Trail F, Chang P K, Cary J, Linz J E
Department of Food Science and Human Nutrition, Michigan State University, East Lansing 48824.
Appl Environ Microbiol. 1994 Nov;60(11):4078-85. doi: 10.1128/aem.60.11.4078-4085.1994.
The nor-1 gene was cloned previously by complementation of a mutation (nor-1) in Aspergillus parasiticus SU-1 which blocked aflatoxin B1 biosynthesis, resulting in the accumulation of norsolorinic acid (NA). In this study, the nucleotide sequences of the cDNA and genomic DNA clones encompassing the coding region of the nor-1 gene were determined. The transcription initiation and polyadenylation sites of nor-1 were located by primer extension and RNase protection analyses and by comparison of the nucleotide sequences of the nor-1 genomic and cDNA clones. A plasmid, pNA51-82, was created for one-step disruption of the nor-1 gene by inserting a functional copy of the nitrate reductase (niaD) gene from A. parasiticus into the coding region of the nor-1 gene. Transformation of A. parasiticus NR-3 (niaD Afl+) with pNA51-82 resulted in niaD+ transformants that accumulated NA and produced reduced levels of aflatoxin as determined by thin-layer chromatography and enzyme-linked immunosorbent assay analyses of extracts from mycelia and the growth medium. Southern analysis of genomic DNA isolated from the NA-accumulating transformants indicated that the wild-type nor-1 gene in the chromosome had been replaced by the nonfunctional allele carried on pNA51-82. This recombinational inactivation event provides direct evidence that the nor-1 gene is functionally involved in aflatoxin biosynthesis. Comparison of the predicted nor-1 amino acid sequence with sequences in the GenBank and EMBL databases suggested that the protein is a member of the family of short-chain alcohol dehydrogenases, consistent with its proposed function as a keto reductase.
nor-1基因先前是通过对寄生曲霉SU-1中的一个突变（nor-1）进行互补克隆得到的，该突变阻断了黄曲霉毒素B1的生物合成，导致诺索尔紫红酸（NA）的积累。在本研究中，测定了包含nor-1基因编码区的cDNA和基因组DNA克隆的核苷酸序列。通过引物延伸和RNase保护分析以及比较nor-1基因组和cDNA克隆的核苷酸序列，确定了nor-1的转录起始和聚腺苷酸化位点。通过将来自寄生曲霉的硝酸还原酶（niaD）基因的功能拷贝插入nor-1基因的编码区，构建了一个质粒pNA51-82，用于一步破坏nor-1基因。用pNA51-82转化寄生曲霉NR-3（niaD Afl+），得到niaD+转化体，这些转化体积累了NA，并且通过对菌丝体和生长培养基提取物的薄层色谱分析和酶联免疫吸附测定分析表明，其产生的黄曲霉毒素水平降低。对从积累NA的转化体中分离的基因组DNA进行Southern分析表明，染色体中的野生型nor-1基因已被pNA51-82上携带的无功能等位基因所取代。这种重组失活事件提供了直接证据，证明nor-1基因在功能上参与黄曲霉毒素的生物合成。将预测的nor-1氨基酸序列与GenBank和EMBL数据库中的序列进行比较，表明该蛋白质是短链醇脱氢酶家族的成员，与其作为酮还原酶的推测功能一致。
