| Objective: The polymorphisms of TLR4 and TLR2 genes in sheep were investigated,and the correlation analysis of Brucella disease was carried out.Then,the haplotype was constructed and analyzed.The SNPs of TLR4 and TLR2 genes were correlated with various diseases of sheep,which could provide a theoretical basis for the selection of Toll-like receptors in order to improve the resistance mechanism of Toll-like receptors.Method: 1.The experiment was carried out on the collection of 480 sheep by serum tiger red plate agglutination experiment to carry out the detection of Brucella bacillus,and use of bioinformatics on the NCBI published on the sheep was used to analyze the polymorphisms of TLR4 and TLR2 exons in sheep.The exon fragments of polymorphic loci were selected and designed to amplify the gene fragments,and then the PCR-SSCP technique Methods The single nucleotide polymorphisms(SNPs)of TLR4 and TLR2 genes were detected,followed by cloning and sequencing of sheep individuals with different alleles,and the results were compared with molecular biology software.All the SNP loci were analyzed and analyzed by SHEsis online software.The correlation between the allele frequency and genotype frequency was analyzed,and the correlation with brucellosis susceptivity was analyzed.2.The PCR products of 80 individual Brucella positive and negative sheep TLR4 were sequenced,and the SNPs were detected by the sequencing results.The software was tested by Hardy-Weinberg equilibrium and the linkage disequilibrium.The haplotypes of SNPs were constructed by SHEsis online software and the haplotypes were studied.The haplotypes associated with brucellosis susceptivity were screened.Result: 1.With the tiger red plate agglutination test,we detected 480 sheep serum,which included 290 China Merino sheep,the results were 80 Brucella positive individuals,210 Brucella negative individuals,The positive rate of Brucella was 27.59%.A total of 100 brucella-positive individuals and 84 brucella-negative individuals were found in 100 rabbits with tiger red plate agglutination test.The positive rate of Brucella was 16%.90 Hu sheep were detected by tiger red plate agglutination test,and 15 brucella positive individuals and 75 brucella negative individuals were found.The positive rate of Brucella was 16.67%.2.TLR4 exon 3 detected a total of 7 SNP 1 site for the nonsense mutation sites,and the remaining 6 for the sense of mutation,which does not exist in the insertion and deletion of the site.There are 8 SNPs in the partial sequence of the TLR2 gene and all of them are nonsense mutations.3.The results showed that there were differences in the SNPs between different sheep breeds,and the SNPs of TLR4 and TLR2 were analyzed by NCBI.4.the TLR4 and TLR2 gene polymorphisms were associated with the susceptibility of brucellosis,respectively,the TLR4 gene can be obtained on the 1180 G / T locus and the Chinese Merino sheep Bruce The susceptibility to pathogenicity was correlated,while the frequency and genotype frequencies of other polymorphic loci were statistically no significant in the brucellosis group and the normal group.And TLR2 8 loci are nonsense mutations and may not be associated with Chinese Merino sheep brucellosis susceptivity.5.To achieve the haplotype requirements TLR4 polymorphic loci linkage disequilibrium heredity analysis of the two LD Block in the TLR4 third exon,the SNPs also have a certain linkage between the imbalance,Which is called Block1-Block2 in sequence and contains two SNPs sites.Block1 includes C1063 T,A1085 G.Block2 includes C 1088 T,C1094 G.6.haplotypes were constructed when the TLR4 gene was constructed in China.The frequencies of Hap2 and Hap4 haplotypes were found to be 20.05% in the case group,And 32.4%,respectively,were significantly higher than those in the normal group and the haplotype frequency distribution was statistically significant(P <0.05),so it was possible to judge that these two haplotypes were likely to be associated with Chinese Merino sheep Brucella The susceptibility of the disease.Conclusion: 1.The sequence analysis of the TLR4 and TLR2 genes of sheep on Gen Bank.The results showed that have diversity of SNPs locus in different sheep breeds.2.Through the analysis of sequence in China Merino TLR4 exon 3 in 7 SNPS were detected 1 loci for nonsense mutations,the remaining 6 for mutation.There are 8 SNP in the partial sequence of TLR2 gene,and all of them are nonsense mutations.That China Merino sheep TLR2 gene exon region may be relatively conservative.3.The sheep TLR4,TLR2 polymorphism and brucellosis susceptibility correlation analysis,found that 1180G/T sites of TLR4 gene may be associated with the susceptibility of sheep brucellosis,and the gene frequency and genotype SNP and other sites may be not associated with susceptibility of brucellosis.4.The linkage disequilibrium analysis of TLR4 polymorphic loci with the requirement of the total Block was obtained,and all the haplotypes were found to be Hap2 and Hap4,which were found in LD... |
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