家蚕丝氨酸蛋白酶抑制剂BmSPI37的克隆、 表达及微生物诱导分析

doi:10.3864/j.issn.0578-1752.2012.23.017

摘要/Abstract

摘要： 【目的】鉴定新的家蚕蛋白酶抑制剂，探讨其在抵御病原微生物入侵方面的功能。【方法】对家蚕蛋白酶抑制剂BmSPI37进行T-A克隆、多序列比对、系统发育树构建、原核表达、RT-PCR时空表达特征及微生物诱导分析。【结果】克隆、表达了1个新的丝氨酸蛋白酶抑制剂，命名为BmSPI37。时空表达特征分析表明，BmSPI37在家蚕5龄幼虫后期表达量很高，而且在中部丝腺中高量表达；在蛹向蛾的变态发育时期，BmSPI37在雌蚕中的表达量显著高于雄蚕。微生物诱导试验表明，BmSPI37在大肠杆菌、黑胸败血菌、球孢白僵菌感染后，强烈上调表达。【结论】推测BmSPI37与防御病原微生物入侵有关。

关键词: 家蚕 , 丝氨酸蛋白酶抑制剂 , 病原微生物诱导 , 防御

Abstract: 【Objective】 The objective of this study is to identify a new protease inhibitor of silkworm (Bombyx mori) and to investigate its function in guarding against invasion of pathogenic microorganisms. 【Method】 T-A cloning, multiple sequence alignment, construction of phylogenetic tree, prokaryotic expression, spatio-temporal expression profile analysis and microbial-induced experiments were performed with BmSPI37. 【Result】 A new serine proteinase inhibitor, named BmSPI37, was cloned and expressed. Spatio-temporal expression profile analysis showed that BmSPI37 expressed highly at the late stage of 5th instar larvae, and expressed highly in the middle silk gland. The expression of BmSPI37 was significantly higher in female than in male during metamorphosis period from pupa to moth. Analysis of the expression of BmSPI37 after infection with Escherichia coli, Bacillus bombysepticus or Beauveria bassiana showed that BmSPI37 was strongly up-regulated.【Conclusion】It is speculated that BmSPI37 is related to defense against pathogenic microorganisms.

Key words: font-size: 10.5pt, mso-bidi-font-size: 10.0pt, mso-font-kerning: 1.0pt, mso-ansi-language: EN-US, mso-fareast-language: ZH-CN, mso-bidi-language: AR-SA, 2012-04-26Bombyx mori ')">mso-fareast-font-family: 宋体" lang="EN-US">2012-04-26Bombyx mori , serine protease inhibitor , pathogenic microbial-induction , defense

李游山, 赵萍, 董照明, 邹勇, 夏庆友, 向仲怀. 家蚕丝氨酸蛋白酶抑制剂BmSPI37的克隆、表达及微生物诱导分析[J]. 中国农业科学, 2012, 45(23): 4898-4908.

LI You-Shan, ZHAO Ping, DONG Zhao-Ming, ZOU Yong, XIA Qing-You, XIANG Zhong-Huai. Cloning, Expression and Microbial-Induction Analysis of Bombyx mori Serine Protease Inhibitor BmSPI37[J]. Scientia Agricultura Sinica, 2012, 45(23): 4898-4908.

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参考文献

[1]Lopez-Otin C, Overall C M. Protease degradomics: a new challenge for proteomics. Molecular Cell Biology, 2002, 3(7): 509-519.

[2]Clynen E, Schoofs L, Salzet M. A Review of the most important classes of serine protease inhibitors in insects and leeches. Medicinal Chemistry Reviews, 2005, 2(3): 197-206.

[3]Zhao Y, Sun W, Zhang P, Chi H, Zhang M J, Song C Q, Ma X, Shang Y, Wang B, Hu Y, Hao Z, Huhmer A F, Meng F, L'Hernault S W, He S M, Dong M Q, Miao L. Nematode sperm maturation triggered by protease involves sperm-secreted serine protease inhibitor (Serpin). Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(5): 1542-1547.

[4]迟庆, 伦永志. Kazal型人类丝氨酸蛋白酶抑制因子研究现状. 中华临床医师杂志, 2010, 4(8): 1331-1333.

Chi Q, Lun Y Z. The research of kazal-type serine protease inhibitors in human. Chinese Journal of Clinicians, 2010, 4(8): 1331-1333. (in Chinese)

[5]Park H J, Seong Y M, Choi J Y, Kang S, Rhim H. Alzheimer's disease-associated amyloid beta interacts with the human serine protease HtrA2/Omi. Neuroscience Letters, 2004, 357(1): 63-67.

[6]Hook G, Hook V, Kindy M. The cysteine protease inhibitor, E64d, reduces brain amyloid-β and improves memory deficits in Alzheimer's disease animal models by inhibiting cathepsin B, but not BACE1, β-secretase activity. Journal of Alzheimer's Disease, 2011, 26(2): 387-408.

