Association Analysis Between The Genetic Polymorphism And The Heat Resistance Of Chinese Holstein Of Ccl3Gene

The optimum temperature of Chinese Holstein is5-20℃. In our country the summer temperature is often higher than35degrees and continue longer, which caused heat stress in Chinese Holstein. We know heat stress may decrease the production performance, reproductive performance and immunity and become an important factor in dairy development. In addition, strong and durable heat stress can cause overheating. The interaction between cytotoxicity and systemic inflammatory response often results in thermal damage and even multiple organ dysfunction syndrome.Chemokine ligand3(CCL3) is an important member of mortar chemokine subfamily (CC chemokine subfamily). It plays an important role in raising the erythroid progenitor cells and forming megakaryocyte colony as well as inflammatory cell. Besides, it is closely related to the multiple myeloma, human immunodeficiency virus infection, acute coronary syndrome, asthma and other diseases. Recent researches suggest that heat-stress of the cows may have some relationship with CCL3. CCL3gene provide new targets and ideas for disease diagnosis, prevention, treatment and animals’resistance breeding.Previous study found that peripheral blood of Chinese Holstein cows by42℃after1h and4h heat shock, CCL3expression appears significantly increased. So we can see peripheral blood of Chinese Holstein can induce other chemokines such as CCL3to express abundantly and cause the immune response. Currently, the process of heat stress on the biological function of CCL3has no relevant reports, but it can be indirect response to cow’s heat capacity and belongs to heat stress-related genes, which have some reference value in breeding Heat-resistant cows.As a candidate gene that can affect cows heat stress, we cloned cows CCL3gene by bioinformatics and screened SNPs of CCL3gene 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 CCL3gene and Chinese Holstein heat stress on rectal temperature, blood, serum biochemical parameters, we conducted the molecular analysis, detailed evaluation by SNPs within the gene for the CCL3.1. The impact of Chinese Holstein heat stress on rectal temperature, blood, serum biochemical parametersThe rectal temperature of Chinese Holstein are significantly lower at15℃than at37℃(P<0.01) and26℃(P<0.01). The blood indexes included WBC, NEU, EOS, BAS, NEU, EOS, BAS, RBC, HGB, HCT, MCV, MCH, MCHC, RDW-CV are significantly lower at15℃than at37℃(P<0.01) and26℃(P<0.01) and LYM, MON are significantly higher at15’C than at37℃(P<0.01) and26℃(P<0.01) MCH is notablely lower at26℃than at37℃(P<0.05). MCHC is significantly lower at26℃than at37℃(P<0.01). The serum in peripheral blood of Chinese Holstein find that MDA is significantly lower at15℃than at26℃(P<0.01) and37℃(P<0.01) and is significantly lower at26℃than at37℃(P<0.01). AKP is significantly higher at15℃than at26℃(P<0.01) and at37℃(P<0.01). T-AOC is significantly higher at15℃than at26℃(P<0.01) and at37℃(P<0.01) and is notablely higher at26℃than at37℃(P<0.05).2. Cloning and bioinformatics analysis of Chinese Holstein CCL3geneAccording to the cDNA of CCL3gene, we designed a pair of primer for cloning cows CCL3gene. Finally, we found that DNA is1874bp in length, containing three exons (1-89bp、945-1061bp、1364-1874bp) exons and two introns (90-944bp、1062-1363bp), which is consistent with other mammals’CCL3gene. In addition, we found that CCL3gene has the homology with European cattle of92.77%and with the human and chimpanzee of53.66%and53.52%by Multi-species comparison.3. PCR-SSCP and polymorphism analysis of CCL3geneIn this study, by PCR-SSCP method, SNPs of the whole cloned CCL3gene was detected in Chinese Holstein. Experiment designed six pairs of primers and detected polymorphism from P2and P6primers. We found512(C→T)、560(C→A)、562(C→T)、1512(T-+C) and1793(A→G) are mutated. Using the Haploview4.0software for the CCL3gene linkage disequilibrium analysis and constructing two single-domain (Haplotype block) in the whole range of CCL3gene by D’values on the95%confidence interval upper and lower limits of the analysis, respectively,512,560, and562SNP sequences represented by the formation of single-domain1,1512and1793with the SNP sequence represented by the formation of single-domain2.We selected two htSNPs (512C→T and1512T→C) by r2values between the single-domain SNP and found htSNP512belongs to a low degree of polymorphism (PIC<0.25), htSNP1512belongs to moderate polymorphism (0.25<PIC<0.5).x2test results showed that two htSNPs of CCL3gene are a significant deviation from Hardy-Weinberg equilibrium (P<0.05).4. Association analysis between the gene polymorphism and rectal temperature, blood, milk traits and biochemical markersThere was a significant correlation between htSNP512and rectal temperature and MCV of Chinese Holstein at heat stress (P<0.05). Multiple comparisons showed that rectal temperature of CT notablely higher than CC(P<0.05) and MCV of CT notablely lower than CC (P<0.05). There was a significant correlation between htSNP1512and LYM and LYM%of Chinese Holstein at heat stress (P<0.05). Multiple comparisons showed that LYM and LYM%of TC notablely higher than TT (P<0.05).