ABI5相互作用蛋白AFP4与AFP5激活/介导ABI5转录活性的机理研究

ABI5 subfamily is a class of leucine zipper transcription factors. They participated in gene expression and regulation during the late embryogenesis, and in responding ABA and stress signal transduction. AFP4 and AFP5 are two ABI5-interacting proteins, they can activate or mediate transcriptional activity of the transcription factor ABI5 in yeast cells. With smaller molecular weight, AFP4 and AFP5 have neither kinase activity nor structural similarity with various types of kinases found to date. Expression of AFP4 gene is induced by ABA in Arabidopsis. It would play as a negative regulator to ABA signal transduction. It is unclear how AFP4 and AFP5 activate / mediate transcriptional activity of ABI5. This project intends to study in depth the interacting sites between AFP4/5 and ABI5 in yeast cells, and which parts of AFP4 and AFP5 are responsible for activating or mediating transcriptional activity of ABI5. In addition, effects of ABI5 phosphorylation by SnRK2 on its transcriptional activity will be investgated and compared to the role of AFP4/5 to ABI5. Transgenic Arabidopsis will be constructed with the AFP5 promoter-driven reporter gene and then assayed for AFP5 spatiotemporal characteristics as well as ABA and stress signal responses. All of these studies are trying to provide new insights into the molecular mechanisms of AFP4 and AFP5 roles.

ABI5亚家族是一类亮氨酸拉链类转录因子,它们参与了植物种子胚胎发育晚期的基因表达调控以及ABA信号转导等生理过程。AFP4与AFP5是2个ABI5相互作用蛋白,它们可在酵母细胞中激活/介导ABI5的转录活性。AFP4与AFP5分子量较小,无激酶活性,也与目前已发现的各类激酶无结构相似性。AFP4在拟南芥中的表达受ABA诱导,可能是ABI5信号转导的负调节子。目前尚不清楚AFP4与AFP5如何激活/介导了ABI5蛋白的转录活性。本项目拟在酵母细胞中深入研究AFP4与AFP5和ABI5相互作用的部位、AFP4与AFP5的哪个区域负责激活/介导ABI5的转录活性、SnRK2磷酸化ABI5蛋白对其转录活性的影响并和AFP4与AFP5的作用相比对、构建AFP5启动子驱动的报告基因转拟南芥植株以了解AFP5的时空表达特性以及对ABA和逆境信号的响应模式。以此了解AFP4与AFP5发挥作用的分子机制。

AFP4和AFP5是一类新发现的转录因子ABI5的相互作用蛋白，目前在拟南芥中发现了5个。这5个蛋白质被认为参与了ABI5介导的一些生理过程，如种子的休眠和萌发、干旱、高盐或低温响应等等。这五个小分子量蛋白质如何通过与ABI5相互作用、进而参与植物生长发育和响应非生物逆境的调控，其分子机制尚不清楚。据此，我们提出了《ABI5相互作用蛋白AFP4与AFP5激活介导ABI5转录活性的机理研究》项目，拟通过分析AFP4/5与ABI5的分子作用模式深入了解AFPs的作用机制。通过本项目四年的研究工作，我们取得了如下研究进展：.① 利用酵母双杂交系统和ABI5蛋白分子N/C端双向缺失分析，确定了AFP1/2/3/4/5结合于ABI5的精确位点，即ABI5蛋白上保守的磷酸化位点。AFP1/2/3/4结合于ABI5的CK2位点TxxD/E，而AFP5结合于SnRKs位点RxxS/T。.② ABI5似乎不结合于AFP4/5的某一小的、连续的序列，而是结合于多处、不连续的序列，即结合于AFP4/5蛋白的较大范围。据此我们认为，AFP4/5是ABI5的结合蛋白，ABI5是被结合蛋白。AFP4/5对ABI5的结合意味着介导某种信号至ABI5。.③ 激酶SnRK2.6、BIN2可与ABI5相互作用，但在酵母菌中共表达时未探测到ABI5的磷酸化。AFP1/2/3/4/5均可通过与ABI5的相互作用增强其转录激活活性。通过精细定位SnRK2.6作用于ABI5的位点，我们认为，不同的SnRKs结合于ABI5的潜在的SnRK位点不同，这种细微差异暗示了不同的SnRKs是如何既分工又协作地调控了ABI5的转录活性。.④ AFP5基因在拟南芥的各个组织中均有表达。过表达AFP5基因可引发转基因拟南芥植株的莲座叶变大、数量变多、种子萌发率上升和开花提前等性状改变。