限制性内切酶介导的串珠镰刀菌插入突变和致病性突变体的分离

 插入突变是研究和分离生物功能基因的重要技术手段,即通过将外源标记基因随即插入生物基因组创建突变体,再利用"质粒拯救"、TAIL-PCR或Reverse-PCR等技术来克隆突变基因。

Inserter mutation by restriction enzyme-mediated integration in Fusarium moniliforme and isolation of pathogenicity mutant

For studying on pathogenicity mechanism of Fusarium moniliforme, REMI was used to transform protoplasts of FT1 strain with the vector pUCATPH, which contained hygromycin B-resistant gene. More than 300 transformants had been obtained, most of them were quite stable after five rounds of successive culture. 25 mutants of morphology and 2 weak pathogenicity mutants were gained by REMI. PCR amplification showed that the hygromycin B-resistant gene had integrated into genomes of the two pathogenicity mutants. The optimum conditions of preparing protoplasts were:the mycelia growing in PDB medium for 14 h, lywallzyme was used to digest the mycelia at 100 r/min, 30℃ for 4 h, and 0.7 mol/L NaCl was used as the osmotic stabilizer.