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【目的】确定rmlB基因在大肠杆菌(O2:K1)L-型鼠李糖合成中的作用。【方法】将基因rmlB进行原核表达并测定酶活;用同源重组的方法将rmlB基因敲除,分析表型变化,并运用质谱,以及核磁共振等手段分析脂多糖O侧链的结构,以确定rmlB在O抗原合成中的作用。【结果】成功对rmlB基因进行了表达并测定了重组蛋白的酶活,确定蛋白RmlB具有dTDP-D-glucose 4,6-dehydratase活性。成功构建了rmlB基因缺失突变株,对突变株进行表型分析发现突变株的表型与野生株相比无变化。对突变株分析发现突变株中的O抗原仍含有L-型鼠李糖,说明在该菌株中可能存在RmlB的同功能酶或者存在其它的L-型鼠李糖合成途径。【结论】rmlB基因编码的蛋白具有dTDP-D-glucose 4,6-dehydratase活性但此基因对于L-型鼠李糖的合成不是必需的。
【Objective】 To determine the role of rmlB gene in the synthesis of L-rhamnose from Escherichia coli (O2: K1). 【Method】 The gene rmlB was prokaryotic expressed and the enzyme activity was determined. The rmlB gene was knocked out by homologous recombination to analyze the phenotypic changes. The structure of lipopolysaccharide O side chain was analyzed by mass spectrometry and nuclear magnetic resonance Determine the role of rmlB in O antigen synthesis. 【Result】 The rmlB gene was successfully expressed and the activity of the recombinant protein was determined. The result showed that the protein RmlB had the activity of dTDP-D-glucose 4,6-dehydratase. The rmlB gene deletion mutant was successfully constructed. Phenotypic analysis of the mutant showed no change in the phenotype of the mutant compared with the wild type. Analysis of mutant strains revealed that the O antigen in the mutant strain still contains L-type rhamnose, indicating that RmlB homofunctional enzymes may exist in the strain or that other L-type rhamnose synthesis pathways exist. 【Conclusion】 The rmlB gene encodes a protein with dTDP-D-glucose 4,6-dehydratase activity but this gene is not essential for the synthesis of L-rhamnose.