胡杨肉质化关键基因PeXTH的启动子分析与转录调控研究

Salt-induced tissue succulence is an important way of adaptation to a prolonged period of salinity stress in Populus euphratica. Our previous study demonstrated that PeXTH gene played crucial roles in the development of leaf succulence. In addition, salt treatment induced higher expression of the PeXTH gene in P. euphratica, while its transcriptional regulatory mechanism remains unknown. Salt-induced promoter of the PeXTH gene had been isolated in our previous study. Bioinformatics analysis showed that the promoter contained several salt-induced cis-elements. In this research, we intend to identify the critical salt-responsive region in the PeXTH promoter using 5’-sequence deletion method, and seek out salinity tolerance-related elements in the salt-responsive region by electrophoretic mobility shift assay and deletion mutation experiments. Then, using these elements as baits, we will isolate candidate transcription factors from P. euphratica leaves by yeast one-hybrid library screening system. At last, we verify the target ones with luciferase activity analysis and electrophoretic mobility shift assay in vivo and in vitro, respectively, which may help us to understand the transcriptional regulatory mechanisms of PeXTH. This study will contribute to a deeper understanding on the molecular mechanisms of salt-induced succulence, and enrich our knowledge in salt tolerance of P. euphratica.

盐胁迫下组织发生肉质化是胡杨适应长期盐渍环境的重要方式。前期研究表明胡杨PeXTH基因在叶片肉质化过程中发挥重要作用，该基因受到盐胁迫诱导转录，但其转录调控的分子机制并不明确。我们前期从胡杨叶片中分离了PeXTH基因的启动子，通过生物信息学预测发现该启动子序列中包含多个盐诱导元件。在此基础上，本项目将采用5’端缺失法确定PeXTH启动子的盐胁迫响应关键区域；再经凝胶迁移分析和缺失突变实验鉴定该区域上的盐诱导元件；进一步利用酵母单杂交系统从胡杨叶片中分离与盐诱导元件相互作用的转录因子，分别通过荧光素酶活性分析和凝胶电泳迁移实验从体内和体外两个方面验证候选转录因子的作用，探讨盐胁迫诱导PeXTH基因转录的调控机理。本项目是前期研究的延续和深入，项目的开展有助于深入理解盐诱导胡杨组织肉质化发生的分子机制，对于丰富胡杨抗盐机理具有重要意义。

盐胁迫下组织发生肉质化是胡杨在形态结构上适应长期盐渍环境的重要表现。我们的前期研究发现胡杨PeXTH基因能够通过调控叶片肉质化发生来提高转基因烟草的抗盐性，但该基因的转录调控机制尚不清楚。本项目从PeXTH基因的启动子入手，对启动子上的盐诱导元件及与其相互作用的转录因子进行研究。我们首先通过5’端缺失法确定了P4（-525~+60）为驱动PeXTH基因盐诱导表达的最有效功能区段。在此基础上，利用反向PCR方法分别对P4（-525~+60）区段上的4个可能的盐诱导元件（WRKY71OS、WBOXATNPR1、MYBCORE、WBOXNTERF3）进行缺失突变，结果显示MYBCORE元件为潜在的盐诱导功能元件。以MYBCORE元件作为诱饵，将该元件的三串联体序列转化到酵母细胞中。同时，构建胡杨叶片cDNA文库。经酵母单杂交实验，筛选出阳性克隆。经测序和序列比对分析，获得了1个与MYBCORE元件相互作用的转录因子，将该转录因子命名为PeMYB1。荧光定量PCR检测结果显示PeMYB1基因受盐胁迫诱导表达上调，后续我们将进一步通过荧光素酶活性分析实验在体内验证该转录因子与PeXTH启动子结合的特异性。本项目揭示了盐诱导PeXTH表达上调的分子机理，为深入探索胡杨组织肉质化发生的机制奠定了理论基础。