Shengyan Xiao, Zhongyuan Shen^*, Xudong Tang, Li Xu, Xuliang Fu, Yajie Yue, Nan Li, Wei Wang

Show more
Jiangsu University of Science and Technology, Zhenjiang, China.
Show less

Abstract: We isolated a microsporidium from the silkworm, Bombyx mori, and classified it as Endoreticulatus sp. Zhenjiang based on morphological characteristics and phylogenetic analyses of ribosomal sequences. This microsporidium causes silkworm pebrine, although its original host and mode of transmission are unknown. To better understand its distribution and transmission mode, it is essential to have species specific molecular markers. Towards this goal, we characterized the alpha tubulin gene from Endoreticulatus sp. Zhenjiang in this study. The full-length alpha-tubulin cDNA from Endoreticulatus sp. Zhenjiang was cloned and sequenced (GenBank ID: KJ784483) using the rapid amplification of cDNA ends (RACE) protocol. The alpha-tubulin cDNA is 1382 bp long with an open reading frame spanning 1320 bp and consisting of a short 20-bp 5’-untranslated region (5’-UTR) and a 42-bp 3’-UTR with a stop codon and a poly (A) tail. This alpha-tubulin cDNA encodes a deduced polypeptide with 439 amino acids, including a complete tubulin domain and a tubulin C domain. This protein has an estimated isoelectric point of 5.1 and a predicted molecular weight of 48.6 kDa.

Keywords: Microsporidium, RACE, Tubulin Gene, Silkworm, Pebrine

Cite this paper: Xiao, S. , Shen, Z. , Tang, X. , Xu, L. , Fu, X. , Yue, Y. , Li, N. and Wang, W. (2014) Molecular Analysis of the Alpha-Tubulin Gene from the Microsporidium, Endoreticulatus sp. Zhenjiang, Isolated from Bombyx mori. Agricultural Sciences, 5, 882-890. doi: 10.4236/as.2014.510095.

References

[1]   Li, Y., et al. (2009) Identification of a Novel Spore Wall Protein (SWP26) from Microsporidia Nosema bombycis. International Journal for Parasitology, 39, 391-398.
http://dx.doi.org/10.1016/j.ijpara.2008.08.011

[2]   Didier, E.S., Snowden, K.F. and Shadduck, J.A. (1998) Biology of Microsporidian Species Infecting Mammals. Advances in Parasitology, 40, 283-320.
http://dx.doi.org/10.1016/S0065-308X(08)60125-6

[3]   Corradi, N. and Keeling, P.J. (2009) Microsporidia: A Journey through Radical Taxonomical Revisions. Fungal Biology Reviews, 23, 1-8.
http://dx.doi.org/10.1016/j.fbr.2009.05.001

[4]   Corradi, N., Haag, K.L., Pombert, J.F., Ebert, D. and Keeling, P.J. (2009) Draft Genome Sequence of the Daphnia Pathogen Octosporeabayeri: Insights into the Gene Content of a Large Microsporidian Genome and a Model for Host-Parasite Interactions. Genome Biology, 10, R106.
http://dx.doi.org/10.1186/gb-2009-10-10-r106

[5]   Cai, S., Lu, X., Qiu, H., Li, M. and Feng, Z. (2011) Identification of a Nosema bombycis (Microsporidia) Spore Wall Protein Corresponding to Spore Phagocytosis. Parasitology, 138, 1102-1109.
http://dx.doi.org/10.1017/S0031182011000801

[6]   Wan, Y., et al. (1995) Separation and Research of Pathogenic Microsporidia SCM (Endoreticulatus. sp), Bombyx mori. Acta Sericologic Sinica, 21, 168-172.

[7]   Xu, X., et al. (2012) Phylogenetic Characterization of a Microsporidium (Endoreticulatus sp. Zhenjiang) Isolated from the Silkworm, Bombyx mori. Parasitology Research, 110, 815-819.
http://dx.doi.org/10.1007/s00436-011-2560-8

[8]   Keeling, P.J. (2003) Congruent Evidence from Alpha-Tubulin and Beta-Tubulin Gene Phylogenies for a Zygomycete Origin of Microsporidia. Fungal Genetics and Biology, 38, 298-309.
http://dx.doi.org/10.1016/S1087-1845(02)00537-6

[9]   Zhu, F., Shen, Z., Xu, L. and Guo, X. (2013) Molecular Characteristics of the Alpha- and Beta-Tubulin Genes of Nosemaphilosamiae. Folia Parasitologica, 60, 411-415.
http://dx.doi.org/10.14411/fp.2013.043

[10]   McKean, P. G., Vaughan, S. and Gull, K. (2001) The Extended Tubulin Superfamily. Journal of Cell Science, 114, 2723-2733.

