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

摘要/Abstract

摘要： 【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℃，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。

关键词: IP-L, 亚细胞定位, 表达, 分析, 番茄花叶病毒

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)

彭浩然，蒲运丹，张永至，薛杨，武改霞，青玲，孙现超. ToMV外壳蛋白互作IP-L蛋白的亚细胞定位及表达分析[J]. 中国农业科学, 2017, 50(17): 3344-3351.

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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参考文献

[1] NIEHL A, HEINLEIN M. Cellular pathways for viral transport through plasmodesmata. Protoplasma, 2011, 248(1): 75-99.

[2] LIU C, NELSON R S. The cell biology of Tobacco mosaic virus replication and movement. Frontiers in Plant Science, 2013, 4(3): Article 12.

[3] NICAISE v. Crop immunity against viruses: outcomes and future challenges. Frontiers in Plant Science, 2014, 5: Article 660.

[4] 周雪平, 钱秀红, 刘勇, 薛朝阳, 李德葆. 侵染番茄的番茄花叶病毒的研究. 中国病毒学, 1996, 11(3): 268-276.

ZHOU X P, QIAN X H, LIU Y, XUE C Y, LI D B. Isolation and characterization of tomato mosaic virus from tomato. Virologica sinica, 1996, 11(3): 268-276. (in Chinese)

[5] 郑贵彬, 徐鹤林, 熊助功. 我国为害番茄的病毒种群与烟草花叶病毒 (TMV) 株系分化的初步鉴定. 中国病毒学, 1988, 3(1): 64-70.

ZHENG G B, XU H L, XIONG Z G. A preliminary study on the virus groups of tomato and strain differentiation of tobacco mosaic virus (TMV) in China. Virologica sinica, 1988, 3(1): 64-70. (in Chinese)

[6] 周雪平, 薛朝阳, 刘勇, 李德葆. 番茄花叶病毒番茄分离物与烟草花叶病毒蚕豆分离物生物学、血清学比较及PCR特异性检测. 植物病理学报, 1997, 27(1): 53-58.

ZHOU X P, XUE C Y, LIU Y, LI D B. Biology and serological comparison of ToMV tomato isoate and TMV broad bean isolate and specific detection with polymerase chain reaction. Acta phytopathologica sinica, 1997, 27(1): 53-58. (in Chinese)

[7] 姜国勇, 杨仁崔. 番茄花叶病毒移动蛋白和番茄抗病基因 Tm-2² 的互作诱导烟草转化体程序性细胞死亡. 微生物学报, 2005, 45(2): 301-304.

JIANG G Y, YANG R C. Tomato R protein Tm-2² gene interacting with ToMV movement protein triggered programmed cell death in tobacco transformations. Acta microbiologica sinica, 2005, 45(2): 301-304. (in Chinese)

[8] YANG G, QIU B S, LIU X G, LI Y, WANG X F. Nonsense mutations of replicase and movement protein genes contribute to the attenuation of an avirulent tomato mosaic virus. Virus research, 2002, 87(2): 119-128.

[9] DOHI K, TAMAI A, MORI M. Insertion in the coding region of the movement protein improves stability of the plasmid encoding a tomato mosaic virus-based expression vector. Archives of virology, 2008, 153(9): 1667-1675.

[10] HIRAI K, KUBOTA K, MOCHIZUKI T, TSUDA S, MESHI T. Antiviral RNA silencing is restricted to the marginal region of the dark green tissue in the mosaic leaves of tomato mosaic virus-infected tobacco plants. Journal of virology, 2008, 82(7): 3250-3260.

[11] YOSHIOKA K, MATSUSHITA Y, KASAHARA M, KONAGAYA K, NYUNOYA H. Interaction of tomato mosaic virus movement protein with tobacco RIO kinase. Molecules and cells, 2004, 17(2): 223-229.

[12] MATSUSHITA Y, DEGUCHI M, YOUDA M, NISHIGUCHI M, NYUNOYA H. The tomato mosaic tobamovirus movement protein interacts with a putative transcriptional coactivator KELP. Molecules and cells, 2001, 12(1): 57-66.

[13] MATSUSHITA Y, MIYAKAWA O, DEGUCHI M, NISHIGUCHI M, NYUNOYA H. Cloning of a tobacco cDNA coding for a putative transcriptional coactivator MBF1 that interacts with the tomato mosaic virus movement protein. Journal of experimental botany, 2002, 53(373): 1531-1532.

