Cloning and characterization of crustinPm5 from the black tiger shrimp penaeus monodon

Abstract

From Penaeus monodon expressed sequence tag database (http://pmonodon. biotec.or.th), six isoforms of crustin (crustinPm1-6 and crustin-likePm) was identified. CrustinPm5 cDNA identified from a gill-epipodite cDNA library contains 510 bp of open reading frame encoding for a 169 amino acid protein. Sequence comparison showed that the deduced amino acid sequence of crustinPm5 shares identity with crustinPm1 and crustin-likePm 38% and 37%, respectively. The genomic sequence of crustinPm5 was obtained by using PCR and genome walking techniques. The crustinPm5 gene contained four exons interrupted by three introns whilst the 5´upstream sequence contains a putative promoter with two potential binding sites for NF-κB, one complete heat-shock regulatory element (HSE) and five putative GATA factor binding sites. The RT-PCR analysis revealed that the transcripts of crustinPm5 were primarily observed in the epipodite and eyestalk and not in hemocytes. Expression analysis revealed that the transcript levels of crustinPm5, crustinPm1 and crustin-likePm in epipodite were up-regulated upon heat treatment and hyperosmotic salinity stress. To further characterize the activity of crustinPm5, the recombinant protein was produced in E. coli system. The recombinant crustinPm5 exhibited antimicrobial activity against some Gram-positive bacteria in vitro, but did not inhibit the growth of any Gram-negative bacteria tested. These results, together with the transcript expression pattern, indicate a diverse function of the proteins in the crustin family particularly crustinPm5 that might function as a stress mediator in addition to its antibacterial action.