ToMV外壳蛋白互作IP-L蛋白的亚细胞定位及表达分析  

Subcellular Localization and Expression Analyses of IP-L Protein Interacting with ToMV Coat Protein

作　　者：彭浩然[1] 蒲运丹 张永至 薛杨 武改霞[1] 青玲[1] 孙现超[1] 

机构地区：[1]西南大学植物保护学院,重庆400716

出　　处：《中国农业科学》2017年第17期3344-3351,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31670148);重庆市社会事业与民生保障创新专项(cstc2015shms-ztzx80012);中央高校基本科研业务费专项资金(XDJK2016A009;2362015xk04)

摘　　要：【目的】前期研究显示To MV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To MV CP与IP-L的共定位情况、IP-L的组织表达和在To MV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p GBKT7-CP和p GADT7-IP-L重组载体上切下To MV CP和IP-L目的片段,构建融合蛋白植物表达载体p PZP-IP-L-N-EGFP和p PZP-CP-N-Ds Red及原核表达载体p EGX-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-Ds Red,共聚焦荧光显微镜下观察IP-L和To MV CP共定位情况。用实时荧光定量RT-PCR(q RT-PCR)分析IP-L在本氏烟各组织部位的表达量。原核表达载体p EGX-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To MV侵染条件下番茄IP-L蛋白表达情况,用q RT-PCR检测IP-L表达变化与蛋白水平是否一致。【结果】To MV 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℃,IPTG诱导浓度为0.3 mmol·L-1条件下表达出大小为42.8 k D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To MV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。q RT-PCR检测结果与Western印迹结果一致,显示IP-L在To MV侵�【Objective】Previous studies showed that the coat protein（CP） of To MV could interact with protein L（IP-L） in Nicotiana benthamiana. The objectives of this study are to understand the collocation of To MV 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 To MV infection. 【Method】The To MV CP and IP-L target fragments were isolated from the recombinant p GBKT7-CP and p GADT7-IP-L recombinant vectors by digestion with corresponded enzymes. Then the plant expression vectors p PZP-IP-L-N-EGFP and p PZP-CP-N-Ds Red and prokaryotic expression vector p EGX-IP-L were constructed. The plant expression vectors were transformed into Agrobacterium tumefaciens EHA105 by heat shock method. The transient expression of To MV CP and IP-L in tobacco epidermal cells was observed under the fluorescent microscope. Real-time quantitative RT-PCR（q RT-PCR） was used to analyze the expression level of IP-L in different tobacco tissues. The recombinant plasmid p EGX-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 To MV were detected by Western blot method and verified it by q RT-PCR. 【Result】The To MV 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 k D was successfully expressed in E. coil BL21 induced with 0.3 mmol·L-1 IPTG at 30℃. About 4.2 mg fusion prote

关 键 词：IP-L 亚细胞定位 表达 分析 番茄花叶病毒 

分 类 号：S432.41[农业科学—植物病理学]