桂花萜烯合酶(TPS)的生物信息学与原核表达分析

Bioinformatic and prokaryotic expression analysis of terpene synthase (TPS) from Osmanthus fragrans

挥发性萜烯类化合物是植物花和果实中重要的香气活性物质,萜烯合酶(TPS)的种类和功能决定物种中萜烯类物质的多样性。桂花作为重要的香花植物,萜烯类物质是其花香的重要成分,但有关桂花萜烯合酶的研究却并不多。为揭示桂花萜烯类物质的生物合成机理,该研究利用生物信息学分析了4个TPS蛋白的理化性质、亚细胞定位及其结构,在原核表达系统中进行4个TPS蛋白的体外表达,并对可溶性的TPS4重组蛋白进行了体外酶反应功能分析。结果表明:(1) 4个TPS蛋白的理化性质差异较小,但仅有TPS4蛋白定位于其他靶向,不含信号肽,延伸链的比例较低,在靠近氨基端的区域内不含延伸链。(2) 4个TPS蛋白在原核系统中均可成功表达,但仅有TPS4获得了可溶性重组蛋白。(3)将纯化的TPS4重组蛋白分别与GPP、NDP和FPP进行反应,均只检测到一种产物,分别为反式-β-罗勒烯、β-水芹烯和α-法呢烯。这为揭示植物萜烯类物质生物合成的分子机理提供了参考,对从蛋白水平研究桂花花香基因功能奠定了基础。

Volatile terpenes are important aroma active compounds in flowers and fruits of plant. Diversities of terpenes are usually determined by type and function of terpene synthase in different species. Osmanthus fragrans is an important fragrant plant, in which terpenes are the important components of floral scent. But there are few studies on terpene synthase in O. fragrans . To reveal the biosynthesis mechanism of terpenes in O. fragrans, we predicted the physicochemical properties, subcellular localization and structure of four TPS proteins by bioinformatics, and expressed them in prokaryotic expression system. Finally, the function of soluble TPS4 recombinant protein was analyzed by enzyme reaction in vitro . The results were as follows:(1) The physicochemical properties of the four TPS proteins had relatively little difference. Only TPS4 protein locating in other targets without signal peptide had low proportional extended strand and no extended strand near amidogen terminal.(2) All of the four TPS proteins were successfully expressed in prokaryotic system, but only TPS4 obtained soluble recombinant protein.(3) The purified TPS4 recombinant protein was reacted with GPP, NDP and FPP respectively, and only one product (trans-β-ocimene,β-phellandrene and α-farnesene) was detected. The results provide reference for functional analysis of floral scent related gene at protein level in O. fragrans and for revealing the molecular mechanism of terpenes biosynthesis in plant.