Cloning And Tissues Distribution Of Pig Lipin And FIT Gene

Fat-induced transcript (FIT) and Lipin are related to fat metabolism gene.FIT is assembled into lipid droplets of fat play a key role of the gene, is a endoplasmic reticulum protein involved in oxidation of lipid metabolism. Lipin is a key regulatory enzymes of fat,it has been identified as a protein that regulates fat metabolism in mammalian cells.In order to study the biological function of Lipin and FIT proteins, EST fragment of this test is based on the principle of the use of cloned sequences primers were designed for a variety of organizations pig of total RNA extracted for RT-PCR, through RT-PCR technology has been cloned FIT (FIT1 and FIT2) and Lipin (lipin1, 2 and 3) cDNA sequences, using bioinformatics tools are available to predict the nature of their biology and distribution of their organizations.The study was divided into two series.Ⅰ.Cloning and tissues distribution of pig Lipin proteins.The cloned sequences containing the open reading frame of Lipin1 gene consist of 2692bp, and ORF is 2-2686bp and encodes 894 amino acids, of which 189 acidic amino acid, basic amino acid for 122, molecular weight 98.91kDa, isoelectric point of 6.40. Forecast containing LNS2 domain, the location of the 678-834, NLS nuclear localization prediction with "KKRRKRRRK" structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the lipin1 nucleotide sequences shared 82.8%, 85.0%, 82.8% homology with that of mouse, human and rat, the deduced amino acid sequences shared 88.8%,89.9% and 88.7% homology with that of mouse, human and rat.The cloned sequences containing the open reading frame of Lipin2 gene consist of 2693bp, and ORF is 11-2686bp and encodes 891 amino acids, of which 190 acidic amino acid, basic amino acid for 126, molecular weight 98.89kDa, isoelectric point of 5.32. Forecast containing LNS2 domain, the location of the 680-836, NLS nuclear localization prediction with " KKKKRRRKK " structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the Lipin2 nucleotide sequences shared 83.0%,86.7%,82.9%, homology with that of mouse, human and rat, the deduced amino acid sequences shared 87.4%,90.3%和87.4%homology with that of mouse, human and rat. The cloned sequences containing the open reading frame of Lipin3 transcript variant1 gene consist of 2872bp, and ORF is 149-2728bp and encodes 859 amino acids, of which 165 acidic amino acid, basic amino acid for 105, molecular weight 93.59kDa, isoelectric point of 5.42. Forecast containing LNS2 domain, the location of the 648-804, NLS nuclear localization prediction with " KKKRRRRKPRRKE " structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the Lipin3 transcript variant1 nucleotide sequences shared 75.6%,83.4%,78.6%, homology with that of mouse, human and rat, the deduced amino acid sequences shared 75.9%,81.6%和77.4% homology with that of mouse, human and rat. The cloned sequences containing the open reading frame of Lipin3 transcript variant2 gene consist of 2848bp, and ORF is 149-2704bp and encodes 851 amino acids, of which 163 acidic amino acid, basic amino acid for 104, molecular weight 92.74kDa, isoelectric point of 5.42. Forecast containing LNS2 domain, the location of the 640-796, NLS nuclear localization prediction with "KKKRRRRKPRRKE " structure, with HAD-like domain "DIDGT" catalytic motif.Identity analysis showed that the Lipin3 transcript variant1 nucleotide sequences shared75.6%,83.2%,78.5%, homology with that of mouse, human and rat, the deduced amino acid sequences shared 76%,80.9%和77.4% homology with that of mouse, human and rat.Tissue distribution results showed that: Lipin1 in a variety of tissues are distributed, but only in Lipin2 and Lipin3 spleen, lung, ileum, and muscle are distributed.Ⅱ.Cloning and tissues distribution of pig FIT proteins.Cloning primers were designed according to the cloning principle of homologous sequence. Total RNA extracted from all kinds of tissue of pig and was amplified by RT-PCR, the PCR products were ligated into the pMD19-T vector, and then transformed into Ecol. JM109 competent cells. The positive clone was identified and the sequence was sequenced and analyzed. The cloned sequences containing the open reading frame of pig FIT1 consist of 1310bp, and ORF is 400-1472bp and encodes 290 amino acids,of which 22 acidic amino acid, basic amino acid for 39, molecular weight 32.17kDa, isoelectric point of 10.47,signal peptide for 1 ~ 35aa, containing six transmembrane domain. Identity analysis showed that the pig FIT1 nucleotide sequences shared 92.1%,93.2%,87.4%和88.1% homology with that of human, bovine, rat and mouse, the deduced amino acid sequences shared 97.2%,96.6%,94.8%和95.5% homology with that of human,bovine, rat and mouse. It provides the experiment basis for further researching its biological function.The cloned sequences containing the open reading frame of pig FIT2 consist of 1776bp, and ORF is 82-870bp and encodes 262 amino acids,of which 28 acidic amino acid, basic amino acid for 31, molecular weight 29.64kDa, isoelectric point of 8.91,signal peptide for 1 ~ 30aa, containing four transmembrane domain. Identity analysis showed that the pig FIT2 nucleotide sequences shared 85.0%,85.7%和88.1% homology with that of mouse, human and rat, the deduced amino acid sequences shared87.4%,87.4%和92.0% homology with that of mouse, human and rat. It provides the experiment basis for further researching its biological function.Tissue distribution results showed that: FIT1 pig is highly expressed in the cerebellum, in the lung, stomach, ileum and a small amount of expression in the brain, FIT2 mainly in the ileum and is highly expressed in the brain.