毛白杨转录因子PtWRKY01调控靶标基因表达的分子机制

Recently, much attention has been focused on the problem about the growth and productivity of the poplars has adversely affected by leaf rust. Our previous studies have demonstrated that WRKY-type transcription factor PtWRKY01 from Populus tomentosa, act as one key defense-related regulator, directly participated in the transcriptional regulation of defense responses against pathogens. In the present study, the anti-PtWRKY01 polyclonal antibody with high specificity, obtained by immunized rabbit with the previously purified fusion protein His-PtWRKY01, can be used for the chromatin immunoprecipitation (ChIP) analysis of PtWRKY01 transcription factor, and the potential target DNAs specifically binding to PtWRKY01 could be obtained on the genome scale by the ChIP coupled with high-throughput sequencing (ChIP-Seq) technique. Through combination with gene ontology analysis and pathway analysis, the transcription factor PtWRKY01-regulated downstream target genes were identified, and the PtWRKY01-mediated possible defense signaling pathways were revealed. Additionally, to elucidate the role of PtWRKY01 transcription factor involved in the regulation of defense signaling pathways during the responses of P. tomentosa to pathogen-resistance, after the previously obtained transgenic P. tomentosa plants of over-expressing and RNAi-mediated suppressing PtWRKY01and wild type were inoculated with Melampsora magnusiana, the infected leaves were used for the level measurement of defense signaling molecules, as well as for the transcriptome analysis of PtWRKY01, disease resistance (R) proteins-related genes, defense-related genes,signaling transduction-related genes and PtWRKY01-regulated target genes by quantitative RT-PCR (qRT-PCR). All these results contribute to a deep probing into the molecular mechanism of PtWRKY01 transcription factor in the regulation of downstream target gene expression involved in defense responses of P. tomentosa to pathogens on the scale of functional genomics, transcriptomics, metabonomics and signaling transduction, and provide us an important foundation for the application of PtWRKY01 to molecular genetic improvement in disease resistance of the crops and tree species with a great potential usefulness.

杨树叶锈病造成巨大经济损失，已引起人们高度关注。本项目以前期研究证实与毛白杨抗锈病密切相关转录因子PtWRKY01为目标，采用染色质免疫沉淀结合高通量测序(ChIP-seq)技术，获得全基因组内与转录因子PtWRKY01互作的DNA序列，进而通过计算分析确定PtWRKY01所调控的靶标基因及其转录表达所涉及的信号通路。同时，对前期已获得未转基因、RNA干扰后以及过表达PtWRKY01毛白杨进行锈病病原菌活体接种实验，开展与抗病密切相关代谢物的测定，并对病程相关蛋白基因、抗病信号转导分子基因以及PtWRKY01所调控靶标基因的转录表达进行检测，从中探讨PtWRKY01在毛白杨抗病免疫系统中的作用机理，进而从功能基因组学、转录组学、代谢组学以及信号通路等层面阐明毛白杨转录因子PtWRKY01调控靶标基因表达的分子机制，这可为PtWRKY01应用于改良某些有经济价值的农作物与林木奠定重要基础。

杨树叶锈病造成巨大经济损失，而WRKY 转录因子广泛参与植物对各种胁迫的应答反应，已引起人们高度关注。本项目以前期研究证实与毛白杨抗锈病密切相关转录因子PtWRKY01为目标，开展病源相关信号分子（SA、MeJA、ABA）诱导、农杆菌胁迫和烟草花叶病毒侵染，采用qRT-PCR技术对毛白杨和转PtWRKY01基因烟草中PtWRKY01和抗病相关蛋白基因的表达特性进行分析，结果发现，病原菌及病原相关信号分子均能诱导PtWRKY01基因表达，但抗病蛋白基因的表达水平和模式不尽相同；同时，对74个毛果杨抗病基因在毛白杨不同组织及不同生物逆境胁迫下的表达特性进行检测后发现，27个抗病基因在毛白杨中呈现组成型表达，但它们表达水平存在差异。这说明PtWRKY01和病源相关蛋白基因的表达可能涉及到不同抗病信号分子介导的转导通路。为了阐明PtWRKY01在毛白杨抗病免疫反应过程中分子作用机制，以毛白杨抗锈病无性系L9为对照，以锈病病原菌处理植株为试材，开展转录组测序研究，拼接后分别得到67,013和52,895条 unigenes, 通过功能注释及分析获得186个受病原菌处理而发生差异表达基因，主要涉及到病程相关蛋白和抗病蛋白及其相关受体蛋白和蛋白激酶，分析它们在锈病病原菌处理不同时间中差异表达谱；并确定85个毛白杨WRKY家族成员。此外，采用染色质免疫沉淀结合ChIP-Seq技术，获得与PtWRKY01互作的DNA序列，通过计算预测与PtWRKY01特异结合位点1198个(预测分值 > 0.85)；结合锈病病原菌处理的毛白杨转录组测序结果，确定PtWRKY01调控的靶基因154个，主要涉及转录调控相关的细胞抗病免疫反应过程。同时，对前期已获得未转基因、RNA干扰及过表达PtWRKY01毛白杨进行病原菌活体接种，并对病程相关蛋白基因、信号转导分子基因以及PtWRKY01所调控靶基因的动态转录进行检测，明确PtWRKY01的锈病病原菌诱导转录对抗病基因表达及信号通路具有明显调控效应。上述研究结果有助于揭示PtWRKY01在毛白杨抗病免疫系统中的作用机理，进而从功能基因组学、转录组学以及信号通路等层面阐明毛白杨转录因子PtWRKY01调控靶基因表达的分子机制，为PtWRKY01应用于改良某些有经济价值的农作物与林木奠定重要基础。