FLO1基因靠近3'端重复序列对酵母菌絮凝蛋白功能的调控

Effect of tandem repeats adjacent to 3'-terminal of FLO1 on the flocculation function of Saccharomyces cerevisiae

【目的】研究絮凝基因FLO1中靠近3'端重复DNA序列缺失对絮凝蛋白Flo1功能的影响,为遗传稳定的最小絮凝功能基因的构建及酵母菌絮凝特性的可控改造提供理论依据。【方法】通过融合PCR的方法得到FLO1内靠近3'端重复DNA序列B、C和D全部缺失的衍生基因FLO1bcd,比较FLO1及FLO1bcd在非絮凝酵母细胞中表达后,菌株在不同条件下絮凝特性的差异。【结果】与表达完整絮凝基因FLO1的酵母菌株YSF1相比,FLO1内靠近3'端重复DNA序列全部缺失的酵母菌株YSF1bcd的絮凝强度仅略有降低,而且其絮凝特性对钙离子的依赖性、金属离子的敏感性及乙醇敏感性均没有明显变化,但对环境pH以及温度的变化表现出更广泛的适应性,受甘露糖抑制的敏感性也有所降低。【结论】FLO1中重复DNA序列B、C和D全部缺失与单独缺失C或D一样可以提高絮凝蛋白的构象和功能稳定性,使酵母细胞在极端酸碱环境下仍然表现出明显的絮凝特性;这些重复序列的完全缺失对其他絮凝特性没有明显影响。因此可以通过重复序列删减策略构建遗传稳定的最小絮凝功能基因,为絮凝特性在发酵工业及环境修复方面的可控应用奠定理论基础。

[Objective]Many tandem repeats exist in FLO1 gene of Saccharomyces cerevisiae,which might have great regulatory effect on the conformation and function of flocculation protein （flocculin）.In this study,we analyzed the effect of 3＇-terminal tandem repeats B,C and D complete deletion on the function of flocculin.[Methods]We constructed the derived gene FLO1bcd with complete deletion of tandem repeats B,C and D of FLO1 by fusion PCR. We then constructed plasmid pYCF1bcd by insertion of FLO1bcd into YCp50,and transformed such plasmid,pYCF1 and YCp50 into S.cerevisiae YS58 separately to generate recombinant strains YSF1bcd,YSF1 and YSP50. We compared the flocculation ability and characteristics of these strains.[Result]Compared to YSF1,YSF1bcd displayed only a slight reduction （4%） in flocculation ability in optimal flocculation buffer （50 mmol /L NaAC,pH 4.5）.Moreover,the dependence of flocculation on Ca^2＋ ,sensitivity to metal ions and ethanol,and the specificity to different sugars showed no obvious difference between strains YSF1 and YSF1bcd.However,strain YSF1bcd displayed much higher flocculation levels than strain YSF1 under conditions with extreme pH,high temperature,or high concentration of mannose.[Conclusion]Combined deletion of tandem repeats B,C and D adjacent to the 3＇-terminal of FLO1 increases the conformation stability of flocculin in response to changes of pH,temperature or concentration of mannose in environment,but does not influence the other characteristics of flocculation.