杜梨钠氢逆向转运蛋白PbrNHX2的抗盐机制及调控网络解析

Our previoius work, PbrNHX2 of Pyrus betulaefolia plays a role in salt tolerance. However, the molecular mechanisms underlying induction of PbrNHX2 by salt and the regulatory network remain unclear at this point. In the current project, critical cis-acting elements required for salt response of PbrNHX2 will be identified in the promoter of PbrNHX2. Meanwhile, methylation status and patterns of the coding region and promoter of PbrNHX2 will be analyzed. These work will help to elucidate the molecular mechanisms underlying salt response of PbrNHX2. Relevant transcriptional factors (TFs) that bind with the identified cis-acting elements will be isolated. Specific binding of the TFs to PbrNHX2 promoter will be verified. Meanwhile, regulation of PbrNHX2 expression and the roles of TFs in salt tolerance will be dissected. In addition, proteins, interacting with the TFs will be explored. Interaction between the interacting proteins (IPs) and the TFs will be verified. Furthermore, implication of the IPs in the TFs-mediated regulation of PbrNHX2 expression and under salt stress will be assessed. The current project will provide clues for illustrating the molecular mechanisms and regulatory network underlying the salt response of PbrNHX2, all these will further our knowledge on the salt response of plants. On the other hand, potential elite genes that can be used for stress tolerance-oriented genetic engineering of pear will be exploited, manifesting the scientific significance and potential application of this project.

前期研究表明，杜梨（Pyrus betulaefolia）钠氢逆向转运蛋白PbrNHX2响应盐胁迫，并具有抗盐功能，但它响应盐胁迫的分子机制及调控网络仍未知。本项目将鉴定PbrNHX2启动子盐应答顺式作用元件，分析PbrNHX2编码区和启动子甲基化模式，以揭示PbrNHX2响应盐的分子机制。在此基础上，分离与盐应答元件结合的转录因子，利用EMSA及ChIP-PCR验证转录因子与PbrNHX2启动子特异性结合，阐明转录因子在调控PbrNHX2中的作用及其抗盐功能。筛选转录因子的互作蛋白(重点蛋白激酶)，验证互作蛋白与转录因子的作用，并鉴定互作蛋白在转录因子调控PbrNHX2表达及抗盐中的功能。本项目是已有研究的深入和延续，一方面将揭示PbrNHX2响应盐胁迫的分子机理和调控网络，丰富盐应答的理论基础，另一方面，为梨抗盐种质改良提供有价值的基因资源，因而具有重要科学意义和潜在应用价值。

丝分裂原激活蛋白激酶（Mitogen-activated protein kinase, MAPK/MPK）是真核生物中的保守的蛋白激酶，并与丝分裂原激活蛋白激酶的激酶的激酶（Mitogen-activated protein kinase kinase kinase, MAPKKK/MEKK）和丝裂原活化蛋白激酶的激酶（Mitogen-activated protein kinase kinase, MAPKK/MEK）一同组成保守的级联，通过磷酸化将上游信号依次传递下去，最终激活MAPK调控多样的下游底物实现信号转导。本研究中，前期，通过盐转录组数据挖掘到抗盐关键基因PbrNHX2，表达模式分析发现其受盐胁迫和脱水诱导，且受盐胁迫更为明显。亚细胞定位显示PbrNHX2定位于液泡质体。超表达PbrNHX2增强了转基因植物的耐盐性，通过病毒诱导基因沉默（VIGS）下调PbrNHX2的表达增强了其对盐胁迫的敏感性。为了探索其抗盐调控机理，我们通过酵母单杂筛库找到了其上游调控因子PbrMYB73，通过EMSA等实验验证了转录因子PbrMYB73能够激活下游靶标基因PbrNHX2。超表达PbrMYB73能够增强植物的抗盐性。在此基础上，进一步通过酵母双杂筛库得到PbrMYB73的互作蛋白PbrMAPK15，且PbrMAPK15能磷酸化转录因子PbrMYB73，从而阐明阐明了PbrMAPK15- PbrMYB73- PbrNHX2的响应盐胁迫的级联信号转导网络，为耐盐基因挖掘奠定了理论基础。