Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (6): 1207-1218.doi: 10.3864/j.issn.0578-1752.2016.06.016

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles    

Characterization and Expression Analysis of Serine Protease BmSP141 from the Silkworm (Bombyx mori ) in Response to Starvation

LIU Hua-wei1, LI You-shan2, TANG Xin1, ZHANG Xiao-lu1, ZHAO Ping1   

  1. 1State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400716
    2Vitamin D Research Institute, Shaanxi University of Technology, Hanzhong 723001, Shaanxi
  • Received:2015-10-30 Online:2016-03-16 Published:2016-03-16

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Abstract: 【Objective】The objective of this study is toanalyze the sequence of BmSP141, clear its spatial and temporal expression patterns, and explore its potential function by starvation and re-feeding in silkworm (Bombyx mori). 【Method】The nucleotide sequence of serine protease gene BmSP141 coding region was obtained by TA cloning. Biology online software was used to analyze the amino acid sequence, molecular weight, functional domain and other information of the coding region of this gene. Sequence alignment and phylogenetic tree analysis of BmSP141 with serine proteases from other species were done by ClustalX1.8 and MEGA5.02 software. Prokaryotic expression vector p28-BmSP141 was constructed and introduced into Rosetta (DE3) to express the target protein, which was purified by nickel affinity chromatography. The expression profile of BmSP141 at mRNA and protein levels in different tissues and development stages were analyzed by semi-quantitative reverse transcription PCR (RT-PCR), quantitative real-time PCR (qPCR) and Western blot. Immunofluorescence localization was used to detect the location of BmSP141 protein in the 4th instar larvae. The impacts of starvation and re-feeding on the expression of BmSP141 mRNA and protein were detected by qPCR and Western blot. 【Result】The BmSP141 encoded a 292 amino acid protein with a 17-residues signal peptide. The predicted molecular mass and isoelectric point of BmSP141 mature protein are 25.9 kD and 7.8, respectively. Multi-sequence alignment showed that BmSP141 share a highly sequence identity (62% and 63%) with the serine proteases of Heliothis virescens and Mamestra configurata,respectively. These homologs have conserved catalytic triad and similar serine protease catalytic motifs, but the substrate specificity pocket sites of BmSP141 are relatively different compared with the conversed pocket sites of chymotrypsin. Furthermore, the recombinant protein BmSP141 was expressed as inclusion body at 37℃, and purified by nickel affinity chromatography. Then, the purified protein was used to generate the BmSP141-specific polyclonal antibodies. RT-PCR analysis of BmSP141 revealed that BmSP141 mRNA was mainly expressed in the larval midgut and highly expressed at the larval feeding stage. Whereas its expression level was lower in the pupae and moth than in the larvae. Western blot analysis showed that BmSP141 protein was only expressed in the midgut and the expression level was increased first and then decreased from 5th instar to wandering stage. Immunohistochemical analysis further confirmed that BmSP141 protein was predominately present in the cytoplasm of midgut epithelium cell. The expression of BmSP141 mRNA and protein was down-regulated after starvation but up-regulated by re-feeding. As a result, the expression of BmSP141 was induced by the food in the midgut.【Conclusion】BmSP141 was highly expressed in the midgut of larval feeding stage and lower expressed in the pupae and moth stages than in the larvae stage. The expression of BmSP141 mRNA and protein was down-regulated after starvation but up-regulated by re-feeding, which suggested that this gene might be involved in food protein digestion in the midgut during larval development.

