禾谷镰孢菌果胶裂解酶PelA的生物学功能分析

Biological Function Analysis of A Pectate Lyase PelA in Fusarium graminearum

果胶裂解酶是果胶酶的重要成员之一,能够通过β-消除作用,催化降解(1→4)-α-D-聚半乳糖醛酸。本研究首次鉴定出禾谷镰孢菌中的一个果胶裂解酶PelA,并在体外检测了其生化特性。PelA基因(FGSG_02386)的cDNA被克隆并进行了原核表达,同时构建了单基因敲除突变体。将重组质粒pET-28a(+)-PelA转化蛋白表达菌株大肠杆菌BL21表达目的蛋白,纯化后的蛋白具有果胶裂解酶活性。酶活性测定结果显示,该酶的活性受到外界环境因素如温度、钙离子浓度和pH条件的影响,其最适反应条件为50℃,CaCl2浓度0.5 mmol·L-1,pH 8.5。在侵染小麦胚芽鞘时PelA单基因突变体的毒力较野生型菌株并没有出现明显变化。

Pectate lyase can eliminate cleavage of （1→4）-α-D-galacturonan by β-elimination as a member of pectinases. This study identified a pectate lyase, PelA, in Fusarium graminearum for the first time, and its biochemical characteristics was verified in vitro. In this study, PelA was expressed in Escherichia coli and knock-out mutants were constructed. Biochemical analysis showed that PelA has pectate lyase activity in vitro. External environment such as temperature, calcium ion concentration and pH affected the enzyme activity, and enzyme activity reached the highest at 50 ℃, 0.5 rnmol·L^-1 CaCl2 and pH 8.5. PelA knock-out strains didn＇t show significant difference in virulence from wild-type strains during infection of wheat coleoptiles.