家蚕感染质型多角体病毒（BmCPV）后中肠组织 差异蛋白质分析

doi:10.3864/j.issn.0578-1752.2013.13.017

摘要/Abstract

摘要： 【目的】分析家蚕感染质型多角体病毒（Bombyx mori cytoplasmic polyhedrosis virus，BmCPV）后中肠组织中蛋白质的差异表达，为从蛋白质分子水平上探索家蚕感染BmCPV后的分子应答机制奠定基础。【方法】通过蛋白质双向凝胶电泳技术（two-dimensional polyacrylamide gel electrophoresis，2-DE）对BmCPV病毒感染组和灭菌水对照组家蚕p50的中肠组织蛋白进行比较，通过基质辅助质量飞行时间质谱技术（MALDI-TOF MS）和家蚕蛋白质数据库检索对所得到的差异蛋白点进行鉴定与分析。【结果】2-DE电泳结果比较分析表明，BmCPV感染组和对照组家蚕的中肠组织有8个差异蛋白斑点，其中对照中肠有3个，感染中肠有5个。经MALDI-TOF MS鉴定结果显示对照家蚕中肠中的3个差异蛋白斑点分别为家蚕的类固醇脱氢酶（Hydroxysteroid dehydrogenase）、线粒体电压依赖的阴离子通道孔蛋白（Voltage-dependent anion-selective channel，VDAC）和1个未知新型蛋白；感染家蚕中肠的5个差异蛋白点分别是家蚕的Ras-鸟嘌呤核苷酸释放因子1（Ras-specific guanine nucleotide- releasing factor 1-like，RASGRF1）、H+转运ATP合成酶亚基、候选肿瘤抑制因子（Putative tumor suppressor protein，PDSS2）、多药耐药蛋白（Multidrug resistance-associated protein，MRP4/ABCC4）、冻坏B样蛋白（Nipped-B-like protein- like，NIPBL）。【结论】BmCPV感染可导致家蚕中肠组织多种蛋白的差异表达，提示家蚕可能通过启动不同的机制来应答病毒的感染，其中VDAC的下调表达以及PDSS2、MRP4/ABCC4和NIPBL的上调表达均与诱导细胞凋亡相关，推测家蚕在BmCPV感染的过程中可能启动了细胞凋亡的抗病毒机制。

关键词: 家蚕 , 质型多角体病毒 , 中肠 , 差异蛋白

Abstract: 【Objective】 The objective of this study is to analyze differentially expressed proteins in the midgut of silkworm strain p50 induced by Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) , and to explore the immune mechanism of silkworm against BmCPV infection, which would also provide valuable information for further study. 【Method】 The differentiated proteins in the midgut of silkworm which induced by BmCPV was investigated by two-dimensional polyacrylamide gel electrophoresis (2-DE), and the characteristics and functions of the differentially expressed proteins were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Silkworm Protein Data Bank.【Result】Between the control and BmCPV infected silkworm midgut, 8 differentiated protein spots (3 in control and 5 in BmCPV infected silkworm) were distinguished. In the control silkworm midgut, the three specific spots were hydroxysteroid dehydrogenase, voltage-dependent anion-selective channel (VDAC) and a new unknown protein. However, in the BmCPV infected silkworm midgut, the five specific spots might be ras-specific guanine nucleotide-releasing factor 1-like (RASGRF1), H+ transporting ATP synthase beta subunit isoform 1, putative tumor suppressor protein (PDSS2), multidrug resistance-associated protein (MRP4/ABCC4) and nipped-B-like protein (NIPBL). 【Conclusion】BmCPV infection induced a variety of differentially expressed proteins in the midgut, suggesting that different mechanisms were activited in the silkworm to response to the virus infection. Of the 8 differnentiated proteins, the down-regulated expression of VDAC and the up-regulated expression of PDSS2, MRP4/ABCC4 and NIPBL could respectively induce apoptosis. It would provide clues for elucidating the apoptosis program of silkworm against BmCPV infection.