Key words: Bombyx mori, serine protease, midgut, starvation, expression analysis

[1]    Lehane S M, Assinder S J, Lehane M J. Cloning, sequencing, temporal expression and tissue-specificity of two serine proteases from the midgut of the blood-feeding fly Stomoxys calcitrans. European Journal of Biochemistry, 1998, 254(2): 290-296.
[2]    Kotani E, Niwa T, Tokizane M, Suga K, Sugimura Y, Oda K, Mori H, Furusawa T. Cloning and sequence of a cDNA for a highly basic protease from the digestive juice of the silkworm, Bombyx mori. Insect Molecular Biology, 1999, 8(2): 299-304.
[3]    Wei Z, Yin Y P, Zhang B S H, Wang Z K, Peng G X, Cao Y Q, Xia Y X. Cloning of a novel protease required for the molting of Locusta migratoria manilensis. Development, Growth and Differentiation, 2007, 49: 611-621.
[4]    Kaji K, Tomino S, Asano T. A serine protease in the midgut of the silkworm, Bombyx mori: Protein sequencing, identification of cDNA, demonstration of its synthesis as zymogen form and activation during midgut remodeling. Insect Biochemistry and Molecular Biology, 2009, 39: 207-217.
[5]    Srinivasan A, Giri A P, Gupta V S. Structural and functional diversities in Lepidopteran serine proteases. Cellular & Molecular Biology Letters, 2006, 11(1): 132-154.
[6]    Klowden M J. Physiological Systems in Insect. San Diego: Academic Press, 2002: 305-364.
[7]    Santos C D, Ferreira C, Terra W R. Consumption of food and spatial organization of digestion in the Cassava hornworm, Erinnyis ello. Journal of Insect Physiology, 1983, 29(9): 707-714.
[8]    do Vale V F, Pereira M H, Gontijo N F. Midgut pH profile and protein digestion in the larvae of Lutzomyia longipalpis (Diptera: Psychodidae). Journal of Insect Physiology, 2007, 53(11): 1151-1159.
[9]    Peterson A M, Fernando G J, Wells M A. Purification, characterization and cDNA sequence of an alkaline chymotrypsin from the midgut of Manduca sexta. Insect Biochemistry and Molecular Biology, 1995, 25(7): 765-774.
[10]   Broehan G, Zimoch L, Wessels A, Ertas B, Merzendorfer H. A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm. The Journal of Experimental Biology, 2007, 210: 3636-3643.
[11]   Sasaki T, Hishida T, Ichikawa K, Asari S. Amino acid sequence of alkaliphilic serine protease from silkworm, Bombyx mori, larval digestive juice. FEBS Letters, 1993, 320(1): 35-37.
[12]   Maki N, Yamashita O. The 30kP protease A responsible for 30-kDa yolk protein degradation of the silkworm, Bombyx mori: cDNA structure, developmental change and regulation by feeding. Insect Biochemistry and Molecular Biology, 2001, 31(4/5): 407-413.
[13]   Chougule N P, Doyle E, Fitches E, Gatehouse J A. Biochemical characterization of midgut digestive proteases from Mamestra brassicae (cabbage moth; Lepidoptera: Noctuidae) and effect of soybean Kunitz inhibitor (SKTI) in feeding assays. Journal of Insect Physiology, 2008, 54: 563-572.
[14]   Sui Y P, Wang J X, Zhao X F. The impacts of classical insect hormones on the expression profiles of a new digestive trypsin-like protease (TLP) from the cotton bollworm, Helicoverpa armigera. Insect Molecular Biology, 2009, 18(4): 443-452.
