Cloning And Bioinformatics Analysis Prokaryotic Expression Of Fat Metabolism Related Gene In Lanzhou Fat-tailed Sheep

In recent years, research on leg, lanzhou sheep mainly concentrated in the population ecology, genetic diversity, breeding, etc., these research results can reflect the sheep leg, lanzhou richness of genetic diversity and genetic uniqueness determine varieties, mianyang, and other varieties of genetic relationship, origin and genetic differentiation, eventually to distinguish the sheep breed or type. Although the current study on germplasm resources protection and development, the tail fat cells developmental changes and blood protein polymorphism is more perfect, but fat deposits at the end of the fat metabolism candidate genes and their correlation and expression rule of the lack of a systematic study, which leads to the sheep leg, in lanzhou tail fat deposition factors lack of the basis of the molecular level. MAPK, PPARG, an HSL, DGAT is one of the important candidate genes related to fat metabolism. Therefore, sheep leg, lanzhou’PPARG, MAPK13gene cloning, analysis of the sequence and its coding protein, biological characteristics, and prokaryotic expression, to research the sheep PPARG, MAPK13gene function and application is of great significance.Sheep leg, Lanzhou, Lanzhou fat-tailed sheep) during the qing dynasty stagnation period (1862～1875) introduced by shaanxi big li area with hybrid breeding sheep and Lanzhou local Mongolian sheep and sheep breeds, because its tail excess fat deposition, cause a large tail, the fat to enrich the name. Sheep MAPK13with less PPARG gene cloning and related function research reports.Peroxidase body growth activated receptor (peroxisome proliferator-activated receptor, PPAR) is to regulate target gene expression within the nuclear receptor transcription factor family members. PPAR molecular weight is controlled in54kda, is mainly expressed in the immune system and adipose tissue, is insulin sensitization agent TZDS drug target molecules, and the body’s immune, closely related to insulin resistance, fat cell differentiation. PPAR according to the structure is divided into three types of alpha, beta, gamma, PPARG gene (gamma) is a major candidate genes of adipocyte differentiation. Peroxidase body growth activated receptor (peroxisome proliferator-activated receptor, PPAR) is to regulate target gene expression within the nuclear receptor transcription factor family members. PPAR molecular weight is controlled in54kda, is mainly expressed in the immune system and adipose tissue, is insulin sensitization agent TZDS drug target molecules, and the body’s immune, closely related to insulin resistance, fat cell differentiation. PPAR according to the structure is divided into three types of alpha, beta, gamma, PPARG gene (gamma) is a major candidate genes of adipocyte differentiation.Promote the mitogen activated protein kinase (mitogen-activated protein kinases, MAPK) chain is one of the important way of eukaryotes signal transmission network. MAPK pathways including ERK, p38MARK and JNK pathway, a major role in fat cells in the organization, its phosphorylation process step by step involved a lipid regulation of differentiation is closely related to insulin resistance, fat cell differentiation. P38lightning delta MAPK also called MAPK13, is the main candidate genes of adipocyte differentiation.According to this study sheep PPARG, CDS MAPK13gene sequence specific primer design, the use of RACE and rt-pcr technology cloned sheep leg, lanzhou PPARG, MAPK13gene sequence, and combined with bioinformatics methods to analyze its biological characteristics.Sheep leg, the results showed that the cloned for lanzhou PPARG gene cDNA sequence is1774bp (GenBank serial number:KF727439), its CDS region fragment length1428bp, encoding475amino acids, coding region two flanks with5’UTR131bp respectively (1-131(nt) and3’UTR214bp (1560-1774nt). Leg, prediction of lanzhou sheep PPARG protein molecular weight54.40kDa, theory of isoelectric point of4.94. Predict PPARG gene encoding protein hydrophobicity maximum value is3.600, the minimum value is2.744, is a transmembrane protein hydrophobicity. Subcellular localization are mainly in the cytoplasm (65.2%) and the nucleus (21.7%), small effects on cell scaffold (4.3%) and peroxidase (4.3%), no signal peptide, do not belong to the secretory proteins. There are26phosphorylation sites, predict its amino acid sequence without the glycosylation sites, contains1ZnF_C4domain structure,1HOLI domain and1the LCR structure domain structure, secondary structure is given priority to with random curl. Homology analysis showed that sheep leg, lanzhou PPARG gene nucleotide sequences and goats, bison, buffalo, sheep, killer whales, wild boar and nucleotide sequence homology between human respectively99%,98%,97%,99%,97%,92%and90%, and the sheep on the leg, lanzhou PPARG amino acid sequence with the goats, bison, buffalo, sheep, killer whales, wild boar and nucleotide sequence homology between human were100%,99.8%,100%,99.8%,98.7%,98.5%and97.5%. Phylogenetic tree showed that leg, lanzhou and sheep and goats, sheep, and the evolution of the buffalo level is more close to, and fish, human and rodents. Its genetic486th base conversion (C please-T),828th base conversion (C please-> A), but the encoded amino acid remains the same.Cloned sheep leg, lanzhou MAPK13gene cDNA sequence length1397bp, the fragment1102bp long CDS area, encoding367amino acids. Leg, prediction of lanzhou sheep MAPK13protein molecular weight42.29kDa, theory of isoelectric point is8.82, is a transmembrane protein hydrophobicity, subcellular localization, mainly in the cytoplasm no signal peptide, do not belong to the secretory proteins. Predict its amino acid sequence has19phosphorylation sites,3glycosylation sites, three phosphorylation function domain structure, four other domain structure,1the LCR segment, secondary structure is given priority to with alpha helix. Homologous analysis showed that sheep leg, lanzhou MAPK13gene sequences similar to most mammals, the852th base conversion (C-A), the loci encoding265amino acid base (T-S), at the same time, the951th base conversion please (G-T), but the encoded amino acid remains the same. Build the genetic evolution of tree analysis results show that the leg, lanzhou sheep and cattle’s closest relative.Analysis shows that lanzhou PPARG leg, sheep and other species, MAPK13genetic similarity is higher in the structure, the gene is highly conservative. Speculated that most PPARG protein synthesis in the free ribosomes, its function is associated with peroxidase body, the prediction results conform to the peroxidase body growth activated receptor status. There are26can become PPARG amino acid sequence of the protein kinase phosphorylation sites, some sites are phosphorylation, dysfunction can lead to a series of obesity related genes and expression changes, such as the hormone insulin sensitization, decrease the expression of adiponectin (adiponectin). Its sequence contains ZnF_C4zinc finger domain structure has the function of combined with lipid HOLI namely LBD ligand structure domain in hormone signal transduction play an important role in the process, including phosphorylation site Ser112PPARG zinc finger domain structure, after being MAPK phosphorylation, can inhibit the PPARG ligands with the combination of reducing the activity of the PPARG protein, both structural domain is associated with a concomitant differentiation process. Sheep leg, lanzhou MAPK13sequence contains S_TKc structure domain gamma phosphoryl ATP can be transferred to the protein serine/threonine residues, results in a series of functional disorder and obesity related gene expression changes. Comprehensive above, for further research, PPARG MAPK13genes and provides reference into the correlation of fat differentiation.