Study On The Genetic Effect Of CAPN1 Gene On Muscle Tenderness In Chicken And It's Expression Characterization

This experiment selects our country Guangdong Qingyuan's high quality chicken variety - Qingyuan partridge chicken and an overseas introduction chicken plants - Recessive White chicken to take the object of study, takes CAPN1 gene as candidate gene for chicken meat tenderness, PCR-SSCP and sequencing methods were used to investigate single nucleotide polymorphisms in CAPN1 gene of the Qingyuan partridge chicken and the Recessive White chicken,also,analyze the relationship between CAPN1 gene and the muscle tenderness; Meanwhile examined the CAPN1 gene' space-time expression rule using SYBR Green I fluorescence quota PCR in the Qingyuan partridge chicken and the Recessive White chicken pectoral muscle and gastrocnemius muscle. The test result is as follows:High quality chicken breeds - Qingyuan partridge chicken (QY) from Qingyuan of Guangdong Province and an exotic breed - Recessive White chicken (RW) were taken in this research, and CAPN1 gene was taken as candidate gene for meat tenderness in chicken, single strand conformation (SSCP) analysis and sequencing reactions were performed to screen single nucleotide polymorphisms (SNPs) in Qingyuan partridge chicken and Recessive White chicken, and the correlation between results of SNPs and meat tenderness was analyzed; further, gene expression of CAPN1 gene in pectoral muscle and gastrocnemius muscle in Qingyuan partridge chicken and Recessive White chicken was investigated by SYBR Green I Real-time PCR technology to explore the expression characteristics, The main results were as follows:1. Polymorphism of CAPN1 genePolymerase chain reaction - single strand conformation (PCR-SSCP) was performed to detect the SNPs of segment sequences in CAPN1 gene in Qingyuan partridge chicken and Recessive White chicken. The result shows three SNPs were detected : T2507C and C2546T from exon 5, G3535A from exon 6,and G9950A from 3'UTR. All the mutations in the coding regions were silence mutations; they didn't cause the alteration of the corresponding amino acid. After Calculated the frequency of genotypes and genes and discovered some differences in the distribution of genotype frequencies among different breeds,A,B alleles and AA,AB,BB genotypes were detected at exon 5 in Recessive White chicken,and AA for wild, BB had an mutation of C2546T, and frequency of B allele (0.752) is higher than A (0.248) obviously, then A, B, C alleles and AA, AB, BB, AC, BC, and CC genotypes were detected in Qingyuan partridge chicken, CC genotype had two mutations of T2507C and C2546T, and the frequency of C allele is 0.188; G, A alleles and GG, GA, AA genotypes were detected at 3'UTR in two chicken breeds, and the frequency of G allele (0.675) was higher than A (0.325) in Qingyuan partridge chicken, but the frequency of G allele (0.484) was lower than A (0.516) slightly in Recessive White chicken; G, A alleles and GG, GA, AA genotypes were detected at exon 6 in two chicken breeds, and the frequency of G allele (0.190) was lower than A (0.810) obviously in Qingyuan partridge chicken, on the contrary, the frequency of G allele (0.840) was higher than A allele frequency(0.160) obviously in Recessive White chicken.2. The relationship between SNPs of CAPN1 gene and the meat qualityThe least square estimation between genotypes,breeds and the shear force indicated:On the SNPC5, it had significant breed effect,genotype effects were significant in Recessive White chicken, AB and BB genotype birds had significant higher shear force than AA genotype birds (P<0.05),AB genotype birds had slight higher shear force than BB genotype birds, but the difference of shear force between AB and BB genotype birds were not significant; Yet genotype effects were not significant in Qingyuan partridge chicken . On the SNPC6, it had significant breed effect,genotype effects were not significant in Recessive White chicken; But GA and AA genotype birds had significant higher shear force than GG genotype birds (P<0.05). On the SNPC3, The breed effect,genotype effect and the interaction effect between breed and genotype are not significant.The least square estimation between genotypes,breeds and the water holding capacity indicated: On the SNPC5, it had significant breed effect,genotype effects were significant in Recessive White chicken, AB genotype birds had significant higher water holding capacity than BB genotype birds (P<0.05). On the SNPC3 and SNPC6, The breed effect,genotype effect and the interaction effect between breed and genotype are not significant.(Note: SNPC3,SNPC5,SNPC6 stand for SNPs site of 3'UTR,exon 5,exon 6 of the CAPN1 gene, the following is by parity of reasoning. )3.The expression research for CAPN1 gene on chicken 's different growth phaseCAPN1 gene mRNA were detected in pectoral muscle and gastrocnemius muscle in different growth phase (0,2,4,6,8,10,12 week's age) of the Qingyuan partridge chicken and the Recessive White chicken. Various ages pectoral muscle expression patterns of Qingyuan partridge chicken and the Recessive White chicken was identical, and the peak of expression both appeared at 6 week's age; But within the gastrocnemius muscle, boundary was taken at 8 week's age, the expression patterns of two chicken breeds was identical 8 week's age later, and opposite tendency existed of two chicken breeds at 0 - 8 week's age, and the peak of Qingyuan partridge chicken expression in gastrocnemius muscle appeared at 2 week's age, but the peak of Recessive White chicken expression appeared at 6 week's age.The contrast results of the expression patterns of different tissue in identical chicken breed indicated: the expression patterns of Qingyuan partridge chicken in pectoral muscle and gastrocnemius muscle was identical at 0 - 2 week's age , and differences existed in two tissues 2 week's age later, and the peak of Qingyuan partridge chicken expression in pectoral muscle appeared at 6 week's age, but the peak of expression in gastrocnemius muscle appeared at 2 week's age. Within the Recessive White chicken, the expression patterns in pectoral muscle and gastrocnemius muscle was identical at 0 - 8 week's age , and differences existed in two tissues 8 week's age later, the peak of expression both appeared at 6 week's age.