[15]   Zhang C, Zhou D, Zheng S, Liu L, Tao S, Yang L, Hu S, Feng Q. A chymotrypsin-like serine protease cDNA involved in food protein digestion in the common cutworm, Spodoptera litura: Cloning, characterization, developmental and induced expression patterns, and localization. Journal of Insect Physiology, 2010, 56(7): 788-799.
[16]   Zhan Q, Zheng S, Feng Q, Liu L. A midgut-specific chymotrypsin cDNA (Slctlp1) from Spodoptera litura: cloning, characterization, localization and expression analysis. Archives of Insect Biochemistry and Physiology, 2011, 76(3): 130-143.
[17]   Ortego F, Farinos G P, Ruiz M, Marco V, Castanera P. Characterization of digestive proteases in the weevil Aubeonymus mariaefranciscae and effects of proteinase inhibitors on larval development and survival. Entomologia Experimentalis et Applicata, 1998, 88(3): 265-274.
[18]   Abdeen A, Virgos A, Olivella E, Villanueva J, Aviles X, Gabarra R, Prat S. Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors. Plant Molecular Biology, 2005, 57(2): 189-202.
[19]   Wu H, Zhang Y, Liu P, Xie J, He Y, Deng C, Clercq P D, Pang H. Effects of transgenic Cry1Ac+CpTI cotton on non-target mealybug pest Ferrisia virgata and its predator Cryptolaemus montrouzieri. PloS One, 2014, 9(4): e95537.
[20]   Zhao P, Wang G H, Dong Z M, Duan J, Xu P Z, Cheng T C, Xiang Z H, Xia Q Y. Genome-wide identification and expression analysis of serine proteases and homologs in the silkworm Bombyx mori. BMC Genomics, 2010, 11: 405.
[21]   罗娟, 陈恩祥, 刘红玲, 彭芷昕, 杨从文, 沈关望, 张海燕, 邢润苗, 林英, 夏庆友. 家蚕卵黄原蛋白受体BmVgR的体外表达. 中国农业科学, 2014, 47(19): 3922-3928.
Luo J, Chen E X, Liu H L, Peng Z X, Yang C W, Shen G W, Zhang H Y, Xing R M, Lin Y, Xia Q Y. Expression of silkworm vitellogenin receptor in vitro. Scientia Agricultura Sinica, 2014, 47(19): 3922-3928. (in Chinese)
[22]   Greer J. Comparative modeling methods: application to the family of the mammalian serine proteases. Proteins: Structure, Function, and Genetics, 1990, 7(4): 317-334.
[23]   Perona J J, Craik C S. Structural basis of substrate specificity in the serine proteases. Protein Sciences, 1995, 4: 337-360.
[24]   Zhu Y C, West S, Liu F X, He Y. Interaction of proteinase inhibitors with Cry1Ac toxicity and the presence of 15 chymotrypsin cDNAs in the midgut of the tobacco budworm, Heliothis virescens (F.) (Lepidoptera: Noctuidae). Pest Management Science, 2012, 68(5): 692-701.
[25]   Erlandson M A, Hegedus D D, Baldwin D, Noakes A, Toprak U. Characterization of the Mamestra configurata (Lepidoptera Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor. Archives of Insect Biochemistry and Physiology, 2010, 75(2): 70-91.
[26]   Nakazawa H, Tsuneishi E, Ponnuvel K M, Furukawa S, Asaoka A, Tanaka H, Ishibashi J, Yamakawa M. Antiviral activity of a serine protease from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Virology, 2004, 321: 154-162.
[27]   Bown D P, Wilkinson H S, Gatehouse J A. Differentially regulated inhibitor-sensitive and insensitive protease genes from the phytophagous insect pest, Helicoverpa armigera, are members of complex multigene families. Insect Biochemistry and Molecular Biology, 1997, 27(7): 625-638.
[28]   Simpson R M, Newcomb R D, Gatehouse H S, Crownhurst R N, Changne D, Gatehouse L N, Markwick N P, Beuning L L, Murray C, Marshall S D, Yauk Y K, Nain B, Gleave A P, Christeller J T. Expressed sequence tags from the midgut of Epiphyas postvittana (Walker) (Lepidoptera: Torticidae). Insect Molecular Biology, 2007, 16(6): 675-690.
