钩吻看家基因筛选及生物碱合成相关酶基因的表达分析

Screening of housekeeping genes in Gelsemium elegans and expression patterns of genes involved in its alkaloid biosynthesis

摘要:

钩吻是我国的一种传统中草药，具有重要的药用价值，萜类吲哚生物碱为其主要的活性组分。为了研究钩吻不同部位最适看家基因，以钩吻4个不同部位根皮、茎段、叶片和花序为材料，通过实时荧光定量PCR技术(quantitative real-time PCR, qRT-PCR)，以及GeNorm、NormFinder、BestKeeper、ΔCT和RefFinder软件对10个候选看家基因(18S、GAPDH、Actin、TUA、TUB、SAND、EF-1α、UBC、UBQ和cdc25)进行表达稳定性评估。结果显示，EF-1α在钩吻的4个部位中均稳定表达，为最适的看家基因。同时，为了研究与生物碱合成相关基因的表达情况，基于“基因组+全长转录组+代谢组”共表达模式筛选出钩吻生物碱合成通路上18个相关基因(AS、AnPRT、PRAI、IGPS、TSA、TSB、TDC、GES、G8H、8-HGO、IS、7-DLS、7-DLGT、7-DLH、LAMT、SLS、STR和SGD)，以看家基因EF-1α为参照，利用qRT-PCR技术对这18个候选基因表达情况进行分析，结果发现这些基因的表达量与钩吻素己含量变化呈相关性，推测其可能参与钩吻生物碱钩吻素己的合成。

关键词:钩吻;实时荧光定量PCR;看家基因;萜类吲哚生物碱;钩吻素己

Abstract:

Gelsemium elegans is a traditional Chinese herb of medicinal importance, with indole terpene alkaloids as its main active components. To study the expression of the most suitable housekeeping reference genes in G. elegans, the root bark, stem segments, leaves and inflorescences of four different parts of G. elegans were used as materials in this study. The expression stability of 10 candidate housekeeping reference genes (18S, GAPDH, Actin, TUA, TUB, SAND, EF-1α, UBC, UBQ, and cdc25) was assessed through real-time fluorescence quantitative PCR, GeNorm, NormFinder, BestKeeper, ΔCT, and RefFinder. The results showed that EF-1α was stably expressed in all four parts of G. elegans and was the most suitable housekeeping gene. Based on the coexpression pattern of genome, full-length transcriptome and metabolome, the key candidate targets of 18 related genes (AS, AnPRT, PRAI, IGPS, TSA, TSB, TDC, GES, G8H, 8-HGO, IS, 7-DLS, 7-DLGT, 7-DLH, LAMT, SLS, STR, and SGD) involved in the Gelsemium alkaloid biosynthesis were obtained. The expression of 18 related enzyme genes were analyzed by qRT-PCR using the housekeeping gene EF-1α as a reference. The results showed that these genes’ expression and gelsenicine content trends were correlated and were likely to be involved in the biosynthesis of the Gelsemium alkaloid, gelsenicine.

Key words:Gelsemium elegans;qRT-PCR;housekeeping gene;terpenoid indole alkaloids;gelsenicine