[11]   Dong, S., Shen, Z., Xu, L. and Zhu, F. (2010) Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia. Current Microbiology, 60, 30-37.
http://dx.doi.org/10.1007/s00284-009-9495-7

[12]   Aurrecoechea, C., Barreto, A., Brestelli, J., Brunk, B.P., Caler, E.V., Fischer, S., et al. (2011) AmoebaDB and MicrosporidiaDB: Functional Genomic Resources for Amoebozoa and Microsporidia Species. Nucleic Acids Research, 39, D612-D619.
http://dx.doi.org/10.1093/nar/gkq1006

[13]   Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The CLUSTAL_X Windows Interface: Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools. Nucleic Acids Research, 25, 4876-4882.
http://dx.doi.org/gka797

[14]   Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. and Kumar, S. (2011) MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 28, 2731-2739.
http://dx.doi.org/10.1093/molbev/msr121

[15]   Yanagawa, N., Tamura, G., Oizumi, H., Endoh, M. and Motoyama, T. (2011) MAGE Expressions Mediated by Demethylation of MAGE Promoters Induce Progression of Non-Small Cell Lung Cancer. Anticancer Research, 31, 171-175.

[16]   Gao, K., Deng, X., Qian, H., Wu, P., Qin, G., Liu, T., et al. (2012) Novel Protein of IBP from Silkworm, Bombyx mori, Involved in Cytoplasmic Polyhedrosis Virus Infection. Journal of Invertebrate Pathology, 110, 83-91.
http://dx.doi.org/10.1016/j.jip.2012.02.011

[17]   Katinka, M.D., Duprat, S., Cornillot, E., Méténier, G., Thomarat, F., Prensier, G., et al. (2001) Genome Sequence and Gene Compaction of The Eukaryote Parasite Encephalitozoon cuniculi. Nature, 414, 450-453.
http://dx.doi.org/10.1038/35106579

[18]   Peyretaillade, E., El-Alaoui, H., Diogon, M., Polonais, V., Parisot, N., Biron, D.G., et al. (2011) Extreme Reduction and Compaction of Microsporidian Genomes. Research in Microbiology, 162, 598-606.
http://dx.doi.org/10.1016/j.resmic.2011.03.004

[19]   Cuomo, C.A., Desjardins, C.A., Bakowski, M.A., Goldberg, J., Ma, A.T., Becnel, J.J., et al. (2012) Microsporidian Genome Analysis Reveals Evolutionary Strategies for Obligate Intracellular Growth. Genome Research, 22, 2478-2488.
http://dx.doi.org/10.1101/gr.142802.112

[20]   Keeling, P.J. and Fast, N.M. (2002) MICROSPORIDIA: Biology and Evolution of Highly Reduced Intracellular Parasites. Annual Review of Microbiology, 56, 93-116.
http://dx.doi.org/10.1146/annurev.micro.56.012302.160854

[21]   Keeling, P.J., Fast, N.M., Law, J.S., Williams, B.A. and Slamovits, C.H. (2005) Comparative Genomics of Microsporidia. Folia Parasitologica, 52, 8-14.
http://dx.doi.org/10.14411/fp.2005.002

[22]   Waller, R.F., Jabbour, C., Chan, N.C., Celik, N., Likic, V.A., Mulhern, T.D., et al. (2009) Evidence of a Reduced and Modified Mitochondrial Protein Import Apparatus in Microsporidian Mitosomes. Eukaryotic Cell, 8, 19-26.
http://dx.doi.org/10.1128/EC.00313-08

[23]   Corradi, N. and Slamovits, C.H. (2011) The Intriguing Nature of Microsporidian Genomes. Briefings in Functional Genomics, 10, 115-124.
http://dx.doi.org/10.1093/bfgp/elq032

[24]   Corradi, N., Gangaeva, A. and Keeling, P.J. (2008) Comparative Profiling of Overlapping Transcription in the Compacted Genomes of Microsporidia Antonospora locustae and Encephalitozoon cuniculi. Genomics, 91, 388-393.
http://dx.doi.org/10.1016/j.ygeno.2007.12.006

[25]   Williams, B.A., Slamovits, C.H., Patron, N.J., Fast, N.M. and Keeling, P.J. (2005) A High Frequency of Overlapping Gene Expression in Compacted Eukaryotic Genomes. Proceedings of the National Academy of Sciences of the United States of America, 102, 10936-10941.
http://dx.doi.org/10.1073/pnas.0501321102

[26]   Grisdale, C.J. and Fast, N.M. (2011) Patterns of 5’ Untranslated Region Length Distribution in Encephalitozoon cuniculi: Implications for Gene Regulation and Potential Links between Transcription and Splicing. Journal of Eukaryotic Microbiology, 58, 68-74.
http://dx.doi.org/10.1111/j.1550-7408.2010.00523.x

[27]   Wang, J., Huang, K., Mao, X., Zhao, Y. and Lu, C. (2001) Small Subunit Ribosomal RNA (ssurRNA) Gene of Microsporidia. Acta Biochiimica et Biophysica Sinica, 33, 229-232.

