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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。
Based on the results of the transmembrane region prediction, two pairs of specific primers, ImpF1 / ImpR1 and ImpF2 / ImpR2, were designed. Four target fragments were obtained by PCR amplification in four combinations of ImpF1 / ImpR1, ImpF1 / ImpR2, Imp-F2 / ImpR1 and ImpF2 / ImpR2, that is, the total length of Imp- (Imp-N); the gene sequence (Imp-C) of the Imp gene that excised the N-terminal transmembrane region; the sequence of the Imp gene that excised N- and C-terminal transmembrane regions Gene sequence (Imp-S). The prokaryotic expression vector Pmal-c2x was ligated into the E.coli BL21 (DE3) PlysS strain. SDS-PAGE electrophoresis showed that only the gene sequence (Imp-S) of Imp gene which excised N- and C-terminal transmembrane regions was abundantly expressed. The results showed that the transmembrane region of wheat blue dwarfism Expression in E. coli. Based on the results of the transmembrane region prediction, two pairs of specific primers, ImpF1 / ImpR1 and ImpF2 / ImpR2, were designed. Four target fragments were obtained by PCR amplification in four combinations of ImpF1 / ImpR1, ImpF1 / ImpR2, Imp-F2 / ImpR1 and ImpF2 / ImpR2, that is, the total length of Imp- (Imp-N); the gene sequence (Imp-C) of the Imp gene that excised the N-terminal transmembrane region; the sequence of the Imp gene that excised N- and C-terminal transmembrane regions Gene sequence (Imp-S). The prokaryotic expression vector Pmal-c2x was ligated into the E.coli BL21 (DE3) PlysS strain. SDS-PAGE electrophoresis showed that only the gene sequence (Imp-S) of Imp gene which excised N- and C-terminal transmembrane regions was abundantly expressed. The results showed that the transmembrane region of wheat blue dwarfism Expression in E. coli.