小麦TaGF14b逆境胁迫相关互作蛋白鉴定及其抗逆调控机制解析

Plants have evolved extraordinary anatomical, physiological and molecular strategies by which they detect and adapt to a multitude of adverse environmental changes. The 14-3-3 protein, a kind of crucial adapter protein, is well known as one of the phosphopeptide-binding proteins and plays significant roles in light signal transduction, plant hormone regulation and plant growth and development. While, there is only a few reports about functional analyses of 14-3-3 in vivo of plants, especially in allopolyploid wheat (Triticum aestivum L.). Our previous studies showed that ectopic expression of TaGF14b in tobacco conferred enhanced tolerance to drought and salt stresses, and that TaGF14b enhances the tolerance of transgenic tobaccos to multiple abiotic stresses through the ABA signaling pathway by altering physiological and biochemical processes. However, the molecular regulation mechanism is still unclear. Identification and characterization analysis of TaGF14b specific interacting proteins under drought and salt stresses, is the key point to understand the signaling mechanism of TaGF14b in plant response to abiotic stresses. In this project, we generated the transgenic lines with GFP and HA tags fused TaGF14b. The direct TaGF14b-interacting proteins under drought and salt stresses will be identified by Co-IP combined with LC-MS/MS assay, and the candidate interactions proteins will be further verified by yeast-two-hybrid and BiFC. Then the necessary region of interaction protein will be identified by yeast two-hybrid deletion mutant assay. Meanwhile, functions of candidate genes will be explored through drought and slat tolerance tests using transgenic lines. Downstream targets of these candidate genes will be identified after transcriptome and proteome analyses. In summary, this project will identify and character new TaGF14b-interacting components involved in plant responding to abiotic stresses, and will lay a foundation for elucidation of the molecular mechanism of TaGF14b in response to drought and salt stresses.

植物通过信号转导系统来检测并响应复杂的环境变化。14-3-3接头蛋白，在多种生命活动中发挥重要调节作用；转录表达数据表明14-3-3参与了多种逆境胁迫的响应。但对其在植物逆境应答过程中的生物学功能研究还很少。申请者前期发现TaGF14b在转基因烟草对干旱和盐胁迫的应答中发挥重要正调控作用，但分子机制仍不清楚。鉴定植物逆境胁迫响应特异的14-3-3互作蛋白是解析其机制的关键。申请人构建了带GFP和HA标签的转基因材料，拟利用Co-IP和液相色谱-质谱联用（LC-MS/MS）技术鉴定TaGF14b的互作蛋白，通过酵母双杂和BiFC验证蛋白间的互作。并通过酵母双杂缺失突变体寻找互作的必需区域。同时，通过转基因技术，鉴定候选基因的抗逆功能；通过组学分析研究该基因对下游信号传导途径的影响。通过研究，期望鉴定出逆境胁迫下TaGF14b的体内互作蛋白及功能，为阐明其逆境响应的分子调控机理奠定基础。

小麦是世界上干旱、半干旱地区的主要粮食作物，但是其生产频繁受到干旱胁迫的威胁。14-3-3作为接头蛋白，可以在转录水平上调控植物对非生物逆境胁迫的响应，但对其在植物逆境应答过程中的生物学功能研究还很少。本研究前期发现一个小麦14-3-3基因，TaGF14b在转基因烟草对非生物逆境胁迫的应答中发挥重要正调控作用，但分子机制仍不清楚。在本研究中，将该基因在小麦中过表达发现，TaGF14b能够显著提高转基因小麦的耐旱性。进一步，我们对TaG14b 调控小麦对干旱胁迫耐受性的机理进行了解析：TaGF14b能够通过增强多种抗氧化酶的活力和基因表达水平的途径提高ROS清除能力，减少过氧化氢积累，以响应干旱胁迫。利用Co-IP结合LC-MS/MS技术分离鉴定到了多条干旱处理下TaGF14b的体内互作蛋白，经酵母双杂交（Y2H）和双分子荧光互补（BIFC）技术验证后，发现三个糖代谢相关蛋白在干旱处理下可以与TaGF14b体内互作的蛋白；进一步分析发现，除了通过与糖代谢蛋白（TaCINV，TaSPS，TaFBA）互作外，TaGF14b还可以增强糖代谢相关酶活力和转录水平等途径，维持可溶性糖的含量，保持细胞代谢水平的稳定，从而提高转基因小麦对干旱胁迫的耐受性。而后，通过转录组和代谢组测序分析对TaGF14b转基因小麦在干旱处理下的调控网络进行了初步探索。本项目的研究结果对明确小麦TaGF14b在干旱胁迫下的生物学功能及其调控机制，具有重要参考意义；并为小麦耐旱的分子育种提供新的基因资源和材料。