[28]   Jiang, Y., Ling, Y. and Zhao, W. (2001) Progress in the Studies on Function of 3’ Untranslated Region on Post-Transcriptional Leval. Chinese Bulletin of Botany, 18, 3-10.

[29]   Pickering, B.M. and Willis, A.E. (2005) The Implications of Structured 5’ Untranslated Regions on Translation and Disease. Seminars in Cell & Developmental Biology, 16, 39-47.
http://dx.doi.org/10.1016/j.semcdb.2004.11.006

[30]   Hughes, T.A. (2006) Regulation of Gene Expression by Alternative Untranslated Regions. Trends in Genetics, 22, 119-122.
http://dx.doi.org/10.1016/j.tig.2006.01.001

[31]   Fang, Y., Xue, J. and Tian, L. (2008) Regulation of Translation Initiation and Targeted Gene Therapy of Tumor. Chinese Journal of Cancer Biotherapy, 15, 396-400.

[32]   Akiyoshi, D.E., Weiss, L.M., Feng, X., Williams, B.A.P., Keeling, P.J., Zhang, Q., et al. (2007) Analysis of the β-Tubulin Genes from Enterocytozoon bieneusi Isolates from a Human and Rhesus Macaque. Journal of Eukaryotic Microbiology, 54, 38-41.
http://dx.doi.org/10.1111/j.1550-7408.2006.00140.x

[33]   Keeling, P.J., Luker, M.A. and Palmer, J.D. (2000) Evidence from β-Tubulin Phylogeny That Microsporidia Evolved from within the Fungi. Molecular Biology and Evolution, 17, 23-31.
http://dx.doi.org/10.1093/oxfordjournals.molbev.a026235

[34]   Keeling, P.J. and Doolittle, W.F. (1996) Alpha-Tubulin from Early-Diverging Eukaryotic Lineages and the Evolution of the Tubulin Family. Molecular Biology and Evolution, 13, 1297-1305.
http://dx.doi.org/10.1093/oxfordjournals.molbev.a025576

[35]   Lee, R.C., Williams, B.A., Brown, A.M., Adamson, M.L., Keeling, P.J. and Fast, N.M. (2008) α- and β-Tubulin Phylogenies Support a Close Relationship between the Microsporidia Brachiola algerae and Antonospora locustae. Journal of Eukaryotic Microbiology, 55, 388-392.
http://dx.doi.org/ 10.1111/j.1550-7408.2008.00348.x

[36]   Haag, K.L., Larsson, J.I., Refardt, D. and Ebert, D. (2011) Cytological and Molecular Description of Hamiltosporidium tvaerminnensis gen. etsp. nov., a Microsporidianparasite of Daphnia magna, and Establishment of Hamiltosporidium magnivora comb.nov. Parasitology, 138, 447-462.
http://dx.doi.org/10.1017/S0031182010001393

[37]   Stine, S.W., Vladich, F.D., Pepper, I.L. and Gerba, C.P. (2005) Development of a Method for the Concentration and Recovery of Microsporidia from Tap Water. Journal of Environmental Science and Health, Part A: Toxic Hazardous Substances and Environmental Engineering, 40, 913-925.
http://dx.doi.org/10.1081/ESE-200056105

[38]   Kokoskin, E., Gyorkos, T.W., Camus, A., Cedilotte, L., Purtill, T. and Ward, B. (1994) Modified Technique for Efficient Detection of Microsporidia. Journal of Clinical Microbiology, 32, 1074-1075.

[39]   Lee, S.H., Joung, M., Yoon, S., Choi, K., Park, W.Y. and Yu, J.R. (2010) Multiplex PCR Detection of Waterborne Intestinal Protozoa: Microsporidia, Cyclospora, and Cryptosporidium. Korean Journal of Parasitology, 48, 297-301.
http://dx.doi.org/10.3347/kjp.2010.48.4.297

[40]   Sanders, J.L. and Kent, M.L. (2011) Development of a Sensitive Assay for the Detection of Pseudoloma neurophilia in Laboratory Populations of the Zebrafish Danio rerio. Diseases of Aquatic Organisms, 96, 145-156.
http://dx.doi.org/10.3354/dao02375

[41]   Taniuchi, M., Verweij, J.J., Sethabutr, O., Bodhidatta, L., Garcia, L., Maro, A., et al. (2011) Multiplex Polymerase Chain Reaction Method to Detect Cyclospora, Cystoisospora, and Microsporidiain Stool Samples. Diagnostic Microbiology and Infectious Disease, 71, 386-390.
http://dx.doi.org/10.1016/j.diagmicrobio.2011.08.012