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| Cloning and Functional Study of the AbWRKY1 Gene from Atropa belladonna |
| PAN Hang, MU De-Hui, YANG Jia, TAN Ai-Juan, MA Bing-Nan, XU Guo-Ju, YANG Li-Hang, QIANG Wei* |
| College of Life Sciences/Research Institute of Agricultural Biological Engineering, Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China |
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Abstract Atropa belladonna is a commercial medicinal plant of tropane alkaloids (TAs) listed in the 《Chinese Pharmacopoeia》, and WRKY transcription factors may regulate the biosynthesis of TAs. In this study, tissue expression profile, phylogenetic tree and inducible expression analyses of the WRKY transcription factor family in A. belladonna were performed to screen out WRKY gene that positively regulated TAs biosynthesis. AbWRKY1 was identified and its function was verified at the biochemical and molecular genetic levels. AbWRKY1 and TAs synthetic pathway genes shared the same tissue and inducible expression characteristics that they were specifically expressed in the adventitious roots and were induced by the calcium channel inhibitor verapamil. AbWRKY1 clustered together with WRKYs from other species that had functions in regulating secondary metabolism. AbWRKY1 protein contained 326 amino acid residues, and localized in nuclear. Yeast one-hybrid result showed that AbWRKY1 could interact with the W-box rich region of hyoscyamine 6β-hydroxylase (H6H) gene promoter. Knockdown of AbWRKY1 expression by virus-induced gene silencing (VIGS) in A. belladonna plants reduced scopolamine and hyoscyamine levels by 44.91% and 79.33%, respectively. AbWRKY1 overexpression raised the amount of scopolamine, anisodamine, and hyoscyamine in A. belladonna's hairy roots, while the expression of the 4 TAs synthesis pathway genes, namely putrescine N-methyltransferase (PMT), tropinone reductaseⅠ(TRⅠ), hyoscyamine dehydrogenase (HDH) and H6H, were increased concomitantly. This study provides a theoretical basis for the elucidation of the regulatory mechanism of TAs biosynthesis and the molecular breeding of A. belladonna.
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Received: 29 January 2024
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Corresponding Authors:
* wayneqiang@126.com
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