【摘要】 茉莉酸甲酯(methyl jasmonate, MeJA)诱导表达的转录因子TwWRKY1基因(GenBank No.GAVZ01022264.1)对雷公藤(Tripterygium wilfordii)吉碱的生物合成有明显的调控作用。为明确其具体的调控机制与关键靶标基因，本研究以雷公藤倍半萜环化酶为研究对象，通过对雷公藤倍半萜环化酶(T.wilfordii sesquiterpene cyclase, Twses-CS)基因的挖掘、启动子顺式作用元件分析并检测TwWRKY1转录因子的表达和生物碱含量变化，结果显示，WRKY转录因子可能通过调控Twses-CS表达影响雷公藤倍半萜类吡啶生物碱的生物合成。酵母单杂交试验分析表明，TwWRKY1与Twses-CS启动子有一定的结合能力。亚细胞定位分析表明，转录因子TwWRKY1定位在细胞核内，属于典型的转录因子。通过电泳迁移率变动分析体外验证结合表明，TwWRKY1和含2个W-box的30 bp探针孵育后可形成DNA-蛋白复合物，能够结合W-box序列。雷公藤吉碱的含量在TwWRKY1过表达发状根中约为对照的1.87倍，达到249.53μg/g DW；雷公藤次碱的含量在TwWRKY1过表达发状根中为对照的1.90倍，达到638.25μg/g DW。结果表明，TwWRKY1可能通过与Twses-CS的启动子上W-box元件结合调控雷公藤倍半萜吡啶生物碱的含量。本研究为进一步解析雷公藤倍半萜类生物碱的生物合成途径及转录因子在转录水平上调控其生物合成机制提供依据，也为利用代谢工程与合成生物学方法解决雷公藤生物碱供应不足提供理论支持。更多还原

【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 Tw WRKY1 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.更多还原