Ji Yunpeng, Xiao Jingwei, Shen Yalin, Ma Dongming, Li Zhenqiu, Pu Gaobin, Li Xing, Huang Lili, Liu Benye, Ye Hechun, Wang Hong
University of the Chinese Academy of Sciences, Beijing 100049, China These authors contributed equally to this work.
National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing 100050, China These authors contributed equally to this work.
Plant Cell Physiol. 2014 Sep;55(9):1592-604. doi: 10.1093/pcp/pcu090. Epub 2014 Jun 26.
Amorpha-4,11-diene synthase (ADS) and Cyt P450 monooxygenase (CYP71AV1) in Artemisia annua L. are two key enzymes involved in the biosynthesis of artemisinin. The promoters of ADS and CYP71AV1 contain E-box elements, which are putative binding sites for basic helix-loop-helix (bHLH) transcription factors. This study successfully isolated a bHLH transcription factor gene from A. annua, designated as AabHLH1, from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AabHLH1 encodes a protein of 650 amino acids containing one putative bHLH domain. AabHLH1 and ADS genes were strongly induced by ABA and the fungal elicitor, chitosan. The transient expression analysis of the AabHLH1-green fluorescent protein (GFP) reporter gene revealed that AabHLH1 was targeted to nuclei. Biochemical analysis demonstrated that the AabHLH1 protein was capable of binding to the E-box cis-elements, present in both ADS and CYP71AV1 promoters, and possessed transactivation activity in yeast. In addition, transient co-transformation of AabHLH1 and CYP71AV1Pro::GUS in A. annua leaves showed a significant activation of the expression of the GUS (β-glucuronidase) gene in transformed A. annua, but mutation of the E-boxes resulted in abolition of activation, suggesting that the E-box is important for the CYP71AV1 promoter activity. Furthermore, transient expression of AabHLH1 in A. annua leaves increased transcript levels of the genes involved in artemisinin biosynthesis, such as ADS, CYP71AV1 and HMGR. These results suggest that AabHLH1 can positively regulate the biosynthesis of artemisinin.
青蒿中的紫穗槐-4,11-二烯合酶（ADS）和细胞色素P450单加氧酶（CYP71AV1）是青蒿素生物合成过程中的两个关键酶。ADS和CYP71AV1的启动子含有E-盒元件，这是碱性螺旋-环-螺旋（bHLH）转录因子的假定结合位点。本研究从青蒿腺毛（GSTs）的cDNA文库中成功分离出一个bHLH转录因子基因，命名为AabHLH1，青蒿素在该腺毛中合成并储存。AabHLH1编码一个含有650个氨基酸的蛋白质，其中包含一个假定的bHLH结构域。AabHLH1和ADS基因受到脱落酸（ABA）和真菌激发子壳聚糖的强烈诱导。AabHLH1-绿色荧光蛋白（GFP）报告基因的瞬时表达分析表明，AabHLH1定位于细胞核。生化分析表明，AabHLH1蛋白能够与ADS和CYP71AV1启动子中存在的E-盒顺式元件结合，并在酵母中具有反式激活活性。此外，AabHLH1与CYP71AV1Pro::GUS在青蒿叶片中的瞬时共转化显示，在转化的青蒿中GUS（β-葡萄糖醛酸酶）基因的表达有显著激活，但E-盒的突变导致激活作用消失，这表明E-盒对CYP71AV1启动子活性很重要。此外，AabHLH1在青蒿叶片中的瞬时表达增加了青蒿素生物合成相关基因的转录水平，如ADS、CYP71AV1和3-羟基-3-甲基戊二酰辅酶A还原酶（HMGR）。这些结果表明，AabHLH1可以正向调控青蒿素的生物合成。
