小麦条锈菌效应蛋白原核表达、纯化及其溶解性研究

摘要

将3个小麦条锈菌效应蛋白基因pst-1374、pst-1178和pst-8713,构建原核表达载体pET-32a-PP-pst-1374、pET-22b-pst-1178/8713,并将其转入大肠杆菌BL21 (DE3),重组阳性克隆经IPTG诱导表达和SDS-PAGE检测.结果表明:效应蛋白pst-1374和pst-8713正确表达且分子质量与理论值一致,而pst-1178基因表达后可能是以多聚体的形式存在,采用镍柱亲和层析、HPLC和离子交换柱进行纯化,获得可溶性的效应蛋白.%Haustorium is the only specialized infection structure of wheat stripe rust fungus(Puccinia striiformis f.sp.tritic).The fungus takes nutrients from the host by haustorium and delivers effectors directly into host cells in order to establish an intimate feeding relationship during infection.In this study,three effectors (pst-1374,pst-1178 andpst-8713) were cloned to pET-32a-PP-pst-1374,pET-22b-pst-1178 /8713 vectors and transformed into E.coli BL21(DE3) cells.The positive recombinant clones were induced by IPTG and the expressed proteins were analyzed by SDS-PAGE.The results indicated the effectorspst-1178 andpst-1374 were expressed correctly and the molecular mass of effectors estimated were in consent with the theoretical values.Meanwhile,preliminary studies suggested the effectorpst-1178 existed as polymer in solution.The target proteins were purified by NiNTA,HPLC and ion-exchange column and the soluble protein were obtained.