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为研究辣椒疫霉(Phytophthora capsici)多聚半乳糖醛酸酶PCIPG2 N-糖基化对其酶活性的影响,从基因组文库中分离克隆到Pcipg2基因,利用定点突变技术突变3个潜在的糖基化位点(N34,N76,N137);构建并表达N-糖基化突变蛋白,并对突变蛋白进行温度和缓冲液体系处理检测其活性。结果发现Pcipg2基因在辣椒疫霉侵染寄主的前期表达并起重要作用。PCIPG2蛋白在30°C,pH5.0(P<0.05)的缓冲液环境下活性最高。单个的Pcipg2糖基化位点N34、N76、N137对PCIPG2的致病性起正调控作用,而3个糖基化位点相互协调的功能抑制PCIPG2的活性,在PCIPG2表达致病过程中起负调控。N-糖基化在PCIPG2酶活性上起直接作用,使得PCIPG2酶在较低水平上保持较高的稳定性。
In order to study the effect of N-glycosylation of Phytophthora capsici polygalacturonase (PCIPG2) on its enzymatic activity, the Pcipg2 gene was isolated from the genomic library and mutated 3 potential glycosylation sites using site-directed mutagenesis (N34, N76, N137). The N-glycosylated muteins were constructed and expressed. The muteins were tested for their activities by temperature and buffer treatment. The results showed that Pcipg2 gene expressed and played an important role in the early stage of Phytophthora capsici infection host. The PCIPG2 protein has the highest activity in a buffer environment at 30 ° C, pH 5.0 (P <0.05). A single Pcipg2 glycosylation site N34, N76, N137 on the pathogenicity of PCIPG2 play a positive role, and coordination of the three glycosylation sites inhibit the activity of PCIPG2 PCIPG2 expression in the pathogenesis of negative Regulation. N-glycosylation plays a direct role in the enzymatic activity of PCIPG2, allowing the PCIPG2 enzyme to maintain high stability at lower levels.