枳SnRK2与ABF互作调控PtrBAM1介导干旱胁迫下可溶糖积累的分子机制及其抗逆功能解析

β-Amylase (BAM) -mediated starch degradation is the most important source of soluble sugar. PtrBAM1, a BAM gene of Poncirus trifoliata that has been shown to be involved in starch degradation, is induced by drought and ABA. However, it remains elusive whether and how ABA signaling regulates PtrBAM1. In this project, two positive regulators of ABA signaling pathway, SnRK2 and ABF, which are herein named SnRK2.n and ABFx, respectively, that are induced by drought and ABA and display expression patterns identical to PtrBAM1 will be identified. Role of the ABRE element within the PtrBAM1 promoter in response to drought and ABA will be elucidated. Phosphorylation features of ABFx by SnRK2.n will be analyzed. Mechanism underlying the regulation of PtrBAM1 by ABFx alone or in combination with its interacting partner SnRK2.n under drought and ABA treatment will be unraveled. Regulatory role of SnRK2.n and ABFx in soluble sugar accumulation under drought stress will be illustrated. In addition, function of SnRK2.n and ABFx in stress tolerance will be characterized. The current work is expected to clarify the gene network associated with the regulation of PtrBAM1 response to drought by ABA signaling pathway and to reveal the molecular mechanism underlying soluble sugar accumulation induced by drought. Meanwhile, the study aims to lay theoretical basis for exploring applied techniques to elevate sugar content in citrus industry and to provide genes of significance for stress and quality-oriented gene engineering in Citrus.

β-淀粉酶（BAM）介导的淀粉分解是可溶性糖形成的最主要来源。我们前期研究表明枳（Poncirus trifoliata）参与淀粉分解的BAM基因PtrBAM1受干旱和ABA诱导，但ABA信号是否以及如何调控PtrBAM1不清楚。本项目将鉴定受干旱和ABA诱导且表达与PtrBAM1最接近的ABA信号途径正调控因子SnRK2和ABF成员（命名为SnRK2.n和ABFx），阐明PtrBAM1启动子ABRE元件在干旱和ABA响应中的作用，分析SnRK2.n磷酸化ABFx特征，解析ABFx及ABFx与SnRK2.n互作调控PtrBAM1响应干旱和ABA的机制，揭示SnRK2.n和ABFx对干旱下可溶性糖积累的调控作用及抗逆功能。研究将阐明ABA信号途径调控PtrBAM1应答干旱的基因网络，揭示干旱诱导可溶性糖积累的分子机制；为产业上增糖技术研发奠定理论基础，为柑橘逆境和品质基因工程提供基因资源。

前期研究表明枳（Poncirus trifoliata）参与淀粉分解的PtrBAM1受干旱和ABA诱导，但ABA信号是否以及如何调控PtrBAM1不清楚，ABA信号转导中的哪些成员参与枳干旱胁迫应答也不清楚。本项目研究结果表明在烟草中超表达PtrBAM1基因后显著提高了转基因植株的抗旱性。通过酵母单杂交筛选，PtrABF4与PtrABR1均可与PtrBAM1启动子互作，EMSA、ChIP-PCR、双荧光素酶实验证明互作性；转基因实验证明ABF4与ABR1过表达可以提高植物的抗旱性，这一过程依赖于激活下游PtrBAM1基因的表达，增加干旱条件下可溶性糖含量。进一步研究发现PtrABF4也可以激活PtrABR1基因上调表达，同时PtrABF4与PtrABR1蛋白之间存在互作，共同存在促进PtrBAM1更高的表达，增强植物干旱胁迫下可溶性糖的含量。研究还发现ABA信号通路的PtrABF2转录因子受到上游PtrSnRK2.4的磷酸化修饰，PtrABF2和PtrSnRK2.4过表达都可以提高植物的抗旱性，反之则降低植物的抗旱能力。以上研究揭示了ABA- ABF4/ ABR1- BAM1模块在枳抗旱中的重要功能，也发掘了ABA调控植物逆境应答的新的信号调控通路。共发表论文11篇，授权国家发明专利5项，获得个人奖项和荣誉称号多项，培养研究生9人，其中博士生7人，硕士生2人。