小麦蓝矮植原体免疫膜蛋白(Imp)的跨膜区影响其在大肠杆菌中的表达  

Influence of the transmembrane region of immunodominant membrane protein (Imp) from wheat blue dwarf phytoplasma on Imp expression in Escherichia coli

作　　者：孙润红[1,2,3] 于祥泉[1] 陶烨[1] 杨洋[2] 吴云锋[1] 张银武 李艳[1] 

机构地区：[1]西北农林科技大学植物保护学院陕西省农业分子生物学重点实验室,杨凌712100 [2]西北农林科技大学生命科学学院,杨凌712100 [3]河南驻马店上蔡县农技站,上蔡463800

出　　处：《植物病理学报》2010年第6期587-592,共6页Acta Phytopathologica Sinica

基　　金：国家自然科学基金(30871625);稻麦重要病毒病株系鉴定和防控技术体系研究(nyhyzx07-051);高等学校学科创新引智计划(B07049)

摘　　要：根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。根据跨膜区预测结果,设计ImpF1/ImpR1和ImpF2/ImpR2两对特异性引物。以ImpF1/ImpR1,ImpF1/ImpR2,Imp-F2/ImpR1和ImpF2/ImpR2共4个组合经PCR扩增得到4个目标片段,即:免疫膜蛋白Imp基因的全长(Imp-B);切除C-端跨膜区的Imp基因的基因序列(Imp-N);切除N-端跨膜区的Imp基因的基因序列(Imp-C);切除N-和C-端跨膜区的Imp基因的基因序列(Imp-S)。经酶切连接到原核表达载体Pmal-c2x,并转入E.coliBL21(DE3)PlysS菌株中。SDS-PAGE电泳验证,只有切除N-、C-端跨膜区的Imp基因的基因序列(Imp-S)得到大量表达,结果表明:小麦蓝矮植原体免疫膜蛋白的跨膜区影响其在大肠杆菌中的表达。Two pairs of specific primers,ImpF1/ImpR1 and ImpF2/ImpR2,were designed according to the prediction result of possible transmembrane helices for immunodominant membrane protein（Imp） of wheat blue dwarf（WBD） phytoplasma.Four sequences,Imp-B,Imp-N,Imp-C and Imp-S were respectively obtained by using combined primers ImpF1/ImpR1,ImpF1/ImpR2,ImpF2/ImpR1 and ImpF2/ImpR2.The overall length of Imp was named Imp-B;the Imp sequence excision of the C-transmembrane region was Imp-N;the Imp excision of the N-transmembrane region was Imp-C and the Imp excision of the C-and N-transmembrane regions was Imp-S.After double enzyme digestion,these four sequences were respectively sub-cloned into the expression vector Pmal-c2x and then transformed into E.coli strain BL21（DE3） PlysS.SDS-PAGE analysis showed that only Imp-S with cleaved N-terminal and C-terminal transmembrane regions could be expressed.The results demonstrated that the transmembrane region can influence the Imp expression of WBD phytoplasma in E.coli.

关 键 词：小麦蓝矮植原表体 免疫膜蛋白 跨膜区 

分 类 号：S432.4[农业科学—植物病理学]