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通过过表达HDR和ADS基因增强转基因黄花蒿中青蒿素的生物合成

[Enhancement of artemisinin biosynthesis in transgenic Artemisia annua L. by overexpressed HDR and ADS genes].

作者信息

Wang Ya-Xiong, Long Shi-Ping, Zeng Li-Xia, Xiang Li-En, Lin Zhi, Chen Min, Liao Zhi-Hua

出版信息

Yao Xue Xue Bao. 2014 Sep;49(9):1346-52.

Abstract

Artemisnin is a novel sesquiterpene lactone with an internal peroxide bridge structure, which is extracted from traditional Chinese herb Artemisia annua L. (Qinghao). Recommended by World Health Organization, artemisinin is the first-line drug in the treatment of encephalic and chloroquine-resistant malaria. In the present study, transgenic A. annua plants were developed by overexpressing the key enzymes involved in the biosynthetic pathway of artemisinin. Based on Agrobacterium-mediated transformation methods, transgenic plants of A. annua with overexpression of both HDR and ADS were obtained through hygromycin screening. The genomic PCR analysis confirmed six transgenic lines in which both HDR and ADS were integrated into genome. The gene expression analysis given by real-time quantitative PCR showed that all the transgenic lines had higher expression levels of HDR and ADS than the non-transgenic control (except ah3 in which the expression level of ADS showed no significant difference compared with control); and the HPLC analysis of artemisinin demonstrated that transgenic A. annua plants produced artemisinin at significantly higher level than non-transgenic plants. Especially, the highest content of artemisinin was found in transgenic line ah70, in which the artemisinin content was 3.48 times compared with that in non-transgenic lines. In summary, overexpression of HDR and ADS facilitated artemisinin biosynthesis and this method could be applied to develop transgenic plants of A. annua with higher yield of artemisinin.

摘要

青蒿素是一种具有内过氧化物桥结构的新型倍半萜内酯,它从传统中草药黄花蒿(青蒿)中提取。青蒿素经世界卫生组织推荐,是治疗脑型疟疾和耐氯喹疟疾的一线药物。在本研究中,通过过表达青蒿素生物合成途径中涉及的关键酶来培育转基因黄花蒿植株。基于农杆菌介导的转化方法,通过潮霉素筛选获得了同时过表达HDR和ADS的转基因黄花蒿植株。基因组PCR分析证实了6个转基因株系,其中HDR和ADS均整合到了基因组中。实时定量PCR进行的基因表达分析表明,所有转基因株系中HDR和ADS的表达水平均高于非转基因对照(除ah3中ADS的表达水平与对照相比无显著差异外);青蒿素的HPLC分析表明,转基因黄花蒿植株产生青蒿素的水平显著高于非转基因植株。特别是,在转基因株系ah70中发现青蒿素含量最高,其青蒿素含量与非转基因株系相比为3.48倍。总之,HDR和ADS的过表达促进了青蒿素的生物合成,该方法可用于培育青蒿素产量更高的转基因黄花蒿植株。

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