酿酒酵母PEP4基因与糖酵解途径关键酶PYK活性关联的作用机制研究

As a substantial experimental model organism, Saccharomyces cerevisiae is very useful for understanding cell-growth, metabolism and metabolic regulations in eukaryotes. Studies showing that PEP4 gene-deficiency and partial delete all did influence on the activities of key enzymes of glycolytic pathway in S.cerevisiae, and further affects the regulations of metabolism and cellular growth performance. However, there is still no answer about how the PEP4 gene influences the key enzymes' activities in glycolytic pathway. This research project is planning to construct S.cerevisiae experimental model strains of higher Proteinase A expression and pep4 gene type using homologous recombination technology, and take the Pyruvate kinase (PYK) as a research objective to discover the mechanism how PEP4 gene affects the activities of key enzymes in glucose metabolic pathway at the molecular level. Firstly, the impact of different PEP4 gene expressions and the metabolism of its expression product on the activity of Pyruvate kinase and the expression of PYK coding gene will be analyzed using the quantitative detection methods for enzyme activities and Real-time RT-PCR for mRNA. And then, we planning to study the effects which PEP4 gene takes on the molecular metabolic dynamics of PYK enzymoprotein through Matrix assisted laser desorption line time of Flight mass spectrometry analysis technology. Eventually, the experimental data gathered from the studies about the effects which PEP4 gene acted on the glucose metabolic flux and energy metabolism will be analyzed. On the basis of the analysis, the molecular model of PEP4 gene acting on the activity of PYK will be construct, lay the theoretical foundations for uncovering the mechanism of PEP4 gene acting on the glucose metabolism.

研究发现，PEP4基因影响酿酒酵母糖酵解途径关键酶的活性及细胞的代谢和生长性能，然而其作用机制目前仍未明确。针对上述问题，本项目拟采用同源重组技术构建pep4基因型和高蛋白酶A表达型酿酒酵母试验模型菌株。首先，以构建的菌株为研究对象，结合Real-time RT-PCR和酶活性的定量方法，分析PEP4基因的不同表达及其编码产物的代谢变化对丙酮酸激酶编码序列的表达和PYK酶活性的影响；然后，综合运用聚丙烯酰胺凝胶电泳（SDS-PAGE）与Western-blot及蛋白分析、分离等技术，通过基质辅助激光解析串联飞行时间质谱分析PYK酶蛋白的分子代谢动态，探索PEP4基因的不同表达对PYK酶蛋白分子代谢动态的影响；最后，综合分析糖代谢流和途径相关能量代谢的实验数据，建立PEP4基因与PYK活性关联的分子作用机制模型，为揭示PEP4基因影响糖代谢的作用机理奠定理论基础。