rolB基因对颠茄表型发育和托品烷生物碱合成的影响  

Effect of the rolB gene on phenotypic development and tropane alkaloids biosynthesis in Atropa belladonna

作　　者：敖雯雯 谭艾娟 马炳南 穆德会 路星星 潘航 张明生 强玮 AO Wen-wen;TAN Ai-juan;MA Bing-nan;MU De-hui;LU Xing-xing;PAN Hang;ZHANG Ming-sheng;QIANG Wei(Key Laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region(Ministry of Education),College of Life Sciences/Institute of Agro-bioengineering,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种质创新教育部重点实验室,贵州贵阳550025

出　　处：《药学学报》2023年第6期1705-1712,共8页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(32200225);贵州省省级科技计划项目(黔科合基础-ZK [2022]一般096);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项目(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pRiA4质粒上的rol基因是促进次生代谢的强效基因,可利用rol基因对颠茄进行分子育种以提升颠茄中托品烷生物碱(tropane alkaloids, TAs)含量。本研究将rolB基因在颠茄植株中进行超表达,为了研究rolB基因对颠茄TAs生物合成的影响,分析了转基因颠茄的表型、TAs含量及TAs合成途径中关键酶基因的表达水平。结果表明,转基因颠茄根系发达,叶片增大、叶片鲜重增加、叶片颜色加深,花朵增大、花形改变,雌蕊高度降低,花粉活力下降。转基因颠茄茎中TAs的含量显著高于对照,东莨菪碱、山莨菪碱、莨菪碱的含量分别为对照的2.11~2.91、1.23~2.37、4.88~5.20倍。与对照组相比, TAs合成途径中的关键酶基因N-甲基腐胺转移酶(PMT)、吡咯烷聚酮合酶(PYKS)、托品酮还原酶I (TRI)、芳香族氨基酸氨基转移酶4 (ArAT4)、UDP-糖基转移酶1 (UGT1)和莨菪碱6-β-羟化酶(H6H)的表达上调,且托品酮还原酶II (TRII)作为代谢分流基因表达下调。研究表明rolB基因通过增强TAs合成途径代谢流和减弱竞争支路代谢分流,增强了颠茄根中TAs的合成能力和茎中的积累。本研究为利用rolB基因进行高产TAs颠茄的分子育种奠定了基础。The rol genes on pRiA4 plasmid of Agrobacterium rhizogenes are potent genes that promote secondary metabolism.Molecular breeding of Atropa belladonna can be conducted by introducing rol genes to increase tropane alkaloids(TAs)content in A.belladonna.In this study,the rolB gene was overexpressed in A.belladonna plants to study the effect of rolB gene on the biosynthesis of TAs.The phenotype,TAs content and expression levels of key enzyme genes in the pathway of TAs biosynthesis of transgenic A.belladonna were analyzed.The results showed that transgenic A.belladonna had developed root system,enlarged leaves,increased leaf fresh weight,deepened leaf color,enlarged flowers,changed flower shape,reduced pistil height and decreased pollen vitality.The content of TAs in the stems of transgenic A.belladonna was significantly higher than that of the control,and the contents of scopolamine,anisodamine,hyoscyamine can reach 2.11-2.91,1.23-2.37 and 4.88-5.20 times of the control,respectively.Compared with the control group,the expressions of key enzymes putrescine N-methyltransferase(PMT),typeⅢpolyketide synthase(PYKS),tropinone reductase I(TRI),aromatic amino acid aminotransferase 4(ArAT4),UDP-glycosyltransferase 1(UGT1)and hyoscyamine 6-β-hydroxylase(H6H)in the TAs biosynthesis pathway were up-regulated,and the expression of tropinone reductaseⅡ(TRⅡ)as a metabolic shunting gene was down-regulated.The results indicated that rolB gene enhanced TAs synthesis ability in roots and accumulation in stems of A.belladonna by enhancing metabolic flow of TAs synthesis pathway and weakening the metabolic shunt of competing pathway.This study laid a foundation for molecular breeding of A.belladonna with high-yield TAs content using rolB gene.

关 键 词：ROLB基因 颠茄 东莨菪碱 山莨菪碱 莨菪碱 

分 类 号：R931[医药卫生—生药学]