同源片段大小对板栗疫病菌同源重组频率的影响

Influence of flanking sequence length on homologous recombination frequency in Cryphonectria parasitica

低毒病毒／板栗疫病菌系统是一个具有独特优点的、崭新的研究病毒与宿主相互作用的模型系统。以同源重组为基础的基因打靶是研究基因功能和病毒与宿主相互作用分子机制的重要手段。为了研究在板栗疫病菌同源重组遗传操作中同源片段大小对同源重组频率的影响，利用一段5．4kb板栗疫病菌染色体DNA序列，构建了以潮霉素抗性基因为遗传转化筛选标记的3个同源双交换片段以及3个同源单交换质粒，分别转化板栗疫病菌EPl55原生质体，检测筛选同源重组转化子并计算发生同源重组频率。结果表明，在板栗疫病菌中3个同源双交换片段FHA1（1．7～2.0kb）、FHA2（1.0～1.2kb）和FHA3（0.5kb）的同源重组频率分别为3．73％、2。06％和0；3个同源单交换质粒pFHl（1．76kb）、pFH2（1．23kb）和pFH3（O．54kb）的同源重组频率分别为0.95％、0和0。这些结果为板栗疫病菌同源重组遗传操作中同源片段大小的设计提供了依据。

Hypovirus/ Cryphonectria parasitica system is a novel model system in virus-host interaction research. Gene disruption based on homologous recombination is a critical strategy for host gene function analysis, and understanding mechanisms of hypovirus and host interaction. To assess the effect of length of flanking sequences on the homologous recombination frequency, a 5.4 kb genomic fragment from C. parasitica was used to construct cassettes with varied lengths in arms that flanked the hph gene, which served as a selectable marker resistant to the antibiotic hygromycin, either for homologous double cross-over or for single cross-over event. These plamid cassettes were used to transform spheroplasts of C. parasitica strain EP155. The homologous recombination frequency for the three homologous double cross-over fragments, FHA1 （1.7~2.0 kb）, FHA2 （1.0~1.2 kb）, FHA3 （0. 5 kb） were 3.73%, 2.06%, and 0, respectively. The homologous recombination frequency for three homologous single cross over plasmids, pFH1 （1.76 kb）, pFH2 （1.23 kb） and pFH3 （0.54 kb） were 0. 95%, 0, and 0, respectively. The findings provide a general guide for designing the franking fragments for homologous recombination in C. parasitica.