[29]   Lenz C J, Kang J, Rice W C, McIntosh A H, Chippendale G M, Schubert K R. Digestive proteinases of larvae of the corn earworm, Heliothis zea: characterization, distribution, and dietary relationships. Archives of Insect Biochemistry and Physiology, 1991, 16: 201-212.
[30]   Prahakar S, Chen M S, Elpidina E N, Vinokurov K S, Smith C M, Marshall J, Oppert B. Sequence analysis and molecular characterization of larval midgut cDNA transcripts encoding peptidases from the yellow mealworm, Tenebrio molitor L.. Insect Molecular Biology, 2007, 16(4): 455-468.
[31]   Botos I, Meyer E, Nguyen M, Swanson S M, Koomen J M, Russell D H, Meyer E F. The structure of an insect chymotrypsin. Journal of Molecular Biology, 2000, 298: 895-901.
[32]   Broehan G, Kemper M, Driemeier D, Vogelpohl I, Merzendorfer H. Cloning and expression analysis of midgut chymotrypsin-like proteinases in the tobacco hornworm. Journal of Insect Physiology, 2008, 54: 1243-1252.
[33]   Dubrovsky E B. Hormonal cross talk in insect development. Trends in Endocrinology & Metabolism, 2005, 16(1): 6-11.
[34]   Rharrabe K, Bouayad N, Sayah F. Effects of ingested 20-hydroxyecdysone on development and midgut epithelial cells of Plodia interpunctella (Lepidoptera, Pyralidae). Pesticide Biochemistry and Physiology, 2009, 93(3): 112-119.
[35]   Bian G, Raikhel A S, Zhu J. Characterization of a juvenile hormone-regulated chymotrypsin-like serine protease gene in Aedes aegypti mosquito. Insect Biochemistry and Molecular Biology, 2008, 38(2): 190-200.
[36]   张翠红, 崔为正, 郭延奎, 王彦文, 牟志美. 家蚕蛹-成虫变态期中肠和涎腺超微结构变化与功能. 昆虫学报, 2007, 50(8): 769-774.
Zhang C H, Cui W Z, Guo Y K, Wang Y W, Mu Z M. Ultrastructure changes and function of the midgut and salivary glands in Bombyx mori during the pupal-adult metamorphism. Acta Entomologica Sinica, 2007, 50(8): 769-774. (in Chinese)
[37]   吴载德. 蚕体解剖生理学. 北京: 农业出版社, 1989: 85.
Wu Z D. Anatomy and Physiology of Silkworm. Beijing: China Agriculture Press, 1989: 85. (in Chinese)
[38]   Eguchi M, Furukawa S. Protease in the pupal midgut of the silkworm, Bombyx mori L.. Journal of Sericultural Science of Japan, 1970, 39(5): 387-392.
[39]   王厚伟, 张翠红, 崔为正, 刘小龙, 周元聪, 蔡幼民, 牟志美. 家蚕蛾溶茧酶分泌器官及蛾吐出液的研究. 蚕业科学, 2005, 31(2): 136-141.
Wang H W, Zhang C H, Cui W Z, Liu X L, Zhou Y C, Cai Y M, Mu Z M. Studies on secretory organs of cocoonase and silkmoth vomiting fluid of silkworm, Bombyx mori. Science of Sericulture, 2005, 31(2): 136-141. (in Chinese)
[40]   祝元锋, 王赵群, 何宁佳, 冯丽春. 家蚕蛾吐出液的酶活性及蛋白质种类鉴定. 2014, 40(3): 452-457.
Zhu Y F, Wang Z Q, He N J, Feng L C. Enzymatic activity and protein species identification of spit liquid from Bombyx mori moths. Science of Sericulture, 2014, 40(3): 452-457. (in Chinese)
[41]   Ponnuvel K M, Nakazawa H, Furukawa S, Asaoka A, Ishibashi J, Tanaka H, Yamakawa M. A lipase isolated from the silkworm Bombyx mori shows antiviral activity against nucleopolyhedrovirus. Journal of Virology, 2003, 77(19): 10725-10729.
[42]   Ponnuvel K M, Nithya K, Sirigineedi S, Awasthi A K, Yamakawa M. In vitro antiviral activity of an alkaline trypsin from the digestive juice of Bombyx mori larvae against nucleopolyhedrovirus. Archives of Insect Biochemistry and Physiology, 2012, 81(2): 90-104.
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