ALFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS PARASITICUS

寄生曲霉中黄曲霉毒素 B1 的生物合成

• 批准号: 2007828
• 负责人: JOHN E LINZ
• 金额: $ 17.54万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2007828
• 关键词: Aspergillus; aflatoxins; enzyme linked immunosorbent assay; fluorescence microscopy; food chain contamination; fungal genetics; gel mobility shift assay; gene deletion mutation; gene expression; genetic promoter element; genetic translation; laboratory rabbit; microorganism toxicology; microorganism toxin; molecular cloning; northern blottings; parasite /microorganism carcinogen; protein biosynthesis; regulatory gene; southern blotting; thin layer chromatography; transcription factor; western blottings

Aflatoxins are biologically active secondary metabolites synthesized by Aspergillus flavus and A. parasiticus. These fungi are ubiquitous and frequently produce aflatoxin contamination in food and feed crops in the US and throughout the world. In animal systems aflatoxin B1 (AFB1) is hepatotoxic, mutagenic, teratogenic, immunotoxic, and carcinogenic. AFB1 is the most potent naturally occurring carcinogen known. Epidemiological studies on human populations suggest that AFB1 is a contributory risk factor in primary liver cancer. The long term goal of this research is to eliminate AFB1 from the food chain. The short term goal of this research proposal is to understand the molecular mechanisms which regulate the expression of key genes (UVM8, nor-1, and ver-1) involved in the biosynthesis of AFB1. The proposed studies are designed to identify several control points in the AFB1 biosynthetic pathway which provide targets for inhibition by compounds synthesized by or introduced onto the host plant. The following specific aims are proposed to develop an in depth understanding of the mechanisms which regulate expression of UVM8, nor-1, and ver-1 at the level of transcription, translation, and protein localization. 1. Identify specific trans-acting factors and their cis- acting sites in the promoters of nor-1, ver-1, and UVM8 which regulate their timing and level of expression. 2. Identify the timing of expression and subcellular localization of the proteins encoded by these three genes. To accomplish specific aim 1, deletion analyses of the nor-1, ver-1 and UVM8 promoters will be conducted on beta glucuronidase (GUS) reporter fusion constructs to determine the number and types of regulatory sites. Gel shift and methylation interference analyses will precisely map these sites and provide a mechanism for purification of trans-acting regulatory factors. To accomplish specific aim 2, antibodies (Ab) raised to nor-1, ver-1 and UVM8 proteins will be utilized to determine the timing of their expression by Western blot analyses of cell extracts. The intracellular localization of these proteins will be determined in cryosections by immunolocalization with fluorescent Ab. Localization of protein expression in whole fungal cells or colonies will be accomplished by localizing GUS reporter protein activity with chromogenic or fluorescent substrates.

黄曲霉毒素是由黄曲霉毒素合成的具有生物活性的次级代谢产物 黄曲霉和寄生曲霉。 这些真菌无处不在 食品和饲料作物经常产生黄曲霉毒素污染 美国和世界各地。在动物系统中，黄曲霉毒素 B1 (AFB1) 是 具有肝毒性、致突变性、致畸性、免疫毒性和致癌性。 AFB1 是已知的最强效的天然致癌物。流行病学 对人群的研究表明 AFB1 是一种促成风险 原发性肝癌的影响因素。 这项研究的长期目标是 从食物链中消除 AFB1。 本研究的短期目标 提议是了解调节的分子机制 参与关键基因（UVM8、nor-1 和 ver-1）的表达 AFB1 的生物合成。 拟议的研究旨在确定 AFB1 生物合成途径中的几个控制点提供 由合成的或引入的化合物抑制的目标 寄主植物。 为深入发展，提出以下具体目标： 了解调节 UVM8、nor-1 表达的机制， 转录、翻译和蛋白质水平的 ver-1 本土化。 1. 识别特定的反式作用因子及其顺式 Nor-1、ver-1 和 UVM8 启动子中的作用位点调节 他们的表达时间和水平。 2. 确定时间 这些编码的蛋白质的表达和亚细胞定位 三个基因。 为了实现具体目标 1，nor-1、ver-1 和 ver-1 的删除分析 UVM8 启动子将在 β 葡萄糖醛酸酶 (GUS) 报告基因上进行 融合构建体以确定调节位点的数量和类型。 凝胶位移和甲基化干扰分析将精确绘制这些 位点并提供纯化反式作用调节的机制 因素。 为了实现特定目标 2，将抗体 (Ab) 提高​​到nor-1， ver-1 和 UVM8 蛋白将用于确定它们的时间 通过细胞提取物的蛋白质印迹分析表达。 细胞内的 这些蛋白质的定位将通过冷冻切片确定 用荧光抗体进行免疫定位。 蛋白质的定位 在整个真菌细胞或菌落中的表达将通过以下方式完成 通过显色或荧光定位 GUS 报告蛋白活性 基材。