[14] FUJISAKI K, ISHIKAWA M. Identification of an Arabidopsis thaliana protein that binds to tomato mosaic virus genomic RNA and inhibits its multiplication. Virology, 2008, 380(2): 402-411.

[15] LI Y, WU M Y, SONG H H, HU X, QIU B S. Identification of a tobacco protein interacting with tomato mosaic virus coat protein and facilitating long-distance movement of virus. Archives of virology, 2005, 150(10): 1993-2008.

[16] ZHANG C Z, LIU Y Y, SUN X C, QIAN W D, ZHANG D D, QIU B S. Characterization of a specific interaction between IP-L, a tobacco protein localized in the thylakoid membranes, and Tomato mosaic virus coat protein. Biochemical and biophysical research communications 2008, 374(2): 253-257.,

[17] SUN X C, LI Y, SHI M D, ZHANG N, WU G X, LI T T, QING L, ZHOU C Y. In vitro binding and bimolecular fluorescence complementation assays suggest an interaction between tomato mosaic virus coat protein and tobacco chloroplast ferredoxin I. Archives of Virology, 2013, 158(12): 2611-2615.

[18] 孙现超, 李勇, 周常勇, 青玲. 普通烟草铁氧还蛋白Ⅰ的原核表达、抗体制备及在ToMV侵染烟草体内表达分析. 中国农业科学,2010, 43(19): 3981-3987.

SUN X C, LI Y, ZHOU C Y, QING L. Prokaryotic expression of Fdn-I, preparation of rabbit anti-Fdn-I antibody and analysis of the Fdn-I levels in ToMV-infected tobacco leaves. Scientia Agricultura Sinica, 2010, 43(19): 3981-3987. (in Chinese)

[19] 刘兆明, 刘宗旨, 白庆武, 方荣祥. Agroinflltration在植物分子生物学研究中的应用. 生物工程学报, 2002, 18(4): 411-414.

LIU Z M, LIU Z Z, BAI Q W, FANG R X. Agroinfiltration, a useful technique in plant molecular biology research. Chinese Journal of Biotechnology, 2002, 18(4): 411-414. (in Chinese)

[20] VOINNET O, PINTO Y M, BAULCOMBE D C. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(24): 14147-14152.

[21] BRADFORD M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry, 1976, 72(1): 248-254.

[22] CALLAWAY A, GIESMAN-COOKMEYER D, GILLOCK E T, SIT T L, LOMMEL S A. The multifunctional capsid proteins of plant RNA viruses. Annual Review of Phytopathology, 2001, 39: 419-460.

[23] VAN REGENMORTEL M H V. The antigenicity of tobacco mosaic virus. Philosophical Transactions of the Royal Society of London, 1999, 354(1383): 559-568.

[24] OHNISHI J, HIRAI K, KANDA A, USUGI T, MESHI T, TSUDA S. The coat protein of Tomato mosaic virus L₁₁Y is associated with virus-induced chlorosis on infected tobacco plants. Journal of General Plant Pathology, 2009, 75(4): 297-306.

[25] CULVER J N. Tobacco mosaic virus assembly and disassembly: determinants in pathogenicity and resistance. Annual review of phytopathology, 2002, 40(1): 287-308.

[26] BANERJEE N, WANG J Y, ZAITLIN M. A single nucleotide change in the coat protein gene of tobacco mosaic virus is involved in the induction of severe chlorosis. Virology, 1995, 207(1): 234-239.

[27] 武改霞, 李婷婷, 孙现超. 与ToMV CP互作基因IP-L的启动子克隆分析//中国植物保护学会2011年学术年会论文集, 2001: 738.

WU G X, LI T T, SUN X C. Promoter cloning analysis of the IP-L interaction with the ToMV CP interaction gene//Chinese Society of Plant Protection 2011 Annual Conference, 2001: 738. (in Chinese)

[28] PARK M R, PARK S H, CHO S Y, KIM K H. Nicotiana benthamiana protein, NbPCIP1, interacting with Potato virus X coat protein plays a role as susceptible factor for viral infection. Virology, 2009, 386(2): 257-269.

[29] 潘琪, 刘旭旭, 彭浩然, 蒲运丹, 张永至, 叶思涵, 吴根土, 青玲, 孙现超. 番茄SYTA的克隆及表达分析. 中国农业科学, 2017, 50(15): 2936-2945.

PAN Q, LIU X X, PENG H R, PU Y D, ZHANG Y Z, YE S H, WU G T, QING L, SUN X C. Cloning, expression analysis of Solanum lycopersicum SYTA. Scientia Agricultura Sinica, 2017, 50(15): 2936-2945. (in Chinese)