白木香倍半萜合酶基因AsVS的克隆与表达分析

Cloning and Expression Analysis of Sesquiterpene Synthase Gene AsVS in Aquilaria sinensis

法尼基焦磷酸(farnesyl diphosphate, FPP)是植物萜类化合物合成途径的重要前体之一,经不同的酶催化可形成各类萜类化合物,Vetispiradiene合酶可催化FPP形成Solavetivone和Lubimin的前体Vetispiradiene。本研究利用白木香转录组数据,首次克隆了编码白木香Vetispiradiene合酶的基因(命名为As VS, Genbank登录号为MH378283),AsVS基因序列全长为1 632 bp,编码543个氨基酸,该氨基酸序列与马来沉香倍半萜合酶聚类于同一分支,具有较近的亲缘关系,该蛋白植物倍半萜的保守性结构域DDXXD,NST/DTE和R(R)X8W,相对分子量为62.73 kD,理论等电点为5.51,包含40个磷酸化位点;二级结构以α螺旋和无规则卷曲为主;不含跨膜结构域。RT-qPCR分析结果表明AsVS基因在结香剂处理过程中于白木香茎干样品中持续上调表达,其第6天及第9天的表达量分别为对照处理的2.36和5.02倍。此结果为进一步研究As VS基因在白木香萜类化合物的合成及沉香致香成分富集中的功能提供了理论基础。

FPP (Famesyl diphosphate), one of most pivotal precursor in plant terpene synthase pathway, can be converted to various terpenes via plant enzyme systems. EAS and its isozyme, VS, can catalyze FFP to 5-epiaristolochene and vetispiradiene. In this study, we cloned a full length of cDNA coding vetispiradiene synthase in Aquilaria sinensis based on the annotation of its transcriptome database. This gene was named it for AsVS and its accession number in GenBank is MH378283. AsVS gene inculding 1 632 base pairs which encoded 543 amino acid residues. There was more homology of protein between lx I S and sesquiterpene synthase from Aquilaria malaccensis and this result was then identified by evolution analysis with MEGA X. In addition, As VS protein contained three conservative structures which were DDXXD, NST/DTE and R(R)X8W. Bioinfbrmatic analysis reveals that MW and pl of 4s VS were 62.73 kD and 5.51, and it is a non-secretory protein and contained 40 phosphorylation sites. The secondary structure of AsVS were mainly mixed a-helix and random coil. The transcript profiles of demonstrated that the relative expression of AsVS was continuous increased in stem of4. sinensis during artificial induce agarwood formation, its relative expression in stem of A. sinensis after treatment with 6 day and 9 day were increased 2.36 and 5.02 than the control treatment. This study offered the functions for indicating AsVS in multi-types terpenes synthesis and regulatory mechanism of aroma components of eaglewood formation in A quilaria spp.