摘要：
[Objective]Previous studies showed that the coat protein (CP) of ToMV could interact with protein L (IP-L) in Nicotiana benthamiana. The objectives of this study are to understand the collocation of ToMV CP and IP-L, investigate the expression of IP-L in different tissues of tobacco, and investigate the IP-L expression in tomato leaves in response to ToMV infection.[Method]The ToMV CP and IP-L target fragments were isolated from the recombinant pGBKT7-CP and pGADT7-IP-L recombinant vectors by digestion with corresponded enzymes. Then the plant expression vectors pPZP-IP-L-N-EGFP and pPZP-CP-N-DsRed and prokaryotic expression vector pEGX-IP-L were constructed. The plant expression vectors were transformed into Agrobacterium tumefaciens EHA105 by heat shock method. The transient expression of ToMV CP and IP-L in tobacco epidermal cells was observed under the fluorescent microscope. Real-time quantitative RT-PCR (qRT-PCR) was used to analyze the expression level of IP-L in different tobacco tissues. The recombinant plasmid pEGX-IP-L was transformed into E. coil BL21 to express the soluble GST-IP-L protein in the optimized condition. GST-IP-L protein was purified with high-affinity GST resin to immunize rabbit for preparing anti-IP-L antibody in a rabbit. The title was determined by enzyme-linked immunosorbant assay (ELISA). The specificity of anti-IP-L antibody and the expression of IP-L in tobacco leaves infected by ToMV were detected by Western blot method and verified it by qRT-PCR. [Result]The ToMV CP was found in cell cytoplasm, cell membrane and chloroplasts, but IP-L only present in leaf epidermal cells membrane. They both co-localized in the plasma membrane. The relative expression of IP-L in the tobacco leaves has the specific high expression, significantly higher than that in stem, root and flower. The soluble GST-IP-L protein with molecular weight 42.8 kD was successfully expressed in E. coil BL21 induced with 0.3 mmol·L-1 IPTG at 30°C. About 4.2 mg fusion protein was purified and used to immunized rabbit. Anti-IP-L antibody with the title of 1/6400 was obtained. Western blot analysis showed that the antibody could specially bind with IP-L. After the tobacco leaves were infected by ToMV for 1, 3 and 7 d, Western blot analysis showed that IP-L was significantly up-regulated in leaves after 7 days of ToMV infection. qRT-PCR also got the same results, the expression level of IP-L was significantly (more than 200%) higher than that in healthy controls. [Conclusion]The ToMV CP and IP-L were co-located in the plasma membrane of the tobacco epidermis cell. IP-L specifically expressed in the tobacco leaves at high level. The high title and specificity antibody of tomato's IP-L were successfully achieved. The results of qRT-PCR and Western blot with the polyclonal antibody showed that the expression of IP-L in tobacco leaves was increased by the infection of ToMV.%[目的]前期研究显示ToMV外壳蛋白(coat protein,CP)可以与烟草蛋白L(CP-interacting protein-L,IP-L)相互作用,本研究旨在明确ToMV CP与IP-L的共定位情况、IP-L的组织表达和在ToMV侵染条件下烟草IP-L及其编码蛋白的表达变化情况,为进一步明确IP-L的功能提供依据.[方法]通过双酶切法从笔者实验室构建保存的pGBKT7-CP和pGADT7-IP-L重组载体上切下ToMV CP和IP-L目的片段,构建融合蛋白植物表达载体pPZP-IP-L-N-EGFP和pPZP-CP-N-DsRed及原核表达载体pEGX-IP-L.通过热激法将融合表达的植物表达载体转化至农杆菌EHA105,在烟草表皮细胞瞬时表达IP-L-N-EGFP和CP-N-DsRed,共聚焦荧光显微镜下观察IP-L和ToMV CP共定位情况.用实时荧光定量RT-PCR(qRT-PCR)分析IP-L在本氏烟各组织部位的表达量.原核表达载体pEGX-IP-L,转化原核表达菌BL21,优化GST-IP-L可溶性表达条件后大量表达该蛋白,用GST亲和层析法纯化获得可溶性GST-IP-L蛋白,免疫家兔制备多克隆抗体.ELISA测定抗体效价,Western印迹法明确抗体的特异性后,利用该抗体分析ToMV侵染条件下番茄IP-L蛋白表达情况,用qRT-PCR检测IP-L表达变化与蛋白水平是否一致.[结果]ToMV CP在本氏烟叶片表皮细胞的细胞质和细胞质膜以及叶绿体均有分布,而IP-L只在本氏烟叶片表皮细胞质膜表达,二者共定位在细胞质膜.IP-L在本氏烟叶片中特异高表达,显著高于茎、根和花中的相对表达量.在温度为30°C,IPTG诱导浓度为0.3 mmol·L-1条件下表达出大小为42.8 kD的可溶性GST-IP-L融合蛋白.纯化获得约4.2 mg可溶性蛋白,免疫家兔制备了效价为1/6400的多克隆抗体.Western印迹结果表明,该抗体可以与IP-L的原核产物特异性结合.在ToMV侵染本氏烟1、3、7 d后,Western印迹分析表明IP-L在叶片内表达量随接种时间呈明显上调趋势.qRT-PCR检测结果与Western印迹结果一致,显示IP-L在ToMV侵染本氏烟第7天后的叶片内表达量是健康对照的3倍多,差异达到显著水平.[结论]ToMV CP和IP-L共定位于本氏烟表皮细胞质膜,IP-L在本氏烟叶片内特异高表达.制备的IP-L多克隆抗体具有良好的免疫反应活性,可以用于IP-L表达量检测;ToMV侵染烟草可诱导IP-L及其编码蛋白表达量显著上调.
