Cloning and Characterization of AabHLH1, a bHLH Transcription Factor that Positively Regulates Artemisinin Biosynthesis in Artemisia annua
Cloning and Characterization of AabHLH1, a bHLH Transcription Factor that Positively Regulates Artemisinin Biosynthesis in Artemisia annua
复制标题
正向调控青蒿素生物合成的 bHLH 转录因子 AabHLH1 的克隆和表征
DOI：
10.1093/pcp/pcu090
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发表时间：
2014-09-01
影响因子：
4.9
通讯作者：
Wang, Hong
中科院分区：
文献类型：
--
作者：
Ji, Yunpeng;Xiao, Jingwei;Wang, Hong
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）是参与青蒿素生物合成的两种关键酶。ADS和CYP71AV1的启动子包含E - 盒元件，它们是碱性螺旋 - 环 - 螺旋（bHLH）转录因子的假定结合位点。本研究从青蒿中成功分离出一个bHLH转录因子基因，将其命名为AabHLH1，该基因来自青蒿素合成和储存的腺毛状分泌腺（GSTs）的cDNA文库。AabHLH1编码一个含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基因可被脱落酸（ABA）和真菌诱导子壳聚糖强烈诱导。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蛋白能够结合ADS和CYP71AV1启动子中存在的E - 盒顺式元件，并在酵母中具有反式激活活性。此外，在青蒿叶片中对AabHLH1和CYP71AV1Pro::GUS进行瞬时共转化，结果显示在转化的青蒿中，GUS（β - 葡萄糖醛酸酶）基因的表达被显著激活，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。再者，AabHLH1在青蒿叶片中的瞬时表达提高了参与青蒿素生物合成的基因（如ADS、CYP71AV1和HMGR）的转录水平。这些结果表明，AabHLH1能够正向调控青蒿素的生物合成。
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 (beta-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.