Cloning,Polymorphism Of Bovine Mastitis Gene And Association Analysis Between Polymorphism And SCS

Mastitis is a common disease in dairy industry, which has high incidence rate and causes serious economic losses. There are so many influencing factors, hereditary factors play an important role in mastitis. Toll family is a newly found receptor family, playing an important role in congenital immunity. It has been proved that some genes of the receptor family have correlations with immunity. In this study, Molecular biology and bioinformatics methods were used to isolate the NFKB1 gene, which is a switch of genes in nucleolus. Meanwhile we use the PCR-RFLP method to analyse the correlations between TLR2 gene polymorphism and SCS. All the results are as follows:1.The mRNA sequence of human and mouse NFKB1 gene were obtained from the NCBI, and were employed to search for bovine expressed sequence tags (ESTs) in the EST-others database through standard BLAST algorithm. Molecular biology and bioinformatics methods were used to isolate the full-length NFKB1 gene, the newly got GenBank ID is DQ464067. The full length is 3837bp, ORF length is 2910 bp, 5' flanking sequence length is 84 bp, 3' flanking sequence length is 814 bp plus 30bp ploy(A). The 2910bp ORF translated into 969 AA. The deduced AA sequence was analysed and the possible Conserved Domains were predicted in NCBI.2. TLR2 gene (NCBI ID:NM₁74197.2) 3 SNPs and associations with 3 Dairy-Cow SCS(1)TLR2 gene 385 locus PCR-RFLP results indicate: 385 locus T/G mutation causes a EcoRV polymorphic locus and aspartic acid/glutamic transition. 3 genotypes AA (241 bp/207bp), AB (448 bp/241 bp/207bp) and BB (448bp) were detected in this polymorphism locus. Allele A in Chinese Holstein and Sanhe cattle.is predominant, Allele B in Chinese Simmental is predominant.(2)TLR2 gene 398 locus PCR-RFLP results indicate: 398 locus G/A mutation causes a Haeâ…¢polymorphic locus. 2 genotypes AA(90 bp/21 bp /19bp) and AB(130bp /90 bp /21 bp /19bp) were detected in this polymorphism locus. Allele A in Chinese Holstein, Sanhe cattle and Chinese Simmental is predominant.(3)TLR2 gene 1884 locus PCR-RFLP results indicate: 1884 locus G/A mutation causes a Hha [polymorphic locus and glycine/serine transition.2 genotypes AA(188bp/22bp) and AB(210bp/188bp/22bp) were detected in this polymorphism locus. Allele A in Chinese Holstein, Sanhe cattle and Chinese Simmental is predominant.(4)The population genetic analyse indicates: All the 3 dairy cows Chinese Holstein, Sanhe cattle and Chinese Simmental reaches Hardy-Weinberg equilibrium, 385 polymorphic locus in 3 dairy cows is moderate polymorphism, 398 and 1884 polymorphic locus are Low polymorphism.(5)This 3 SNPs were associated with SCS. The TLR1 gene 398-SNP and 1884-SNP were not significantly realated to SCC(P＞0.05), 385-SNP is significantly realated to SCC(P＜0.05). The BB genotype of 385-SNP reached significant compare to genotype AB and AA(P＜0.05), BB genotype was assumed to be the predominant genotype to mastitis.3.The first-strand cDNA was synthesized by RT-PCR using poly-A primer, 12 different tissues including galactophore, ovary, liver, muscle, small intestine, fattiness, uterus, kidney, heart, lung, pancreas and testis were obtained from a Chinese Holstein for genes expression study of NFKB1 and TLR2 gene. Expression analysis was performed to determine the relative mRNA level of NFKB1 and TLR2 gene in various tissues. Theβ-actin gene was used for endogenous control. Expression data demonstrate that the bovine TLR1 and NFKB1 gene expressed in all 12 tissues of interest, it could be the base for further real-time PCR study.