ANALYSIS OF ZEIN GENE EXPRESSION IN MAIZE

玉米玉米醇溶蛋白基因表达分析

• 批准号: 3291735
• 负责人: BRIAN A LARKINS
• 金额: $ 11.95万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3291735
• 关键词: DNA; corn; developmental genetics; electroporation; gene expression; genetic library; genetic manipulation; genetic recombination; genetic transcription; genetically modified plants; messenger RNA; molecular cloning; mutant; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; plant genetics; plant proteins; protein biosynthesis; radiotracer; regulatory gene; seeds

The zein genes of maize encode the seed storage proteins. Previous analyses have shown that zeins consist of three distinct protein types designated alpha, beta, and gamma. The alpha zeins account for approximately 70% of the total storage protein and are encoded by a multigene family. The beta and gamma zeins, which account for approximately 10% and 20% of the zein fraction, respectively, are each encoded by only one or two genes. Zein genes are actively expressed during development of normal endosperms, but their expression is quantitatively and qualitatively inhibited in several mutant genotypes. Our preliminary studies indicate that there are differences in the relative expression of genes among zein gene families that correlate with differences in the amount of cytoplasmic mRNA. To define the mechanisms regulating the expression of zein genes during endosperm formation, we propose to characterize the DNA sequences controlling their transcription and developmental regulation. Hybridization of 32P-labeled nuclear RNA to well characterized genomic clones will establish sites of transcription, timing of transcription, and whether transcription or post-transcriptional processing play a major role in the regulation of the zein gene expression. This analysis will also determine the extent to which these reactions are affected by the mutant genotypes. To define DNA sequences regulating transcription, gene flanking sequences will be ligated to a chloramphenicol acetyltransferase (CAT) gene and transferred to plant protoplasts by electroporation. Sequences required for transcription will be monitored by assaying CAT activity in protoplast extracts. We will also analyze transcriptional regulatory sequences following transformation of zein genes into tobacco and petunia plants with a Ti-plasmid vector. In this case we will monitor mRNA and protein synthesis in seeds from regenerated plants to delineate DNA sequences controlling developmental expression. Through labeling of endosperm proteins and differential screening of cDNA libraries we will attempt to identify gene products whose expression is modified in mutants that reduce the level of zein gene expression.

玉米醇溶蛋白基因编码种子贮藏蛋白。 先前 分析表明玉米醇溶蛋白由三种不同的蛋白质类型组成 分别命名为阿尔法贝塔和伽马 α玉米醇溶蛋白 约占总贮藏蛋白的70%，由一个 多基因家族 β和γ玉米醇溶蛋白， 分别约10%和20%的玉米醇溶蛋白级分各自 由一两个基因编码 玉米醇溶蛋白基因表达活跃， 正常胚乳的发育，但其表达是定量的 并且在几种突变基因型中被定性抑制。 我们的初步 研究表明，在不同的细胞中， 玉米醇溶蛋白基因家族中的基因， 细胞质mRNA的量。 确定管理 玉米醇溶蛋白基因在胚乳形成过程中的表达，我们建议， 表征控制其转录的DNA序列， 发育调节 32P标记的核RNA与孔的杂交 特征化的基因组克隆将建立转录位点， 无论是转录还是转录后 加工在玉米醇溶蛋白基因的调节中起主要作用 表情 这一分析还将确定这些因素在多大程度上 反应受突变基因型的影响。 来定义DNA序列 为了调节转录，基因侧翼序列将连接到 氯霉素乙酰转移酶（CAT）基因并转入植物 原生质体电穿孔。 转录所需的序列将 通过测定原生质体提取物中的CAT活性来监测。 我们还将 分析转录调控序列， 用Ti质粒载体将玉米醇溶蛋白基因导入烟草和矮牵牛植物。 在 在这种情况下，我们将监测种子中的mRNA和蛋白质合成， 再生植物描绘DNA序列控制发育 表情 通过胚乳蛋白质标记和差异表达， 筛选cDNA文库，我们将尝试鉴定基因产物， 在降低玉米醇溶蛋白基因水平的突变体中， 表情