北卡罗来纳州立大学谢德玉课题组在木质素和青蒿素代谢关联方面取得进展

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内容来源于Front. Plant Sci.

黄花蒿是唯一能够产生治疗疟疾的青蒿素的药用植物。作者从一个黄花蒿自交品系中克隆了一个肉桂醇脱氢酶（AaCAD）基因并报道了该基因对木质素和青蒿素产量的影响。通过异源表达系统得到纯化了AaCAD蛋白。酶活实验证明AaCAD能够将香菜酰、松香酰、辛纳酰基醛转变为它们响应的醇，这些醇是木质素合成途径中重要的中间产物。对这三个底物的Km, Vmax和 Vmax/Km 值都进行了计算。为了验证AaCAD在体内的功能，将其超表达到了黄花蒿自交品系植物体内。用乙酰溴化定量的方法测定了转基因植株中木质素含量，发现明显高于野生型。通过GC-MS的方法也发现转基因植株中香豆素的含量明显升高。通过HPLC-MS的方法发现转基因植株中青蒿素的含量明显下降，进一步通过GC-MS的方法发现转基因植株中的青蒿素B和其他6种倍半萜的含量也明显减少。共焦显微镜观察到AaCAD定位在细胞质中。这些数据表明AaCAD同时参与到木质素和青蒿素的合成途径，一方面促进木质素的合成另一方面抑制青蒿素的合成。青蒿素和其他合成途径交叉关系的研究有利于以后研究怎样提高青蒿素的代谢流从而为高产青蒿素品种的产生奠定基础。

Front. Plant Sci., 19 June 2018

Artemisia annua is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) A. annua cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function in planta, AaCAD was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wild-type (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.

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