乙烯响应因子PhERF2调控矮牵牛耐涝的分子机理研究

Petunia (Petunia × hybrida) as a model plant, is also an important landscape flowers. Waterlogging become to one of the major abiotic stresses constraining flowers' plantation and application in the landscape. The ethylene responsive factors (ERF) has been demonstrated to be widely involved in regulation of various responses to different abiotic stresses. It has great potential in the application of transgenic breeding. PhERF2 belongs to subfamily VII of ERF transcription factors, and plays a very important role in plant stress responses. We have generated the transgenic plants of over-expression or RNA interference (RNAi)-suppression of PhERF2 previously. This project is designed to elucidate the regulatory of waterlogging mechanisms by dissecting the roles of the ethylene responsive factors pathway. ①We will explore the molecular mechanism of PhERF2 regulation of waterlogging tolerance in petunia. All transgenic plants and their wildtype controls will be subjected to a period of waterlogging treatments. Morphological alterations and changes in the ethylene pathway and the anaerobic respiration pathway will be determined. Programmed cell death and aerenchyma formation will be detected by laser confocal fluorescence microscopy observation and DAPI (4',6-Diamidino-2-phenylindole dihydrochloride) staining. ② Downstream gene network regulated by PhERF2 in response to waterlogging will be generated. We plan to study the root transcriptome under waterlogging using the RNA-sequencing technology and digital gene expression profiling and differential gene expression identification. ③We will study the function of downstream target gene response to PhERF2 in regulation waterlogging in petunia. Based on bioinformatics analysis, we will construct models for gene co-expression networks in response to the waterlogging. Our objectives are to uncover the potential regulatory mechanism(s) of an ethylene responsive factor (PhERF2) in waterlogging tolerance in petunia at physiological and molecular levels. Information obtained from this study will provide the foundation for molecular breeding of petunia resistance to waterlogging in the future.

矮牵牛为模式植物，也是重要的园林景观花卉，涝渍胁迫成为制约花卉生产应用的主要因素。ERF(ethylene responsive factor)转录因子广泛参与植物逆境胁迫反应的调控，在转基因育种应用中潜力巨大。矮牵牛乙烯响应因子PhERF2属于ERF型转录因子VII亚族，在植物抗逆反应中起重要作用。申请人前期研究已获得PhERF2的过表达、RNAi植株，本申请以此为基础材料，拟通过①乙烯响应因子PhERF2的耐涝功能分析：进行涝渍胁迫处理后，观察植株涝害表型，检测乙烯途径和无氧呼吸途径变化；DAPI染色镜检根系细胞程序性死亡及通气组织情况；②PhERF2调控的下游基因网络构建：利用转录组测序和数字表达谱测序方法，进行根响应涝渍胁迫的转录组分析；③PhERF2调控的下游靶标基因功能验证，建立基因间共表达网络，阐明PhERF2对矮牵牛响应涝渍胁迫的调控机理，为耐涝分子育种奠定基础。

涝渍胁迫成为制约花卉生产的主要因素，乙烯响应因子ERF(ethylene responsive factor)广泛参与植物逆境胁迫反应的调控，在转基因育种应用中潜力巨大。ERF2属于ERF型转录因子VII亚族，在植物抗逆反应中起重要作用。以矮牵牛乙烯响应因子PhERF2过表达株系、PhERF2-RNAi沉默株系及其野生型WT为材料，进行涝渍胁迫处理，通过观察涝害表型症状和生理生化特性，揭示了PhERF2通过诱导根系细胞程序性死亡（PCD），促进通气组织形成；增强植物抗氧化系统，提高植株对活性氧的清除能力；减轻乙醇、乳酸等代谢产物对细胞的毒害作用，从而提高了植物的耐涝性。采用RNA-seq技术结合生物信息学法，筛选出涝渍胁迫相关ERF转录因子进行蛋白理化性质及高级结构分析、系统进化树构建、磷酸化修饰位点预测、保守基序分析和亚细胞定位预测。对涝渍处理前后进行RNA-Seq高通量测序研究，共获得115.49G的Clean Data，各样本的有效数据量分布在6.04~6.66G，Q30碱基分布在81.34~91.55%，平均GC含量为43.99%，比对率为92.16~94.49%，通过转录组数据库比较，筛选出6788个差异表达基因，进行GO分类、KEGG富集分析，从其涉及的四条生化代谢通路：①植物激素信号转导代谢通路②无氧呼吸途径③抗氧化酶系统④乙烯及其信号途径，筛选到PhERF2转录因子调控的下游关键候选基因：ADH1, LDH, PDC4, SODA, CAT3, ACO1, ACS3, SnRK2, ABF, EBF1/2和ERF1/2共11个。通过qRT-PCR技术，分析候选基因的表达模式进行验证。推测PhERF2通过在转录水平上调控上述候选基因，从而促进植物光合作用、减轻无氧呼吸产物对细胞的毒害作用并增强植物抗氧化能力，进而提高了植物的耐涝性。着重筛选重要差异表达基因ADH1-2，通过EMSA和LUC实验进一步研究PhERF2对矮牵牛耐涝性的调控机制，解析了PhERF2通过与ADH1-2启动子中的ATCTA顺式作用元件结合，调控矮牵牛耐涝的分子机理。.项目执行期间，共培养研究生3名，发表标注该基金资助论文6篇，其中SCI论文2篇，刚被《分子植物育种》录用1篇（论文编码：M24416），另有2篇正在整理，申请国家发明专利2项，获得国家级和省部级奖项3个。