequence of AsSXS was aligned with those of the other plant sesquiterpene synthase proteins that exhibit high sequence identities. AsSXS maybe a novel member of sesquiterpene synthase family.The791bp and1066bp promoter region of AsSGS and AsSXS were isolated using PCR-based DNA Walking. TATA box and other core configurations were found in promoter regions. Several sequences similar to eukaryotic m-regulatory element, such as TATA box, CAAT-box, GARE-motif, G-Box, TGA-element, ABRE, W-box, Box I, were found in the AsSGS and AsSXS promoter regions. A promoter deletion analysis revealed that the AsSGS and AsSXS drive specific expression of the GUS gene using Agrobacterium-mediated transient expression system. The AsSGS promoter was positively regulated expression by MeJA, GA, ABA and SA. The AsSGS promoter was up-regulated expression by MeJA, Eth and IAA.