建鲤催乳素受体基因全长cDNA的克隆、序列分析与组织表达

Full length cDNA cloning and tissue expression of PRLR in Cyprinus carpio var. jian

为了解催乳素受体(Prolactin Receptor,PRLR)的基因序列及其在建鲤(Cyprinus carpio var.jian)渗透调节组织中的表达情况,采用同源克隆和末端快速扩增(rapid amplication of cDNA ends,RACE)的方法分离克隆了建鲤PRLR基因全长cDNA,得到2 440 bp的全长cDNA,包括1 821 bp的开放阅读框(ORF),213 bp的5′末端非编码区(UTR)以及406 bp的3′末端非编码区(UTR)。对该基因序列和推测的氨基酸序列进行同源性比对和系统分析显示:建鲤与其他硬骨鱼类该基因的氨基酸序列相似度在37.46%～87.25%,与鲤(Cyprinus carpio)的氨基酸相似度最高(87.25%),鲫(Carassius auratus)次之(86.86%),和金头鲷(Sparus aurata)的相似度较低(41.87%),和人(Homo sapiens)最低(37.46%),表明不同物种间的PRLR的氨基酸序列具有较高的保守性。用实时定量PCR(RT-PCR)检测该基因在建鲤脑、肠、鳃、性腺、肝、脾、肾、皮肤中催乳素受体的相对表达量,其中鳃、肾、肠3个主要渗透调节组织的表达量较高,这表明建鲤的渗透调节组织是PRLR的主要表达场所,从定量角度提示了催乳素(prolactin,PRL)通过PRLR的表达,作用于渗透调节组织从而行使其渗透压调节功能。本研究旨在为进一步探索建鲤的渗透调控机制提供基础依据。

Prolaetin is a single peptide hormone secreted from the animal pituitary. It has a variety of functions in teleosts and is thought to be one of the primary hormones regulating the balance of the osmotic pressure and ions. PRL exerts its biological effects by binding with the prolaetin receptor （PRLR）. Thus, our understanding of the physiological functions and mechanism of action of PRL relies on a thorough understanding of the PRLR. Prolactin receptor is a single transmembrane protein, belonging to type I of the eytokine receptor superfamily. PRLR mRNA has been detected in several organs associated with osmotic regulation. Changes in the concentration of ions or salinity in the environment are associated with changes in the expression of PRLR, which is thought to represent the molecular mechanism of PRL regulation. We investigated the gene sequences of PRLR and the tissue specific expression pattern in C carpio var. jian. The full-length eDNA encoding of gene PRLR was cloned from C carpio vat. jian using homology cloning and RACE PCR. The PRLR was 2 440 bp in length, including a 213 bp 5＇terminal UTR, 1 821 bp encoding region, and 406 bp 3＇terminal UTR. Alignment of the deduced amino acid sequences of the PRLR and those of other vertebrates demonstrated that they shared many protein features common in fish. Phylogenetic analysis revealed that the putative PRLR amino acid sequencehas high similarity with the sequence in other teleosts （range： 37.46%-87.25%）. For example, the PRLR amino acid sequence of C. carpio var.jian and that of C. carpio shared 87.25% similarity. For other species, the similarity was 86.86%, 82.72%, 57.43%, 49.67%, 49.16%, 42.37%, and 41.87%, with. auratus, D.rerio, O.mykiss, O.niloticus, P.olivaceus, T. rubripes, and S.aurata, respectively. Real-time quantitative PCR suggested that the PRLR genes expressed in the brain, liver, kidney, intestine, gonad, skin, spleen, and gill. The PRLR transcript was detected at a high level in gill, kidney, and intestine suggesting that the primary function of PRLR in C. carpio var. jian is in osmotic regulation. The PRLR transcript was detected in the gonads, liver, and spleen, suggesting that prolactin has a range of physiological functions in fish. Our results have provided valuable insight into the mechanisms of osmotic regulation in C. carpio var. jian.