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目的构建红色糖多孢菌ΔSACE_0065、ΔSACE_2559和ΔSACE_2565三个突变体并研究其功能。方法将待失活目的基因的上下游同源片段依次连接到pUCTSR质粒Thio抗性基因(tsr)两侧,利用PEG介导的原生质体转化法将线性DNA同源片段转入红色糖多孢菌A226内,通过同源重组使目的基因失活,最后将构建出的三个突变株与出发菌株A226及ΔbldD突变株进行比较,观察孢子生长有无差异。结果与结论成功构建ΔSACE_0065、ΔSACE_2559和ΔSACE_2565三个突变株;与A226相比,ΔSACE_0065和ΔSACE_2559气生菌丝和孢子形成推迟,而ΔSACE_2565突变株的生长情况则无明显变化,提示红色糖多孢菌SACE_0065、SACE_2559基因参与其形态分化的调控。
Objective To construct three mutants of ΔSACE_0065, ΔSACE_2559 and ΔSACE_2565 and study their functions. Methods The upstream and downstream homologous fragments of the target gene to be inactivated were sequentially ligated to both sides of the Thio resistance gene (tsr) of pUCTSR plasmid. The linear DNA homologous fragment was transformed into Saccharomyces cerevisiae A226, the target gene was inactivated by homologous recombination. Finally, three constructed mutants were compared with the original strain A226 and ΔbldD mutant to observe whether the spores grow with or without differentiation. RESULTS AND CONCLUSIONS Three mutant strains, ΔSACE_0065, ΔSACE_2559 and ΔSACE_2565, were successfully constructed. Compared with A226, the formation of aerial mycelium and sporulation of ΔSACE_0065 and ΔSACE_2559 was delayed, while the growth of ΔSACE_2565 mutant did not change significantly, SACE_0065, SACE_2559 genes involved in the regulation of their morphological differentiation.