FLO1基因内重复序列对酿酒酵母絮凝能力及稳定性的影响

Regulatory effect of FLO1 tandem repeats on the flocculation characteristics and genetic stability in Saccharomyces cerevisiae

【目的】了解絮凝基因FLO1中重复DNA序列A对酵母菌絮凝能力及其遗传稳定性的影响,为构建遗传性能稳定、工业应用前景优良的最小絮凝功能基因奠定理论基础。【方法】通过融合PCR方法构建了FLO1中全部重复DNA序列A发生缺失的衍生基因FLO1a,以含有FLO1基因的大肠杆菌为筛选模型,通过连续传代培养及质粒快速分析获得FLO1内重复DNA序列A不同程度、不同位点缺失的系列衍生基因FLO1a1-FLO1a5。完整FLO1基因和上述衍生基因转化非絮凝型酵母YS58,得到重组菌株YSF1、YSF1a及YSF1a1-YSF1a5,分析了上述不同酵母菌株絮凝特性及其遗传稳定性。【结果】絮凝基因FLO1中重复DNA序列A完全缺失使酵母细胞完全失去絮凝能力,部分重复DNA序列A发生缺失导致絮凝能力降低,絮凝基因中重复DNA序列A的个数与细胞絮凝能力成正相关,但不是简单的比例关系。其中衍生基因FLO1a3含有的重复DNA序列A是FLO1基因的33.3%,但菌株YSF1a3的絮凝能力可达YSF1絮凝能力的71.4%。而且菌株YSF1a3的絮凝特性比菌株YSF1的絮凝特性具有更好的环境适应性和遗传稳定性。【结论】重复DNA序列A是絮凝基因中非常活跃的序列,是导致絮凝特性遗传不稳定的关键因素,该序列的部分缺失不但可以使酵母细胞呈现适度的絮凝能力,而且使絮凝特性具有更好的环境适应性和遗传稳定性。该研究为通过对絮凝基因内衔接重复序列的合理调控,促进酵母絮凝特性在发酵工业及其他生物化工过程和环境修复中的广泛应用提供了重要的理论依据和解决策略。

[Objective] There are a large number of tandem repeats in FLO1,which are highly dynamic components in genome leading to the unstable flocculation profiles in Saccharomyces cerevisiae.The effects of complete or partial deletion of repeated DNA sequence A in FLO1 on the flocculation characteristics and genetic stability in yeast were studied to provide theoretical guide for construction genetically stable flocculation gene with minimal size.[Methods] We constructed the derived gene FLO1a with complete deletion of repeated DNA sequence A in the central domain by fusion PCR,and isolated the derived genes FLO1a1-FLO1a5 with partial deletion of repeated DNA sequence A at different sites using E.coli DH5α carrying the FLO1 gene as selective model.We analyzed the physiological characteristics and genetic stability of flocculation in yeast strains YSF1,YSF1a,and YSF1a1-YSF1a5 containing FLO1,FLO1a and FLO1a1-FLO1a5 respectively.[Results] No obvious flocculation was observed for yeast strain YSF1a,but various levels of flocculation were observed for strains YSF1a1-YSF1a5.Flocculation of YSF1a3,YSF1a4 and YSF1a5 were more tolerant to environmental changes than that of strain YSF1,and displayed more genetic stability.[Conclusion] Repeated DNA sequence A is important for the function of flocculation protein.