ALFLATOXIN B1 BIOSYNTHESIS IN ASPERGILLUS PARASITICUS

黄曲霉毒素是生物活性的次级代谢产物， 黄曲霉（Aspergillus flavus）和A.寄生虫 这些真菌无处不在， 经常产生黄曲霉毒素污染的粮食和饲料作物， 美国和世界各地。在动物系统中，黄曲霉毒素B1（AFB1） 肝毒性、致突变、致畸、免疫毒性和致癌。 AFB1 是已知最强的自然致癌物流行病学 对人群的研究表明，AFB1是一种促成风险， 原发性肝癌的病因 这项研究的长期目标是 将AFB1从食物链中清除。 本研究的短期目标是 建议是要了解调节的分子机制， 表达的关键基因（UVM8，nor-1和ver-1）参与了 AFB1的合成。 拟议的研究旨在确定 AFB1生物合成途径中的几个控制点， 通过合成的或引入的化合物抑制的靶点 寄主植物 建议以下具体目标，以深入发展 了解调节UVM8，nor-1， 和ver-1在转录、翻译和蛋白质水平的表达 本地化 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， 表达的时间和程度。 2.确定的时间 这些蛋白质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，对nor-1、ver-1和 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，以确定调控位点的数量和类型。 凝胶位移和甲基化干扰分析将精确地映射这些 位点，并提供了纯化反式作用调节蛋白的机制。 因素 为了实现特定目的2，产生针对去甲-1的抗体（Ab）， ver-1和UVM8蛋白将被用来确定它们的表达时间。 通过Western印迹分析细胞提取物的表达。 细胞内 这些蛋白质的定位将在冷冻切片中确定， 用荧光抗体免疫定位。 蛋白质定位 在整个真菌细胞或菌落中的表达将通过以下方式实现： 用显色或荧光定位GUS报告蛋白活性 印刷受体.

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.