Molecular Cloning And Tissue Expression Analysis Of Leptin Gene In Freshwater Cultured Fishes

In order to study the structural and functional relationship of vertebrate Leptin gene,we cloned partial liver Leptin cDNA sequences of Chinese perch,silver carp,bighead carp,grass carp and mud carp as well as the full-length cDNA sequences of Chinese perch and silver carp Leptin gene by RACE.After isolating Chinese perch and silver carp genomic DNA and constructing the library,we cloned the introns sequences of Chinese perch and silver carp Leptin gene by PCR.The Chinese perch Leptin gene consists of 2 exons and 1 intron and was 1398 bp in length,which contained a complete open reading frame of 486 bp encoding a predicted 17.9 kDa protein with a 20 aa signal peptide and a 141 aa mature peptide.The silver carp Leptin gene consists of 3 exons and 2 introns,with the coding sequence in exons 2 and 3, and was 2194 bp in length,which contained a complete open reading frame of 519 bp encoding a predicted 19.6 kDa protein with a 20 aa signal peptide and a 152 aa mature peptide.Using genome walker method,the 5'-flanking regions of Chinese perch and silver carp Leptin gene were obtained and several potential regulatory elements were identified in the promoter regions. Despite the significantly low amino acid sequence identity of Leptin gene between different species,in phylogenetic analyses that include other members of the classâ… helical cytokine family,all vertebrate Leptins cluster together.Further more,the branching pattern with the Leptin cluster is in accordance with the established patterns of vertebrate evolution because the teleostean Leptins branch off before the separation of the amphibian and mammalian Leptin cluster.At the same time,the CNTF and GH sequences cluster together.respectively,and form a root tree together with Leptin cluster indicating that they are all evolved from an ancestral gene.Using Real-Time PCR and Northern blotting,we analysed the Leptin gene tissue expression patterns of Chinese perch and silver carp.The mRNA expression of Chinese perch Leptin gene was detected in all the examined tissues:highest in liver,followed by brain,and at low levels in other tissues including adipose tissue,intestine,spleen,and muscle.At the same time,the mRNA expression of silver carp Leptin gene was detected in liver by Northern blotting analysis.These results are useful for better understanding of the molecular mechanism in relation to fish Leptin gene regulating ingestion,digestion,metabolism-related genes.