| Tibetan sheep is one of the oldest breed of Chinese sheep, Living in the altitude over 2500 m in Qinghai Tibet Plateau and its adjacent area, Is the main livestock resources and tools of production and life on the Qinghai Tibet Plateau. In recent years, due to the natural and man-made factors, Tibetan sheep production performance, disease resistance and resistance and other high altitude adaptation fall characteristics show degradation trend. Therefore, the research on adaptive evolution mechanism of development of Tibetan sheep and genetic diversity of different geographical and ecological groups take is very necessary.Major histocompatibility complex is one of the functions of gene family plays an important role in the existence and the immune system in vertebrates, the MHC molecules encoded with antigen presenting and trigger the immune function and the ability to maintain body generation to adapt to environmental changes, also is the best marker of adaptive evolution of vertebrates at present. The study of Tibetan sheep(3 groups of Gansu and 2 groups of Qinghai) as the research object, cloning and determination of Tibetan sheep DQA and DQB gene sequence analysis of loci and bioinformatics, the relative expression by real-time fluorescent quantitative PCR technique to detect DQA1, DQA2, DQB1 and DQB2 gene m RNA in eighteen months at the age of Gannan Tibetan sheep 17 organizations in quantity, and use PCR-SSCP technology analysis of DQA1, DQA2, DQB1 and DQB2 gene polymorphisms and analyzes its adaptability and evolution of the plateau of Tibet sheep.The main research results and conclusions of this study are as follows:1.Tibetan sheep DQA and DQB gene sequence cloning and bioinformatics prediction(1)The DQA1 gene and DQA2 gene of Tibetan sheep CDS length is 768 bp, encoding 255 amino acids, the molecular weight of the protein was 28.23 KD and 29.83 KD respectively, theoretical isoelectric point(PI) were 5.20 and 4.76. Prediction found DQA genes are secreted protein, and has strong hydrophilicity, mainly located in the endoplasmic reticulum, Golgi and cell membrane; found 27 phosphorylation sites in DQA protein, but not found that glycosylation sites in DQA protein; forecast also found that there are 3 domains and 1 transmembrane domains in the DQA protein; the two stage structure is mixed with random coil and alpha helix, cattle, goat and horse of amino acid sequence has low homology and phylogenetic relationship the distance.(2) The DQB1 gene and DQB2 gene of Tibetan sheep CDS length is 786 bp, encoding 261 amino acids, the molecular weight of the protein is 29.91 KD and 30.05 KD respectively, theoretical isoelectric point(PI) is 5.52 and 8.83. Bioinformatics prediction found DQB loci are secreted protein, and has strong hydrophilicity, mainly distribution in the outside of the cell, cell membrane and endoplasmic reticulum; 38 phosphorylation sites in DQB protein was found, 4 glycosylation sites were found also; there are 4 domains and 2 transmembrane helical region in the DQA protein found; two stage structure is mixed with random coil and alpha helix, the amino acid sequence of the bovine, horse mountain, such as the low homology, and their genetic relationship Department of far and near uniform.2. DQA1, DQA2, DQB1 and DQB2 gene m RNA expression in the different tissues of Tibetan SheepThe DQA and DQB locus in Tibetan sheep longissimus muscle, brain, lung and liver tissues, but the expression level was different. A high expression of DQA1 gene in longissimus muscle, spleen, adrenal, kidney, thyroid and ovary tissues, moderate expression in the lung, heart, omasum, reticulum and rumen tissue; expression level of DQA2 gene in spleen, heart and the highest expression in the ovary, moderate in liver, hind legs muscle, ileum and rumen tissues; the high expression of DQB1 gene in liver, ileum and ovarian tissues, expressed in the adrenal gland and heart tissues in moderate amount; the high expression of DQB2 gene in the omasum, spleen, thyroid, cerebellum and pancreatic tissues in moderate amount in the back, expression in muscle tissue.3.Adaptive evolution of DQA and DQB genes in Tibetan sheep(1) The DQA and DQB loci genetic characteristics of Tibetan sheep.â‘ The DQA1 gene of Tibetan sheep exons 2 have 16 alleles, designated as OLA-DQA1*A, *B to *O and *P.And sheep DQA1 gene sequence has high homology. Thay have a total of 54 nucleotide sequence polymorphic loci, accounting for about 20.07% of the total sequence. In the amino acid sequence in a total of 26(32.50%) amino acid mutation.â‘¡ The DQA2 gene of Tibetan sheep exons 2 have 17 alleles,designated as OLA-DQA2*A, *B to *P and *Q. The OLA-DQA2*I to *Q was first discovered in Tibetan sheep, OLA-DQA2*A to *H is the published; And sheep DQA2 gene sequence has high homology. Thay have a total of 71 nucleotide sequence polymorphic loci, accounting for about 29.34% of the total sequence.In the amino acid sequence in a total of 41(56.90%) amino acid mutations.â‘¢The DQB1 gene of Tibetan sheep exons 2 have 5 alleles, respectively designated as OLA-DQB1*A to *E.And sheep DQB1 gene sequence has high homology. Thay have a total of 61 nucleotide sequence polymorphic loci, accounting for about 21.79% of the total sequence. In the 89 amino acid sequence in a total of 36(40.45%) amino acid mutation.â‘£The DQB2 gene of Tibetan sheep exons 2 have 21 alleles, which were named OLA-DQB2*A, *B to*T and *U.And sheep DQB2 gene sequence has high homology. Thay have a total of 48 nucleotide sequence polymorphic loci, accounting for about 18.68% of the total sequence. In the amino acid sequence in a total of 31(38.75%) amino acid mutation.(2) The DQA and DQB loci gene selection of Tibetan sheepBalancing selection is one of the main mechanisms to maintain Tibetan sheep DQA and DQB loci polymorphisms.DQA1, DQA2, DQB1 and DQB2 genes of the antigen binding site(ABS) of the non synonymous mutation rate is higher than the rate of synonymous mutation rate, that the 4 gene of Tibetan sheep DQA and DQB loci are subject to balancing selection function.(3) The DQA and DQB gene of recombinant in Tibetan sheep.Reorganization is one of the main evolutionary patterns of Tibetan sheep DQA genes, but not found in the DQB genes. In the DQA1 allele sequences, 2 recombination events were detected by the Max Chi method, and 4 recombination events were detection in DQA2 gene, 2 events were detected by Max Chi and Si Scan two kinds of methods.(4) The DQA and DQB gene of Tibetan sheep genetic adaptations.Based on the DQA and DQB gene differentiation coefficient show, Ganjia type of Tibetan sheep and other 4 groups significantly. According to the results of AMOVA analysis, the 5 Tibetan sheep groups according to geographic location is divided into 2 regions, namely Gansu population(OLX, QKX and GJX) and Qinghai population(QHOLX and QHGYX), show that the Gansu and Qinghai exchange between the 5 ecological groups of Tibetan sheep area larger than the same region variation among different groups, the results also support the division of this region. |
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