Construction Of Alopex Lagopus Spleen CDNA Library And Cloning For Melatonin Receptor 1A

Melatonin is a small hydrophobic molecule, which is secreted rhythmically by vertebrate pineal glands as a kind of hormone. In the past, it was thought that melatonin could only regulate physiological periodic rhythms. In recent years, however, with the intensive study of Mel, it becomes clear that it also adjusts reproduction, immunity, anti-oxidation, anti-tumor and so on. The melatonin receptor 1A (MTNR1A) is one of the necessary receptors for melatonin exerting its biological function.A cDNA library was constructed with the Alopex lagopus spleen and a positive clone of MTNR1A was isolated from the cDNA library. The main results were as following:1.Construction of the cDNA libraryThe total RNA was extracted from Alopex lagopus spleen using Trizol method, and the quality of the total RNA isolated by using the optimized method could satisfy the requirement of cDNA library construction. The cDNA library was constracted with SMARTTM cDNA library construction kit. The first strand cDNA was synthesized from the total RNA using the one point mutant of Moloney marine leukemia virus (MMLV) reverse transcriptase, and the dscDNA was Amplified by LD PCR. The dscDNA were then digested by SfiI and fractionated by CHROMA SPIN-400 columns. The cDNA fragments longer than 400bp were collected and ligated toλTripIEx2 vector. Thenλphage packaging reaction and library amplification were performed. Quality of the original and the amplified cDNA libraries was strictly checked by using conventional titer determination. Ten plaques were randomly picked up and tested using PCR with universal primers derived from the sequence flanking the vector. The results showed that titer of the primary library was 3.0×106pfu/ml, and the recombination rate was 90%. The sizes of the PCR amplified bands were 0.6~1.8kb, with an average of 1.2kb. Hence the high quality of cDNA Library of Alopex lagopus spleen were obtained. The construction of cDNA Library has laid the foundation for further screening and cloning new and specific genes of Alopex lagopus spleen.2. Cloning of MTNR1ABased on the known Alopex lagopus MTNR1A partial gene sequence and full length of dog MTNR1A gene sequence, MTNR1A specific primers were designed using Primer5.0 primer design software, combined with PCR primer design optimum conditions. Subsequently, a positive clone was isolated through PCR based 96-hole library screening method. the clone had 100% homology with the known Alopex lagopus MTNR1A partial gene, and 98%,88%,85% homology with MTNR1A of dog,house and Rhesus monkey. Acquirement of the Alopex lagopus MTNR1A cDNA will provide the opportunity for the further study of the mechanism underlying the interactions of ligand-receptor of Mel, and its biological function.