Key words: Bombyx mori , cytoplasmic polyhedrosis virus , midgut , differentiated expressed proteins

高坤, 邓祥元, 裘智勇, 覃光星, 郭锡杰. 家蚕感染质型多角体病毒（BmCPV）后中肠组织差异蛋白质分析[J]. 中国农业科学, 2013, 46(13): 2796-2807.

GAO Kun, DENG Xiang-Yuan, QIU Zhi-Yong, QIN Guang-Xing, GUO Xi-Jie. Comparative Analysis of Differential Proteins from Midgut of Silkworm Induced by Cytoplasmic Polyhedrosis Virus Infection[J]. Scientia Agricultura Sinica, 2013, 46(13): 2796-2807.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.13.017

https://www.chinaagrisci.com/CN/Y2013/V46/I13/2796

参考文献

[1]Tan Y R, Sun J C, Lu X Y, Su D M, Zhang J Q. Entry of Bombyx mori cypovirus 1 into midgut cells in vivo. Journal of Electron Microscopy (Tokyo), 2003, 52(5): 485-489.

[2]Sun J C, Chen D N, Yang Y F, Zhang Q F, Tan P C, Xu X Y, Qiu B L. Penetration and replication of Bombyx mori cytoplasmic polyhedrosis virus in vivo. Acta Scientiarum Naturalium Universitatis Sunyatseni, 2006, 45(2): 78-82.

[3]张建红, 高玮, 张立人. 家蚕质型多角体病毒在蚕体内的形态发生及其细胞病理变化. 蚕业科学, 1990, 16(3): 140-144.

Zhang J H, Gao W, Zhang L R. Morphogenesis of cytoplasmic polyhedrsis virus of Bombyx mori in vivo and cytopathologic changes. Acta Sericologica Sinica, 1990, 16(3): 140-144. (in Chinese)

[4]钟杨生, 徐秋云, 林健荣, 王叶元, 范兰芬. 高温干燥对家蚕胚胎发育影响的蛋白质表达谱分析//中国蚕学会第六届青年学术研讨会论文集 (2), 2009.

Zhong Y S, Xu Q Y, Lin J Y, Wang Y Y, Fan L F. Analysis on protein expression of silkworm embryonic development under high temperature and dry condition//The Sixth Youth Symposium of Chinese Society of Sericultural Science (2), 2009. (in Chinese)

[5]裘智勇, 李木旺, 沈兴家, 郭锡杰. 家蚕对浓核病毒 (镇江株)抵抗性和感受性品种的中肠组织蛋白比较分析. 蚕业科学, 2008, 34(2): 244-249.

Qiu Z Y, Li M W, Shen X J, Guo X J. Comparative analysis of proteins extracted from midgut of silkworm strains susceptible and non-susceptible to Bomby mori Densovirus (Zhenjiang strain). Acta Sericologica Sinica, 2008, 34(2): 244-249. (in Chinese)

[6]刘晓勇, 陈克平, 姚勤, 李军, 蔡克亚. 家蚕中肠组织抗核型多角体病毒病的相关蛋白分析. 昆虫学报, 2008, 51(4): 443-448.

Liu X Y, Chen K P, Yao Q, Li J, Cai K Y. Proteome analysis of silkworm (Bombyx mori) midgut proteins related to BmNPV infection resistance or susceptibility. Acta Entomologica Sinica, 2008, 51(4): 443-448. (in Chinese)

[7]Qin L G, Xia H C, Shi H F, Zhou Y J, Chen L, Yao Q, Liu X Y, Feng F, Yuan Y, Chen K P. Comparative proteomic analysis reveals that caspase-1 and serine protease may be involved in silkworm resistance to Bombyx mori nuclear polyhedrosis virus. Journal of Proteomics, 2012, 75(12): 3630-3638.

[8]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(1/2): 248-254.

