桔梗DXS基因的原核表达、亚细胞定位和酶活性分析

Prokaryotic expression,subcellular localization and enzymatic activity analysis of DXS gene from Platycodon grandiflorum

1-脱氧-D-木酮糖-5-磷酸合酶(1-deoxy-D-xylulose-5-phosphatesynthase,DXS)是甲基赤藓醇磷酸(2-methyl-D-erythritol-4-phosphate,MEP)途径中催化3-磷酸甘油醛和丙酮酸缩合生成1-脱氧-木酮糖-5-磷酸(DXP)的第一个关键酶。本研究通过反转录·聚合酶链反应(RT-PCR)从桔梗根中克隆得到PgDXS1、PgDXS2、PgDXS3基因,开放阅读框(ORF)全长分别为2160、2208和2151 bp,分别编码719、735、716个氨基酸,同源氨基酸序列分析表明桔梗与橡胶树、曼陀罗及甜菊的DXS基因编码的氨基酸序列具有较高同源性;进一步构建原核表达载体pET-28a-PgDXSs,转化至大肠杆菌BL21(DE3)感受态细胞,成功诱导3个DXS蛋白的表达。亚细胞定位结果显示,PgDXS1和PgDXS2蛋白主要定位于叶绿体中,PgDXS3蛋白定位于叶绿体、细胞核和细胞质中。运用实时荧光定量PCR检测3个DXS基因在2个产区不同组织中的表达水平,结果表明3个DXS基因均在太和桔梗叶中高表达。在茉莉酸甲酯(MeJA)诱导处理下,PgDXS1、PgDXS2、PgDXS3基因表达水平在不同诱导的时间点(3~48 h)呈现先降低后增高的趋势;酶活性检测结果显示:桔梗的3个组织中DXS酶活性在MeJA处理后不同的时间点也呈现先增加后降低的趋势。本研究为进一步阐明桔梗中萜类化合物合成途径PgDXS基因的生物学功能提供参考。

1-Deoxy-D-xylulose-5-phosphate synthase(DXS),the first key enzyme in 2-methyl-D-erythritol-4-phosphate(MEP)pathway,catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxyxylose-5-phosphate(DXP).In this study,PgDXS1,PgDXS2,and PgDXS3 genes were cloned from the root of Platycodon grandiflorum(P.grandiflorum).The open reading frame(ORF)of PgDXS1,PgDXS2,and PgDXS3 were 2160,2208,and 2151 bp in full length,encoding 719,735,and 716 amino acids,respectively.Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis,Datura stramonium and Stevia rebaudiana.The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli(E.coli)BL21(DE3)cells,and the induced proteins were successfully expressed.Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts,and PgDXS3 was located in chloroplasts,nucleus and cytoplasm.The expression of three DXS genes in different tissues of two producing areas of P.grandiflorum were assayed via real-time fluorescence quantitative PCR,and the results showed that all of them were highly expressed in leaves of P.grandiflorum from Taihe.Under methyl jasmonate(MeJA)treatment,the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points(3-48 h),and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P.grandiflorum.This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P.grandiflorum.