中国野生华东葡萄抗白粉病转录因子VpRFP1基因原核表达、纯化及多克隆抗体制备  

Fusion Expression and Purification of the Transcription Factor VpRFP1 Gene Resistance to Uncinula necator from Chinese Wild Vitis pseudoreticulata and Preparation of Its Polyclonal Antibodies

作　　者：余义和[1] 徐伟荣[1] 李树秀[1] 黎涛[1] 王跃进[1] 

机构地区：[1]西北农林科技大学园艺学院,农业部西北园艺植物种质资源利用重点开放实验室,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2011年第1期85-92,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30771493)资助

摘　　要：为揭示中国野生华东葡萄抗病转录因子基因在与葡萄白粉病互作过程中的作用机制,本研究以中国野生华东葡萄(Vitis pseudoreticulata W.T.Wang)高抗葡萄白粉病株系白河35-1为材料,接种葡萄白粉病菌(Unicinula necator(Schw.)Burr.)病原菌诱导后7个不同时期的反转录产物为模板,用RT-PCR扩增得到V.pseudoreticulata RING-finger protein1基因(VpRFP1)的开放阅读框1053bp;将其克隆到pMD19-T载体经测序后,亚克隆到pGEX-4T-1原核表达载体并转化大肠杆菌(Escherichia.coli)BL21;经IPTG诱导表达出64kD的融合蛋白GST-VpRFP1,主要以包涵体表达形式存在;采用电透析法纯化融合蛋白,并以纯化产物免疫新西兰大白兔获得抗血清,Western blot检测免疫-抗原反应良好。本研究结果表明,在27℃、0.1mmol/L的IPTG诱导4h融合蛋白GST-VpRFP1的表达量最大,免疫大白兔获得的抗血清能够用于VpRFP1基因的功能分析。To reveal the disease-resistant mechanism of Chinese wild grape transcription factor in the process of grape resistant to powdery mildew,the highly resistant line Baihe-35-1 of Chinese wild Vitis pseudoreticulata W.T. Wang was used as the materials,and a 1 053 bp full-length cDNA encoding V. pseudoreticulata RING-finger protein1 gene （VpRFP1） was obtained using the transcript product induced by grape powdery mildew （Unicunula necator （Schw.） Burr.） at seven different stages as templates. The resultant PCR products were cloned to pMD19-T vector,sequenced and subcloned into pGEX-4T-1 vector. The recombinant expression clone were transformed into Escherichia.coli BL21 and induced expression by IPTG. A 64 kD fusion protein GST-VpRFP1 was obtained with the form of inclusion body. The fusion protein was purified by electrodialysis,and immunized the rabbit to obtain the polyclonal antibodies. The immunity-antigen reaction was tested by Western blot,showing a specific antigen-antibody recognition feature. The results suggested that the fusion protein GST-VpRFP1 was expressed at high levels 4 h after 0.1 mmol/L of IPTG induction （27℃）. And the polyclonal antibodies obtained by immunized rabbit with the purified fusion protein can be used for the functional analysis of VpRFP1 gene.

关 键 词：华东葡萄 融合表达 转录因子 VpRFP1 多克隆抗体 

分 类 号：S436.631.1[农业科学—农业昆虫与害虫防治]