Cloning And Expression Analysis Of Three Somatostatins And Two Somatostatin Receptors In Siniperca Chuatsi

Somatostatins (SS) play important physiological functions in vertebrate’s growthand development, which belong to a multiple gene family. Combining with thesomatostatin receptor (SSTR), they could regulate individual development and growth,though the growth hormone-insulin-like growth factor-I (GH-IGF-Ⅰ) axis at multiplelevels. In this study, the complete cDNA sequences of three distinct preprosomatostatins(PSSⅠ, PSSⅡand PSSⅢ) were isolated from Siniperca chuatsi brain and cloned bymeans of rapid amplification of cDNA ends (RACE). The full length of PSSⅠ cDNAwas647bp, which contained369bp open reading frame (ORF), and encoded122amino acids with a signal peptides of26amino acids, it contained the conservativeSS-14sequence at the C-terminal extremity, which is identical to that of othervertebrates. The full length of PSSⅡ cDNA was655bp, which contained387bp openreading frame (ORF), encoded128amino acids with a signal peptides of25amino acids,it contained [Tyr7, Gly10]SS-14at the C-terminal extremity. The full length of PSSⅢcDNA was823bp, which contained333bp open reading frame (ORF), encoded110amino acids with a signal peptides of21amino acids, it contained [Pro2]SS-14at itsC-terminal extremity. PSS mRNA expression in different adult tissues were analyzed byRT-PCR technique, all PSS mRNA were found in brain, while PSSⅠ mRNA wasn’tfound in others peripheral tissues, high levels of PSSⅡ mRNA were also found inesophagus and stomach, PSSⅢ in kidney and gonad, which suggested that all PSSsparticipate in neurological regulation in brain, whereas PSSⅡ can also regulatedigestion activity, PSSⅢ also regulate reproduction activity. Cellular localization ofPSS mRNAs in brain were determined by in situ hybridization, PSSⅠ mRNA wasexpressed in hypothalamus and medulla oblongata, PSSⅡ mRNA was expressed inhypothalamus, PSSⅢ mRNA was expressed in hypothalamus, optic tectun, medullaoblongata and spinal cord. Since all PSS mRNA were detected in the hypothalamus, they could regulate fish development and growth as hormones. Distinct expression inother brain parts suggested somatostatins may also be involved in various neurologicaland physiological regulations as neurotransmitters or hormones, and exhibited differentbiological effects.The complete cDNA sequences of two distinct somatostatin receptors (SSTR2andSSTR3) were isolated from S. chuatsi brain and cloned by means of rapid amplificationof cDNA ends (RACE). The full length of SSTR2cDNA was1820bp, which contained1146bp open reading frame and encoded382amino acids. The full length of SSTR3cDNA was1874bp, which contained1458bp open reading frame and encoded486amino acids. Five domains were detected in SSTRs: the N-terminal domain, sevenputative transmembrane domains (TMD), three extracellular loops (ECLs), fourintracellular loops (ICLs) and the C-terminal domain. NJ phylogenetic tree showed thatSSTR2and SSTR3formed a relatively independent branch respectively; the amino acidsequence similarity between SSTR2and SSTR3was51.2%, which indicated that theyare encoded by two different genes. SSTR2and SSTR3mRNA expression in differentadult tissues were analyzed by real-time quantitative RT-PCR technique. The highestexpression of SSTR2mRNA was found in liver, medium expression in brain, spleen,gonads and pyloric caeca, and low expression in heart, esophagus, stomach andintestinal. The highest expression level of SSTR3mRNA was revealed in stomach,medium expression in brain, esophagus, kidney and gonad, low expression in gill andpyloric caeca. SSTR2and SSTR3are expressed in the brain, suggested that the twoSSTRs participation in the neural and hormonal regulation of SS in the brain. The highexpression level of SSTR2mRNA in the liver suggested that may be involved inmetabolic regulation in the liver. The high expression level of SSTR2mRNA in thestomach, suggested that may be involved in the regulation of SS in the stomach.