洋金花托品酮还原酶Ⅰ基因的克隆与表达分析

Cloning and Expression Analysis of Tropinone Reductase Ⅰin Daturametel L.

洋金花的入药成分是莨菪碱和东莨菪碱,托品酮还原酶Ⅰ(tropinone reductase I, TRI)是托品烷类生物碱合成途径中的关键酶,该酶的编码基因是TAs代谢工程研究重要的靶标基因。本研究从洋金花中克隆TRⅠ基因,命名为Dm TRⅠ(GenBank登录号:MW455085)。DmTRⅠ基因包含一个822 bp的完整开放阅读框,编码273个氨基酸。生物信息学分析发现,DmTRⅠ为稳定的亲水性蛋白,是DmTRⅠ作为辅酶NADPH的结合位点,有TGXXXGXG保守结构域。从生物进化关系上发现,DmTRⅠ与同为茄科的颠茄、三分三等5种植物的TRⅠ聚为一支,且与曼陀罗属的木本曼陀罗TRI亲缘关系最近。进一步以ACTIN和PGK双基因为内参基因,DmTRⅠ基因表达分析显示,DmTRⅠ在根、茎、叶、花、果实各器官中均有表达,其中根的表达量最高,花,茎、叶的表达量较低,在果实中表达量最低。DmTRⅠ基因的获得及表达分析为进一步研究洋金花TAs的生物合成提供理论依据。

The medicinal ingredients of Datura metel L.are hyoscyamine and scopolamine.Tropinone reductase Ⅰ is a key enzyme in the synthesis of tropane alkaloids.The coding gene of this enzyme is an important target gene for TAS metabolic engineering research.This study cloned the TR Ⅰ gene from Datura metel L.and named it DmTR Ⅰ(GenBank accession number:MW455085).The DmTR Ⅰ gene contains a complete open reading frame of 822 bp,encoding 273 amino acids.Bioinformatics analysis showed that DmTRI was a stable hydrophilic protein;which contained the binding site of the coenzyme NADPH and a conserved domain of TGXXXGXG.From the biological evolutionary relationship,DmTR Ⅰ was clustered with the TR Ⅰ of five species ofAtropa belladonna L.and Anisodus acutangulus,which belong to the Solanaceae,and was closely related to BaTR Ⅰ of the genus Datura.Furthermore,ACTIN and PGK genes were used as internal reference genes.The analysis of DmTR I gene expression showed that DmTR I was expressed in root,stem,leaf,flower,and fruit.The expression level ofroot was the highest,followed by flower,stem and leaf.The expression level was the lowest in the fruit.The acquisition and expression analysis of DmTR Ⅰ gene provide a theoretical basis for further study of TAs biosynthesis in Datura metel L.