1. Cloning and characterization of AabHLH1, a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua.
- Author
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Ji Y, Xiao J, Shen Y, Ma D, Li Z, Pu G, Li X, Huang L, Liu B, Ye H, and Wang H
- Subjects
- Alkyl and Aryl Transferases genetics, Alkyl and Aryl Transferases metabolism, Amino Acid Sequence, Artemisia annua chemistry, Artemisia annua cytology, Artemisia annua enzymology, Basic Helix-Loop-Helix Transcription Factors metabolism, Binding Sites, Biosynthetic Pathways, Cloning, Molecular, Cytochrome P-450 Enzyme System genetics, Cytochrome P-450 Enzyme System metabolism, Gene Expression, Gene Expression Regulation, Enzymologic, Gene Library, Genes, Reporter, Molecular Sequence Data, Plant Leaves chemistry, Plant Leaves enzymology, Plant Leaves genetics, Plant Proteins genetics, Plant Proteins metabolism, Plants, Genetically Modified, Promoter Regions, Genetic genetics, Sequence Alignment, Sequence Analysis, DNA, Artemisia annua genetics, Artemisinins metabolism, Basic Helix-Loop-Helix Transcription Factors genetics, Gene Expression Regulation, Plant
- Abstract
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., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Published
- 2014
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