Identification of genes involved in osmoregulation of the black tiger shrimp Penaeus monodon

Abstract

Differential Display Polymerase Chain Reaction technique (DD-PCR) was used for the identification of genes involved in osmoregulation in antennal gland, epipodite and gill tissues of the salinity stressed Penaeus monodon. Shrimp reared at 25 ppt salinity as a control group were transferred to 3 and 40 ppt salinities for 6, 24 h and 2 weeks (stressed groups). Parallel comparison of stress and control DD-PCR profiles derived from 26 combinations of arbitrary and oligo-dT primer were performed to identify the differentially expressed genes. A total of 97 differentially expressed bands could be identified from 17 primer combinations. These bands were successfully reamplified, cloned and sequenced. A total of 129 unique sequences were found. Homology search showed that 37 different sequences significantly matched the GenBank database. Among the matched sequences, 22, 6 and 9 gene homologues were known genes, ribosomal proteins and hypothetical proteins, respectively. Twenty-three of 129 unique sequences including carbonic anhydrase, corin isoform 2, sarcolemmal associated protein 2, NIMA-family kinase Nek 7, karyopherin alpha 4, vacuolar protein sorting 18, CHK1 CHECKPOINT, Rps 16 protein, integrin alpha, 5 hypothetical proteins and 9 unknown gene products, were selected for confirmation of their differential expression patterns using semi-quantitative RT-PCR. The RT-PCR results confirmed that 21 out of 23 cDNA fragments were regulated by salinity stress. Eight cDNA fragments of unknown genes regulated by salinity from RT-PCR analysis were predicted for domain homologies using SMART program and Kyte-Doolittle hydropathy plots. The results showed that the encoded peptide of S5, S114 and S126 cDNA fragments contained transmembrane regions. In summary, the salinity-responsive genes that were identified from this study are potentially involved in osmoregulation of P. monodon.