白桦BplPCF5转录因子调控高盐胁迫的分子机制

TCPs are plant specific transcription factors (TF), which play important roles in abiotic stress tolerance in plants. We had identified the TCP family TFs from birch (Betula platyphylla), and found a gene, BplPCF5, that is highly induced by salt and confers high salt tolerance to transgenic Arabidopsis plants overexpressing BplPCF5. Based on these studies, we are going to investigate the salt tolerance mechanism of BplPCF5 in depth. The different truncated promoters of BplPCF5 are seperately fused with a reporter gene to construct reporter constructs, transiently transformed into birch, and studied the expression of reporter gene to determine the salt stress responsive DNA region present in promoter, and this DNA region is further used for identify the upstream regulator of BplPCF5 using yeast one hybrida. The transgenic birch overexpression and knock out/knock down of BplPCF5 will be generated for study. RNA-seq is performed to identify the target genes of BplPCF5, and some important target genes will be studied in salt stress tolerance using a quick method on identification of the stress tolerance of genes that we previously built. ChIP-Seq will be performed to determine the DNA sequences (peaks) bound by BplPCF5, and their roles in gene expression will be further studied.Metabonomics and physiloigcal studies are used to investigate the salt tolerance mechanism of BplPCF5, and RNA-seq is further combined to reveal the genes that related with metabonomic and physiloigcal salt stress tolerance. Co-Immunoprecipitation will be used to identify the protein interacting with BplPCF5, and the protein complex on transcriptional activition and recognizing motif will be further studied.

TCP是植物特有转录因子，在调控抗逆中起重要作用。我们鉴定了东北白桦TCP家族基因，并筛选出一个高度受盐诱导且显著提高转基因拟南芥耐盐能力的基因BplPCF5 (简称PCF)，说明该基因在耐盐调控中起作用。有鉴于此，本项目拟系统研究其调控耐盐机制。将该基因启动子分段，分别驱动报告基因，瞬时转化白桦，盐胁迫下检测报告基因的表达以确定其响应盐胁迫区段，并用酵母单杂技术调取其上游调控基因。培育PCF过表达及敲除或抑制表达的转基因白桦，通过转录组鉴定其调控靶基因，从中筛选出重要基因，利用我们建立的抗逆基因快速鉴定技术研究其耐盐性。ChIP-Seq技术研究PCF识别的DNA序列及这些序列在基因表达调控中的作用。通过代谢组及生理学分析明确其调控耐盐的代谢及生理机制，结合转录组及靶基因抗逆研究阐明决定抗逆代谢及生理机制的基因。Co-IP鉴定与PCF互作蛋白，并研究蛋白互作对转录激活及元件识别的作用。

植物TCP转录因子家族是连接胁迫信号和发育信号的关键节点基因，但对其研究较少，尤其是其抗逆调控机制尚不清晰。本项目对白桦TCP家族的一个耐盐能力强的成员BplPCF5(以下简称PCF)进行了耐盐机制研究。包括其上游调控因子、识别元件、翻译后修饰、互作蛋白，下游靶基因及耐盐的生理学机制等。研究表明，PCF被白桦的WRKY32所调控，并获得了WRKY32转基因过表达植株。获得PCF过表达株系7个，基因编辑株系3个。PCF通过调控脯氨酸合成基因，ROS清除相关基因以及离子转运基因的表达来提高渗透势，减少活性氧积累，减少K+的流失和Na+的积累，进而提高耐盐能力。通过ChIP-Seq鉴定了4个PCF结合的顺式作用元件，这些元件在PCF调控耐盐基因表达过程中起主要作用。通过RNA-seq和ChIP-Seq联合分析鉴定PCF直接调控基因359个，其中上调表达基因142个，下调表达基因217个。PCF在盐胁迫下通过乙酰化修饰来响应胁迫，CoIP发现，其与乙酰化酶Dihydrolipoamide acetyltransferase互作。提示PCF可能通过与该蛋白的互作来进行乙酰化修饰。此外，我们建立了两个技术，一个是染色质免疫共沉淀技术(ChIP)，分别从蛋白和DNA的交联、解交联、免疫共沉淀，染色质的浓缩和纯化等方面进行了创新和优化，将白桦ChIP富集效率较经典技术提高14倍以上，使得白桦ChIP-seq研究顺利完成。为克服CRISPR的CAS切割靶序列的选择问题，建立了一种高效的CAS蛋白靶位点切割效率检测技术(TCEP)，其可准确检测CAS蛋白对不同靶点的切割效率，还能用来研究影响基因编辑的各种因素，从而用于白桦基因编辑体系的优化。以上研究阐明了PCF调控白桦耐盐机制，发现其具有育种价值，尤其是鉴定到了PCF的上游调控基因WRKY32，因其作为更上层的调控基因，在耐盐调控中起关键作用，可能具有重要研究与应用价值。