[9]Gordaliza M, Castro M A, Miguel del Corral J M, Lopez-Vazquez M L, Garcia P A, Garcia-Gravalos M D, San Feliciano A. Synthesis and antineoplastic activity of cyclolignan aldehydes. European Journal of Medicinal Chemistry, 2000, 35: 691-698.

[10]Hoogenboom B W, Suda K, Engel A, Fotiadis D. The supramolecular assemblies of voltage-dependent anion channels in the native membrane. Journal of Molecular Biology, 2007, 370: 246-255.

[11]Mannella C A. The ‘ins’ and ‘outs’ of mitochondrial membrane channels. Trends in Biochemical Sciences, 1992, 17: 315-320.

[12]Dihanich M. The biogenesis and function of eukaryotic porins. Experientia, 1990, 46: 146-153.

[13]Forte M, Guy H R, Mannella C A. Molecular genetics of the VDAC ion channel: structural model and sequence analysis. Journal of Bioenergetics and Biomembranes, 1987, 19(4): 341-350.

[14]Hiller S, Abramson J, Mannella C, Wagner G, Zeth K. The 3D structures of VDAC represent a native conformation. Trends in Biochemical Sciences, 2010, 35: 514-521.

[15]Shimizu S, Konishi A, Kodama T, Tsujimoto Y. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(7): 3100-3105.

[16]Vander Heiden M G, Chandel N S, Schumacker P T, Thompson C B. Bcl-XL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Molecular Cell, 1999, 3: 159-167.

[17]Vander Heiden M G, Li X X, Gottleib E, Hill R B, Thompson C B, Colombini M. Bcl- XL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane. Journal of Biological Chemistry, 2001, 276(22): 19414-19419.

[18]Shimizu S, Shinohara Y, Tsujimoto Y. Bax and Bcl- xL independently regulate apoptotic changes of yeast mitochondria that require VDAC but not adenine nucleotide translocator. Oncogene, 2000, 19(38): 4309-4318.

[19]Tan W, Lai J C, Miller P, Stein C A, Colombini M. Phosphorothioate oligonucleotides reduce mitochondrial outer membrane permeability to ADP. American Journal of Physiology-Cell Physiology, 2007, 292: 1388-1397.

[20]Tan W, Loke Y H, Stein C A, Miller P, Colombini M. Phosphorothioate oligonucleotides block the VDAC channel. Biophysical Journal, 2007, 93: 1184-1191.

[21]Lai J C, Tan W, Benimetskaya L, Miller P, Colombini M, Stein C A. A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(19): 7494-7499.

[22]Tan W, Colombini M. VDAC closure increases calcium ion flux. Biochimica et Biophysica Acta, 2007, 1768: 2510-2515.

[23]Peng M, Falk M J, Haase V H, King R, Polyak E, Selak M, Yudkoff M, Hancock W W, Meade R, Saiki R, Lunceford A L, Clarke C F, Gasser D L. Primary coenzyme Q deficiency in Pdss2 mutant mice causes isolated renal disease. PLoS Genetics, 2008, 4(4): e1000061.

[24]Ziegler C G, Peng M, Falk M J, Polyak E, Tsika E, Ischiropoulos H, Bakalar D, Blendy J A, Gasser D L. Parkinson's disease-like neuromuscular defects occur in prenyl diphosphate synthase subunit 2 (Pdss2) mutant mice. Mitochondrion, 2012, 12: 248-257.

[25]Chen P, Zhao S H, Chu Y L, Xu K, Zhu L, Wu Y, Song J, Cao C X, Xue X, Niu Y Y. Anticancer activity of PDSS2, prenyl diphosphate synthase, subunit 2, in gastric cancer tissue and the SGC7901 cell line. Anticancer Drugs, 2009, 20(2): 141-148.

[26]Borst P, Evers R, Kool M, Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins. Journal of the National Cancer Institute, 2000, 92(16): 1295-1302.

