Biochemical and Molecular Characterization of The Gliadin Multigene Family: Endosperm-Specific Nuclear Binding Factors

醇溶蛋白是小麦的醇溶蛋白，是胚乳组织的主要蛋白质。从普通小麦和二倍体小麦中分离到两个醇溶蛋白基因组克隆，并测定了它们的结构。目前的研究表明，存在胚乳特异的核因子，它与几个胚乳基因中保守的醇溶蛋白启动子元件特异结合，冈田博士将其称为CACA盒。在这项资助中，他建议通过凝胶延迟、Southwestrn blotting和DNase I足迹分析来研究“CACA”的确切DNA序列结合域和其他醇溶蛋白启动子结合活性。这些DNA结合活性对转录的影响将通过将具有DNA结合活性的醇溶蛋白基因结构物电穿孔到单子叶悬浮培养物或胚乳细胞的原生质体中和/或通过异源体外转录实验来评估。将利用FPLC和DNA亲和层析广泛纯化“CACA”结合因子。利用纯化的结合因子，可以获得抗体探针，以评价该因子的组织特异性和积聚的发育模式，并最终介导其基因的分离和分子鉴定。这些研究将有助于理解特定的植物基因在植物发育过程中是如何被控制的，并确定负责这种控制的分子事件。醇溶蛋白基因是一个高度多态的蛋白质家族，作为小麦主要的胚乳贮藏蛋白，在很大程度上决定了小麦的营养品质。

The gliadins, the prolamines of wheat, are the predominant proteins of endosperm tissue. Two gliadin genomic clones from common and diploid wheat have been isolated and their structures determined. Present studies now show that there are endosperm-specific nuclear factors that bind specifically to a gliadin promoter element conserved in several endosperm genes which Dr. Okita has termed the "CACA" box. In this grant he proposes to study the exact DNA sequence binding domain of the "CACA" and other gliadin-promoter binding activities by gel retardation, Southwestrn blotting, and DNase I footprinting assays. The effect of these DNA binding activities on transcription will be evaluated in vivo by electroporation of gliadin gene constructs complexed with the DNA binding activities into protoplasts of monocot suspension cultures or endosperm cells and/or by employing a heterologous in vitro transcription assay. The "CACA" binding factor will be extensively purified utilizing FPLC and DNA affinity chromatography. With the purified binding factor, antibody probes can be obtained to evaluate the tissue-specificity and developmental pattern of accumulation of the factor and eventually to mediated the isolation and molecular characterization of its gene. These studies will contribute to an understanding of how specific plant genes are controlled during plant development, and a determination of the molecular events which are responsible for this control. The gliadin genes are a highly polymorphic family of proteins, which as the major endosperm storage protein of wheat, are largely responsible for the nutritional quality of this crop.