GENETIC-TRANSFORMATION SYSTEM FOR THE AFLATOXIN-PRODUCING FUNGUS ASPERGILLUS-FLAVUS
GENETIC-TRANSFORMATION SYSTEM FOR THE AFLATOXIN-PRODUCING FUNGUS ASPERGILLUS-FLAVUS
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DOI：
10.1128/aem.55.1.86-90.1989
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发表时间：
1989-01-01
影响因子：
4.4
通讯作者：
ADKINS, CR
中科院分区：
文献类型：
--
作者：
WOLOSHUK, CP;SEIP, ER;ADKINS, CR
建立了黄曲霉异源转化体系，转化效率大于20个稳定转化子/微克DNA。用含有粗链孢子菌PYR-4基因的质粒和粘粒载体转化该真菌需要尿嘧啶的菌株的原生质体。转化子的选择是因为它们在缺乏尿嘧啶的培养基上生长和产生孢子的能力。载体DNA似乎随机整合到黄曲霉的基因组中，并有多个串联插入的趋势。单次或多次插入的转化子在含有尿嘧啶的培养基上连续转移5次后是稳定的。通过紫外线照射分生孢子和筛选5-氟罗丹酸抗性菌落，或通过无性重组将突变的PYR基因座转移到菌株中，获得了需要尿嘧啶的受体菌株。这是黄曲霉毒素产生菌转化系统的首次报道。黄曲霉毒素阴性突变体的转化系统和可获得性为研究黄曲霉毒素的生物合成和调控提供了新的途径。
A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per .mu.g of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vector containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoro-orotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.