The Study On Sequence Characterization, Polymorphisms And Developmental Expression Of Three Candidate Genes Correlated With Sheep Meat Quality Traits

Small-tailed Han sheep,Tan sheep and Inner Mongolia sheep are the major sheep breedsin north China,and also are the main genetics resources for cultured the high quality meatproductive sheep breeds in the future.The aim of this study was to scan reliable meat qualitymolecular markers in three Chinese native sheep breeds and provide theoretical basis forselective breeding of meat sheep.The encoding sequence of sheep TNNC2,FABP4 andDGAT1 genes were isolated.The associations between polymorphisms of the three candidategenes and meat quality traits were analyzed extensively in 286 lambs by means of PCR-SSCPand PCR-RFLP methods.Additionally,the expression pattems of candidate genes wereexamined in four stages of Tan sheep by real time RT-PCR.The results are as following:1.The encoding sequence of sheep TNNC2 gene was cloned(GenBank accessionnumber EU239357),and the length of sequence was 483bp which contained an open readframe(ORF)coded 160 amino acids.The analysis demonstrated that the amino acid sequenceof sheep TNNC2 protein showed 99%,99%,98%,98% and 89% identity,with Bos taurus(NP₀01069841),Homo sapiens(CAG46682),Mus musculus(NP₀33420)and Gallusgallus(NP₉90781),respectively.It implied the sheep TNNC2 gene is conserved inevaluation.2.The full length cDNA of sheep FABP4 gene was cloned.The 399bp fragmentcontained an ORF coded 132 amino acids.Alignment indicated the identity of amino acidsequence of sheep FABP4 protein was 86%,86%,93%,84%,72% and 74%,compared withMus musculus(CAJ18597),Homo sapiens(CAG33184),Bos taurus(NP₇76739),Porcine(ABK58164),Gallus gallus(NP₉89621)and Anas platyrnynchos(ABC96712),respectively.3.The full length cDNA of sheep DGAT1 gene was cloned.The 1470bp fragmentcontained an ORF encoded 489 amino acids.Compared with Homo sapiens(NP₀36211),Mus musculus(AAH03717),Porcine(AAM66767)and Bos taurus(NP₇77118),the identityscore of sheep DGAT1 protein sequence was 88%,84%,92% and 98%,respectively.4.A deletion(T/-)of T base pair was detected at 7bp in intron 1 of sheep TNNC2 gene,which was named allele D.The frequency of allele D deleted was 0.78,0.77 and 0.71 in Small-tailed Han sheep,Tan sheep and Inner Mongolia sheep,respectively,and thedistribution of gene and genotype were no significant difference among the three sheeppopulations by X² test(p＞0.05).The association analysis between the genotypes and meatquality traits indicated the genotypes significantly influenced sheep meat quality.Comparedwith TT genotype,the DD and TD genotypes demonstrated higher carcass weight,drip lossrate and share force(p＜0.05),and significantly lower in marbling score and pH value(p＜0.01),but there was no significant difference in all meat traits between DD and TDgenotype.5.A mutation(A＞G)was detected at 219bp in intron 1 of sheep FABP4 gene byPCR-SSCP and sequencing.Frequency of allele A was 0.68,0.57 and 0.60 in Small-tailedHan sheep,Tan sheep and Inner Mongolia sheep,respectively.The gene and genotypefrequencies have no significant difference among the three sheep populations after X² tested(p＞0.05)..Analysis of association of genotypes and meat quality traits indicated the AA andAG genotype lambs have higher intramuscular fat content(p＜0.05;IMF),compared with GGgenotype.The shear force of AG and GG genotypes were higher than AA genotype but nosignificant difference was observed between AG and GG genotypes.The longissimus dorsi(LT)marbling score of AA genotype was higher than GG genotype,significantly.Thegenotypes of FABP4 gene did not influence the body weight,carcass weight,loin-eye area,net meat percentage,drip loss rate,pH value and fat thickness in 7th rib of 90 days old lambs(p＞0.05).6.A silent mutation(T＞C)was detected in exon 17 at 1461bp of sheep DGAT1,themutation created/deleted a restriction site of Alu I restriction endonuclease and the mutationhas been confirmed by PCR-RFLP.The frequency of allele T was 0.62,0.59 and 0.74 inSmall-tailed Han sheep,Tan sheep and Inner Mongolia sheep,respectively.The gene andgenotype distribution of DGAT1 gene have no significant difference among the threeexperimental sheep populations(p＞0.05).The genotype effect of meat quality traits andanalysis demonstrated that the IMF and marbling score of TT genotype in sheep were higherthan those of CC genotype in sheep,but the drip loss rate and share force of TT genotype waslower compared with CC genotype(p＜0.05).The locus did not affect the LT meat color,pH,90-days body weight,loin-eye area,GR value,fat thickness in 7th and net meat percentage ofsheep(p＞0.05).7.The examination of candidate gene expression patterns indicated that TNNC2 washigh expression in sheep LT muscle,but the FABP4 and DGAT1 were low.The mRNAcontent of FABP4 and DGAT1 gene in LT muscle were only about 1% ofβ-actin mRNAcontent. 8.Among stages of 120,160 and 200 days old,the LT muscle mRNA content of TNNC2and FABP4 gene were increased with the sheep growth(p＜0.05).The mRNA content ofDGAT1 gene was no obvious regular in 90,120,160 and 200 days old,there were nosignificant difference of mRNA content in all stages except for between 120～160 days old.