National Key Laboratory of Plant Molecular Genetics and National Center for Plant Gene Research, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Mol Plant. 2012 Mar;5(2):353-65. doi: 10.1093/mp/ssr087. Epub 2011 Nov 20.
Plants of Artemisia annua produce artemisinin, a sesquiterpene lactone widely used in malaria treatment. Amorpha-4,11-diene synthase (ADS), a sesquiterpene synthase, and CYP71AV1, a P450 monooxygenase, are two key enzymes of the artemisinin biosynthesis pathway. Accumulation of artemisinin can be induced by the phytohormone jasmonate (JA). Here, we report the characterization of two JA-responsive AP2 family transcription factors--AaERF1 and AaERF2--from A. annua L. Both genes were highly expressed in inflorescences and strongly induced by JA. Yeast one-hybrid and electrophoretic mobility shift assay (EMSA) showed that they were able to bind to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs present in both ADS and CYP71AV1 promoters. Transient expression of either AaERF1 or AaERF2 in tobacco induced the promoter activities of ADS or CYP71AV1, and the transgenic A. annua plants overexpressing either transcription factor showed elevated transcript levels of both ADS and CYP71AV1, resulting in increased accumulation of artemisinin and artemisinic acid. By contrast, the contents of these two metabolites were reduced in the RNAi transgenic lines in which expression of AaERF1 or AaERF2 was suppressed. These results demonstrate that AaERF1 and AaERF2 are two positive regulators of artemisinin biosynthesis and are of great value in genetic engineering of artemisinin production.
青蒿植物产生青蒿素，一种广泛用于疟疾治疗的倍半萜内酯。青蒿烯合酶（ADS）和 CYP71AV1 是青蒿素生物合成途径中的两种关键酶，CYP71AV1 是一种 P450 单加氧酶。青蒿素的积累可以被植物激素茉莉酸（JA）诱导。在这里，我们报告了来自青蒿 L. 的两种 JA 响应 AP2 家族转录因子 AaERF1 和 AaERF2 的特征。这两个基因在花序中高度表达，并被 JA 强烈诱导。酵母单杂交和电泳迁移率变动分析（EMSA）表明，它们能够结合到 ADS 和 CYP71AV1 启动子中存在的 CRTDREHVCBF2（CBF2）和 RAV1AAT（RAA）基序。在烟草中瞬时表达任一个 AaERF1 或 AaERF2 都能诱导 ADS 或 CYP71AV1 的启动子活性，并且过表达这两种转录因子的转基因青蒿植物表现出 ADS 和 CYP71AV1 的转录水平升高，导致青蒿素和青蒿酸的积累增加。相比之下，在抑制 AaERF1 或 AaERF2 表达的 RNAi 转基因系中，这两种代谢物的含量降低。这些结果表明 AaERF1 和 AaERF2 是青蒿素生物合成的两个正调控因子，在青蒿素生产的遗传工程中具有重要价值。
