家蚕丝氨酸蛋白酶BmSP141的表达特征及对饥饿的响应

doi:10.3864/j.issn.0578-1752.2016.06.016

摘要/Abstract

摘要： 【目的】分析家蚕(Bombyx mori)丝氨酸蛋白酶基因BmSP141序列信息，明确其时空表达模式，结合饥饿与重新喂食处理对其表达的影响，探究该基因在家蚕中的功能。【方法】对家蚕丝氨酸蛋白酶基因BmSP141进行T-A克隆，得到其编码区核苷酸序列；应用生物信息学在线网站对该基因编码区推导的氨基酸序列、分子量、结构域等信息进行分析；利用ClustalX1.8和MEGA5.02软件对BmSP141与其他物种的丝氨酸蛋白酶进行多序列比对和系统发生树分析；构建原核表达载体p28-BmSP141，并转化到Rosetta（DE3）菌株中诱导表达，利用镍柱亲和层析纯化重组蛋白。利用半定量RT-PCR和实时荧光定量PCR方法对其组织和时期表达特征进行分析；采用蛋白质免疫印迹（Western blot）技术，分别分析BmSP141蛋白在家蚕5龄第3天各组织的表达情况和5龄幼虫中肠的表达变化；通过免疫荧光定位分析BmSP141蛋白在4龄幼虫中肠的分布情况；利用实时荧光定量PCR和Western blot方法对饥饿处理和再喂食后BmSP141的表达情况进行分析。【结果】BmSP141编码含有292个氨基酸的蛋白，其中第1—17位氨基酸为信号肽，其成熟体蛋白预测分子量为25.9 kD，等电点为7.8。同源序列比对表明，BmSP141与烟芽夜蛾丝氨酸蛋白酶和蓓带夜蛾丝氨酸蛋白酶序列同源性较高，分别达到62%和63%。这些同源序列具有保守的催化三联体，与活性相关的基序也很保守，但与胰凝乳蛋白酶保守的底物特异性位点相比，BmSP141的底物特异性位点发生改变。在16和37℃条件下诱导后，重组蛋白均以包涵体形式表达，经镍柱亲和层析纯化后得到了较纯的重组蛋白，并用该蛋白制备了效价较高的多克隆抗体。组织和时期表达特征分析表明，该基因主要在家蚕幼虫的中肠组织特异性表达, 且在幼虫起蚕到食桑期表达逐渐增加，但眠期表达下降，蛹期和成虫期表达水平较低。Western blot分析也表明，BmSP141蛋白仅在家蚕中肠组织表达，且从5龄起蚕到上蔟表达量先增加后降低。免疫荧光定位结果表明，BmSP141定位于中肠上皮细胞的细胞质中，进一步证实BmSP141在中肠特异表达。在转录和翻译水平，BmSP141饥饿处理后表达量显著下调，而重新喂食后其表达量上调，表明BmSP141的表达受到消化道食物的诱导。【结论】家蚕丝氨酸蛋白酶BmSP141在中肠组织表达，在幼虫期食桑期高表达，蛹期和成虫期表达水平较低，受消化道食物的诱导而上调表达，推测该基因可能参与家蚕中肠的消化过程。

关键词: 家蚕, 丝氨酸蛋白酶, 中肠, 饥饿, 表达分析

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

刘华伟，李游山，唐新，张晓璐，赵萍. 家蚕丝氨酸蛋白酶BmSP141的表达特征及对饥饿的响应[J]. 中国农业科学, 2016, 49(6): 1207-1218.

LIU Hua-wei, LI You-shan, TANG Xin, ZHANG Xiao-lu, ZHAO Ping. Characterization and Expression Analysis of Serine Protease BmSP141 from the Silkworm (Bombyx mori ) in Response to Starvation[J]. Scientia Agricultura Sinica, 2016, 49(6): 1207-1218.

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.0578-1752.2016.06.016

https://www.chinaagrisci.com/CN/Y2016/V49/I6/1207

参考文献

[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.