3'-甲氧基葛根素生物合成途径中关键甲基转移酶基因的克隆与功能分析

3'-methoxy puerarin is mainly originated from the roots of Pueraria lobata and shows significant activities on protecting the cerebral ischemia-reperfusion injury. However, the key gene encoding the methyltransferase involved in the biosynthesis of 3'-methoxy puerarin has never been isolated so far. Through RNA-sequencing of the roots and leaves from P. lobata, seven kinds of partial cDNA sequences encoding putative methyltransferases were identified in our preliminary experiments. Using RACE-PCR technique, the full-length cDNAs of these candidates were amplified. In this study, the gene overexpression and RNAi strategy will be applied to screen the functions of these gene candidates in P. lobata cells in vivo using the established transformation system in our lab. The specific gene encoding the methyltransferase in the step to the biosynthesis of 3'-methoxy puerarin will be identified; the relevance of the transcript levels of the target gene candidate to the productions of 3'-methoxy puerarin will be investigated in different developmental stages of the plant; the biochemical functions of the targeted methyltransferase will be further characterized through transferring the full-length cDNA of the targeted gene into Escherichia coli or yeast cells. The success of this study will provide the essential gene resource for the production of 3'-methoxy puerarin in heterologous hosts, and it is also very important to understand the biosynthetic pathway of 3'-methoxy puerarin in P. lobata.

3'-甲氧基葛根素主要来源于野葛的根，对脑缺血再灌注损伤具有明显的保护作用，但其生物合成途径中的关键甲基转移酶基因至今未被分离。通过野葛根与叶的转录组测序分析，获得7个候选甲基转移酶基因的部分序列信息，利用RACE-PCR技术，项目组已获得了这些基因的全长cDNAs。本项目将借助已建立的野葛细胞遗传转化体系，通过基因的过量表达和RNA干扰技术，在野葛植物细胞体内对这些基因的功能进行分析，筛选出3'-甲氧基葛根素生物合成途径中的关键甲基转移酶基因；研究侯选基因的时空表达模式与目标化合物3'-甲氧基葛根素合成之间的相关性；通过原核生物蛋白表达、纯化以及酵母表达系统，对侯选甲基转移酶基因的生化功能进行分析。本项目的成功开展将为异源系统生产3'-甲氧基葛根素提供必需的基因资源，对于解析野葛中3'-甲氧基葛根素的生物合成途径具有重要意义。

3'-甲氧基葛根素主要来源于野葛的根，对脑缺血再灌注损伤具有明显的保护作用，但其生物合成途径中的关键甲基转移酶基因至今未被分离。通过对野葛根与叶的转录组测序数据分析，获得9个候选的甲基转移酶基因，均被成功克隆，命名为PlOMT1-PlOMT9。通过酿酒酵母表达系统对这9个候选基因进行功能分析，成功筛选出一个具有异黄酮3'-O-甲基转移酶活性的基因PlOMT4，PlOMT4能催化3'-羟基大豆苷元生成3'-甲氧基大豆苷元，但是不能利用3'-羟基葛根素为底物生成3'-甲氧基葛根素，以上实验结果表明野葛异黄酮甲基化的修饰先于糖基化修饰；对PlOMT4的体外酶活性进行了测定，PlOMT4对3'-羟基大豆苷元的活性最高，对木犀草素和槲皮素有较弱的活性；还研究了该基因在野葛不同器官的表达与相关代谢产物3'-甲氧基大豆苷元分布的相关性，实验结果表明PlOMT4基因的表达与相关代谢产物3'-甲氧基大豆苷元的分布具有相关性。以上实验结果表明，我们成功地从野葛中分离出一个异黄酮3'-O-甲基转移酶基因。此外我们还发现PlOMT9主要具有异黄酮4'-O-甲基转移酶活性，体外酶活分析显示PlOMT9以大豆苷元为底物，不以以往报道的2,7,4'-三羟基异黄烷酮为底物合成芒柄花素，揭示出一条新的芒柄花素合成路径。本项目的成功开展将为异源系统生产3'-甲氧基葛根素提供必需的基因资源，对于解析野葛中3'-甲氧基葛根素和芒柄花素的生物合成途径具有重要意义。