家蚕半胱氨酸蛋白酶抑制剂BmCPI40鉴定及时空表达特征

doi:10.3864/j.issn.0578-1752.2015.13.016

Abstract

Abstract: 【Objective】 The objective of this study is to identify a newly discovered cysteine protease inhibitor of silkworm (Bombyx mori) and to investigate the expression characteristic and regulation mechanism, thereby providing data for further investigation on the inhibitor functions in the development process of molting and metamorphosis in the silkworm. 【Method】 Primers were designed using Primer Premier 5.0 software according to the Silkworm Genome Database, and were used to amplify the sequence of BmCPI40 that the putative signal peptide was eliminated. The multiple sequence alignment and phylogenetic analysis of BmCPI40 was performed by bioinformatics software Clustal X and MEGA 4.0. The recombinant BmCPI40 protein was expressed in prokaryotic expression system, and then was used to produce the polyclonal antibody after purification. The spatial and temporal expression pattern of BmCPI40 was investigated using RT-PCR and quantitative real-time PCR (qPCR), and the protein distribution and localization in different tissues and different developmental stages was analyzed using Western blot and immunofluorescence. In addition, the 20E-induced expression of BmCPI40 was also studied after injection using qPCR. Furthermore, the promoter activity of BmCPI40 was analyzed in silkworm cells with Dual Glo Luciferase Assay System. 【Result】A new cysteine protease inhibitor of silkworm, named BmCPI40, was cloned and expressed. The open reading frame of BmCPI40 was 366 bp that encoded a protein with 121 amino acid residues, the first 18 residues of which was predicted as signal peptides, and the isoelectric point was 4.43. Phylogenetic analysis indicated that this inhibitor showed high sequences identity with the propeptides of several microbes cysteine proteinases. Soluble protein was produced from recombinant E. coli cell. The spatial expression results suggested that BmCPI40 was expressed high in the epidermis and weakly in the head. The temporal expression indicated that the expression of BmCPI40 in the larvae stage was significantly higher than that in the pupal stage, while the expression was significantly reduced during molting and metamorphosis. Immunofluorescence experiments showed that BmCPI40 was synthesized in the epithelial cells. Moreover, it was found that BmCPI40 could be inhibited by ecdysone, because that its expression level decreased after injection with 20E in the silkworm and promoter activity also decreased after induction with 20E in the BmE cell. 【Conclusion】Ecdysone induced the decrease of expression level of BmCPI40, which may be involved in the molting and metamorphosis in the silkworm.

Key words: Bombyx mori, cysteine protease inhibitor, molting, metamorphosis

LI Jian-wei, LI Yi, ZHOU Xiao-ying, LI Zhi-lang, CHEN Shi-da, TIAN Sha, HOU Yong, XIA Qing-you. Identification and Analysis of the Characteristics of Spatial and Temporal Expression of Bombyx mori Cysteine Protease Inhibitor BmCPI40[J].Scientia Agricultura Sinica, 2015, 48(13): 2645-2655.

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Identification and Analysis of the Characteristics of Spatial and Temporal Expression of Bombyx mori Cysteine Protease Inhibitor BmCPI40

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