AS  Vol.2 No.4 , November 2011
Molecular cloning of a phosphotriesterase-related protein gene of silkworm and its expression analysis in the silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus
ABSTRACT
Bombyx mori cytoplasmic polyhedrosis virus is one of the major viral pathogens for the silkworm. The immune response of silkworm to the virus infection is obscure. A phosphotriesterase-related protein gene of silkworm, Bombyx mori (BmPTERP) was found in our previous microarry analysis of the midgut infected with the virus. In the present study, we cloned and analyzed the full-length cDNA of BmPTERP gene by means of rapid amplification of complementary DNA ends (RACE) and bioinformatic analysis for exploring its functions in interaction between the silkworm and the virus. The nucleotide sequence of the gene is 1349-bp and contains a 131 bp 5’UTR and a 165 bp 3’UTR. The 1053 bp open reading frame encodes a 350 amino acid protein. The deduced protein contains specific hits of phosphotriesterase-related proteins and belongs to the amidohydrolase superfamily. RTPCR analysis revealed that BmPTERP gene was expressed in all the tissues tested, including midgut, hemocyte, gonad, fat body and silk gland. Real-time quantitative polymerase chain reaction analysis indicated that the relative transcript of BmPTERP gene in the infected midgut was 19.32 fold lower than that in normal midgut at 72 hours post inoculation.

Cite this paper
nullWang, X. , Gao, K. , Wu, P. , Qin, G. , Liu, T. and Guo, X. (2011) Molecular cloning of a phosphotriesterase-related protein gene of silkworm and its expression analysis in the silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus. Agricultural Sciences, 2, 406-412. doi: 10.4236/as.2011.24052.
References
[1]   Roodveldt, C. and Tawfik, D.S. (2005a) Shared promiscuous activities and evolutionary features in various members of the amidohydrolase superfamily. Biochemistry, 44, 12728-12736. doi:10.1021/bi051021e

[2]   Chen, S.L., Fang, W.H. and Himo, F. (2007) Theoretical study of the phosphotriesterase reaction mechanism. The Journal of Physical Chemistry, 111, 1253-1255. doi:10.1021/jp068500n

[3]   Porzio, E., Merone, L., Mandrich, L., Rossi, M. and Manco, G. (2007) A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfata. Biochimie, 89, 625- 636. doi:10.1016/j.biochi.2007.01.007

[4]   Merone, L., Mandrich, L. and Rossi, M. (2005) A thermostable phosphotriesterase from the archaeon Sulfolobus solfataricus: Cloning, overexpression and properties. Extremophile, 9, 297-305. doi:10.1007/s00792-005-0445-4

[5]   Griffiths, A.D. and Tawfik, D.S. (2003) Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. The EMBO Journal, 22, 24-35. doi:10.1093/emboj/cdg014

[6]   Roodveldt, C. and Tawfik, D.S. (2005b) Directed evolution of phosphotriesterase from Pseudomonas diminuta for heterologous expression in Escherichia coli results in stabilization of the metal-free state. Protein Engineering, Design & Selection, 18, 51-58. doi:10.1093/protein/gzi005

[7]   Buchbinder, J.L., Stephenson, R.C., Dresser, M.J., Pitera, J.W., Scanlan, T. S. and Fletterick, R.J. (1998) Biochemical characterization and crystallographic structure of an Escherichia coli protein from the phosphotriesterase gene family. Biochemistry, 37, 5096-5106. doi:10.1021/bi971707+

[8]   Scanlan, T.S. and Reid, R.C. (1995) Evolution in action. Chemistry & Biology, 2, 71-75. doi:10.1016/1074-5521(95)90278-3

[9]   Hou, X.Y., Maser, R.L., Magenheimer, B.S. and Calvet, J.P. (1996) A mouse kidney- and liver-expressed cDNA having homology with a prokaryotic parathion hydrolase (phosphotriesterase)-encoding gene: Abnormal expression in injured and polycystic kidneys. Gene, 168, 157- 163. doi:10.1016/0378-1119(95)00746-6

[10]   Ikeda, K., Nagaoka, S., Winkler, S., Kotani, K., Yagi, H., Nakanishi, K., Miyajima, S., Kobayashi, J. and Mori, H. (2001) Molecular characterization of Bombyx mori cytoplasmic polyhedrosis virus genome segment 4. Journal of Virology, 75, 988-995. doi:10.1128/JVI.75.2.988-995.2001

[11]   Qanungo, K.R., Kundu, S.C., Mullins, J.I. and Ghosh, A.K. (2002) Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9. Journal of General Virology, 83, 1483-1491.

[12]   Wu, P., Wang, X., Qin, G.X., Liu, T., Jiang, Y.F., Li, M.W. and Guo, X.J. (2011) Microarray analysis of gene expression profile in the midgut of silkworm infected with cytoplasmic polyhedrosis virus. Molecular Biology Reports, 38, 333-341. doi:10.1007/s11033-010-0112-4

[13]   Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2?ΔΔCT Method. Methods, 25, 402-408. doi:10.1006/meth.2001.1262

[14]   Watanabe, H. (2002) Genetic resistance of the silkworm, Bombyx mori to viral diseases. Current Science, 83, 439-446.

[15]   Sun, Y., Wu, A., Dai, R. and Shen, X. (1982) Synthesis of structural proteins in a cell free system directed by silkworm cytoplasmic polyhedrosis virus mRNA synthesized in vitro. Scientia Sinica, 24, 685-690.

[16]   Tuovinen, K., Kaliste-Korhonen, E., Raushel, F.M. and Hanninen, O. (1996) Protection of organophosphate-in- activated esterases with phosphotriesterase. Fundamental and Applied Toxicology, 31, 210-217. doi:10.1006/faat.1996.0093

[17]   Davies, J.A., Buchman, V.L., Krylova, O. and Ninkina, N.N. (1997) Molecular cloning and expression pattern of rpr-1, a resiniferatoxin-binding, phosphotriesterase-re- lated protein, expressed in rat kidney tubules. FEBS Letters, 410, 378-382. doi:10.1016/S0014-5793(97)00614-5

[18]   Vaughan, A., Rodriguez, M. and Hemingway, J. (1995) The independent gene amplification of electrophoretically indistinguishable B esterases from the insecticide-resistant mosquito Culex quinquefasciatus. Biochem. Journal, 305, 651-658. PMid:7530448

[19]   Yildirim, M., Colak, A., Col, M. and Canakci, S. (2009) A new recombinant phosphotriesterase homology protein from Geobacillus caldoxylosilyticus TK4: An extremely thermo- and pH-stable esterase. Process Biochemistry, 44, 1366-1373. doi:10.1016/j.procbio.2009.07.014

 
 
Top