Molecule Clone, Expression And The Genetic Effects In Meat Quality Of The Chicken Calpain 1, 2, 3 And Calpastatin Genes

The calpain system is ubiquitous in cells which mainly including calpain and calpastatin. Calpains(CAPN) are intracellular cysteine proteases activated in a Ca²⁺-dependent manner and are involved in a variety of cellular processes,and play a key role in muscle protein degradation in living tissue and in postmortem tenderization.Calpastatin(CAST) is a specific,endogenous inhibitor of the calpains activated by Ca²⁺ inhibiting both forms of calpains(low calcium-requiring form called u-calpain and a high calcium-requiring form called m-calpain),and inhibits the decomposability of protein in muscle and reduces the growth speed of muscle cell.Meanwhile,it can inhibit the activity of calpain and depress the hydrolyzation of protein after slaughter.Researches showed that the calpain system gene taken part in the renovation of protein during muscle growth.And it has been found that calpain system gene was associated with muscle growth and meat tenderness in livestock.In this research,Sichuan Mountainous Black-Bone chickens were used.We successfully obtained the coding sequence of CAPN1,CAPN2,CAPN3 and CAST gene. GenBank assigned them accession numbers FJ232589,FJ232590,FJ497056 and FJ232592, respectively.Based on these four cDNA sequences,four amino acid sequences were obtained,their lengths were 705aa,700aa,810aa and 768aa.We found CAPN1,CAPN2, CAPN3 and CAST protein maybe a drophilic and transmembrane protein,respectively. Meanwhile,they were of catalysis domain and common characteristic for calpain family. The chicken CAPN1,CAPN2,CAPN3 amino acid sequences had high identity with that of other animal by bioinformative analysis,but CAST was opposite.Sichuan Mountainous Black-Bone chicken,Tibetan chicken and line S01 of Dahen chicken were used.We adopted the real-time PCR(SYBR greenâ… ) method to test the expression quantity of CAPN1,CAPN2,CAPN3 and CAST gene in the breeds mentioned above at the first day and the 70th day after birth.And we also tested the expression quantity of CAPN1,CAPN2,CAPN3 and CAST gene in different tissues at certain growth point,that in different growth points at certain tissue and had correlation analysis of these relative quantities and traits in Sichuan Mountainous Black-Bone chicken.The results indicated that CAPN1,CAPN2,CAPN3 and CAST gene expression in all tissues at all growth points and there was advantageous tissue for gene expression at each growth point. CAPN1,CAPN2,CAPN3 and CAST gene expression in a certain tissue was different in different breeds.The CAPN1,CAPN2,CAPN3 and CAST gene expression in each tissue in Tibetan chicken and line S01 was higher than that of Sichuan Mountainous Black-Bone chicken except for CAST gene expression in Glandular and Muscular stomach in Tibetan. The results also indicated that the developmental change of CAPN1 gene expression quantity in breast muscle and leg muscle was positively related with the cumulative values of living weight(P<0.05),and the developmental change of CAPN2,CAPN3 and CAST gene expression quantity in breast muscle and leg muscle were not related with the cumulative values of living weight(P>0.05),but there was also advantageous growth point in breast muscle tissue for CAPN1,CAPN3 and CAST gene expression.The expression level of CAPN1 gene in breast muscle had "rise" trend in total,CAST gene was opposite, and CAPN3 gene had "rise-decline" trend in total.It was similar with the theoretical expression of CAPN1,CAPN3 and CAST gene.Study on the ratio of the expression quantity of CAPN1 gene to CAST and CAPN3 gene along with the growth points in Sichuan Mountainous Black-bone chicken discovered that the ratio in breast muscle increased firstly,then deceased and increased in last,respectively.It was concluded that the developmental change of CAPN1,CAPN3 and CAST gene expression quantity were tightly correlated with the development change of muscle growth,and them may have key role in turns in control chicken muscle protein metabolism.In this study,Dahen high-quality chicken in Sichuan province,Huxu chicken, Xinghua chicken,Qingyuan ma chicken,Xiayang chicken in Guangxi province,totally 310 samples were used.We screened all the extrons and part of intron sequences of CAPNI gene and four single nucleotide polymorphisms(SNP) were obtained.They were located in the 5th exon,the 6th exon,the 16th exon and 3'-untraslated region.SNP1(C/T) was at position 2546nt,SNP2(G/A) was at position 3535nt,SNP3(C/A) was at position 7198nt and SNP4(G/A) was at position 9950nt(GenBank Accession NO:NC₀06090.1).All the SNPs detected in the current study were novel and synonymous SNPs.Sixteen halplotypes were constructed from the 4 SNPs.Three main haplotypes were found including H1 (C-G-C-G),H2(C-G-C-A)and H5(C-A-C-G).The effect of halplotype on carcass traits in chickens were analyzed,the results showed that halplotype had significant genetic effect on living weight,carcass weight,semi-eviscerated percentage,the percentage of breast muscle,leg muscle weight and the percentage of leg muscle(P<0.05).H1 had a negative effect on muscle growth,H9(T-G-C-G) and H13(T-A-C-G) had a positive effect on muscle growth.We screened all the extrons and part of intron sequences of CAPN3 gene and two single nucleotide polymorphisms(SNP) were obtained.They were located in the 8th intron and 10th exon.SNP1(T/A) was at position 11818nt and SNP2(T/G) was at position 12814nt(GenBank Accession NO:NC₀06092.2).All the SNPs detected in the current study were novel and synonymous SNPs.Four halplotypes were constructed from the 2 SNPs and four haplotypes ineluding H1(T-G),H2(T-T),H3(A-G) and H4(A-T) were accounted for 100%of all the observations.The effect of halplotype on carcass traits in chickens were analyzed,the results showed that halplotype had significant genetic effect on semi-eviscerated percentage,the percentage of breast muscle and abdominal fat weight (P<0.05).Haplotype H1(T-G) had a negative effect on muscle growth,Haplotype H2 (T-T) had a positive effect on muscle growth and had a negative effect on abdominal fat weight.We screened all the extrons and part of intron sequences of CAST gene and three single nucleotide polymorphisms(SNP) were obtained.They were located in the 8th exon, 11th exon and 11th intron.SNP1(T/C) was at position 36127nt,SNP2(A/T) was at position 37752nt and SNP3(G/A) was at position 37868nt(GenBank Accession NO: NC₀06127.2).All the SNPs detected in the current study were novel and synonymous SNPs.Eight halplotypes were constructed from the 3 SNPs.Four main haplotypes were found including H1(T-A-G),H2(T-A-A),H3(T-T-G) and H4(T-T-A).The effect of halplotype on carcass traits in chickens were analyzed,the results showed that halplotype had significant genetic effect on living weight,carcass weight,semi-eviscerated percentage,eviscerated percentage,breast muscle weight,leg muscle weight,the percentage of breast muscle and the percentage of leg muscle(P<0.05).H3 had a positive effect on muscle growth,H5(C-A-G) and H6(C-A-A) had a negative effect on muscle growth.So it was concluded that the CAPN1,CAPN3 and CAST gene may be three major gene affecting carcass traits of chicken or it is linked with the major gene.The effect of single locus on meat quality traits in chickens were analyzed,the results showed that CAPN1 gene had significant genetic effect on warner-bratzler shear force and water depletion rate,CAPN3 gene had significant genetic effect on water depletion rate and CAST gene had significant genetic effect on inosine monophosphate(P<0.05).So further it is essential to confirm the association between the alleles or haplotypes of CAPN1, CAPN3 and CAST polymorphisms and carcass and meat quality traits in chicken.