ALFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS PARASITICUS

黄曲霉毒素是一种具有生物活性的次生代谢物，由 黄曲霉和寄生曲霉。这些真菌无处不在，而且 经常在食品和饲料作物中产生黄曲霉毒素污染 我们和整个世界。在动物体内，黄曲霉毒素B1(AFB1)是 肝毒性、致突变性、致畸性、免疫毒性和致癌性。黄曲霉毒素1 是已知的最强的天然致癌物质。流行病学 对人群的研究表明，黄曲霉毒素B 1是一种致病风险 原发性肝癌的致病因素。这项研究的长期目标是 将黄曲霉毒素1从食物链中消除。这项研究的短期目标是 建议理解调节细胞周期的分子机制。 相关关键基因(UVM8、NOR-1和VER-1)的表达 黄曲霉毒素B_1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 由合成的或引入到其上的化合物的抑制靶标 寄主植物。 提出以下具体目标，以深入发展 了解UVM8、NOR-1表达的调控机制， 和VER-1在转录、翻译和蛋白质水平上的差异 本地化。1.确定特定的反式作用因子及其顺式作用因子- 调控NOR-1、VER-1和UVM8启动子的作用位点 他们的时机和表达水平。2.确定时间安排 这些编码蛋白的表达和亚细胞定位 三个基因。 为实现具体目标1，对NOR-1、VER-1和 UVM8启动子将对β葡萄糖苷酸酶(GUS)进行报道 融合构建以确定调控位点的数量和类型。 凝胶漂移和甲基化干扰分析将精确地绘制出这些 并提供了一种纯化反式作用调节蛋白的机制 各种因素。为了实现特定的目标2，抗体(Ab)提高到NOR-1， VER-1和UVM8蛋白将被用来确定其 通过细胞提取物的Western印迹分析进行表达。细胞内 这些蛋白质在冰冻切片中的定位将通过以下方式确定 荧光抗体免疫定位。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 发色法或荧光法定位GUS报告蛋白活性 底物。

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.