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莨菪碱6β-羟化酶(hyoscyamine 6 beta-hydroxylase,H6H)是托品烷类生物碱(tropane alkaloids,TAs)合成途径中的最后一个限速酶,直接催化东莨菪碱的生物合成,同时也是TAs代谢工程改造的首要靶标基因。本研究克隆了木本曼陀罗(Datura arborea)H6H基因的全长c DNA和g DNA序列(命名为Da H6H,c DNA Gen Bank登录号为KR006981,g DNA Gen Bank登录号为KR006983),对该基因进行了组织和诱导表达并在大肠杆菌中进行了重组表达。Da H6H基因c DNA全长1 375 bp,编码347个氨基酸残基,其g DNA含有4个外显子和3个内含子,和曼陀罗(Datura stramonium)的H6H基因相似性最高。Da H6H编码蛋白也与曼陀罗的H6H序列一致性最高(90.5%),具备保守的2-oxoglutarate结合基序和两个iron结合基序。q PCR检测Da H6H在老叶中表达量最高,其次为须根,主根中不表达,同时其表达受Me JA的抑制。大肠杆菌重组诱导表达成功诱导出了约39 k D的重组Da H6H蛋白。对比了不同TAs资源植物发根和根中的东莨菪碱和莨菪碱含量,表明木本曼陀罗是少有的东莨菪碱占优势的植物。Da H6H基因的克隆和重组表达为深入研究不同植物中TAs生物合成的分子调控机制奠定了基础,同时也为开展东莨菪碱代谢工程研究提供了新的候选基因。
Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme in the synthetic pathways of tropane alkaloids (TAs). It directly catalyzes the biosynthesis of scopolamine and also the metabolism of TAs. The engineered primary target gene. In this study, the full-length cDNA and g DNA sequence of the H6H gene of Datura arborea (named Da H6H, cDNA Gen Bank accession number KR006981, g DNA Gen Bank accession number KR006983) was cloned. The gene was organized and induced and expressed recombinantly in E. coli. Da H6H gene c DNA has a full length of 1 375 bp and encodes 347 amino acid residues. Its g DNA contains 4 exons and 3 introns, and has the highest similarity to the H6H gene of Datura stramonium. The Da H6H-encoded protein also has the highest identity (90.5%) to the H6H sequence of Datura stramonium, and possesses a conserved 2-oxoglutarate binding motif and two iron-binding motifs. q PCR showed that Da H6H had the highest expression in old leaves, followed by fibrous roots, but not in main roots. Meanwhile, its expression was inhibited by Me JA. Recombinant inducing expression of E. coli successfully induced a recombinant Da H6H protein of about 39 kD. The contents of scopolamine and scopolamine in roots and roots of plants with different TAs were compared, indicating that Datura stramonium is a rare plant dominated by scopolamine. The cloned and recombinant expression of Da H6H gene laid a foundation for further study on the molecular regulation mechanism of TAs biosynthesis in different plants, and also provided a new candidate gene for the research of scopolamine metabolism engineering.