赤霉病菌特异抗体-防御素融合蛋白的表达和功能鉴定

Expression and Functional Assessment of a Fusion Protein Comprising a Defensin and Single-chain Antibody Gene Specific for Wheat Scab Fungus

选择对赤霉病菌具有高度特异性和亲合力的单链抗体,与植物防御素构建成融合蛋白基因,将融合蛋白及防御素基因分别与细菌表达载体pGEX和植物表达载体pMBL4构建成重组载体,在细菌中经IPTG诱导表达GST融合蛋白,亲和层析纯化蛋白;经根癌农杆菌介导的真空渗入法在烟草叶片中瞬间表达,提取叶片总蛋白用于鉴定。抑菌活性分析表明,烟草叶片瞬间表达的防御素和抗体-防御素融合蛋白,均对赤霉病菌有强烈的抑菌作用;从细菌中纯化的防御素蛋白,也能有效抑制赤霉病菌生长。说明赤霉病菌抗体-防御素融合蛋白基因,可作为抗赤霉病资源,用于改良植物的抗赤霉病性。

An single-chain antibody gene with a high specificity and affinity to wheat scab fungus was selected to construct a fusion gene with a plant defensin sequence. The antibody-defensin fusion gene and defensin gene alone were cloned into bacterial expression vector pGEX and plant expression vector pMBL, respectively. Bacterial expression was induced upon addition with IPTG, and the antibody-defensin fusion and defensin protein as GST fusions were affinity-purified. Both the antibody-defensin fusion and defensin protein were transiently expressed in tobacco leaves via vacuum infiltration of Agrobacterium tumefaciens. Antifungal assays of total proteins extracted from infiltrated tobacco leaves revealed that the antibody-defensin fusion and defensin proteins displayed a clear inhibitory effect on the fungal growth of wheat scab fungus in vitro. The defensin protein affinity-purified from bacteria also inhibited this fungus. This result indicated that the antibody-defensin fusion constructed in this study could be used as resistance germplasm for genetically improving resistance of plants to this fungus.