Regulation of Aflatoxin Production in A. Parasiticus

寄生黄曲霉毒素产生的调控

• 批准号: 6727033
• 负责人: SHUBHA K IRELAND
• 金额: $ 9.61万
• 依托单位: XAVIER UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727033
• 关键词: Aspergillus; SDS polyacrylamide gel electrophoresis; aflatoxins; biosynthesis; fungal genetics; gene expression; genetic transcription; microarray technology; northern blottings; polymerase chain reaction; protein sequence; proteomics; regulatory gene; southern blotting; spores; structural genes; two dimensional gel electrophoresis

Filamentous fungi are well-known producers of secondary metabolites (natural products) such as antibiotics, toxins, and pigments. These compounds often demonstrate no apparent advantage to the producing organisms and have complex biosynthetic pathways. Aflatoxins (AFs) are secondary metabolites produced by only certain Aspergillus species such as A. parasiticus and A. flavus and are highly toxic, mutagenic and carcinogenic to animals and humans. Many structural genes of the AF pathway have been cloned and characterized, but only one, the af/R gene, has been shown to positively regulate the transcription of the pathway structural genes. Mechanisms that govern af/R function however, remain largely unknown. Several recent studies includingours have suggested that aspects of fungal development and secondary metabolism are correlated. The main goals of this project are to identify new factors that act separately from, or in conjunction with af/R in controlling AF production and to determine the extent of correlation between AF regulation and fungal development. The present proposal is centered upon previously isolated A. parasiticus sec- (for secondary metabolism minus) strains, also displaying altered morphology and sporuiation. Recent work from our laboratory has shown that a mutation in af/R coding or promoter region is not responsible for the sec- phenotype. Yet, the af/R expression is lowered in the sec- strains, there is no expression of the AF pathway genes and af/R overexpression does not reverse the sec- phenotype. Preliminary results have also revealed clear differences between the sec- and their parental sec+ (for secondary metabolism plus) total protein-profiles. In this study, the isogenic sec+/sec- pairs will be used in proteomics, specifically to identify and characterize the 'up' regulated proteins in the sec-strains since these are suggestive of negative regulators of af/R/AF biosynthesis. Through protein sequencing, identified genes will studied for their role in AF production and/or fungal development. Other experiments will include testing specific homologous and heterologous developmental genes [latter from a related sterigmatocystin (ST) pathway of A. nidulans] known to have a positive influence on secondary metabolism, for their possible role in the sec-reversal. Lastly, constructed A. flavus DNA microarrays (98 % homologous to A. parasiticus) will be used to identify the possible transcription-level differences between the isogenic sec+/sec- pairs and candidate genes will be isolated in future. Results obtained from this study will contribute toward its long-term goal of preventing preharvest AF contamination of susceptible crops by developing effective strategies such as 'designer' biocompetitive strains.

丝状真菌是著名的二级代谢产物（天然产物）的生产者，例如抗生素，毒素和颜料。这些化合物通常对生产生物没有明显的优势，并且具有复杂的生物合成途径。黄曲霉毒素（AFS）是仅由某些曲霉物种（如寄生虫和黄曲曲霉）产生的继发代谢产物，对动物和人类具有剧毒，诱变和致癌性。 AF途径的许多结构基因已被克隆和表征，但只有一个AF/R基因已被证明可以积极调节途径结构基因的转录。但是，控制AF/R功能的机制仍然在很大程度上未知。最近的一些研究包括旅局，表明真菌开发和 次级代谢是相关的。该项目的主要目标是确定与AF/R在控制AF生产中分开或与AF/R结合起作用的新因素，并确定AF法规与真菌开发之间的相关程度。本提案以先前隔离的寄生虫sec-（用于继发代谢减去）菌株为中心，也显示出变化的形态和孢子形成。我们实验室的最新工作表明，AF/R编码或启动子区域中的突变对表型概不负责。然而， AF/R表达在分节菌株中降低，没有AF途径基因的表达，而AF/R过表达不会逆转SEC型。初步结果还揭示了SEC和其父母SEC+（用于继发代谢加的）总蛋白质profiles之间的明显差异。在这项研究中，ISEGENIC SEC+/sec-将用于蛋白质组学，专门用于识别和表征SEC-strains中的“ UP”调节蛋白 由于这些暗示了AF/R/AF生物合成的负调节剂。通过蛋白质测序，确定的基因将研究其在AF生产和/或真菌发育中的作用。其他实验将包括测试特定的同源性和异源发育基因[后者来自植曲菌的相关sterigmatocystin（st）途径]，该基因已知对次级代谢有积极影响，因为它们在SEC-逆转中可能的作用。 最后，将使用构建的拟建的Flavus DNA微阵列（98％与寄生虫同源）来确定ISEGONIC SEC+/sec-pairs之间可能的转录级差异，并且将来将分离候选基因。从这项研究中获得的结果将有助于其长期目标，即通过制定诸如“设计师”生物竞争菌株之类的有效策略来防止易感农作物的预先进行AF污染。