STUDIES ON STRUCTURE AND FUNCTION OF ACOCADO CYTOHROME P450

ACOCADO CYTOHROME P450结构与功能的研究

• 批准号: 6240650
• 负责人: KRISTIN R BOZAK
• 金额: $ 14.59万
• 依托单位: CALIFORNIA STATE POLY U POMONA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240650
• 关键词: DNA binding protein; DNA footprinting; chemical structure function; cytochrome P450; developmental genetics; ethylenes; fruit; gel mobility shift assay; gene expression; gene induction /repression; genetic regulatory element; genetically modified plants; northern blottings; plant extracts; plant genetics; plant growth /development; polymerase chain reaction; southern blotting; tobacco; western blottings; wound

Regulatory mechanisms controlling expression of cytochrome P450 genes from mammals have been well deliniated, whereas the analysis of plant P450 gene regulation has just recently begun. The description of plant P459 inducers is documented in the literature, yet no single plant P450 gene's controlling element(s) has been defined. This grant request proposed studies which will further understanding of the mechanism by which an avacado cytochrome P450 gene (cyp71A1) is regulated during developmental processes, and analyzes specific upstream segments which respond to ethylene evolved either as a result of wounding or ripening. This will further the understanding of the mechanism by which this plant gene whose product is involved in oxidative metabolism responds to different stimuli via a hormone mediated signal transduction apparatus. Aim 1 To determine if ethylene evolved either during ripening or wounding is required and/or sufficient to activate cyp71A1. Initial studies will define temporal expression of P450 antigen and cyp71a1 mRNA following mechanical wounding of avocado leaves (1A). Additionally the role of ethylene during either ripening or wounding will be assayed (1B,1C,1D). Aim 2 To identify where the ethylene-responsive elements of cyp71A1 are located. The upstream region of cyp71A1 has been studied in the context of ri[pe avocado fruit mesocarp protoplasts through a transient expression assay. Proposed studies will further narrow the previously identified broad segments critical for ethylene regulation, and will differentiate between wounding and ripening-specific elements, and those which directly respond to ethylene. The proposed studies include both functional assays assessing activity of various promoter fragments in ripe avocado fruit protoplasts (2A), transgenic tobacco either wounded (2B) or gassed with ethylene (2C) and gel mobility shifts to reveal whether these identify the same element(s) (2D). Aim 3 To train students in contemporary molecular biology techniques (analyzing hormone-mediated gene regulation) that are used in research conducted at biotechnology companies, hospitals and universities. Understanding the regulation of this first fully-analyzed plant (P450 will serve as a basis for comparison with other plant and animal P450 regulatory mechanisms.

细胞色素P450基因表达的调控机制 哺乳动物已经很好地阐明，而植物P450基因的分析 监管刚刚开始。 植物P459诱导剂的描述 在文献中有记载，但没有单一的植物P450基因的 已定义控制元素。 这份赠款申请建议 这些研究将进一步了解 鳄梨细胞色素P450基因（cyp 71 A1）在发育过程中受到调控 流程，并分析特定的上游环节， 乙烯的形成是受伤或成熟的结果。 这将 进一步了解这种植物基因的机制， 产物参与氧化代谢，对不同刺激做出反应 通过激素介导的信号转导装置。 目的1确定乙烯是否在成熟或受伤过程中产生 需要和/或足以激活cyp 71 A1。 初步研究将确定P450抗原和cyp 71 a1的时间表达 鳄梨叶机械损伤后的mRNA（1A）。 另外 将测定乙烯在成熟或损伤过程中的作用 (1B，1C，1D）。 目的2鉴定cyp 71 A1的乙烯反应元件在哪里， 所在 cyp 71 A1的上游区域已经在ri[pe] 鳄梨果实中果皮原生质体通过瞬时表达测定。 拟议的研究将进一步缩小以前确定的广泛的 乙烯调节的关键环节，并将区分 伤害和成熟的具体因素，以及那些直接回应 乙烯。 拟议的研究包括两种功能测定， 不同启动子片段在成熟鳄梨果实原生质体中的活性 (2A)，转基因烟草受伤（2B）或用乙烯充气（2C） 和凝胶迁移率的变化，以揭示这些识别是否相同， 元素（2D）。 目标3培养学生掌握现代分子生物学技术 （分析酶介导的基因调控），用于研究 在生物技术公司、医院和大学进行。 了解这第一个全面分析的工厂的调节（P450将 作为与其他植物和动物P450调节的比较的基础 机制等