GASA6互作蛋白的筛选及其互作调控种子萌发的机理研究

Seed germination, determining the initiation of the plant life cycle, is of immense significance for agriculture and has been studied for centuries. Yet, our understanding of the molecular mechanisms underlying regulation of germination is still in its infancy. Our previous works have indicated that the GASA6 protein, a small protein with 12 cysteines in its highly conserved GASA domain, functions as a positive regulator in regulating the transcriptional level of EXPA1, resulting in the regulation of seed germination through a promotion of cell elongation. To uncover the mechanism of GASA6, as a protein located in the cell wall, regulating the EXPA1 expressions, more GASA6 Interacting Proteins (GBPs) need further investigation. Therefore, in this proposal, firstly, we are going to identify GBPs through the Co-IP and confirm their interactions by bimolecular fluorescence complementation (BiFC) and Pull-down experiments. Secondly, to confirm whether GBPs regulate the expressions of EXPA1, the analysis of genes expression in mutants, sequencing, seed germination phenotype analysis, subcellular location analysis, and the genetic relationship analysis will be performed. Finally, phosphorylation levels of GBPs regulated by GASA6 will be detected in vivo and in vitro, as well as the gene expressions of EXPA1 regulated by GBPs after CIP treatments. Our researches will shed light onto the mechanism of GASA proteins in the regulation of seed germination by regulating the expressions of downstream genes.

种子是高等植物特有的保证种族生存和繁衍的器官，种子萌发是整个植物生命周期的起始，阐明其分子调控机理具有重要的科学意义和潜在的应用价值。本项目前期研究发现小分子蛋白GASA6通过调控EXPA1基因表达来调节种子萌发中胚轴细胞的伸长。探究作为细胞壁蛋白GASA6调控EXPA1转录水平的作用机理，分离鉴定其互作蛋白至关重要。本项目拟进行以下研究：1.利用Co-IP筛选互作蛋白GBPs，通过BiFC和Pull-down验证其互作；2.通过基因表达检测或表达谱测序、表型分析、亚细胞定位、遗传关系分析等研究GASA6与GBPs是否参与调控EXPA1表达；3.通过体内和体外实验检测GASA6对GBPs磷酸化水平影响，以及检测CIP处理GBPs调控EXPA1或其他下游基因表达水平等研究GASA6与GBPs互作调控下游基因表达的分子机理。研究结果将为阐明GASA类小分子蛋白调控种子萌发的分子机理奠定基础。