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| Overexpression of Transcription Factor TwWRKY1 Gene Increased the Content of Wilforgine and Wilforine in Tripterygium wilfordii |
| YANG Li-Li1,*, XI Wen-Jing1,*, HE Ze-Dong1, CHENG Ling1, MA Zhi-Qing1,2, ZHANG Bin1,2, ZHU Chuan-Shu1,2,** |
1 College of Plant Protection, Northwest A&F University, Yangling 712100, China;
2 Engineering and Technology Centers of Bio-pesticidein Shaanxi, Yangling 712100, China |
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Abstract The methyl jasmonate (MeJA)-induced TwWRKY1 (GenBank No. GAVZ01022264.1) gene apparently regulated the biosynthesis of wilforgine in Tripterygium wilfordii. To elucidate the regulation mechanism and key target gene, the T. wilfordii sesquiterpene cyclase (Twses-CS) was selected for further exploring, the Twses-CS was screened as the key enzyme involved in the sesquiterpene biosynthesis pathway, the cis-acting element was analyzed in the Twses-CS promoter, and the expression of the TwWRKY1 and alkaloid content changes were detected. The results showed that the WRKY transcription factor might regulate the biosynthesis of sesquiterpene-pyridine alkaloids through regulating Twses-CS gene in T. wilfordii. Yeast one hybrid (Y1H) assays revealed that TwWRKY1 binded to the promoter of Twses-CS. Subcellular localization analysis indicated that the TwWRKY1 was located in the nucleus, consistent with its role as a typical transcription factor. Electrophoretic mobility shift assays (EMSA) further confirmed that TwWRKY1 formed a DNA-protein complex with a 30 bp probe containing 2 W-box sequences, indicating its binding ability to the W-box element. Overexpression of TwWRKY1 in hairy roots resulted in a significant increase in wilforgine and wilforine content about 1.87 and 1.90 times compared with the control, the content of wilforgine and wilforine was 249.53 and 638.25 μg/g DW, respectively. These results suggested that TwWRKY1 likely regulated the content of sesquiterpene-pyridine alkaloids in T. wilfordii by binding to the W-box element in the promoter of Twses-CS. This study provides basis for further exploration of alkaloid biosynthesis pathway and the transcription regulatory mechanism for sesquiterpene alkaloids, and provides a theoretical basis for using metabolic engineering and synthetic biology ways to solve the alkaloid shortage.
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Received: 14 November 2024
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Corresponding Authors:
**zhchshjhch@nwsuaf.edu.cn
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| About author:: *These authors contributed equally to this work |
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