降解赤霉病菌毒素Tri101基因的原核表达及抗体制备  

Prokaryotic expression and antiserum preparation of toxicity reduction gene Tri101 from Fusarium graminearum Schwabe

作　　者：闫海霞[1,2] 杨丽荣[2] 薛保国[2] 段立清[1] 孙虎[2] 全鑫[2] 

机构地区：[1]内蒙古农业大学林学院,呼和浩特010019 [2]河南省农业科学院植物保护研究所,郑州450002

出　　处：《植物病理学报》2010年第5期469-474,共6页Acta Phytopathologica Sinica

基　　金：国家自然科学基金项目(30771439);农业部948项目(2008-Z22)

摘　　要：采用RT-PCR方法克隆了编码禾谷镰刀菌单端孢酶烯3-O-乙酰转移酶Tri101基因的cDNA序列,并连接到原核表达载体pGEX-4T2上,将获得的重组载体pGEX-4T2/Tri101转化大肠杆菌BL21后用IPTG进行诱导表达。SDS-PAGE和Western blot分析表明,经IPTG诱导后,Tri101基因在大肠杆菌BL21中获得了高效表达,融合蛋白GST-Tri101分子量为75.45 kDa。将该融合蛋白切胶纯化后免疫家兔,制备兔抗GST-Tri101多克隆抗体。经ELISA法测定抗体效价大于1∶256 000。Western blot分析表明制备的抗体与原核细胞体外表达的Tri101蛋白可以特异性结合,表明该抗体的特异性良好。应用该抗体验证了感赤霉病小麦中Tri101基因的表达。兔抗GST-Tri101抗体的成功制备,为进一步研究Tri101的生物学功能、细胞定位以及在其它植物中的表达等奠定了基础。The Tri101 gene of Fusarium graminearum Schw.was amplified by RT-PCR and ligated to the expression vector,pGEX-4T2.The recombinant plasmid,pGEX-4T2/Tri101 was then transformed into E.coli BL21 strain and induced by IPTG.The results of SDS-PAGE and Western blot analysis showed that the Tri101 gene was highly expressed.The molecular weight of the recombinant fusion protein is 75.45 kDa,consistent with the predicted result.Polyclonal anti-GST-Tri101 rabbit antibody was successfully prepared by using purified Tri101 protein as immunogen.The ELISA titer of antiserum against GST-Tri101 is greater than 1∶256 000.Western blot analysis showed that the antiserum could specifically bind to the expressed Tri101 protein.Trichothecene 3-O-acetyltransferase encoded by Tri101 gene in Fusarium-damaged kernels of wheat was successfully detected with the antibody.The rabbit antibody against GST-Tri101 can be further used to study the biological function,localization and expression in other plants of Tri101 gene.

关 键 词：Tri101基因 原核表达 多克隆抗体 WESTERN BLOT 

分 类 号：S432.1[农业科学—植物病理学]