Phenolic acids and tanshinones are the two groups of pharmaceutically active metabolites in Salvia miltiorrhiza Bunge. Their contents are the key quality indicator to evaluate S. miltiorrhiza. bHLH transcription factors have important roles in regulation of plant specialised metabolism. In this study, an endogenous bHLH transcription factor, SmbHLH3, was identified and functionally analyzed. SmbHLH3 was presented in all the six tissues and mostly expressed in fibrous roots and flowers. It was localized to the nucleus. Overexpression of SmbHLH3 decreased both phenolic acids and tanshinones contents. Contents of caffeic acid and rosmarinic acid were both decreased to 50% of the control. And accumulation of salvianolic acid B was decreased as much as 62%. Content of cryptotanshinone, dihydrotanshinone I, tanshinone I and tanshinone IIA in SmbHLH3-overexpression lines were reduced 97%, 62%, 86% and 91%, respectively. In the transgenic lines, expression of C4H1, TAT and HPPR in phenolic acids pathways were reduced to about 43%, 66% and 77% of the control, respectively. For tanshinone biosynthetic pathways, transcripts of DXS3, DXR, HMGR1, KSL1, CPS1 and CYP76AH1 were reduced to 46%, 65%, 78%, 57%, 27% and 62% of the control, respectively. There was an E/G-box specific binding site in SmbHLH3, which may bind the E/G-box present in promoter region of these biosynthetic pathway genes. Y1H results indicated that SmbHLH3 could bind the promoter of TAT, HPPR, KSL1 and CYP76AH1. These findings indicated that SmbHLH3 downregulate both phenolic acids and tanshinone accumulation through directly suppressing the transcription of key enzyme genes.

中文翻译：

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SmbHLH3 作为丹参毛状根中酚酸和丹参酮生物合成的转录抑制因子

酚酸和丹参酮是丹参中的两组药学活性代谢物。它们的含量是评价丹参的关键质量指标。bHLH 转录因子在植物特殊代谢的调节中具有重要作用。在这项研究中，鉴定了内源性 bHLH 转录因子 SmbHLH3 并对其进行了功能分析。SmbHLH3 存在于所有六种组织中，主要在须根和花中表达。它定位于细胞核。SmbHLH3 的过表达降低了酚酸和丹参酮的含量。咖啡酸和迷迭香酸的含量均降至对照的50%。丹酚酸 B 的积累减少了 62%。隐丹参酮、二氢丹参酮Ⅰ、SmbHLH3 过表达株系中的丹参酮 I 和丹参酮 IIA 分别降低了 97%、62%、86% 和 91%。在转基因品系中，酚酸途径中 C4H1、TAT 和 HPPR 的表达分别降低至对照的 43%、66% 和 77%。对于丹参酮生物合成途径，DXS3、DXR、HMGR1、KSL1、CPS1 和 CYP76AH1 的转录物分别减少到对照的 46%、65%、78%、57%、27% 和 62%。SmbHLH3中存在一个E/G-box特异性结合位点，可能与这些生物合成途径基因的启动子区存在的E/G-box结合。Y1H结果表明SmbHLH3可以结合TAT、HPPR、KSL1和CYP76AH1的启动子。这些发现表明 SmbHLH3 通过直接抑制关键酶基因的转录来下调酚酸和丹参酮的积累。