Single Nucleotide Polymorphisms Of LAP3 Gene In Bovine And Its Correlation With Milk Production Traits

Leucine aminopeptidases (LAPs) is exopeptidase which catalyses the removal of N-terminal amino acids and is a part of family of aminopeptidases that have been found in many tissues including lens, kidney, pancreas, muscle, liver, mammary and subcellular locations in a diverse of species. LAPs are often viewed as cell maintenance enzymes with critical roles in turnover of peptides. In mammals, LAPs contribute to processing of bioactive peptides (oxytocin, vasopressin, enkephalins), and vesicle trafficking to the plasma membrane and have a role in MHC I antigen presentation. In the present study, we investigated the polymorphisms of LAP3 gene in three kinds of Chinese cattle breeds and assess their associations with milk performance traits in Chinese Holstein cows. Our finding demonstrated that the LAP3 gene possibly contributed to conducting association analysis and can be used as molecular marker in milk production traits and other performance for animal breeding. The results were as followed:1. In this study, we detected the polymorphisms of LAP3 gene by PCR-SSCP, CRS-PCR, PCR-RFLP and DNA sequencing methods in three Chinese cattle breeds including 743 Chinese Holstein,135 Luxi Yellow and 38 Bohai Black. One novel single nucleotide polymorphism (SNP) (g.24564G>A ss196003366) and four previously deposited SNPs in the GenBank database (g.24 794T>G, g.24 803T>C, g.24 846T>C, g.25 415T>C) were detected. Three of the SNPs (g.24794T>G, g.24803T>C, g.24846T>C) were firstly found to be linked completely and regarded as a SNP g.24794 M>N by PCR-SSCP and DNA sequencing in the tested breeds, but they were not found in Luxi Yellow. The alleles T, T, T, G and T were the dominant alleles at position 24794(CH0.579/LY 1/BB 0.722),24803(CH 0.579/LY 1/BB 0.722),24856(CH 0.579/LY 1/BB 0.722),24564(CH 0.584/LY0.775/BB 0.500) and 25415 (CH 0.596/LY 0.796/BB 0.750)in the three breeds. The PIC of the five SNPs in the three breeds all belong to the midrange polymorphism (0.25C-CC genotype had higher protein rate than ones with TT genotype (P<0.05). In addition, eight haplotypes and 23 combined haplotypes were identified based on the nine genotypes and the association between combined haplotypes and milk production traits were analyzed. Statistic results showed that the cows with haplotype combination MAT/MGC (H2H3) have higher protein and fat rate and lower SCS.2. Genetic polymorphisms of LAP3 gene 5'Flanking and haplotype combinations and its correlation with milk performance traits in Chinese Holsteins. The results showed two SNPs were found in 5'Flanking. And the cows with genotype CC and GA had higher fat percentage than genotype TT and GG (P<0.05); and the cows with TC and GG genotype had lower SCS than ones with CC and GA genotype (P<0.05). The cows with haplotype combination H2H1 have higher fat rate than H4H4. The cows with haplotype combination H4H2 have lower SCS than H2H1.3. In this research,we cloned the 433bp DNA sequence of bovine LAP3 5'flanking region. We constructed a Luciferase reporter gene constructs containing 433bp of the bovine LAP3 promoter. The promoter activity analysis was performed by dual-luciferase reproter assaysystem. The result showed the core promoter of the gene was located at the -2315bp～-2 747bp. At the same time, the SNP at loci -2545 was influence in activity of LAP3 promoter.4. Fluorescence quantitative PCR was used to detect the LAP3 mRNA expression level in the tissues of China Holsteins. The result showed that the highest expression level was in the kidney. At the same time, lower expression level in the other tissues, such as heart, liver, spleen, lung, muscle, mammary gland and intestinal. The result is the same as the result of PT-PCR.