Li Jin-Di, Qin Bai-Fu, Yang Chun-Xian, Lan Xiao-Zhong, Wu Neng-Biao, Liao Zhi-Hua
Chongqing Engineering and Technology Research Center for Sweetpotato, Southwest University, Chongqing 400715, China.
Zhongguo Zhong Yao Za Zhi. 2013 Jun;38(11):1719-24.
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 Kan(r) 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 x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x 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.
通过代谢工程培育出了含有高含量东莨菪碱的转基因颠茄。参与东莨菪碱生物合成途径中限速酶h6h的一个功能基因通过农杆菌介导在颠茄中过量表达。通过微繁殖和驯化培养转基因植物直至结果。通过基因组聚合酶链反应（PCR）分析证实h6h基因整合到转基因植物的基因组DNA中。使用SPSS软件对株高、冠幅、茎直径、叶长、叶宽、分枝数和鲜重的差异进行分析。通过高效液相色谱法（HPLC）测定根、茎、叶和果实中东莨菪碱和莨菪碱的含量。通过定量PCR（qPCR）研究Hnh6h的表达水平。在五个转基因颠茄植株系（A8、A11、A12、C8和C19）中检测到了卡那霉素抗性（Kan(r)）基因和Hnh6h基因，但在对照中未检测到。与非转基因植株相比，转基因植株的株高、冠幅、茎直径、叶长、叶宽、分枝数和鲜重没有降低，而且转基因植株的一些农艺性状优于对照。在转基因颠茄的叶片中发现东莨菪碱含量最高，并且叶片中东莨菪碱的含量也高于莨菪碱。不同转基因株系叶片中东莨菪碱的含量依次为：C8＞A12＞C19＞A11＞A8，特别是转基因株系C8中东莨菪碱的含量为2.17mg·g(-1)干重，是非转基因植株（0.42mg·g(-1)干重）的4.2倍。在所有转基因植株中检测到了转基因Hnh6h的表达，但在对照中未检测到。在转基因叶片中发现Hnh6h的表达水平最高。Hnh6h的过量表达能够打破东莨菪碱生物合成下游途径中的限速步骤，从而促进代谢流流向东莨菪碱的生物合成，提高转基因植物中东莨菪碱的生物合成能力。结果，培育出了东莨菪碱含量更高的转基因颠茄植株。
