Molecular engineering of isoquinoline alkaloid biosynthesis in higher plant using Coptis O-metheyl-transferases cDNAs

利用黄连 O-甲基转移酶 cDNA 进行高等植物异喹啉生物碱生物合成的分子工程

• 批准号: 08456172
• 负责人: SATO Fumihiko
• 金额: $ 2.69万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456172/
• 关键词: Molecular engineering; Isoquinoline alkaloid biosynthesis; O-methyltransferese; Eschscholtzia; alkaloid composition; scoulerine-9-O-methyltransferase; tetrahydrocolumbamine; テトラハイドロコロンバシン; モルフィナンアルカロイド; 形質転換; Agrobacterium; パーティクルカン; モルフィキンアルカ

The molecular engineering of isoquinoline alkaloid biosynthesis in plant cells was investigated. Using cauliflower mosaic virus 35S promoter with enhancer elements, high expression vectors for 3 OMTs of berberine biosyntheis were constructed and introduced into 3 different plants ; Coptis (homologous plant), Eschscholtzia (a plant which produces benzophenanthridine alkaloid derived from scoulerine, an intermediate for berberine) and Nicotiana tabacum (nicotine alkaloid plant which does not produce isoquinoline type alkaloid). Although transformation of Coptis cells was very difficult, at least transformants with SMT (scoulerine 9-0-methyltransferase) expression were established for all host plant species. Transformed Coptis cells showed increased SMT activity and modified alkaloid composition ; I.e. more berberine and less coptisine (different end product from scoulerine). This result suggest the modification of branching point enzyme (I.e. SMT) would be a useful method to modify the composition of alkaloid, whereas modification was limited. Transformed Eschscholtzia cells also showed clear SMT activity and modified metabolite composition ; high accumulation of tetrahydrocolumbamine, a product of SMT reaction. On the other hand, the transformants of tobacco did not show SMT activity, whereas SMT transcripts were detected. Effect of host plant species on molecular engineering of alkaloid biosynthesis is discussed.

对异喹啉生物碱在植物细胞中生物合成的分子工程进行了研究。利用带有增强子元件的花椰菜花叶病毒35S启动子，构建了3种黄连素生物合成OMT的高效表达载体，并将其导入3种不同的植物：黄连(同源植物)、石杉(从黄连提取的苯并菲并菲生物碱的中间体)和烟草(不产生异喹啉类生物碱的尼古丁生物碱植物)。虽然黄连细胞的转化非常困难，但至少为所有寄主植物建立了表达SMT(Soulerine 9-0-甲基转移酶)的转化子。转化的黄连细胞表现出SMT活性增强和生物碱成分的修饰，即更多的黄连碱和更少的黄连碱(不同于黄连碱的最终产物)。这一结果表明，分支点酶(SMT)的修饰是一种有效的生物碱组成修饰方法，但修饰效果有限。转化细胞也表现出明显的SMT活性和修饰的代谢物组成；SMT反应产物四氢柱胺的高度积累。另一方面，烟草的转化子没有显示SMT活性，但检测到SMT转录本。讨论了寄主植物种类对生物碱生物合成分子工程的影响。

项目成果

S. Kitajima, F. Sato et al.: "Constitutive expression of the neutral PR-5 (OLP, PR-5d) gene in roots and cultured cells of tobacco is mediated by ethylene-responsive cis-element AGCCGCC sequences"Plant Cell Reports. 18. 173-179 (1998)

S. Kitajima、F. Sato 等人：“烟草根和培养细胞中中性 PR-5 (OLP、PR-5d) 基因的组成型表达是由乙烯响应顺式元件 AGCCGCC 序列介导的”植物细胞报告

Kitajima, S.: "Plant pathogeniesis-related proteins: molecular mechanisms of gene expression and protein function." J.Biochem. 125. 1-8 (1991)

Kitajima, S.：“植物发病机制相关蛋白质：基因表达和蛋白质功能的分子机制。”

H.Koiwa: "Purification and characterization of tobacco pathogenesis-related protein PR-5d,an antifungal thaumatin-like protein." Plant Cell Physiology. 38. 783-791 (1997)

H.Koiwa：“烟草发病机制相关蛋白 PR-5d（一种抗真菌索马甜样蛋白）的纯化和表征。”

S.Kitajima、F.Sato et al: "Constitutive expression of the neutral PR-5 COLP,PR-5dy gene in roots and cultured cells of tobacco is mediated by ethylene-responsive cis-element AGCCGCC Sequences."Plant Cell Rep.. 18. 173-179 (1998)

S.Kitajima、F.Sato 等人：“烟草根和培养细胞中中性 PR-5 COLP、PR-5dy 基因的组成型表达是由乙烯响应顺式元件 AGCCGCC 序列介导的。”植物细胞代表。 18. 173-179 (1998)

S. Kitajima & F. Sato: "Plant pathogeniesis-related proteins : molecular mechanisms of gene expression and protein function"J. Biochem.. 125(1). 1-8 (1999)

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