Data_Sheet_1_TcMYB29a, an ABA-Responsive R2R3-MYB Transcriptional Factor, Upregulates Taxol Biosynthesis in Taxus chinensis.PDF

Paclitaxel (Taxol), a highly modified diterpene agent mainly obtained from Taxus species, is the most widely used anticancer drug. Abscisic acid (ABA) is a well-known stress hormone that plays important roles in the secondary metabolism of plants, and it can also induce the accumulation of taxol in Taxus cell suspension cultures. However, the mechanism behind the regulation of taxol biosynthesis by ABA remains largely unknown. In previous research, a R2R3 MYB transcription factor (TF) TcMYB29a was observed to show a significant correlation with taxol biosynthesis, indicative of its potential role in the taxol biosynthesis. In this study, the TcMYB29a encoded by its gene was further characterized. An expression pattern analysis revealed that TcMYB29a was highly expressed in the needles and roots. Overexpression of TcMYB29a in Taxus chinensis cell suspension cultures led to an increased accumulation of taxol, and upregulated expression of taxol-biosynthesis-related genes, including the taxadiene synthase (TS) gene, the taxane 5α-hydroxylase (T5OH) gene, and the 3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase (DBTNBT) gene as compared to the controls. Chromatin immunoprecipitation (ChIP) assays, yeast one-hybrid (Y1H) assays, electrophoretic mobility shift assays (EMSAs), and dual-luciferase reporter assays verified that TcMYB29a could bind and activate the promoter of TcT5OH. Promoter sequence analysis of TcMYB29a revealed that its promoter containing an AERB site from -313 to -319 was a crucial ABA-responsive element. Subsequently, the ABA treatment assay showed that TcMYB29a was strongly upregulated at 6 h after ABA pretreatment. Furthermore, TcMYB29a was strongly suppressed at 3 h after the methyl jasmonate (MeJA) treatment and was depressed to the platform at 12 h. Taken together, these results reveal that TcMYB29a is an activator that improves the accumulation of taxol in Taxus chinensis cells through an ABA-medicated signaling pathway which is different from JA-medicated signaling pathways for the accumulation of taxol. These findings provide new insights into the potential regulatory roles of MYBs on the expression of taxol biosynthetic genes in Taxus.

紫杉醇（Paclitaxel, Taxol）是一种经过高度修饰的二萜类制剂，主要提取自红豆杉属（Taxus）植物，是目前临床应用最广泛的抗肿瘤药物。脱落酸（Abscisic acid, ABA）是一类经典的胁迫激素，不仅在植物次生代谢过程中发挥关键调控作用，还可诱导红豆杉细胞悬浮培养体系中紫杉醇的积累。然而，脱落酸调控紫杉醇生物合成的具体分子机制目前仍未完全阐明。既往研究发现，R2R3型MYB转录因子（Transcription factor, TF）TcMYB29a的表达水平与紫杉醇生物合成存在显著相关性，提示其可能参与紫杉醇生物合成的调控过程。本研究针对该基因编码的TcMYB29a展开了进一步的功能表征。表达模式分析结果显示，TcMYB29a在中国红豆杉（Taxus chinensis）的针叶与根中表达量较高。在该细胞悬浮培养体系中过表达TcMYB29a，可显著提升紫杉醇的积累量，并上调紫杉醇生物合成相关基因的表达水平，包括紫杉二烯合酶（taxadiene synthase, TS）基因、紫杉烷5α-羟化酶（taxane 5α-hydroxylase, T5OH）基因以及3'-N-脱苯甲酰基-2'-脱氧紫杉醇-N-苯甲酰基转移酶（3′-N-debenzoyl-2′-deoxytaxol-N-benzoyltransferase, DBTNBT）基因，与对照组相比差异显著。染色质免疫共沉淀（Chromatin immunoprecipitation, ChIP）实验、酵母单杂交（yeast one-hybrid, Y1H）实验、电泳迁移率变动分析（electrophoretic mobility shift assay, EMSA）以及双荧光素酶报告基因实验均证实，TcMYB29a可结合并激活TcT5OH基因的启动子区域。对TcMYB29a启动子序列的分析显示，其启动子内位于-313至-319位的AERB元件是关键的ABA应答顺式作用元件。后续的ABA处理实验结果表明，经ABA预处理6小时后，TcMYB29a的表达量被显著上调。此外，经茉莉酸甲酯（methyl jasmonate, MeJA）处理3小时后，TcMYB29a的表达被显著抑制，并在12小时后趋于稳定平台期。综上，本研究结果表明，TcMYB29a是一种转录激活因子，可通过ABA介导的信号通路促进中国红豆杉细胞中紫杉醇的积累，这一调控途径与茉莉酸（JA）介导的紫杉醇积累通路存在差异。本研究结果为阐明MYB类转录因子对红豆杉属植物紫杉醇生物合成基因的调控作用提供了新的理论视角。