中国中药杂志

2013, v.38(11) 1719-1724

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过表达莨菪h6h基因提高颠茄东莨菪碱含量的研究
Enhanced biosynthesis of scopolamine in transgenic Atropa belladonna by overexpression of h6h gene

李金弟;秦白富;杨春贤;兰小中;吴能表;廖志华;
LI Jin-di1,2,QIN Bai-fu1,2,YANG Chun-xian1,2,LAN Xiao-zhong3,WU Neng-biao1,LIAO Zhi-hua1,2(1.Chongqing Engineering and Technology Research Center for Sweetpotato,Southwest University,Chongqing 400715,China; 2.Key Laboratory of Eco-environments in Three Gorges Reservoir Region,Ministry of Education, Southwest University,Chongqing 400715,China; 3.Tibet Agricultural and Animal Husbandry College,Nyingchi 860000,China)

摘要(Abstract):

文章采用代谢工程方法培育东莨菪碱高产转基因颠茄。根癌农杆菌遗传转化颠茄,过量表达东莨菪碱合成途径中限速酶基因h6h(Hnh6h),卡那霉素筛选获得转基因植株,快繁炼苗后,田间栽培至果期。基因组PCR鉴定转Hnh6h基因颠茄。SPSS软件分析转基因与非转基因颠茄株高、顶宽、主茎粗、叶长、叶宽、分支数和鲜重等性状的差异;HPLC测定其根、茎、叶、果中莨菪碱及东莨菪碱含量;采用qPCR技术测定各组织Hnh6h基因表达量。结果所获得的5个转基因颠茄株系(A8,A11,A12,C8,C19)都能同时检测到Kanr和Hnh6h基因,在非转基因颠茄中没有检测到这2个基因;与非转基因颠茄比较,转基因颠茄株系的株高、顶宽、主茎粗、叶长、叶宽、分支数和鲜重等都没有降低,且部分性状优于对照。转基因颠茄叶片中东莨菪碱含量最高,且高于莨菪碱;不同转基因颠茄株系叶片中东莨菪碱含量依次为C8>A12>C19>A11>A8,C8的东莨菪碱质量分数(2.17 mg.g-1DW)比非转基因颠茄(0.42 mg.g-1DW)提高4.2倍;在转基因颠茄根、茎、叶和果中都检测到Hnh6h基因表达,且在叶片中表达量最高,非转基因颠茄没有检测到Hnh6h基因表达。因此,过表达Hnh6h基因能够打破颠茄的东莨菪碱生物合成的限速反应,从而推动代谢流向东莨菪碱合成方向流动,提高了东莨菪碱合成能力,最终获得了东莨菪碱高含量的转基因颠茄。
Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering.A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A.belladonna via Agrobacterium-mediation.The transgenic plants were culturing till fruiting through micropropogating and acclimating.The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction(PCR) analysis.Analysis of the difference of plant height,crown width,stem diameter,leaf length,leaf width,branch number and fresh weight was carried out using SPSS software.The content of hyoscyamine and scopolamine in roots,stems,leaves and fruits was determined by HPLC.The investigation of the expression levels of Hnh6h by qPCR.Both Kanr and Hnh6h genes were detected in five transgenic lines of A.belladonna plants(A8,A11,A12,C8 and C19),but were not detected in the controls.The plant height,crown width,stem diameter,leaf length,leaf width,branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones,and furthermore some agronomic characters of transgenic plants were better than those of the controls.The highest level of scopolamine was found in leaves of transgenic A.belladonna,and the content of scopolamine was also higher than that of hyoscyamine in leaves.The contents of scopolamine of leaves in different transgenic lines were listed in order: C8>A12>C19>A11>A8,especially,the content of scopolamine in transgenic line C8 was 2.17 mg·g-1 DW that was 4.2 folds of the non-transgenic ones(0.42 mg·g-1 DW).The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control.The highest level of Hnh6h expression was found in transgenic leaves.Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis,and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants.As a result,transgenic plants of A.belladonna with higher level of scopolamine were developed.

关键词(KeyWords): 颠茄;莨菪碱6β-羟化酶;东莨菪碱;转基因
Atropa belladonna;hyoscyamine 6β-hydroxylase;scopolamine;transgene

Abstract:

Keywords:

基金项目(Foundation): 国家高技术研究发展计划(863)项目(2011AA100605);; 重庆市科技攻关项目(CSTC2012GGYYJS80013)

作者(Author): 李金弟;秦白富;杨春贤;兰小中;吴能表;廖志华;
LI Jin-di1,2,QIN Bai-fu1,2,YANG Chun-xian1,2,LAN Xiao-zhong3,WU Neng-biao1,LIAO Zhi-hua1,2(1.Chongqing Engineering and Technology Research Center for Sweetpotato,Southwest University,Chongqing 400715,China; 2.Key Laboratory of Eco-environments in Three Gorges Reservoir Region,Ministry of Education, Southwest University,Chongqing 400715,China; 3.Tibet Agricultural and Animal Husbandry College,Nyingchi 860000,China)

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