Huo Yanbo, Zhang Jing, Zhang Bin, Chen Ling, Zhang Xing, Zhu Chuanshu
College of Plant Protection, Northwest A&F University, Yangling 712100, China.
Shaanxi Research Center of Biopesticide Engineering & Technology, Northwest A&F University, Yangling 712100, China.
Plants (Basel). 2021 Apr 1;10(4):679. doi: 10.3390/plants10040679.
Triptolide, an important bioactive diterpenoid extracted from the plant , exhibits many pharmacological activities. MYC2 transcription factor (TF) plays an important role in the regulation of various secondary metabolites in plants. However, whether MYC2 TF could regulate the biosynthesis of triptolide in is still unknown. In this study, two homologous MYC2 TF genes, and , were isolated from hairy roots and functionally characterized. The analyses of the phylogenetic tree and subcellular localization showed that they were grouped into the IIIe clade of the bHLH superfamily with other functional MYC2 proteins and localized in the nucleus. Furthermore, yeast one-hybrid and GUS transactivation assays suggested that TwMYC2a and TwMYC2b inhibited the promoter activity of the miltiradiene synthase genes, and , by binding to the E-box (CACATG) and T/G-box (CACGTT) motifs in their promoters. Transgenic results revealed that RNA interference of significantly enhanced the triptolide accumulation in hairy roots and liquid medium by upregulating the expression of several key biosynthetic genes, including (), (), , and . In summary, our findings show that TwMYC2a and TwMYC2b act as two negative regulators of triptolide biosynthesis in hairy roots and also provide new insights on metabolic engineering of triptolide in the future.
雷公藤甲素是从该植物中提取的一种重要的生物活性二萜类化合物，具有多种药理活性。MYC2转录因子（TF）在植物各种次生代谢产物的调控中起重要作用。然而，MYC2 TF是否能调控雷公藤甲素在[植物名称未给出]中的生物合成仍不清楚。在本研究中，从[植物名称未给出]毛状根中分离出两个同源的MYC2 TF基因，即[基因名称未给出]和[基因名称未给出]，并对其进行了功能鉴定。系统发育树分析和亚细胞定位表明，它们与其他功能性MYC2蛋白一起被归入bHLH超家族的IIIe分支，并定位于细胞核。此外，酵母单杂交和GUS反式激活分析表明，TwMYC2a和TwMYC2b通过与雷公藤二烯醇合酶基因[基因名称未给出]和[基因名称未给出]启动子中的E-box（CACATG）和T/G-box（CACGTT）基序结合，抑制了它们的启动子活性。转基因结果显示，[基因名称未给出]的RNA干扰通过上调包括[基因名称未给出]（[基因名称未给出]）、[基因名称未给出]（[基因名称未给出]）、[基因名称未给出]和[基因名称未给出]在内的几个关键生物合成基因的表达，显著提高了毛状根和液体培养基中雷公藤甲素的积累。总之，我们的研究结果表明，TwMYC2a和TwMYC2b是[植物名称未给出]毛状根中雷公藤甲素生物合成的两个负调控因子，也为未来雷公藤甲素的代谢工程提供了新的见解。
