rolC基因对颠茄托品烷生物碱合成的影响  

Effect of the rolC gene on biosynthesis of tropane alkaloids in Atropa belladonna

作　　者：路星星 谭艾娟 周浩 敖雯雯 吕世明[2] 满建民 张明生 强玮 LU Xing-xing;TAN Ai-juan;ZHOU Hao;AO Wen-wen;LU Shi-ming;MAN Jian-min;ZHANG Ming-sheng;QIANG Wei(Ministry of Education,Collaborative Innovation Center for Mountain Ecology&Agro-Bioengineering(CICMEAB),College of Life Sciences/Institute of Agro-bioengineering,Guizhou University,Guiyang 550025,China;College of Animal Science,Guizhou University,Guiyang 550025,China)

机构地区：[1]贵州大学生命科学学院/农业生物工程研究院,山地植物资源保护与保护种质创新教育部重点实验室,山地生态与农业生物工程协同创新中心,贵州贵阳550025 [2]贵州大学动物科学学院,贵州贵阳550025

出　　处：《药学学报》2022年第2期533-540,共8页Acta Pharmaceutica Sinica

基　　金：贵州省科技计划重大专项课题(黔科合平台人才[2017]5411-06);贵州大学培育项目(贵大培育[2019]51号);贵州大学引进人才科研项目(贵大人基合字[2017]58);贵州省中药材现代产业技术体系建设项目(GZCYTX-02);国家喀斯特石漠化防治工程技术研究中心建设(2012FU125X13);贵州省生物学一流学科建设项目(GNYL[2017]009)。

摘　　要：发根农杆菌浸染植物后产生的发根可合成高水平次生代谢产物,这种代谢增强作用是由rol基因诱导激发的,因此可利用rol基因提高颠茄中托品烷生物碱(tropane alkaloids,TAs)的合成能力。本研究以颠茄为实验材料,将发根农杆菌pRiA4质粒上的rolC基因通过CaMV35S启动子驱动,导入颠茄中超表达。分析转基因颠茄的表型、TAs含量以及TAs合成途径关键酶基因转录表达水平表化。结果表明,转基因颠茄表现出根系发达、雄性不育、雄蕊柱长高于雌蕊、提前开花、节间缩短、花小且多、腋芽和侧芽增多、顶端优势下降和叶片狭长等表型;转基因颠茄的TAs含量均显著高于对照,莨菪碱、山莨菪碱和东莨菪碱含量最高可达对照的2.58、3.59和15.77倍;TAs合成途径中的3个关键酶基因腐胺N-甲基转移酶(PMT)、托品酮还原酶I(TRⅠ)和莨菪碱6-β-羟化酶(H6H)的表达量与对照相比表达上调。以上研究表明rolC基因通过上调颠茄TAs合成关键酶基因的表达提高了颠茄中TAs的合成能力,为利用rolC基因进行高TAs含量的颠茄分子育种奠定了基础。Hair roots induced by Agrobacterium rhizogene produce higher levels of secondary metabolites than non-induced plants,and the enhanced metabolic capacity is driven by the rol gene.We hypothesized that rol genes can be utilized to improve the biosynthesis of tropane alkaloids(TAs)in Atropa belladonna.In this study,the rolC gene from Agrobacterium rhizogene pRiA4 plasmid,driven by a CaMV35 S promoter,was overexpressed in A.belladonna.The phenotypes,TAs content and transcriptional expression of key genes in TAs biosynthesis were analyzed in transgenic A.belladonna plants.Results show that transgenic A.belladonna exhibited a well-developed root system,male sterility,higher stamen column length than pistil,early flowering,internode shortening,smaller but more flowers,increased axillary buds and lateral buds,decreased apical dominance,and long and narrow leaves as compared to wild-type plants.Transgenic A.belladonna produced more TAs than wild-type plants,with the content of hyoscyamine,anisodamine and scopolamine reaching 2.58,3.59 and 15.77-fold that of the control group,respectively.The gene expression of putrescine N-methyltransferase(PMT),tropinone reductase Ⅰ(TRⅠ)and hyoscyamine 6-β-hydroxylase(H6H),key enzymes in TAs biosynthesis,were up-regulated compared with the control group.The above results indicate that the rolC gene enhances TAs biosynthesis in A.belladonna by up-regulating the expression of key enzymes in the TAs biosynthesis pathway,laying a foundation for genetic manipulation of A.belladonna to increase TAs content by increasing rolC gene expression.

关 键 词：颠茄 ROLC基因 莨菪碱 东莨菪碱 山莨菪碱 

分 类 号：R931[医药卫生—生药学]