农业生物技术学报2025，Vol.33Issue(9)：1921-1934,14.DOI:10.3969/j.issn.1674-7968.2025.09.005
转录因子基因TwWRKY1过表达提高雷公藤吉碱和次碱含量
Overexpression of Transcription Factor TwWRKY1 Gene Increased the Content of Wilforgine and Wilforine in Tripterygium wilfordii
摘要
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.关键词
雷公藤/雷公藤吉碱/雷公藤次碱/雷公藤倍半萜环化酶(Twses-CS)/TwWRKY1/发状根Key words
Tripterygium wilfordii/Wilforgine/Wilforine/T.wilfordii sesquiterpene cyclase(Twses-CS)/TwWRKY1/Hairy roots分类
农业科技引用本文复制引用
杨莉莉,惠文静,何泽东,陈玲,马志卿,张斌,祝传书..转录因子基因TwWRKY1过表达提高雷公藤吉碱和次碱含量[J].农业生物技术学报,2025,33(9):1921-1934,14.基金项目
陕西省自然科学基础研究计划重点项目(2023-JC-ZD-11) （2023-JC-ZD-11）
国家重点研发计划(2020YFA0907900) （2020YFA0907900）
国家自然科学基金(32102260) （32102260）
西北农林科技大学科研启动费项目(2452023050) （2452023050）
