色氨酸脱羧酶基因TDC参与调节芍药耐高温能力的分子机制解析

Herbaceous peony is a traditional famous flower and new high-grade cut flower in China, and its large-scale production and application in the south are severely restrained by summer high temperature. Our previous studies based on the transcriptomic analysis and transgenic tobacco plants have ascertained that tryptophan decarboxylase gene (TDC) controlling the melatonin biosynthesis plays an important role in regulating herbaceous peony resistance to high temperature, but the molecular mechanisms of what upstream transcription factors regulating TDC gene expression, what downstream gene networks affected by TDC gene in response to high temperature stress is unclear yet. On this basis, over-expression of TDC gene in herbaceous peony and its comparative transcriptome analysis will be used to reveal the downstream molecular regulation network of TDC gene responding to high temperature stress in this project. On the other hand, genome walking, yeast one-hybridization, chromatin immunoprecipitation and electrophoretic mobility shift assay technologies will be used to search the upstream key transcription factors regulating herbaceous peony TDC gene expression. Moreover, gene expression pattern, subcellular localization, transcriptional activation activity and transgenic validation will be systematically used to explore the mechanism of key transcription factors regulating TDC gene expression. Eventually, these results would systematically elucidate the molecular mechanism of herbaceous peony responding to high temperature stress centered on TDC gene, which would provide theoretical basis for the molecular improvement of high temperature resistance in herbaceous peony.

芍药是我国传统名花和新兴的高档切花，夏季高温严重制约了芍药在南方的规模化生产与应用。申请者的前期工作中，基于转录组分析和转基因烟草植株研究发现，涉及褪黑素合成的上游基因—色氨酸脱羧酶基因（TDC）在调节芍药耐高温能力上具有重要作用，但对于TDC基因在其上游受何种转录因子调控表达、在其下游又是通过影响哪些基因网络来响应高温胁迫的分子机制尚不清楚。基于此，本项目拟一方面通过芍药同源转基因研究和比较转录组学分析来构建TDC基因响应高温胁迫的下游分子调控网络；另一方面，采用染色体步移、酵母单杂交、染色质免疫沉淀、凝胶阻滞电泳等技术来寻找调控芍药TDC基因表达的关键转录因子，再分别从表达模式、亚细胞定位、转录激活活性、转基因验证等方面入手系统地探究该转录因子对芍药TDC基因表达的上游调控机制。最终解析以TDC基因为核心的芍药高温胁迫响应的分子机制，为芍药耐高温能力的分子改良提供理论依据。

芍药是我国传统名花和新兴的高档切花，夏季高温严重制约了芍药在南方的规模化生产与应用。前期研究初步表明，调控褪黑素生物合成的TDC基因可能在调节芍药耐高温能力方面发挥重要作用。本项目首先采用芍药VIGS和烟草超表达技术获得了转TDC基因植株，并通过高温胁迫表型观察以及相关生理指标与褪黑素含量测定明确了TDC基因在参与调节芍药耐高温能力中的重要作用，这一结果也同样得到了酵母异源系统的验证。在此基础之上，对转TDC基因植株开展了转录组学比较分析，明确了芍药TDC基因不仅影响褪黑素生物合成，还能通过影响植株苯丙氨酸代谢、光合作用和类黄酮生物合成来响应高温胁迫。随后，采用染色体步移技术获得了2402 bp的TDC基因启动子序列，它含有多种顺式作用元件。在此基础之上，采用DNA-pull down技术筛选到1个TDC基因上游转录调控因子AP2，全长1935 bp，编码524个氨基酸，与拟南芥TOE3亲缘关系最为接近，属于AP2亚家族，被命名为芍药TOE3。接着，采用酵母单杂交和双荧光素酶系统对芍药TOE3与TDC基因启动子的结合能力进行验证，明确了两者之间具有特异靶向调控关系。进一步研究发现芍药TOE3定位于细胞核中，并且能够响应高温胁迫进行转录。此外，通过芍药VIGS和烟草超表达等实验揭示了TOE3通过靶向调控TDC基因表达来增强耐高温能力的分子机制。这一结果最终解析了以TDC基因为核心的芍药响应高温胁迫的分子机制，为芍药耐高温能力的分子改良提供理论依据。