澳洲香水石斛花香成分及DeTPS6的克隆与表达

Volatile Profiling,Cloning and Expression Analysis of DeTPS6 in Dendrobium Aozhouxiangshui

花香是评价石斛商业价值的重要性状之一。为探究石斛花香物质的形成机理,本研究采用顶空固相微萃取-气相色谱-质谱联用法(HS-SPME-GC-MS)、转录组测序和PCR技术分析了不同花期澳洲香水石斛花香物质变化及其与萜类合成关键酶基因表达间的关系。结果表明,澳洲香水石斛中共检测到17种花香物质,其中1,4-二甲氧基苯、α-蒎烯和苯乙醇的含量相对较高,盛花期的花香释放量呈现明显的昼夜节律变化和组织特异性。基于转录组数据,筛选到77个与萜类合成相关的差异表达基因(DEGs),并成功克隆DeTPS6的全长,为1749 bp。DeTPS6蛋白具有萜类合成酶家族的保守结构域,属于TPS-b亚家族。亚细胞定位分析结果显示,DeTPS6定位于胞质。实时荧光定量PCR(qRT-PCR)分析结果显示,DeTPS6在澳洲香水石斛的盛花期中高表达,且呈现显著的昼升夜降的变化趋势,与花香物质的含量呈正相关。本研究为DeTPS6生物学功能的探讨提供了理论基础。

Floral aroma is a key trait for evaluating the commercial value of Dendrobium.To unveil the formation mechanism of floral aroma in Dendrobium Aozhouxiangshui during different florescences,the change of volatile compounds and its relation to terpenoid synthase gene expression were investigated by headspace solid-phase microextraction-gas chromatography-mass spectrometry(HS-SPME-GC-MS),transcriptomes,and PCR technology.The results showed that a total of 17 volatile compounds were identified.Among them,the contents of 1,4-dimethoxybenzene,alpha-pinene,and phenylethyl alcohol were relatively high.The release of floral aroma during the blooming stage exhibited significant circadian rhythm and tissue specificity.Based on RNA sequencing analysis,77 differentially expressed genes related to terpenoid synthesis were screened,and the full length of DeTPS6(1749 bp)was successfully cloned.DeTPS6 contained the conserved domains of the terpenoid synthase family and belonged to the TPS-b subfamily.Subcellular localization analysis revealed that DeTPS6 was localized in the cytoplasm.Quantitative real-time PCR(qRT-PCR)analysis showed that DeTPS6 was highly expressed at the flowering stage with a significant circadian rhythm,which also showed a positive correlation with aroma components content.This study lays the theoretical basis for exploring the biological functions of DeTPS6.