[27]Schuetz J D, Connelly M C, Sun D, Paibir S G, Flynn P M, Srinivas R V, Kumar A, Fridland A. MRP4: A previously unidentified factor in resistance to nucleoside-based antiviral drugs. Nature Medicine, 1999, 5(9): 1048-1051.

[28]Wielinga P R, van der Heijden I, Reid G, Beijnen J H, Wijnholds J, Borst P. Characterization of the MRP4- and MRP5-mediated transport of cyclic nucleotides from intact cells. Journal of Biological Chemistry, 2003, 278(20): 17664-17671.

[29]Jedlitschky G, Burchell B, Keppler D. The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. Journal of Biological Chemistry, 2000, 275(39): 30069-30074.

[30]Guo Y, Kotova E, Chen Z S, Lee K, Hopper-Borge E, Belinsky M G, Kruh G D. MRP8, ATP-binding cassette C11 (ABCC11), is a cyclic nucleotide efflux pump and a resistance factor for fluoropyrimidines 2',3'-dideoxycytidine and 9'- (2'-phosphonylmethoxyethyl) adenine. Journal of Biological Chemistry, 2003, 278(32): 29509-29514.

[31]Karin M. Signal transduction from the cell surface to the nucleus through the phosphorylation of transcription factors. Current Opinion in Cell Biology, 1994, 6: 415-424.

[32]Copsel S, Garcia C, Diez F, Vermeulem M, Baldi A, Bianciotti L G, Russel F G, Shayo C, Davio C. Multidrug resistance protein 4 (MRP4/ABCC4) regulates cAMP cellular levels and controls human leukemia cell proliferation and differentiation. Journal of Biological Chemistry, 2011, 286(9): 6979-6988.

[33]Hammond C L, Marchan R, Krance S M, Ballatori N. Glutathione export during apoptosis requires functional multidrug resistance- associated proteins. Journal of Biological Chemistry, 2007, 282(19): 14337-14347.

[34]Hammond C L, Lee T K, Ballatori N. Novel roles for glutathione in gene expression, cell death, and membrane transport of organic solutes. Journal of Hepatology, 2001, 34: 946-954.

[35]Coppola S, Ghibelli L. GSH extrusion and and the mitochondrial pathway of apoptotic signalling. Biochemical Society Transactions, 2000, 28(2): 56-61.

[36]Tagami M, Kusuhara S, Imai H, Uemura A, Honda S, Tsukahara Y, Negi A. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells. Biochemical Biophysical Research Communications, 2010, 400: 593-598.

[37]Hannivoort R A, Dunning S, Vander Borght S, Schroyen B, Woudenberg J, Oakley F, Buist-Homan M, van den Heuvel F A, Geuken M, Geerts A, Roskams T, Faber K N, Moshage H. Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells. Hepatology, 2008, 48: 624-634.

[38]Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Nasmyth K. Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Molecuar Cell, 2000, 5: 243-254.

[39]Gillespie P J, Hirano T. Scc2 couples replication licensing to sister chromatid cohesion in Xenopus egg extracts. Current Biology, 2004, 14: 1598-1603.

[40]Takahashi T S, Yiu P, Chou M F, Gygi S, Walter J C. Recruitment of Xenopus Scc2 and cohesin to chromatin requires the pre-replication complex. Nature Cell Biology, 2004, 6(10): 991-996.

[41]Watrin E, Schleiffer A, Tanaka K, Eisenhaber F, Nasmyth K, Peters J M. Human Scc4 is required for cohesin binding to chromatin, sister-chromatid cohesion, and mitotic progression. Current Biology, 2006, 16(9): 863-874.

[42]单士刚, 姚青, 彭建新, 洪华珠. 动物病毒诱导的细胞凋亡及其生理意义. 病毒学报, 2006, 22(3): 233-236.

Shan S G, Yao Q, Peng J X, Hong H Z. Physiological significance of apoptosis in animal virus infection. Chinese Journal of Virology, 2006, 22(3): 233-236. (in Chinese)