过表达内源PMT和H6H基因对颠茄发根托品烷类生物碱合成的影响  

Enhancement of tropane alkaloids production in transgenic hair roots of Atropa belladonna by overexpressing endogenous genes AbPMT and AbH6H

作　　者：龙世平[1] 卢衍[1] 王亚雄[1] 杨春贤[1] 兰小中[2] 廖志华[1] 

机构地区：[1]西南大学生命科学学院,重庆市甘薯工程技术研究中心,重庆400715 [2]西藏农牧学院生物技术教研室,西藏林芝860000

出　　处：《药学学报》2013年第2期243-249,共7页Acta Pharmaceutica Sinica

基　　金：国家高技术研究发展计划(863计划)资助项目(2011AA100605)

摘　　要：颠茄是我国药典规定的托品烷类生物碱包括东莨菪碱和莨菪碱最主要的商业栽培的药源植物。本研究采用基于发根的基因表达系统,农杆菌介导遗传转化颠茄,潮霉素筛选获得转颠茄自身PMT和H6H基因发根;基因组PCR鉴定转基因发根;液体摇床培养后,HPLC测量莨菪碱和东莨菪碱含量;qPCR检测颠茄PMT和H6H基因表达水平。基因组PCR检测获得5个双基因共转化颠茄发根系,其中2个发根系(PH2和PH14)的总生物碱含量得到显著提高,最高的PH2总生物碱含量为非转基因发根的2.7倍;另外3个转基因发根系总生物碱含量与对照相比没有显著性差异。4个转基因发根系(PH2、PH32、PH14和PH20)东莨菪含量都有提高,其中PH2东莨菪碱含量比对照提高了8.2倍。基因表达量与生物碱含量结果基本一致,说明颠茄发根中PMT和H6H两者同时维持高水平表达可以极大程度的促进东莨菪碱积累。Atropa belladonna L. is the officially medicinal plant species and the main commercial source of scopolamine and hyoscyamine in China. In this study, we reported the simultaneous overexpression of two functional genes involved in biosynthesis of scopolamine, which respectively encoded the upstream key enzyme putrescine N-methyltransferase （PMT; EC 2.1.1.53） and the downstream key enzyme hyoscyamine 6β-hydroxylase （H6H; EC 1.14.11.11） in transgenic hair root cultures of Atropa belladonna L. HPLC results suggested that four transgenic hair root lines produced higher content of scopolamine at different levels compared with non- transgenic hair root cultures. And scopolamine content increased to 8.2 fold in transgenic line PH2 compared with that of control line; and the other four transgenic lines showed an increase of scopolamine compared with the control. Two of the transgenic hair root lines produced higher levels of tropane alkaloids, and the content increased to 2.7 fold in transgenic line PH2 compared with the control. The gene expression profile indicated that both PMT and H6H expressed at a different levels in different transgenic hair root lines, which would be helpful for biosynthesis of scopolamine. Our studies suggested that overexpression of A. belladonna endogenous genes PMT and H6H could enhance tropane alkaloid biosynthesis.

关 键 词：东莨菪碱 颠茄 N-甲基-腐胺-转移酶 莨菪碱6β-羟化酶 

分 类 号：R931[医药卫生—生药学]