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

麦醇溶蛋白是小麦的主要蛋白质 胚乳组织。 普通小麦和二倍体小麦醇溶蛋白基因组克隆 小麦已经被分离出来并确定了它们的结构。本 现在的研究表明，有胚乳特异性核因子， 特异性地结合到在几种中保守的麦醇溶蛋白启动子元件上， Okita博士称之为“CACA”盒的胚乳基因。 在这 格兰特，他建议研究确切的DNA序列结合域的 “CACA”和其它通过凝胶阻滞的麦胶蛋白-启动子结合活性， Southwestern印迹和DNA酶I足迹分析。 的影响 将在体内评价这些DNA对转录的结合活性 通过电穿孔与DNA复合的麦醇溶蛋白基因构建体， 结合到单子叶植物悬浮培养物的原生质体中的活性，或 胚乳细胞和/或通过采用异源体外 转录测定“CACA”结合因子将广泛地 利用FPLC和DNA亲和层析纯化。 与 纯化的结合因子，可以获得抗体探针以评估结合因子的活性。 组织特异性和发育模式的积累， 因子，并最终介导分离和分子 其基因的特征。这些研究将有助于 了解特定的植物基因是如何在植物生长过程中被控制的 发展，并确定分子事件， 负责这个控制。 麦醇溶蛋白基因是一种高度 多态性蛋白质家族，作为胚乳的主要贮藏物质， 小麦的蛋白质，在很大程度上负责的营养质量， 这个作物。

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.