ToMV外壳蛋白互作IP-L蛋白的亚细胞定位及表达分析

doi:10.3864/j.issn.0578-1752.2017.17.009

Abstract

Abstract: 【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℃. About 4.2 mg fusion protein was purified and used to immunized rabbit. Anti-IP-L antibody with the title of 1/6 400 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.

Key words: IP-L, subcellular localization, expression, analysis, Tomato mosaic virus (ToMV)

PENG HaoRan, PU YunDan, ZHANG YongZhi, XUE Yang, WU GaiXia, QING Ling, SUN XianChao. Subcellular Localization and Expression Analyses of IP-L Protein Interacting with ToMV Coat Protein[J].Scientia Agricultura Sinica, 2017, 50(17): 3344-3351.

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

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