薰衣草DXS基因的克隆、表达分析及原核表达

Cloning,Expression Analysis and Prokaryotic Expression of Lavender DXS Gene

【目的】研究拟克隆薰衣草DXS基因,并分析其表达,为揭示该基因在调控薰衣草萜类物质合成中的分子机理提供研究基础。【方法】以薰衣草杂花为试材,同源克隆薰衣草DXS基因,进行基因序列分析、表达量比较和原核表达。【结果】(1)薰衣草DXS基因开放阅读框长为2181 bp,编码由726个氨基酸组成的蛋白质序列;薰衣草DXS蛋白等电点为6.57,分子量约为78.39 KDa,具有高度的保守性,与狭叶薰衣草、冬凌草、毛喉鞘蕊花的DXS蛋白亲缘关系相近;(2)DXS基因在杂花花器官的衰败期表达量最高,在法国蓝花器官的盛开期表达量最高,DXS基因在杂花花器官五个不同发育时期的表达量均高于法国蓝;DXS基因在杂花花萼中表达量最高,在法国蓝雄蕊中表达量最高,DXS基因在杂花花器官5个不同组织表达量均高于法国蓝(雌蕊、雄蕊除外);(3)在37℃、IPTG 0.8 mM条件下诱导4 h后,DXS蛋白表达量最大。【结论】DXS基因表达量与薰衣草精油产量存在正相关关系。

【Objective】This study intends to clone the lavender DXS gene and analyze its expression preliminarily in the hope of providing a research basis for revealing the molecular mechanism of this gene in regulating lavender terpenoid synthesis.【Methods】Lavender"Zahua"was taken as the testing material to clone lavender DXS gene,and then gene sequence analysis was conducted,expression comparison performed and prokaryotic expression finished.【Results】(1)The open reading frame length of lavender DXS gene was 2,181 bp,encoding a protein sequence consisting of 726 amino acids,with an isoelectric point of 6.57 and a molecular weight of about 78.39 KDa.Lavender DXS protein was highly conserved and closely related to the DXS protein of Lavandula angustifolia,Isodon rubescens,and Plectranthus barbatus.(2)The expression level of DXS gene was the highest in the decaying period of Zahua organs and the highest in the blooming period of French blue flower organs.The expression level of DXS gene in the five different developmental stages of Zahua organs was higher than that of French blue.The expression level of DXS gene was the highest in the calyx of Zahua and the highest in the stamens of French blue.The expression level of DXS gene in five different tissues of hybrid flowers was higher than that in French blue(except pistil and stamen).(3)The results of prokaryotic expression analysis showed that the expression of DXS protein was the largest after 4 h induction at 37℃and IPTG 0.8 mM.【Conclusion】There is a positive correlation between DXS gene expression and lavender essential oil production.The results of this study provide material basis and experimental conditions for further confirming the function of DXS gene in the metabolism of lavender terpenoids.