Cloning And Expression Analysis Of The Prolactin And Prolactin Receptor Of Cyprinus Acutidorsalis Wang

Prolactin(PRL) is a single peptide hormone secreted from animal pituitary and considered to be a main hormone in regulating the balance of osmotic pressure and ion of teleosts. Prolactin receptor(PRLR) is a single transmembrane protein, belong to I type of cytokine receptor superfamily. PRL has a variety of functions in teleosts and plays its biological effects through prolactin receptor. Researches about PRL and PRLR gene focus more on the euryhaline and migrating teleosts. We cloned the gene sequences of PRL and PRLR, detected their expression levels in different tissues of Cyprinus acutidorsalis Wang under different salinity waters.To investigate the gene expression of PRL and PRLR and molecular mechanisms of the osomregulation in C. acutidorsalis Wang, the full-length cDNA encoding Cyprinus acutidorsalis Wang PRL and PRLR were cloned from C. acutidorsalis Wang using homology cloning and RACE PCR approach. The PRL was877bp in length, including633bp ORF encoding210amino acids which contined one transmembrane-spanning region, one signal peptide structure and one growth hormone-prolactin and related hormone signal site was detected in this gene. The PRLR was2309bp in length, including1815bp encoding region and and coding604amino acids and there have two transmembrane-spanning regions, at the same time,one signal peptide structure and one erythropoietin receptor single family signal site was detected in this gene.Alignment of deduced amino acid sequences of the PRL and PRLR and those of other vertebrates demonstrated that they shared many protein features common in fish. Phylogenetic analysis using Mega5software showed that the putative PRL amino acid sequence and other teleosts have the high similarity for52.86%-96.19%. The PRL amino acid sequences of C. acutidorsalis Wang and that of C. carpio and C. carpio var. Jian were closest to each other with96.19%similarity. The similarity was95.24%、94.74%、89.52%、74.16%、67.62%、67.62%、65.71%、52.86%between C. acutidorsalis Wang with Ctenopharyngodon idella, Schizothorax prenanti, Danio rerio, Silurus meridionalis, Oncorhynchus keta, Oncorhynchus mykiss, Plecoglossus altivelis, Takifugu rubripes, respectively. The similarity between PRL of C. acutidorsalis Wang and some mammalians was more lower between26.44%-30.43%. PRLR amino acid sequence and other teleosts have the similarity for43.05%-96.36%. The PRLR amino acid sequence of C. acutidorsalis Wang and that of C. carpio were close to each other in similarity of96.36%. For other animals, the similarity was92.00%、88.08%、81.41%、43.05%with Carassius auratus, C. carpio var.Jian, Danio rerio, Oreochromis niloticus, respectively. The similarity between PRLR of C. acutidorsalis Wang and three kinds of amphibians and some mammalians was more lower.Real-time quantitative PCR results indicated that the PRL and PRLR genes were expressed in all the9tissues-pituitary, brain, gill, intestine, liver, kidney, spleen, skin, and gonad. The PRL and PRLR transcript was detected in in all the9tissues. At the same time, when the concentration of salinity of living environment changed, the expression of PRL and PRLR would also change. The two gene expressions still existed tissue specificity. The PRL transcript was detected at a high level in pituitary and the PRLR transcript was detected at a high level in gill, kidney and intestine. The expression quantity of other organizations were obviously lower than these organizations.Then, the expression quantity of PRL in pituitary and PRLR in gill, kidney, intestine was becoming lower with water salinity rising, and this shows the high consistency of the two gene expression variation. This suggests that the osmoregulation organization is the main target tissue and PRL through the PRLR signal transduction in the role of osmoregulation organization to exercise its osmoregulation function in C. acutidorsalis Wang.This results showed that the PRL and PRLR of C. acutidorsalis Wang have higher conservative in evolution and the amino acid homology related to its classification status and genetic relationship. The expression of PRL and PRLR have specificity in different tissue, and different salinity have a certain degree of influence to the gene expression level. This had laid certain foundation to clarify the osmoregulation mechanism of C. acutidorsalis Wang from the molecular level. In addition, the results of experiments showed that the C. acutidorsalis Wang have strong adaptive capacity in different salinity water, and this had provided the theoretical support for aquaculture of C. acutidorsalis Wang in the seawater, brackish water and fresh water.