Nucleotide Sequence Analysis of Phylogenetic and Evolutionary Status of Leptin Gene in Camel

Leptin is a pleiotropic protein best known for regulation of appetite and fat storage in mammals. While many leptin orthologs have been identified among vertebrates, an authentic leptin in birds hasremained elusive and controversial. Leptin, the ob gene product, is a 167 amino acid polypeptide known to play a major role in regulating the fat stores of the body and is found in all eukaryotes, including mammals and in invertebrates. In mammals, leptin functions as an adiposity signal: circulating leptin fluctuates in proportion to fat mass, and it acts on the hypothalamus to suppress food intake. However, little is known about the molecular evolution of the leptin sequence gene. Therefore, the aim of this study, we conducted an analysis of the evolutionary and phylogenetic of the mammalian’s Leptin nucleotide sequences in native camel of Sistan and Baluchistan province and compare with other species in NCBI gene bank. In this study, blood samples were collected from 50 camels, randomly from two stock of Sistan and Baluchistan province. DNA is extracted from whole blood with phenol-chloroform method. PCR amplification of 2000 bp from of partial ob gene including intron2 and xon3 of Leptin gene was performed using one pairs of special primers. PCR product was digested with endonuclease Asuhpi enzyme and purified on the agarose gel. Then, the sequencing of the digestion products was performed by the Sanger method. Data sequence for other species was achieved and aligned by searching its genome database (NCBI). The nucleotide substitution rate of the sequences (transition and transversion substitution rate) and molecular evolution (including polymorphism site, conservation site and gene conversion) of the Leptin were calculated by maximum likelihood and neighbor-joining (NJ) method respectively and phylogenetic tree was based on nucleotide sequences constructed. Evolutionary and phylogenetic tree analysis was performed by using MEGA6 and Dnasp v5 software's. Finally, a fundamental measure of the elative importance of selection and genetic drift in causing amino-acid substitutions is the dN/dS ratio. In this study was calculated with online package HIV_SNAP v2.1.1. Results of alignment of DNA sequencing of two populations of camel in Sistan and Baluchistan showed 99% similarity, but diversity showed distinct from other mammalian species. The results showed that the transitional substitution was more than transversional substitution and ratio these was 1.58. Totally, there were 591 mutations including insert, deletion and polymorphism sites at the DNA level of leptin gene (ob gene) in different species but sequence alignment of the Leptin gene fragment revealed only 309 polymorphic sites and conservation area of ob gene was very small. Evolutionary pressures on proteins are often quantified by the ratio of substitution rates at non-synonymous and synonymous sites. The dN/dS ratio was originally developed for application to distantly diverged sequences, the differences among which represent substitutions that have fixed along independent lineages. Nevertheless, the dN/dS measure is often applied to sequences sampled from a single population, the differences among which represent segregating polymorphisms. The dN/dS ratio of the Leptin sequences in this study (0.76) indicated that negative selection was accrued during evolution. Phylogenetic tree for the leptin gene in different organisms show that seven categories in the mammals such as camel, cows and buffalo, sheep and goat, pork's, bats, cats and marine. Phylogenetic analysis of leptin gene using Neighbor-Joining method showed that Baluchi camel population in this study has the highest similarity with Lama among the Camelidae family. Phylogenetic analysis of DNA sequence has played an important role in the study on evolution of life. However, recent researches suggest in some cases phylogenetic trees based on the analyses of DNA sequences may be misleading and that based on trees protein-based trees from amino acid sequences may be more reliable. Similarities of between the two populations of Baluchi camel in this study showed that common ancestry and genetic similarity. Mutations and natural selection resulted in the development of new varieties, new proteins and also stabilizes their performance during the evolution and advance progress toward their performance has been purified. According to this study, protected areas make up a small part of leptin gene sequence insures that this reflects the polymorphism of this gene as well as being susceptible to variations and mutations, respectively. The results of dN/dS suggested that evolution in camel is different of other species.