广藿香FPPS基因原核表达及茉莉酸甲酯对FPPS表达量的影响

Prokaryotic expression of FPPS gene from Pogostemon cablin and the effect of methyl jasmonate on the expression of FPPS

关键词:  广藿香, 法尼基二磷酸, 无缝克隆, 茉莉酸甲酯, 荧光定量PCR

Key words:  Pogostemon cablin, farnesyl pyrophosphate, seamless cloning, methyl jasmonate, qPCR

摘要:

法尼基焦磷酸合酶(farnesyl diphosphate synthase, FPPS)是广藿香甲羟戊酸途径中萜类物质生物合成的关键酶,其催化异戊二烯焦磷酸(IPP)和二甲基烯丙基焦磷酸(DMAPP)合成萜类物质前体法尼基焦磷酸。为了进一步研究广藿香萜类合成途径的分子机制,该文通过逆转录聚合酶链式反应获得FPPS基因的cDNA序列,利用生物信息学软件预测FPPS编码蛋白的理化性质、结构和功能。结果表明:(1)该序列的开放阅读框全长1 050 bp,编码349个氨基酸,预测分子量为40 KD,等电点为5.43,存在一个结构域,参与异戊二烯化合物的合成,不存在信号肽,亚细胞定位于细胞质; 系统发育分析结果显示,广藿香FPPS氨基酸序列和丹参(Salvia miltiorrhiza)、撒尔维亚(S. officinalis)的氨基酸序列亲缘关系最近。(2)使用无缝克隆技术构建pET-32b-FPPS原核表达载体,并导入菌株BL21(DE3)中,考察不同浓度异丙基-β-D-硫代半乳糖苷(IPTG)对诱导融合蛋白的表达量的影响。结果发现融合表达蛋白以包涵体形式存在沉淀中,4个浓度的IPTG诱导蛋白表达效果差异不明显。(3)采用荧光定量技术分析0.10、0.25 mmol·L^-1 MeJA对FPPS基因表达水平的影响,发现0.10 mmol·L^-1MeJA诱导后FPPS基因的表达量趋势是先升高后降低再升高再降低; 0.25 mmol·L^-1 MeJA诱导后FPPS基因的表达量趋势是先降低后升高再降低。因此,推测植物体内MeJA浓度的变化能影响FPPS基因的表达,高浓度具抑制作用,低浓度具促进作用。本研究为广藿香萜类合成途径的研究奠定基础,以及为后续基因功能验证提供理论参考。

Abstract:

Farnesyl diphosphate synthase(FPPS), a key enzyme for terpene biosynthesis in patchouli mevalonate pathway, catalyzes isoprene pyrophosphate(IPP)and dimethylallyl diphosphate pyrophosphate(DMAPP)into farnesyl pyrophosphate. In order to study the molecular mechanism of terpenoid synthesis pathway in patchouli, this study obtained the cDNA sequence of the FPPS gene by reverse transcription, and used bioinformatics softwares to predict the physicochemical properties, structure and function of the protein encoded by FPPS. The results were as follows:(1)The open reading frame of the sequence is 1 050 bp in length and encodes 349 amino acids. The predicted molecular weight of the expressed protein is 40 KD. The isoelectric point of the protein is 5.43. And there is a domain involved in the synthesis of isoprene compounds. There is no signal peptide in the protein, and its subcellular localization is in the cytoplasm. Phylogenetic analysis showed that the amino acid sequence of patchouli FPPS is closest to the amino acid sequence of Salvia miltiorrhiza and S. japonica.(2)In order to study the expression of FPPS protein, the effects of different concentrations of isopropyl-β-D-thiogalactoside on the expression of the fusion protein were examined. The results indicated that fusion-expressed protein was present in the form of inclusion bodies. And there was no significant differences in the expression of protein induced by four concentrations of IPTG.(3)In order to study the effects of methyl jasmonate(MeJA)on the expression of FPPS, this experiment used fluorescence quantitative technology to analyze the effects of 0.10 and 0.25 mmol·L^-1 MeJA on the expression level of FPPS gene. The results found that the expression of FPPS gene after 0.10 mmol·L^-1 MeJA induction was first increased, then decreased, then increased and then decreased. However, after 0.25 mmol·L^-1 MeJA induction, the expression of FPPS gene tended to decrease first, then increase and then decrease. It is speculated that high concentrations of MeJA have an inhibitory effect, and low concentrations of MeJA have a promoting effect. This study lays the foundation for the research on terpenoid synthesis pathways of patchouli and provides a theoretical reference for subsequent gene function verification.