赤霉病菌特异抗体-防御素融合蛋白的表达和功能鉴定

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。

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

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.