杜鹃花TPS基因家族鉴定及与萜类物质代谢的关系分析

Identification of terpene synthase gene family members in Rhododendron and its relationship with terpenoid metabolism

摘要:

萜烯合成酶（terpene synthase，TPS）能催化不同的前体物质生成不同的萜类化合物，是合成萜类物质的关键酶。为探究杜鹃花TPS基因家族成员在萜类物质代谢过程中的表达模式，本文基于杜鹃花基因组数据库，利用生物信息学方法对杜鹃花TPS基因（TPS）进行家族成员鉴定；通过云锦杜鹃和诺娃杜鹃两种不同种高山杜鹃的转录组测序结果，结合qRT-PCR、顶空固相微萃取和气相色谱-质谱联用技术，分析两种杜鹃不同发育时期花瓣中TPS家族成员表达水平和代谢物含量变化关系。结果表明，从杜鹃花基因组数据库中共鉴定获得47个RsTPS成员，RsTPS家族成员长度在591-2 634 bp之间，含有3-12个外显子不等，编码196-877个氨基酸；RsTPS家族成员主要分布在叶绿体和细胞质；系统进化分析结果显示RsTPS基因分为5个亚组。通过分析转录组数据得到7个功能注释为TPS的基因家族成员，发现TPS1、TPS10、TPS12和TPS13的表达量在4个时期中呈现出先上升，到盛开期达到顶峰后再下降的趋势。对基因表达量变化与萜类物质含量变化进行相关性分析，发现TPS1、TPS4、TPS9、TPS10、TPS12和TPS13表达量与云锦杜鹃不同时期花瓣中萜类物质含量变化呈显著性正相关，推测这6个基因家族成员可能是参与云锦杜鹃花香调控的关键基因。

关键词:杜鹃花;TPS基因家族;生物信息学;表达分析;挥发性萜类物质

Abstract:

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron ‘Nova Zembla’. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.

Key words:Rhododendron;TPS gene family;bioinformatics;expression analysis;volatile terpenoids