Brown J S, Aufauvre-Brown A, Holden D W
Department of Infectious Diseases, Imperial College School of Medicine, Hammersmith Hospital, London, UK.
Mol Gen Genet. 1998 Aug;259(3):327-35. doi: 10.1007/s004380050819.
We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.
我们研究了用异源DNA进行转化作为烟曲霉插入诱变的一种方法。采用了两种方法，即聚乙二醇介导的原生质体转化和萌发孢子的电穿孔，以建立能使转化DNA在不同基因组位点单拷贝整合的条件。我们评估了限制酶介导整合（REMI）对这两种方法的效果。非REMI原生质体转化导致大多数转化体中转化DNA多拷贝整合。原生质体转化的REMI结果因所用酶而异。几种限制酶的低浓度刺激了转化，但在所研究的十种酶中，只有用XhoI和KpnI进行的REMI能使大多数转化体中转化DNA单拷贝整合。对于基于XhoI或KpnI的REMI原生质体转化，分别有50%和76%的插入是由于在与用于REMI的酶相对应的基因组酶切位点整合。不添加限制酶的孢子电穿孔导致高转化效率，高达67%的转化体含有单拷贝的转化DNA。与原生质体转化不同，在限制酶存在下的孢子电穿孔并未提高转化效率，也未导致在基因组限制位点插入。Southern分析表明，对于用KpnI或XhoI进行REMI的原生质体转化以及不添加限制酶的孢子电穿孔，在高比例的转化体中，转化DNA插入到不同的基因组位点。
