香雪兰萜类合酶基因FhTPS4的克隆及功能鉴定

发布时间：2018-05-20 16:22

本文选题：香雪兰 + 萜类化合物　；参考：《东北师范大学》2017年硕士论文

【摘要】：植物挥发性成分组成复杂,主要由萜类化合物及其衍生物构成。萜类物质的合成和释放对植物在进化过程中适应复杂多变的环境具有重要的意义。在挥发性萜类物质的生物合成过程中,萜类合酶(terpene snythases,TPS)起到至关重要的作用。它们可以催化香叶基二磷酸(geranyl diphoshate,GPP)、法尼基二磷酸(farnesyl diphoshate,FPP)、香叶基香叶基二磷酸(geranylgeranyl diphoshate,GGPP)等底物,生成种类丰富的萜类化合物。目前,对于植物TPS基因家族及萜类物质代谢的研究主要集中在双子叶模式植物以及部分重要的农作物上,而对揭示进化过程中TPS基因功能多样性有重要作用的单子叶植物却研究较少。香雪兰(Freesia hybrida)属鸢尾科香雪兰属,是重要的单子叶园艺花卉。其花香浓郁,馥郁芬芳,花期较长,是研究植物挥发性萜类物质代谢的优良材料。本研究基于香雪兰常见栽培种(Red River?)转录组数据,利用RACE-PCR技术成功从中克隆得到一个萜类合酶基因FhTPS4,cDNA全长为1701 bp,编码566个氨基酸。生物信息学分析表明,其氨基酸序列具有萜类合酶家族典型的DDXXD和NSE/DET保守结构域。进化树分析发现,FhTPS4聚类于TPS-g亚家族。TPS-g亚家族的TPS多具有双功能的萜类合酶催化性质,既能催化GPP合成单萜物质,又能催化FPP合成倍半萜物质。研究表明,TPS-g亚家族的TPS多数定位于叶绿体,本研究通过原生质体瞬时转染技术证明FhTPS4也定位于叶绿体中,暗示其参与了控制单萜物质合成的质体代谢途径(MEP pathway)。此外,体外酶活实验结果显示FhTPS4可催化GPP和FPP底物分别生成沉香醇和橙花叔醇,即FhTPS4同时具有单萜合酶和倍半萜合酶两种功能。为了进一步探究FhTPS4在香雪兰植物体内的功能,通过Real-time PCR分析FhTPS4基因时空表达模式,结合前期对香雪兰花朵挥发性成分分析证明,香雪兰体内FhTPS4的功能是催化花托和花萼中沉香醇的合成。
[Abstract]:The volatile components of plants are complex, mainly composed of terpenoids and their derivatives. The synthesis and release of terpenoids play an important role in the evolution of plants. Terpene synthase (TPS) plays an important role in the biosynthesis of volatile terpenes. They can catalyze the substrates of geranyl diphoshatetate, farnesyl diphoshatefen, geranylgeranyl diphoshatetate (GGPP) and other substrates to form terpenoids of rich species. At present, the studies of plant TPS gene family and terpene metabolism are mainly focused on dicotyledonous model plants and some important crops. However, there are few studies on monocotyledonous plants that play an important role in revealing the functional diversity of TPS gene during evolution. Freesia hybrida is an important monocotyledonous horticultural flower. It is an excellent material for studying the metabolism of volatile terpenes in plants. This study is based on Red Rivera, a common cultivated species of Xanthoxylum sinensis. Transcriptome data were used to clone a terpene synthase gene FhTPS4 cDNA with a length of 1701 BP encoding 566 amino acids. Bioinformatics analysis showed that its amino acid sequence had the conserved domains of DDXXD and NSE/DET of terpene synthase family. Phylogenetic tree analysis showed that the TPS clustered in the TPS-g subfamily. TPS-g subfamily had bifunctional terpene synthase catalytic properties, which could catalyze the synthesis of monoterpenes from GPP and the synthesis of sesquiterpenes by FPP. The results showed that most of TPS of TPS-g subfamily was located in chloroplast. In this study, we demonstrated that FhTPS4 was located in chloroplast by protoplast transient transfection technique, suggesting that FhTPS4 was involved in the plastid metabolic pathway that controlled the synthesis of monoterpenes. In addition, the results of enzyme activity in vitro showed that FhTPS4 could catalyze GPP and FPP substrates to produce amethanol and nerolol, that is, FhTPS4 had two functions: monoterpene synthase and sesquiterpene synthase. In order to further explore the function of FhTPS4 in the plant, the expression pattern of FhTPS4 gene in time and space was analyzed by Real-time PCR. The function of FhTPS4 is to catalyze the synthesis of amarol in receptacle and calyx.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

【参考文献】