药学学报, 2015, 50(10): 1346-1355
引用本文:
强玮, 侯艳玲, 李笑, 夏科, 廖志华. 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[J]. 药学学报, 2015, 50(10): 1346-1355.
QIANG Wei, HOU Yan-ling, LI Xiao, XIA Ke, LIAO Zhi-hua. Cloning and expression of the key enzyme hyoscyamine 6 beta-hydroxylase gene (DaH6H) in scopolamine biosynthesis of Datura arborea[J]. Acta Pharmaceutica Sinica, 2015, 50(10): 1346-1355.

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
强玮, 侯艳玲, 李笑, 夏科, 廖志华
西南大学生命科学学院, 重庆 400715
摘要:
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cDNA和gDNA序列 (命名为DaH6H, cDNA GenBank登录号为KR006981, gDNA GenBank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1 375 bp, 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 (Datura stramonium) 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39 kD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。
关键词:   
Cloning and expression of the key enzyme hyoscyamine 6 beta-hydroxylase gene (DaH6H) in scopolamine biosynthesis of Datura arborea
QIANG Wei, HOU Yan-ling, LI Xiao, XIA Ke, LIAO Zhi-hua
School of Life Science, Southwest University, Chongqing 400715, China
Abstract:
Hyoscyamine 6 beta-hydroxylase (H6H) is the last rate-limiting enzyme directly catalyzing the formation of scopolamine in tropane alkaloids (TAs) biosynthesis pathway. It is the primary target gene in the genetic modification of TAs metabolic pathway. Full-length cDNA and gDNA sequences of a novel H6H gene were cloned from Datura arborea (DaH6H, GenBank accession numbers for cDNA and gDNA are KR006981 and KR006983, respectively). Nucleotide sequence analysis reveals an open reading frame of 1 375 bp encoding 347 amino acids in the cDNA of DaH6H, while the gDNA of DaH6H contains four exons and three introns, with the highest similarity to the gDNA of H6H from D. stramonium. DaH6H also exhibited the most identity of 90.5% with DsH6H in amino acids and harbored conserved 2-oxoglutarate binding motif and two iron binding motifs. The expression level of DaH6H was highest in the mature leaf, followed by the secondary root, and with no expression in the primary root based on qPCR analysis. Its expression was inhibited by MeJA. DaH6H was expressed in E. coli and a 39 kD recombinant protein was detected in SDS-PAGE. Comparison of the contents of scopolamine and hyoscyamine in various TAs-producing plants revealed that D. arborea was one of the rare scopolamine predominant plants. Cloning of DaH6H gene will allow more research in the molecular regulatory mechanism of TAs biosynthesis in distinct plants and provide a new candidate gene for scopolamine metabolic engineering.
Key words:   
收稿日期: 2015-03-26
基金项目: 教育部新世纪人才支持计划资助项目 (NCET-12-0930); 国家“863”计划资助项目 (2011AA100605); 国家自然科学基金资助项目 (31370333); 重庆市科技攻关项目 (CSTC2012GGYYJS80013); 西南大学中央高校基本科研业务费专项资助项目 (XDJK2013A024).
通讯作者: 廖志华
Email: zhliao@swu.edu.cn
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