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OALib Journal期刊
ISSN: 2333-9721
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木本曼陀罗中催化东莨菪碱生物合成关键步骤的h6h基因克隆与表达分析
, PP. 1346-1355
Keywords: 木本曼陀罗,莨菪碱6β-羟化酶,东莨菪碱,代谢工程
Abstract:
莨菪碱6β-羟化酶(hyoscyamine6beta-hydroxylase,h6h)是托品烷类生物碱(tropanealkaloids,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(daturaarborea)h6h基因的全长cdna和gdna序列(命名为dah6h,cdnagenbank登录号为kr006981,gdnagenbank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。dah6h基因cdna全长1375bp,编码347个氨基酸残基,其gdna含有4个外显子和3个内含子,和曼陀罗(daturastramonium)的h6h基因相似性最高。dah6h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。qpcr检测dah6h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meja的抑制。大肠杆菌重组诱导表达成功诱导出了约39kd的重组dah6h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。dah6h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。
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