小麦干旱应答基因TaPP2A调节亚基的作用机理及功能分析

Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation events and play a prominent role in the regulation of specific signal transduction cascades in plants. The predominant form of PP2A consists of catalytic subunit C, scaffold subunit A, and highly variable regulatory subunit B, which determines the substrate specificity of PP2A holoenzymes. To further understand how plant responds to drought stress, our group had cloned the wheat protein phosphatase 2A (PP2A) and named respectively as TaPP2Aa, TaPP2Ac and TaBβ-1. Our previous results showed that transgenic Arabidopsis plants over-expressing TaPP2Aa and TaPP2Ac could improve tolerance to drought. The expression of TaBβ-1 was up-regulated by PEG and NaCl treatments. This project plan to analysis the interaction way and the functional domain of regulatory subunit with the other two subunits through BiFC, pull-down and yeast two hybrid, and also identify the physiological function of TaBβ-1 by transgenic Arabidopsis plants. These research may contribute to further understanding the roles of PP2A in wheat, and provide molecular theoretical basis for obtain the drought-tolerant crops in the future.

干旱缺水是当今的世界性难题，培育作物抗逆新品种已成为现代农业研究的重要目标。为了深入研究小麦响应干旱的分子机制，项目组克隆了逆境应答调节基因小麦蛋白磷酸酶2A(TaPP2A)。蛋白磷酸酶不仅能够使底物去磷酸化，在细胞代谢、信号传递以及对干旱等胁迫的应答中也发挥着重要作用。TaPP2A由结构亚基A、催化亚基C以及决定底物特异性的调节亚基B组成，编码这三个亚基的基因分别命名为TaPP2Aa、TaPP2Ac和TaBβ-1。我们的研究显示TaBβ-1的表达受PEG和NaCl诱导，过表达TaPP2Ac和TaPP2Aa可增强转基因拟南芥的抗旱性。本项目以TaPP2A亚基间的相互作用为切入点，拟采用BiFC、Pull-down、酵母双杂交等方法重点分析调节亚基TaBβ-1与其他亚基的互作方式，明确其功能域，同时结合转基因拟南芥功能分析，系统阐释TaPP2A的功能，为小麦抗旱研究提供理论依据。

小麦是我国重要的粮食作物，干旱、盐碱、高温等逆境条件不仅严重制约着小麦的生产，也对抗逆新品种的培育提出了更高的要求。植物蛋白磷酸酶2A(protein phosphatase 2A，PP2A)由结构亚基A、催化亚基C以及调节亚基B组成，在逆境胁迫应答途径中发挥着重要作用。本项目以调节亚基B为研究对象，该家族成员众多，功能多样，决定着PP2A底物的特异性。项目组研究表明，过表达调节亚基PR55亚家族基因TaBβ-1增强了转基因拟南芥在幼苗期和成株期对干旱、盐和冷胁迫的抗性，提高了细胞膜稳定性和光合效率。调节亚基B′亚家族基因TaPP2Ab-B′1响应渗透胁迫，过表达TaPP2Ab-B′1提高了转基因拟南芥对逆境胁迫的耐受性，胁迫下转基因株系主根长显著高于对照株系，胁迫应答基因的表达量升高。调节亚基B"亚家族基因TaPP2AbB"-α在苗期根部表达量明显高于叶片，并可以响应渗透胁迫和低温胁迫，同时可被短时间内ABA处理所诱导。过表达TaPP2AbB"-α转基因拟南芥株系在胁迫下叶片的长势优于对照，主根长和侧根密度显著高于对照株系，提高了对逆境胁迫的耐受性。互作研究显示，TaBβ-1与A/C亚基在洋葱表皮细胞的细胞膜和细胞核内发生互作，TaPP2Ab-B′1与A/C亚基在洋葱表皮细胞的细胞膜发生互作，TaPP2AbB"-α定位于小麦原生质体的细胞核、细胞质与细胞膜上，在酵母与烟草中可与A/C亚基发生互作。上述研究结果揭示了TaPP2A调节亚基的生理功能，初步分析了不同调节亚基与A/C亚基的相互作用模式，为阐释小麦抗逆机制提供了理论依据，同时为培育抗逆新品种提供了优异基因资源。