| Polymorphism of seven genes from AMPK family on630individuals from four cattle breeds (Qinchuan, Nanyang, Luxi and Angus) were detected by PCR-SSCP, PCR-RFLP, ACRS-PCR-RFLP, MALDI-TOF to analyze genes genetic structures and varieties. We also analyzed the association of the different genotypes with200Qinchuan cattle body measurements and meat quality traits, and further analyzed the gene and protein structure characters of the seven genes via bioinformation in order to profoundly understand their regularization and mechanism in molecular level.The objectives of the present study were to select candidate functional genes or gene markers which were important to beef cattle economic traits, to set up efficient breeding and molecular markers data base and to provide useful genetic information for cattle germplasm protection and usage. The main results of our study were as follows.1. Systematical analysis on nucleotide sequences and their protein sequences of7gengs including PRKAA1, PRKAA2, PRKAB1, RKAB2, PRKAG1, PRKAG2and PRKAG3. By theories and methods of bioinformation, the tissue expression patterns of7genes were analyzed and induced. We predicted the secondary and tertiary structure, hydrophobic nature, signal peptide, transmembrane helices region, functional domain and subcellular localization of the seven genes coding protein. Moreover, we compared the amino acid sequences of target genes with other corresponding proteins from other species, and further constructed the phylogenetic trees of them. It is demonstrated that AMPK family gene are homologous and conservative highly and plays a significant role in the origin of species evolution process.2. PCR-SSCP and DNA sequencing method were used to study genetic variations of exon5, exon7, exon8, exon9, and exon10of PRKAA1gene on four bovine breeds, including Qinchuan, Nanyang, Luxi as well as Angus.213T>A mutation were detected in exon7affected the intramuscular fat content (IFC) of beef in Qinchuan cattle (P<0.01) significantly. No SNPs were found in PRKAA1gene exon5, exon8, exon9and exon10.3. Two SNPs (27G>C,60T>C) located in exon4and two SNPs (1046G>C,1188T>A) in intro8were detected in PRKAA2gene by random DNA sequencing method and MALDI-TOF technology. To analyze genetic structures and varieties on the detected4SNPs in four cattle populations and association with body measurement or meat quality traits in Qinchuan cattle, the27G>C SNP were associated with Back Fat thickness (BF) and Loin Muscle Area (LMA)(P<0.05) significantly. The60T>C SNP were associated with Body Length (BL), Withers Height (WH), Rump Length (RL), Hip Width (HW), Pin Bone Width (PBW) and LMA (P<0.05) significantly. The1046G>C SNP were associated with BF and Chest Depth (CD)(P<0.05) significantly. The1188T>A SNP affected PBW, BF and LMA (P<0.05) significantly. Haplotype analysis results showed there were5genotypes from the4SNPs had the haplotype frequencies over0.03. Combined27G>C with60T>C, we obtained six genotypes, including CCTT, CGCT, CGTT, GGCC, GGCT, GGTT, and we found the cattle individuals of GGCC genotype had superior body measurements and meat quality traits, and maybe can be selected as the candidate genotype. Combined1046G>C with1188T>A, we obtained six genotypes, including CCTT、CGA、CGTT、GGAA、GGA、GGTT, and we found the cattle individuals of GGAT genotype had superior body measurements and meat quality traits.4. Eighteen SNPs were detected in PRKABI gene of Qinchuan cattle population by PCR-SSCP and DNA sequencing respectively. All the mutations found in the three exons caused amino acids synonymous mutations. SNP129T>C effected IMC and LMA of Qinchuan cattle (P<0.05) significantly, SNPs63T>C were associated with BL, HW and LMA of Qinchuan cattle (P<0.05) significantly.12SNPs of the detected15SNPs in intron3, exon4and intron4were associated with WH, HW, RL, CD and Heart Girth (HG) of Qinchuan cattle (P<0.05) significantly. Only In444G/A SNP affected LMA (P<0.05) significantly. SNP57C>T in intron5had significant differences on HG and LMA (P<0.01).The analysis results of detected15SNPs in intron3, exon4and intron4haplotype showed only5haplotypes frequencies were higher than0.03. Based on the association results between In423A/G and In424C/T with cattle related production traits, we selected AACT as candidate genotype.5. Through PCR-SSCP and DNA sequencing technology, ten SNPs have been found in intron3and intron5of PRKAB2gene. The association analysis suggested that In336C/A and In385T/G in intron3no relationship with body measurement traits and meat quanlity traits, In561A/G、In5130C/G, In5410T/C SNPs of the8SNPs in intron5had no relationship with body measurement traits in Qinchuan cattle population (P>0.05); whereas, the other5SNPs (In548T/C、In560A/G、In5118A/G、In5396A/G、In5421A/C) were associated with body measurement traits (P<0.05); In548T/C, In560A/G, In561A/G, In5118A/G, In5130C/G, and In5421A/C SNPs were associated with IMF (P<0.05) significantly. The possibility of forming haplotypes of the8SNPs in intron5was carried out by the online software SHEsis,5 haplotypes frequencies were higher than0.03.After analyzing the combined effects of Ins60A/G and Ins61A/G SNPs, AAAA and AAAG were found to be the best genotype.6. Throuth DNA sequencing and MALDI-TOF,11746T>G SNP was found in intron2of PRKAG1gene. Via DNA sequencing and ACRS-PCR-RFLP technology,12482T>C SNP was detected in intron5of PRKAG1gene. The two SNPs were both in medium polymorphism level. In Qinchuan cattle population, they were at Hardy-Weinberg equilibrium.11746T>G SNP had no association with body measurement and meat quality traits (P>0.05).However,12482T>C SNP was associated with Withers height (WH), Hip width (HW), Heart girth (HG), Pin bone width (PBW), LMA and IMF (P<0.05) significantly.7. Eight SNPs were found in PRKAG2gene, only En3223A>G was missense mutation with the change of Ser into Gly. Bioinformatics analysis suggested that this missense mutation led to great change in tertiary structure of PRKAG2gene. En3223A>G introduced a restriction enzyme cutting site for Msp I. Association analysis suggested that En3204G>C, En3223A>G, and In39T>C SNPs were related to WH, HH, HW, RL, CD, HG and LMA (P <0.05);En13112T>C SNP was associated with WH, RL, and HW (P<0.05).8. By DNA sequencing, MALDI-TOF and ACRS-RFLP-PCR,45SNPs were found in PRKAG3gene,10in coding region,18in3’UTR and17in introns. The SNPs in coding region did not lead to amino acid change, except1794T/C,4002C/T, and5359C/T mutations. The association analysis suggested that4SNPs were associated with BL significantly;8SNPs were associated with WH significantly;21SNPs were associated with HH and RL;17SNPs were associated with HW and CD;6SNPs were associated with HG;5SNPs were associated with PBW;2SNPs were associated with IMF;7SNPs were associated with LMA;3SNPs were associated with BF;7SNPs had no association with body measurement or meat quality traits in Qinchuan cattle. |
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