Cloning and molecular characterization of PRL and PRLR from turbot (Scophthalmus maximus) and their expressions in response to short-term and long-term low salt stress.

Abstract

The pituitary hormone prolactin (PRL) regulates salt and water homeostasis by altering ion retention and water uptake through peripheral osmoregulatory organs. To understand the role of PRL and its receptor (PRLR) in hypoosmoregulation of turbot (Scophthalmus maximus), we characterized the PRL and PRLR gene and analyzed the tissue distribution of the two genes and their gene transcriptional patterns in the main expressed tissues under long-term and short-term low salt stress. The PRL cDNA is 1486bp in length, incorporating an ORF of 636bp with a putative primary structure of 211 residues. And the PRLR cDNA is 2849bp in length, incorporating an ORF of 1944bp with a putative primary structure of 647 residues. The deduced amino acid sequences of these two genes shared highly conserved structures with those from other teleosts. Quantitative real-time PCR results showed that PRL transcripts were strongly expressed in the pituitary and very weakly in brain, but were hardly expressed in other tissues. PRLR transcripts were most abundant in the kidney, to a lesser extent in the gill, intestine, brain, and spleen, and at low levels in the pituitary and other tissues examined. The expression of PRL in the pituitary increased after short-term or long-term low salt stress, and the highest expression level appeared 12h after stress (P < 0.05). And there is no significant difference between both low salt group (5ppt and 10ppt) at each sampling point. The variation of PRLR expression in gill under short-term low salt stress is similar to that of PRL gene in pituitary, with highest value in 12h (P < 0.05). However, the expression under long-term low salt stress was significantly higher than control group even than 12h group under 5ppt (P < 0.05). The expression of PRLR in the kidney increased first and then decreased after low salt stress, and the highest value also appeared in 12h after stress and there was no significant difference between the salinity groups. After long-term low salt stress, the expression level also increased significantly (P < 0.05), but it was flat with 24h, which was lower than 12h. The variation of PRLR expression in the intestine was basically consistent with that in the kidney. The difference was that the expression level of 24h after stress in the 5ppt group was significantly higher than that of the 10ppt group (P < 0.05). After a comprehensive analysis of the expression levels of the two genes, it can be found that the expression level increased and peaked at 12h after short-term low salt stress, indicating that this time point is the key point for the regulation of turbot in response to low salt stress. This also provides very important information for studying the osmotic regulation of turbot. In addition, our results also showed that the expression of PRLR was stable in the kidney and intestine after long-term low salt stress, while the expression in the gill was much higher than short-term stress. It suggested that PRL and its receptors mainly exert osmotic regulation function in the gill under long-term low salt stress. At the same time, such a result also brings a hint for the low salt selection of turbot, focusing on the regulation of ion transport in the gill.