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通过对辣椒全基因组数据库分析获得黄灯笼辣椒eIF4E基因家族的四个基因,设计两端带有Eco RⅠ和XhoⅠ酶切位点的引物分别扩增四个基因的ORF,克隆到LexA-酵母双杂交系统的激活域载体pB42AD中,并构建pB42AD-eIF4E、pB42AD-eIF(iso)4E、pB42AD-eIF4E-Xm、pB42AD-n CBP9090重组载体。将重组质粒导入酵母菌株EGY48(p8op-Lac Z)中,进行重组激活域载体的毒性检验和自激活验证。结果表明,扩增获得了正确的黄灯笼辣椒Cc-eIF4E、Cc-eIF(iso)4E、Cc-eIF4E-Xm、Cc-n CBP9090基因ORF,并成功克隆到pB42AD载体中,同时转化有激活域载体的EGY48(p8op-Lac Z)在SD/-Trp/-Ura营养缺陷型平板上生长良好,在SD/-His/-Ura平板上不生长,说明重组质粒表达产物对该酵母细胞无毒性,对下游报告基因无自激活作用。本研究结果证明pB42AD-eIF4E、pB42AD-eIF(iso)4E、pB42AD-eIF4E-Xm、pB42AD-n CBP9090重组载体可用于该系统的互作蛋白筛选,为下一步通过酵母双杂交系统筛选与黄灯笼辣椒eIF4E家族蛋白相互作用的病毒蛋白奠定基础。
Four genes of eIF4E gene family were obtained by analyzing the whole genome database of pepper. The ORFs of four genes were amplified by PCR with EcoRI and XhoI ends, and cloned into LexA-yeast two-hybrid System activation vector pB42AD and pB42AD-eIF4E, pB42AD-eIF (iso) 4E, pB42AD-eIF4E-Xm, pB42AD-n CBP9090 recombinant vector was constructed. The recombinant plasmid was introduced into the yeast strain EGY48 (p8op-Lac Z) for toxicity testing and self-activation verification of recombinant activation domain vectors. The results showed that the correct ORF of Cc-eIF4E, Cc-eIF (iso) 4E, Cc-eIF4E-Xm and Cc-n CBP9090 genes were amplified and cloned into pB42AD vector, The vector EGY48 (p8op-Lac Z) grew well on the SD / -Trp / -Ura auxotrophic plate and did not grow on the SD / -His / -Ura plate, indicating that the recombinant plasmid expression product was non-toxic to the yeast cell, No downstream self-activation of the reporter gene. The results of the present study demonstrate that the recombinant vectors of pB42AD-eIF4E, pB42AD-eIF (iso) 4E, pB42AD-eIF4E-Xm and pB42AD-n CBP9090 can be used for the screening of the interacting proteins of the system. Peptide eIF4E family of protein interactions lay the foundation for viral proteins.