冬凌异冬草二萜类生物合成途径两个关键酶的分子克隆与特性研究

Xiuhong Su, Lei Yin, Sui-qin Chen
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引用次数: 1

摘要

冬凌草是一种重要的药用植物,含有多种萜类化合物。这种植物的活性成分主要是具有抗肿瘤特性的冬凌草甲素。作为草甲素生物合成的前体,通过MEP途径合成。在前期研究的基础上,我们分离并克隆了MEP途径中催化二萜类生物合成的两个重要基因。2- c -甲基- d -赤藓糖醇2,4-环二磷酸合成酶和4-羟基-3-甲基-丁-2-烯基二磷酸还原酶分别是甲基赤藓糖醇磷酸(MEP)途径的第五酶和最后一步关键酶,对类异戊二烯生物合成具有重要的调控作用。序列分析显示,DcIspF (accession no. 5)为。KT948057)全长966 bp,包含708 bp的基因开放阅读框(ORF),属于mecdp -合成酶超家族;DcIspH (accession no KT948058)包含1389 bp的ORF,编码462个氨基酸多肽,是lytB_ispH超家族的成员。推导出的DcIspF和DcIspH氨基酸序列分别与其他植物的DcIspF和DcIspH具有较高的相似性,每个序列都具有一个n端传递肽和保守的氨基酸位点。实时荧光定量PCR分析表明,DcIspF在叶片中的表达量较高,在愈伤组织中表达量最低。这些结果表明,我们已经鉴定出具有功能的DcIspF和DcIspH酶,它们可能在冬凌草二萜类化合物的生物合成中发挥关键作用。
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Molecular Cloning and Characterization of Two Key Enzymes involved inthe Diterpenoid Biosynthesis Pathway of Isodon rubescens
Isodon rubescens, an important medical plant, contains various terpenoids. This plant’s active compounds are primarily oridonin with antitumor properties. As the precursor for oridonin biosynthesis, are synthesized by MEP pathway. On the basis of our earlier studies, we isolated and cloned two important genes catalyzing diterpenoid biosynthesis in the MEP pathway. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and 4-hydroxy-3- methylbut-2-enyl diphosphate reductase are the fifth enzymes and the last step key enzyme for the methylerythritol phosphate (MEP) pathway, respectively, which is important for the regulation of isoprenoid biosynthesis. Sequence analysis revealed that DcIspF (accession no. KT948057) was 966 bp, contains a gene open reading frame (ORF) of 708 bp belonging to the MECDP-synthase superfamily and DcIspH (accession no KT948058) contains a 1389 bp ORF encoding a predicted 462 amino acid polypeptides as a member of the lytB_ispH superfamily. The deduced DcIspF and DcIspH amino acid sequences shared high similarity with DcIspF and DcIspH of other plant respectively, each of them exhibiting an N-terminal transit peptide and conserved amino acid sites. Quantitative real-time PCR analysis showed that the expression of DcIspF was considerably higher in leaves, the lowest in callus. These results indicate that we have identified functional DcIspF and DcIspH enzymes, which may play a pivotal role in the biosynthesis of diterpenoid in I. rubescens.
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