bHLH96的克隆及其在薄荷萜烯生物合成调控中的功能

Cloning of bHLH96 Gene and Its Roles in Regulating the Biosynthesis of Peppermint Terpenes

【目的】薄荷精油是日化、医药和食品工业中的重要原料,bHLH转录因子在调控植物挥发性次生代谢产物合成中具有重要作用。探究bHLH蛋白在薄荷挥发性物质合成调控中的作用,为通过代谢或基因工程改良薄荷种质提供重要基因资源,拓展有关植物挥发性次生代谢产物合成途径与调控机制的认识。【方法】利用转录组测序和RT-qPCR技术分析bHLH96在薄荷根、茎、叶中的表达,并通过RT-PCR法克隆其全长序列,再利用DNA重组技术构建植物表达载体pBI121-bHLH96,利用农杆菌介导法在薄荷叶片中过表达,检测过表达bHLH96对薄荷叶片萜烯化合物含量和合成相关基因表达的影响。【结果】薄荷bHLH96的编码区全长861 bp,编码286个氨基酸残基。薄荷bHLH96是一个细胞核定位蛋白,植物bHLH96蛋白间的序列相似性为45.45%-73.68%,薄荷bHLH96与薰衣草LaMYC4和丹参SmbHLH94等唇形科植物bHLH蛋白的亲缘关系较近。在薄荷叶片中瞬时过表达bHLH96,显著影响15种萜烯化合物的相对含量。过表达bHLH96显著上调萜烯合成相关基因OC2、SM1、KS1、ND和EAO1的表达,显著下调NLS1、LS1和iPR的表达。【结论】薄荷bHLH96可能通过调控萜烯合成相关基因的表达,影响相关萜合成酶的活性,从而调控薄荷萜烯物质的合成。

【Objective】Peppermint essential oil is an important raw material in the cosmetics,pharmaceutical,and food industries.bHLH transcription factors(TFs)play a crucial role in regulating the biosynthesis of volatile secondary metabolites(VSMs)in plants.Exploring the role of bHLH TFs in the regulation of VSM biosynthesis in peppermint may provide important gene resources for improving peppermint germplasm through metabolic or genetic engineering,and expand our understanding of the pathways and regulatory mechanisms of plant VSM biosynthesis.【Method】The expression pattern of bHLH96 in peppermint roots,stems,and leaves was analyzed using transcriptome sequencing and qRT-PCR techniques.The full-length of bHLH96 coding sequence(CDS)was cloned using the RT-PCR method.Subsequently,a plant expression vector,pBI121-bHLH96,was constructed using DNA recombination technology,and the overexpression of bHLH96 in the peppermint leaves was achieved through Agrobacterium-mediated transformation.The effects of bHLH96 overexpression on the content of terpenoid compounds and the expression of related genes involved in terpenoid biosynthesis in the peppermint leaves were examined.【Result】The results showed that the full length of bHLH96 CDS was 861 bp encoding 286 amino acids.Peppermint bHLH96 protein localized at the nucleus,and the sequence similarity among plant bHLH96 proteins ranged from 45.45%to 73.68%.Peppermint bHLH96 had a close relationship with plant bHLH proteins from the Labiatae family such as Lavandula angustifolia LaMYC4 and Salvia miltiorrhiza SmbHLH94.Transient overexpression of the bHLH96 gene in peppermint leaves significantly influenced relative contents of 15 terpenoid compounds.bHLH96 overexpression significantly upregulated the expressions of 5 terpene synthesis-related genes,such as OC2,SM1,KS1,ND,and EAO1,while downregulated the expressions of NLS1,LS1 and iPR.【Conclusion】Peppermint bHLH96 might regulate terpene synthesis by modulating the expressions of terpene synthesis-related genes and influencing the activities of terpene synthases.