两步融合PCR法构建烟曲霉ssk1基因敲除株

The application of the two-step fusion PCR method in construction of the ssk1 gene knockout strain of Aspergillus fumigatus

目的通过两步融合PCR法构建烟曲霉ssk1基因敲除株。方法通过在ssk1基因上下游的侧翼序列设计引物时引入15 bp的融合片段的互补的同源区,使用两步融合PCR的方法将长度约为4.3 kb的目的片段融合扩增,并直接使用PCR产物通过传统的原生质体法进行烟曲霉的转化,得到转化子运用PCR和Southern blot进行相应的验证。结果通过融合PCR法较快的得到大约4.3 kb的融合的目的基因片段,运用原生质体法成功进行了烟曲霉的转化,得到5个转化子。经过PCR和Southern blot经验证有4株是正确的转化子。结论在合适的引物设计及适当的融合PCR反应条件下,两步融合PCR法是一种简单高效的构建烟曲霉基因敲除株的方法,值得推广。

Objective To construct the sskl knockout strain of Aspergillus fumigatus using a two-step fusion PCR. Methods Prim- ers were designed with diverse homologous regions of 15 bp only in flanking regions of the sskl sequence and amplified with 4.3 kb- long fragment by two-step fusion PCR, The PCR product was transformed into A. fumigatus by traditional protoplasts method and then certificated by the PCR and Southern blot. Results The 4.3 kb-long fragment was successfully amplified by two-step fusion PCR. To- tally five transformants were obtained, and PCR and Southern blot showed that four transformants were correct. Conclusion With the appropriate primer and PCR reaction conditions, the two-step fusion PCR might be a highly efficient tool in gene knockout of A. fumiga- tus,and this approach should be promoted.