Bovine ISG15Gene IL8Gene Polymorphism And Genetic Effects On Milk Production Traits And Somatic Cell Score

The results of the Wenzhou buffalo Affymetrix Microarray showed that numerous genes expression changed between early lactation and mid-lactation period and between mid-lactation and late lactation period. We found that the expression change trends of ISG15gene and IL8gene was decreased after an initial increase.The two gengs expression trends is similar and2times up and down regulation. Because of the important roles of ISG15and IL8genes in innate immunity and acquired immunity,we choose them as the candidate genes that can affect bovine disease resistance.We cloned Wenzhou buffalo ISG15gene and IL8gene by rapid amplification of cDNA ends and designed primers for cloning Wenzhou buffalo ISG15gene and IL8gene,then we screened SNPs of the two genes comprehensively by polymerase chain reaction-Single strand conformation polymorphism (Polymerase Chain Reaction-Single Strand Conformation Polymorphism, PCR-SSCP) analysis.Through the association analysis between the single nucleotide polymorphisms of ISG15gene and IL8gene and different varieties bovine lactation traits,we can provide a theoretical basis to improve the performance of bovine disease and improve lactation performance.1Wenzhou Buffalo ISG15gene cloning and bioinformatics analysisBased on the use of RNA5’ RACE technology transcriptional jumping mechanisms we get the full sequence of Wenzhou buffalo ISG15gene.The gene is629bp in length,including5’ UTR72bp (1-72bp),open reading frame465bp (73-535bp),coding154amino acids,and3’ UTR92bp (536-629bp).The cloned DNA conteined two exons (1458-1460bp and1897-2424bp) and one intron (1461-1896bp), which is consistent with other mammals’ ISG15gene. The Wenzhou buffalo ISG15protein Molecular Weight was17333.1Da. Multiple alignment of mammalian ISG15protein showed, Wenzhou buffalo ISG15protein sequence had99%identity with the buffalo ISG15protein. ISG15protein subcellular localization predicted the possibility of the protein has a signal peptide was13.8%.2Wenzhou Buffalo IL8gene cloning and bioinformatics analysisWe get the full sequence of Wenzhou buffalo IL8gene.The gene is1522bp in length,including5’UTR82bp (1-82bp),open reading frame303bp (83-388bp),coding101 amino acids,and3’UTR1134bp (389-1522bp).The cloned DNA conteined four exons (74-137bp,1690-1839bp,2113-2196bp and2639-2660bp) and three introns (138-1689bp,1840-2112bp and2197-2638bp), which is consistent with other mammals’IL8gene. The Wenzhou buffalo IL8protein Molecular Weight was11305.4Da. Multiple alignment of mammalian IL8protein showed, Wenzhou buffalo IL8protein sequence had100%identity with the buffalo(AAW67481.1) and bovine(ACI23534.1) IL8protein. IL8protein subcellular localization predicted that Wenzhou buffalo IL8putative protein domains, domain SCY found in its31-92amino acid residues.3PCR-SSCP and polymorphism analysis of bovine ISG15geneThe blood is in caudal vein. Chinese Holsteins’are from Yikang farm in Zhejiang Jinhua.Wenzhou buffaloes’ are from Zhejiang Wenzhou Pingyang and Hubei Jinmen Shayang.Wenling humped cattles’are from Wenling Animal Husbandry and Veterinary Station.Tiantai bovines’are from Tiantai Animal Husbandry and Veterinary Station.The DNA of Luxi bovine are given by Li Junya in Beijing Institute of Animal Chinese Academy of Agricultural Sciences.In this study, by PCR-SSCP method, SNPs of the whole cloned ISG15gene was detected in Wenzhou buffalo and Chinese Holstein. Experiment designed fifteen pairs of primers and detected polymorphism from U2D2,U7D7and U9D9primers. We found374(G→A),433(C→T),1806(G→C) and2104(G→A) are mutated. We selected two htSNPs (512C→T and1512T→C) by r2values between the single-domain SNP and found g.374G→A and g.2104G→Abelongs to a low degree of polymorphism (PIC<0.25), g.433C→T and g.1806G→C belongs to moderate polymorphism (0.25<PIC<0.5).χ2test results showed that g.433(C→T) and g.1806(G→C) are a significant deviation from Hardy-Weinberg equilibrium (P<0.05).General Linear Models analysis between SNPs and milk production traits indicated that there were significant correlation between three genotypes on milk protein and somatic cell score.4PCR-SSCP and polymorphism analysis of bovine IL8geneIn this study, by PCR-SSCP method, SNPs of the whole cloned IL8gene was detected in Wenzhou buffalo and Chinese Holstein. Experiment designed seventeen pairs of primers and detected polymorphism from UODO,U2D2,U6D6,U7D7,U9D9,U1OD10,U11D11and U13D13primers. We found-63(G→A),249(C→T),311(C-T),349(A→G),1247(C→T),1501(G→T),1939(A-C),1986(G→A),2256(T→G),2269(G-A),2420(G→A),2831(A→G),2904(T→C) and3030(G→A) are mutated. We found all SNPs belong to moderate polymorphism (0.25<PIC<0.5).χ2test results showed that g.1247C→T and g.1501G→T gene are a significant deviation from Hardy-Weinberg equilibrium (P<0.05).Using the Haploview4.0software for the IL8gene linkage disequilibrium analysis and constructing four single-domains (Haplotype block) in the whole range of IL8gene by D ’values on the95%confidence interval upper and lower limits of the analysis, g.249C→T,g.1939A→C,g.2256T→G and g.2831A→G are selected htSNPs.The r2value is1between g.249C→T,g.311C→T and g.349A→G.So do be the value between g.1939A→C and g.1986G→A, g.2256T→G and g.2269G→A, g.2831A→G, g.2904T→C and g.3030G→A. g.249C→T’s effect can replace g.311C→T’s and g.349A→G’s. g.1939A→C’s effect can replace g.1986G→A’s. g.2256T→G’s effect can replace g.2269G→’s. g.2831A→G’s effect can replace g.2904T→C’s and g.3030G→A’s.General Linear Models analysis between SNPs and milk production traits indicated that there was a significant correlation between g.-25G→A and305days milk yield (P<0.05),g.249C→T and305days milk yield and milk fat (P<0.05), g.1247C→T and somatic cell score (F<0.05), g.1501G→T and305days milk yield (P<0.05), g.2256T→G and milk fat (P<0.05), g.2831A→G and305days milk yield (P<0.01) and milk fat (P<0.05).