Assoiciation Of Polymorphisms In MHC-DR Gene With Immune Trait In Qianbei Ma Goat

4-6 month Qianbei Ma Goat as material,we used PCR method to screened SNPs in MHC-DR gene to explore Qianbei Ma Goat MHC-DR subregion gene Polymorphism and its immune trait correlation.And association analysis,we screened genetic genetic marker,further found the corresponding Qianbei Ma Goat;when the corresponding goat grown up,we slaughted 3 Qianbei Ma Goat without low significance(group A)and 3 Qianbei Ma Goat with high significance(group B)to obtained 10 kinds of tissues,including Heart,liver,spleen,lung,kidney,pancreas,jaws,lymphatic,submandibular gland,neck lymph and thymus,then extracted RNA from those tissues for real-time qPCR to observe expession difference of MHC-DR gene in the difference tissue of the two groups,so as to provide scientific basis for disease resistance breeding assisted selection of Qianbei Ma Goat.1.Screened for genetic polymorphism of MHC-DRgene in Qianbei Ma GoatUsing PCR method,we screened 67 SNPs from MHC-DRgene in Qianbei Ma Goat.The DRA gene had no SNPs;exon3 of DRB1 gene was screeened 8 SNPs,respectively A8858G(Thr?Ala),C8859 T,G8969A(Gly?Ser),C8974 T,G8989A,G9094T(Glu?Asp),C9100 T and G9124 A,of which has there missense mutation;DRB2 gene has 14 SNPs,5 SNPs of which are in Exon3,respectively G13286 T,C13405T(Pro?Leu),G13406 A,A13469G,T13481 C,and 9 SNPs of which are in Exon 4,respectively A15573G(Ile?Val),C15574T(Thr?Ile),G15636A(Val?Met),A15642 G,G15753A(Val?Met),G15779C(Gln?His),G15780A(Gly?Ser),C15797 T,C15831A(Leu?Ile),inclonding 8;DRB3 gene was screened 45 SNPs,34 SNPs are in Exon2,respectively A8925C(Lys?Thr),C8929A(Ser?Arg)A8937G(His?Arg),C8953 G,C8966T(Leu?Phe),T8984C(Tyr?His),T8999A(Tyr?Asn),A9000T(Tyr?Phe),T9009A(Phe?Tyr),T9030 A,G9037A,G9041A(Ala?Thr),G9059A(Ser?Gly),A9060G(Ser?Asn),C9061 T,A9065G(Lys?Glu),C9088G(Glu?Asp),C9091 T,A9099G(Asp?Gly),C9100G(Asp?Glu),G9103C(Arg?Ser),C9104 A,A9107G(Thr?Ala),G9110 T,C9111A(Ala?Glu),G9112C(Glu?Asp),C9120A(Thr?Lys),T9122G(Tyr?Asp),A9123T(Tyr?Phe),C9124 G,C9131T(His?Tyr),T9147 G,A9158G(Tyr?Ala),C9159G(Tyr?Ser),11 SNPs of which is in Exon3,respectively A11244G(Tyr?Ala),G11262A(Ala?Tyr),A11264 G,T11312C,A11315 T,G11355A(Gly?Ser),C11360 T,G11450C(Gln?His),A11451G(Ser?His),T11480G(Asp?Glu),C11486 T,in total 31.2.Analysis of genetic characteristics and Antigen epitope predictionAfter analysis of genetic characteristics,we found the PIC(polymorphism information content)of MHC-DR gene is 0.25~0.5,belonged moderate polymorphism which shown the most SNPs reasonably provied genetic infromation;heterozygosity analysis result shown heterozygosity of the SNPs is about 0.5,expect exon 3 of DRB3 gene 0.25,which shown the Qianbei Ma Goat exsited a certain degree of genetic mutation;advantage allele analysis shown the wild type(in accordance with reference sequence)is advantage allele gene;linkage disequilibrium analysis shown no complete linkage phenomenon.Used antigen epitope prediction online software,we found SNPs of MHC-DR gene change antigen epitope:A8858G site of DRB1gene;C15831A and G15779 C site of DRB2;A8937G and A11244 of GDRB3 gene result in the change of amino acid residures in antigen epitope.3.Assoiciation of Polymorphisms in MHC-DR Gene with Immune Trait in Qianbei Ma gaotAfter assoiciation with immune trait,we found 26 signficence sites,14 obvious shnificence sites in 67 SNPs.After assoiciation with blood routine index,we found A8858 G of DRB1 gene Exon3,A15573 G and G15636 A of DRB2 gene Exon4,A8937 G ofDRB3gene Exon2,T11480 G of DRB3 gene Exon3 obvious significence and significence with beyond there blood routine indexs;and found G13286 T and T13481 C of DRB2 gene Exon3,G9059 A and G9112 C of DRB3 gene Exon2 obvioussignificence and significence with two blood routine indexs;found G8989 A of DRB1 gene Exon3,T13481 C of DRB2 gene Exon3,G15636 A and G15780 A of DRB2 gene Exon4,A8937 G,G9059A,C9100 G and G9103 C of DRB3 gene Exon2,A11315 T,G11450C and T11480 G of DRB3 gene Exon3 obvious significence and significence with one blood routine index;found T13481 C of DRB2 gene Exon3,G15636 A of DRB2 gene Exon4,A8937 G and G9059 A of DRB3 gene Exon2,A11451 G and T11480 G of DRB3 gene Exon3.4.Qianbei Ma gaot MHC-DR gene expression researchUsing real-time qPCR method to analsis expression differnce of MHC-DR gene,DRB2 gene and DRB3 gene in heart,liver,lung,kidney,and immune tissues,the result shown MHC-DR gene,DRB2 gene and DRB3 gene expreeeson in all tissues,higher in immune tissues.DRA,DRB2 and DRB3 gene is highest in slpeen of the low significance group(group A)and the corresponding group(group B),and has obvious increase in immune tissues of B group relative to A group;expression differnce of DRB1 gene has no obvious significence in A group and B group.Thus it is concluded that MHV-DR subregion has high amount of gene expression in immune tiussue of Qianbei Ma Goat,and preliminary validation Qianbei Ma Goat immune character has validity,belongs to the most significant correlation of genetic markers.To sum up,Qianbei Ma Goat MHC-DR subregion gene belonged to the moderate polymorphism,found no complete chain phenomenon;Screened out 67 polymorphic sites,of which associated with a significant immune traits of site has 26,extremely significant correlation with 14;Qianbei Ma Goat MHC-DR subregion gene expression in immunoe tissue is higher;we found 8 SNPs genetic marker which have significant correlation in the immune traits in MHC-DR gene of Qianbei Ma Goat,respectlyG15636 A of DRB2 gene Exon4,A8937 G and G9059 A of DRB3 gene Exon2,A11244 G,A11451G and T11480 G of DRB3 gene Exon3,so as to provide scientific basis for disease resistance breeding assisted selection of Qianbei Ma Goat.