[7]Kim J T, Song E Y, Chung K S, Kang M A, Kim J W, Kim S J, Yeom Y I, Kim J H, Kim K H, Lee H G. Up-regulation and clinical significance of serine protease kallikrein 6 in colon cancer. Cancer, 2011, 117(12): 2608-2619.

[8]Krowarsch D, Cierpicki T, Jelen F, Otlewski J. Canonical protein inhibitors of serine proteases. Cellular and Molecular Life Science, 2003, 60(11): 2427-2444.

[9]Hibbetts K, Hines B, Williams D. An overview of proteinase inhibitors. Journal of Veterinary Internal Medicine, 1999, 13(4): 302-308.

[10]Magdolen V, de Prada N A, Sperl S, Muehlenweg B, Luther T, Wilhelm O G, Magdolen U, Graeff H, Reuning U, Schmitt M. Natural and synthetic inhibitors of the tumor-associated serine protease urokinase-type plasminogen activator. Advanced in Experimental Medecine and Biology, 2000, 477: 331-341.

[11]Roose J P, Van Noorden C J. Synthetic protease inhibitors: promising compounds to arrest pathobiologic processes. The Journal of Laboratory and Clinical Medecine, 1995, 125(4): 433-441.

[12]Engles L. Review and application of serine protease inhibition in coronary artery bypass graft surgery. American Journal of Health-System Pharmacy, 2005, 62(18 Suppl. 4): 9-14.

[13]Dunse K M, Stevens J A, Lay F T, Gaspar Y M, Heath R L, Anderson M A. Coexpression of potato type I and II proteinase inhibitors gives cotton plants protection against insect damage in the field. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(34): 15011-15015.

[14]王亚伟, 欧阳学农, 余宗阳. TRA IL联合蛋白酶抑制剂MG132诱导肺癌A549细胞凋亡的实验研究. 临床肿瘤学杂志, 2011, 16(4): 294-297.

Wang Y W, Ouyang X N, Yu Z Y. An experimental study of the effect of TRA IL combined with proteasome inhibitor MG132 on lung adenocarcinoma cell line A549. Chinese Clinical Oncology, 2011, 16(4): 294-297. (in Chinese)

[15]叶兼菱, 李荣贵, 郭道森, 赵博光. 半胱氨酸蛋白酶抑制剂防治桑天牛的室内生测. 林业科技开发, 2011, 25(1): 40-42.

Ye Q L, Li R G, Guo D S, Zhao B G. Bioassay of cysteine protease inhibitor for controlling Apripona germari (Hope). China Forestry Science and Technology, 2011, 25(1): 40-42. (in Chinese)

[16]Eguchi M. Inhibition of fungal protease by haemolymph protease inhibitors of the silkworm, Bombyx mori. Applied Entomology and Zoology, 1982, 17(4): 589-590.

[17]Kanost M R. Serine proteinase inhibitors in arthropod immunity. Development and Comparative Immunology, 1999, 23(4/5): 291-301.

[18]Yoshida S, Yamashita M, Yonehara S, Eguchi M. Properties of fungal protease inhibitors from the integument and haemolymph of the silkworm and effect of an inhibitor on the fungal growth. Comparative Biochemistry and Physiology, 1990, 95B(3): 559-564.

[19]Eguchi M, Itoh M, Chou L Y, Nishino K. Purification and characterization of a fungal protease specific protein inhibitor (FPI-F) in the silkworm heamolymph. Comparative Biochemistry and Physiology, 1993, 104B(3): 537-543.

[20]Zhao P, Dong Z, Duan J, Wang G, Wang L, Li Y, Xiang Z, Xia Q. Genome-wide identification and immune response analysis of serine protease inhibitor genes in the silkworm, Bombyx mori. PLoS One, 2012, 7(2): e31168.

[21]Schagger H. Tricine-SDS-PAGE. Nature Protoccols, 2006, 1(1): 16-22.

[22]Pham T N, Hayashi K, Takano R, Itoh M, Eguchi M, Shibata H, Tanaka T, Hara S. A new family of serine protease inhibitors (Bombyx family) as established from the unique topological relation between the positions of disulphide bridges and reactive site. Journal of Biochemistry, 1996, 119(3): 428-434.

[23]Bania J, Stachowiak D, Polanowski A. Primary structure and properties of the cathepsin G/chymotrypsin inhibitor from the larval hemolymph of Apis mellifera. European Journal of Biochemistry, 1999, 262(3): 680-687.

[24]Fogaca A C, Almeida I C, Eberlin M N, Tanaka A S, Bulet P, Daffre S. Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases. Peptides, 2006, 27(4): 667-674.

[25]Huang K, Strynadka N C, Bernard V D, Peanasky R J, James M N. The molecular structure of the complex of Ascaris chymotrypsin/ elastase inhibitor with porcine elastase. Structure, 1994, 2(7): 679-689.

