Gene Structure, Evolution, And Expression Analysis Of Leptin-B Gene In Siniperca Chuatsi(Basilewsky)

We screened Siniperca chuatsi (Basilewsky) transcriptome assemblies for Leptin-b gene and found partial cDNA sequence of Leptin-b. Blast was conducted in the NCBI website to identify its accuracy. Primers were designed to confirm the partial cDNA sequence. Then to obtain the complete cDNA sequence of Leptin-b gene, the PCR primers for the5’-and3’-RACE were designed based on the partial cDNA sequence. To understand the regulation of Leptin-b gene, the5’-flanking region of Leptin-b gene was obtained using genome walker method. And the intron was identified by PCR method.The cDNA of S. chuatsi Leptin-b was698bp, containing a471bp open reading frame (ORF) with a coding potential for a157-amino acid protein, a130bp5’-untranslated region (UTR) and a94bp3’-UTR. The5’-flanking region of Leptin-b gene was418bp. Analysis of the promoter regions of Leptin-b gene revealed a typical TATA box, a cAMP response element binding (CREB) site, an activator protein2(AP2) site, an activator protein1(AP1) site, GAT A binding factors, a CCAAT/enhancer-binding protein (C/EBP) site, located at31bp,155bp,209bp,242bp,286bp,305bp and325bp upstream of the transcriptional start site, respectivelyWe confirmed that S. chuatsi Leptin-b and Leptin-a gene had only one intron after comprehensive detection of S. chuatsi Leptins genomic sequences. Comparing with the gene structure of other species, the only one intron of S. chuatsi Leptins appeared at similar positions as intron2in other Leptins suggest the loss of the first intron in S. chuatsi. The3D modeling structure of the S. chuatsi Leptins protein showed strong conservation of tertiary structure that is characteristic of vertebrate Leptin.To detect the evolution of Leptins in S. chuatsi, multiple sequence alignments were carried out based on the amino acid sequences of Leptins and a phylogenetic tree was constructed. Although there was a polytomy among fish Leptins, mammalian Leptins clustered with the zebrafish (Danio rerio) and grass carp (Ctenopharyngodon idellus) Leptin-b. Thus mammalian Leptin was more likely the ortholog of cyprinidae Leptin-b, more identification of Leptin-b in other teleosts was needed for confirming it.Tissue distribution analysis of Leptins (Leptin-a and Leptin-b) and Leptin receptor (Lepr) were conducted in S. chuatsi and grass carp, two teleost species of totally different feeding habit. In S. chuatsi, Leptin-a was prominently expressed in the liver. While the expression of Leptin-a in grass carp was abundant in liver and every part of the brain. The expression level of S. chuatsi Leptin-b was luxuriant in every part of the brain. However, trace amount of grass carp Leptin-b was detected in in every part of the brain except cerebellum, the most amount of grass carp Leptin-b was present in spleen and kindey. The function of Leptin-b may be more concentrated in S. chuatsi, while Leptin-a may be more concentrated in grass carp. The S. chuatsi Lepr was detected in all tested organs, and a similar Lepr expression pattern was detected in grass carp. Different tissue distribution pattern of Leptins in S. chuatsi and grass carp suggests different physiological roles of these two genes.