Key Lab of Chinese Medicine Resources Conservation, State Administration of Traditional Chinese Medicine of the People's Republic of China, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing, 100193, China.
College of Chinese Materia Medica, Shanxi University of Chinese Medicine, Jinzhong, 030619, China.
Plant Mol Biol. 2019 May;100(1-2):83-93. doi: 10.1007/s11103-019-00845-7. Epub 2019 Mar 7.
The novel AP2/ERF transcription factor SmERF128 positively regulates diterpenoid tanshinone biosynthesis by activating the expression of SmCPS1, SmKSL1, and SmCYP76AH1 in Salvia miltiorrhiza. Certain members of the APETALA2/ethylene-responsive factor (AP2/ERF) family regulate plant secondary metabolism. Although it is clearly documented that AP2/ERF transcription factors (TFs) are involved in sesquiterpenoid biosynthesis, the regulation of diterpenoid biosynthesis by AP2/ERF TFs remains elusive. Here, we report that the novel AP2/ERF TF SmERF128 positively regulates diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza. Overexpression of SmERF128 increased the expression levels of copalyl diphosphate synthase 1 (SmCPS1), kaurene synthase-like 1 (SmKSL1) and cytochrome P450 monooxygenase 76AH1 (SmCYP76AH1), whereas their expression levels were decreased when SmERF128 was silenced. Accordingly, the content of tanshinone was reduced in SmERF128 RNA interference (RNAi) hairy roots and dramatically increased in SmERF128 overexpression hairy roots, as demonstrated through Ultra Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Furthermore, SmERF128 activated the expression of SmCPS1, SmKSL1, and SmCYP76AH1 by binding to the GCC box, and to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs within their promoters during in vivo and in vitro assays. Our findings not only reveal the molecular basis of how the AP2/ERF transcription factor SmERF128 regulates diterpenoid biosynthesis, but also provide useful information for improving tanshinone production through genetic engineering.
新型 AP2/ERF 转录因子 SmERF128 通过激活丹参 SmCPS1、SmKSL1 和 SmCYP76AH1 的表达正向调控二萜丹参酮生物合成。AP2/ERF 家族的某些成员调控植物次生代谢。虽然明确记载了 AP2/ERF 转录因子（TFs）参与倍半萜生物合成，但 AP2/ERF TFs 对二萜生物合成的调控仍不清楚。在这里，我们报告新型 AP2/ERF TF SmERF128 正向调控丹参中二萜丹参酮的生物合成。SmERF128 的过表达增加了焦磷酸香叶酯合酶 1（SmCPS1）、贝壳杉烯合酶样 1（SmKSL1）和细胞色素 P450 单加氧酶 76AH1（SmCYP76AH1）的表达水平，而当 SmERF128 沉默时，它们的表达水平降低。因此，通过超高效液相色谱（UPLC）和液相色谱-串联质谱（LC-MS/MS）分析，SmERF128 RNAi 毛状根中的丹参酮含量降低，SmERF128 过表达毛状根中的丹参酮含量显著增加。此外，SmERF128 通过结合 GCC 盒在体内和体外试验中激活 SmCPS1、SmKSL1 和 SmCYP76AH1 的表达，并在其启动子内结合 CRTDREHVCBF2（CBF2）和 RAV1AAT（RAA）基序。我们的研究结果不仅揭示了 AP2/ERF 转录因子 SmERF128 调控二萜生物合成的分子基础，而且为通过遗传工程提高丹参酮产量提供了有用信息。
