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

麦醇溶蛋白多基因家族的生化和分子特征：胚乳特异性核结合因子

• 批准号: 8702182
• 负责人: Thomas Okita
• 金额: $ 26.5万
• 依托单位: Washington State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=8702182&HistoricalAwards=false
• 关键词: Biochemical; Molecular; Characterization; Gliadin; Multigene

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.

麦胶蛋白，小麦的脯胺，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出两个麦胶蛋白基因组克隆并确定了它们的结构。目前的研究表明，有一些胚乳特异性的核因子可以与一些保存在胚乳基因中的麦胶蛋白启动子元件特异性结合，Okita博士将其称为“CACA”盒子。在这项资助中，他建议通过凝胶阻滞、西南印迹和DNA酶I足迹测定来研究“CACA”和其他麦胶蛋白启动子结合活性的确切DNA序列结合域。这些DNA结合活性对转录的影响将在体内通过将麦胶蛋白基因结构与DNA结合活性结合到单子叶悬浮培养或胚乳细胞的原生质体中和/或采用异源体外转录试验来评估。“CACA”结合因子将广泛纯化利用FPLC和DNA亲和层析。纯化后的结合因子可获得抗体探针，用于评估该因子的组织特异性和积累的发育模式，并最终介导其基因的分离和分子鉴定。这些研究将有助于了解植物发育过程中特定的植物基因是如何被控制的，并确定负责这种控制的分子事件。麦胶蛋白基因是一个高度多态性的蛋白家族，是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。