Morioka Luiz Rodrigo I, Furlaneto Márcia C, Bogas Andréa C, Pompermayer Patrícia, Duarte Rubens T D, Vieira Maria Lúcia C, Watanabe Maria Angélica Ehara, Fungaro Maria Helena P
Centro de Ciências Biológicas, Universidade Estadual de Londrina, PO Box 6001, 86051-990 Londrina, PR, Brazil.
Curr Microbiol. 2006 Jun;52(6):469-72. doi: 10.1007/s00284-005-0402-6. Epub 2006 Apr 17.
Aspergillus carbonarius is a potent ochratoxin A producer that has been found in products such as grapes, coffee, spices, and cocoa. Ochratoxin A has nephrotoxic effect, and it has been classified as a possible carcinogenic substance for humans. Here we describe for the first time a transformation system for A. carbonarius, providing an important step towards its genetic manipulation. Conidia were transformed to acquire hygromycin B resistance using the AGL-1 strain of Agrobacterium tumefaciens. Genetic transformation was evaluated growing A. tumefaciens cells in induction medium containing or not acetosyringone prior to co-cultivation. The mean transforming efficiencies in IM+AS and IM-AS conditions were 62.2 and 44.5 transformants per 10(5) conidia, respectively. The hph gene was random integrated into the genome of A. carbonarius. Fungal sequences flanking the insertion site could be amplified by TAIL-PCR.
黑曲霉是一种强效的赭曲霉毒素A产生菌，已在葡萄、咖啡、香料和可可等产品中被发现。赭曲霉毒素A具有肾毒性作用，并且已被归类为对人类可能的致癌物质。在此我们首次描述了黑曲霉的转化系统，这为其基因操作迈出了重要一步。使用根癌农杆菌AGL-1菌株将分生孢子转化以获得潮霉素B抗性。在共培养之前，通过在含有或不含有乙酰丁香酮的诱导培养基中培养根癌农杆菌细胞来评估遗传转化。在IM+AS和IM-AS条件下，平均转化效率分别为每10⁵个分生孢子62.2个和44.5个转化体。hph基因随机整合到黑曲霉的基因组中。插入位点侧翼的真菌序列可通过热不对称交错PCR（TAIL-PCR）进行扩增。
