GST-MtDef4融合蛋白的原核表达及体外抑菌活性的检测  

The Purification of GST-MtDef4 Fusion Protein and Detection of Its Antifungal Activity in vitro

作　　者：景晓辉[1] 吴伦英[1] 沈雁[2,3] 赵辉[4] 吴琳[5] 

机构地区：[1]海南省热带作物资源可持续利用重点实验室海南大学农学院,海南海口570228 [2]热带作物种质资源保护与开发利用教育部重点实验室海南大学园艺园林学院,海南海口570228 [3]中国热带农业科学院椰子研究所,海南文昌571339 [4]农业部热带作物生物学与遗传资源利用重点实验室中国热带农业科学院热带生物技术研究所,海南海口571101 [5]中国热带农业科学院环境与植物保护研究所,海南海口571101

出　　处：《热带作物学报》2014年第4期718-723,共6页Chinese Journal of Tropical Crops

基　　金：农业部948项目(No.2013-Z15);海南省自然基金项目(No.809037;312039);海南省研究生创新科研课题(No.Hyb2012-1);国家公益性行业农业科研专项(No.200903049)

摘　　要：为研究苜蓿(Medicago truncatula)防卫素基因MtDef4的抑菌功能,以含有该基因片段的质粒pAND-MtDef4为模板,扩增出相应的目的基因,测序正确后将MtDef4基因片段连接到带有GST标签的原核表达载体pGEX6P-1中,转化大肠杆菌TransB(DE3),经IPTG诱导,Glutathione Sepharose 4B亲和纯化,通过SDS-PAGE电泳及Western blot确定融合蛋白的表达。体外抑菌试验表明,MtDef4能够抑制病原真菌Corynespora cassiicola而非Collectotrichum gloeosporioides的生长。本研究结果可为进一步培育转基因橡胶抗病品种奠定基础。MtDef4 was amplified from pAND-MtDef4 by PCR, and cloned into vector pGEX-6P-1 with glutathione- S-transferase （GST） tag. After identified by restriction endonuclease digestion and sequencing, the recombinant plasmids pGEX-6P-1-MtDef4 were transformed into Escherichia coli TransB （DE3）, an efficient strain to guarantee the correct fold of disulfide bond. The recombined cells were grown and induced by IPTG. After purification using glutathione sepharose 4B affinity chromatography, the expressed protein was separated by SDS-PAGE and detected by western blot. The results showed that the MtDef4-GST fusion protein was successfully expressed. Antifungal assay indicated that MtDef4-GST fusion protein could inhibit the growth of a filamentous fungus Corynespora cassiicola but not Collectotrichum gloeosporioides.

关 键 词：苜蓿防卫素基因 原核表达 抗真菌活性 多主棒孢菌 胶孢炭疽菌 

分 类 号：Q782[生物学—分子生物学]