Back
 AJMB  Vol.2 No.3 , July 2012
Expression and subcellular localization of a novel gene Bm-X of the silkworm, Bombyx mori
Abstract: According to the large-scale sequencing of cDNA library from silkworm pupae, the cDNA of a novel gene with blank research background was identified and temporarily named Bm-X. The length of this cDNA is 778 bp. We obtained its ORF for further study by bioinformatics analysis. It is 444 bp and encodes 147 amino acid residues, with a predicted molecular weight (MW) of 16.4 kD and an isoelectric point (pI) of 3.69. In this study, we successfully constructed a recombinant plasmid pET-28a(+)-Bm-X and expressed it in Escherichia coli. We used the fusion protein rBm-X which purified by Niaffinity chromatography to produce polyclonal antibodies against Bm-X for Western blot analysis. The analysis revealed that Bm-X was expressed in the larval midgut, the epidermis and the silk gland. In addition, the subcellular localization analysis of silkworm ovary epithelial cells (BmN cells) showed that Bm-X protein was located both in cytoplasm and nucleus, and the signal was stronger in cytoplasm than in nucleus. Our findings indicate that Bm-X gene is a novel species-specificity gene and its expression product can be detected in tissues of the fifth silkworm instar larvae and BmN cells.
Cite this paper: Lv, Z. , Liu, Y. , Chen, Y. , Li, B. , Liu, L. , Chen, J. , Nie, Z. , Sheng, Q. , Yu, W. and Zhang, Y. (2012) Expression and subcellular localization of a novel gene Bm-X of the silkworm, Bombyx mori. American Journal of Molecular Biology, 2, 224-231. doi: 10.4236/ajmb.2012.23023.
References

[1]   Zhang, Y., Huanga, J.H., Jia, S.H., et al. (2007) SAGE tag based cDNA microarray analysis during larval to pupal development and isolation of novel cDNAs in Bombyx mori. Genomics, 90, 372-379.

[2]   Huang, N., Clem, R.J. and Rohrmann, G.F. (2011) Characterization of cDNAs encoding p53 of Bombyx mori and Spodoptera frugiperda. Insect Biochemistry and Molecular Biology, 41, 613-619.

[3]   Xia, Q.Y., Zhou, Z.Y., Lu, C., et a1. (2004) A draft sequence for the genome of the domesticated silkworm Bombyx mori. Science, 306, 1937-1940.

[4]   Zhang, Y.Z., Chen, J., Nie, Z.M., et al. (2007) Expression of open reading frames in silkworm pupal cDNA library. Appl Biochem Biotechnol, 136, 327-343.

[5]   Chen, J.Q., Chen, J., Gai, Q.J., et al. (2007) Molecular characterization and immunohistochemical localization of a novel troponin C duiing silkworm development. Cell and Tissue Research, 331, 725-738.

[6]   Wang, X.J., Chen, J., Lv, Z.B., et al. (2007) Expression and functional analysis of the cellular retinoic acid binding protein from silkworm pupae (Bombyx mori). Journal of Cellular Biochemistry, 102, 970-979.

[7]   Hattori, K., Hirayama, M., Suzuki, H., et al. (2007) Cloning and Expression of a Novel Sulfotransferase with Unique Substrate Specificity from Bombyx mori. Biosci. Biotechnol. Biochem., 71, 1044-1051.

[8]   Bao, Y.Y., Yamano, Y. and Morishima, I. (2005) A novel lebocin-like gene from eri-silkworm, Samia Cynthia ricini, that does not encode the antibacterial peptide lebocin. Comparative Biochemistry and Physiology, 140, 127-131.

[9]   Suzuki, Y., Matsuoka, T., Iimura, Y., et al. (2002) Ecdysteroid-depedent expression of a novel cuticle protein gene BmCPG1 in the silkworm, Bombyx mori. Insect Biochemistry and molecular Biology, 32, 599-607.

[10]   Daimon, T., Hamada, K., Mita, K., et al. (2003) A Bombyx mori gene, Bmchih, encodes a protein homologous to bacterial and baculovirus chitinases. Insect Biochemistry and Molecular Biology, 33, 749-759.

[11]   Zhang, H.B., Liu, M.Y., Tian, Y.J., et al. (2011) Comparative Characterization of Chitinases from Silkworm (Bombyx mori) and Bollworm (Helicoverpa armigera). Cell Biochemistry and Biophysics, 61, 267-275.

[12]   Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254.

[13]   Altschul, S.F., Madden, T.L., Schaffer, A.A., et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res, 25, 3389-3402.

[14]   Wilkins, M.R., Lindskog, I., Gasteiger, E., et al. (1997) Detailed peptide characterization using PEPTIDEMASS-a world-wide-web-accessible tool. Electrophoresis, 18, 403-408.

[15]   Joseph, S. and David, W.R. (2002) Molecular cloning: a laboratory manual. CSHL Press.

 
 
Top