[26]Kellenberger C, Boudier C, Bermudez I, Bieth J G, Luu B, Hietter H. Serine protease inhibition by insect peptides containing a cysteine knot and a triple-stranded β-sheet. The Journal of Biological Chemistry, 1995, 270(43): 25514-25519.

[27]Nirmala X, Mita K, Vanisree V, Zurovec M, Sehnal F. Identification of four small molecular mass proteins in the silk of Bombyx mori. Insect Molecular Biology, 2001, 10(5): 437-445.

[28]Kurioka A, Yamazaki M, Hirano H. Primary structure and possible functions of a trypsin inhibitor of Bombyx mori. European Journal of Biochemistry, 1999, 259(1/2): 120-126.

[29]Nirmala X, Kodrik D, Zurovec M, Sehnal F. Insect silk contains both a Kunitz-type and a unique Kazal-type proteinase inhibitor. European Journal of Biochemistry, 2001, 268(7): 2064-2073.

[30]Shiba H, Uchida D, Kobayashi H, Natori M. Involvement of cathepsin B- and L-like proteinases in silk gland histolysis during metamorphosis of Bombyx mori. Archives of Biochemistry and Biophysics, 2001, 390(1): 28-34.

[31]Fullaondo A, Garcia-Sanchez S, Sanz-Parra A, Recio E, Lee S Y, Gubb D. Spn1 regulates the GNBP3-dependent Toll signaling pathway in Drosophila melanogaster. Molecular and Cellelar Biology, 2011, 31(14): 2960-2972.

[32]Soderhall K, Cerenius L. Role of the prophenoloxidase-activating system in invertebrate immunity. Current Opinion in Immunology, 1998, 10(1): 23-28.

[33]Ishii K, Hamamoto H, Imamura K, Adachi T, Shoji M, Nakayama K, Sekimizu K. Porphyromonas gingivalis peptidoglycans induce excessive activation of the innate immune system in silkworm larvae. Journal of Biological Chemistry, 2010, 285(43): 33338-33347.

[34]An C, Kanost M R. Manduca sexta serpin-5 regulates prophenoloxidase activation and the Toll signaling pathway by inhibiting hemolymph proteinase HP6. Insect Biochemistry and Molecular Biology, 2010, 40(9): 683-689.

[35]Tang H, Kambris Z, Lemaitre B, Hashimoto C. A serpin that regulates immune melanization in the respiratory system of Drosophila. Developmental Cell, 2008, 15(4): 617-626.

[36]Travis J, Potempa J, Maeda H. Are bacterial proteinases pathogenic factors? Trends of Microbiology, 1995, 3(10): 405-407.

[37]Leger R J S. The role of cuticle-degrading proteases in fungal pathogenesis of insects. Canidian Journal of Botany, 1995, 73(Suppl. 1): S1119-S1125.

[38]Donpudsa S, Tassanakajon A, Rimphanitchayakit V. Domain inhibitory and bacteriostatic activities of the five-domain Kazal-type serine proteinase inhibitor from black tiger shrimp Penaeus monodon. Developmental and Comparative Immunology, 2009, 33(4): 481-488.

[39]Wang Z H, Zhao X F, Wang J X. Characterization, kinetics, and possible function of Kazal-type proteinase inhibitors of Chinese white shrimp, Fenneropenaeus chinensis. Fish and Shellfish Immunology, 2009, 26(6): 885-897.

[40]Augustin R, Siebert S, Bosch T C G. Identification of a kazal-type serine protease inhibitor with potent anti-staphylococcal activity as part of Hydra's innate immune system. Developmental and Comparative Immunology, 2009, 33(7): 830-837.

[41]Zou Z, Picheng Z, Weng H, Mita K, Jiang H. A comparative analysis of serpin genes in the silkworm genome. Genomics, 2009, 93(4): 367-375.

[42]Zhu L, Song L, Chang Y, Xu W, Wu L. Molecular cloning, characterization and expression of a novel serine proteinase inhibitor gene in bay scallops (Argopecten irradians, Lamarck 1819). Fish and Shellfish Immunology, 2006, 20(3): 320-331.

[43]Kong H J, Cho H K, Park E M, Hong G E, Kim Y O, Nam B H, Kim W J, Lee S J, Han H S, Jang I K, Lee C H, Cheong J, Choi T J. Molecular cloning of Kazal-type proteinase inhibitor of the shrimp Fenneropenaeus chinensis. Fish and Shellfish Immunology, 2009, 26(1): 109-114.

[44]Tanaka H, Ishibashi J, Fujita K, Nakajima Y, Sagisaka A, Tomimoto K, Suzuki N, Yoshiyama M, Kaneko Y, Iwasaki T, Sunagawa T, Yamaji K, Asaoka A, Mita K, Yamakawa M. A genome-wide analysis of genes and gene families involved in innate immunity of Bombyx mori. Insect Biochemistry and Molecular Biology, 2008, 38(12): 1087-1110.