Studies On Pork Tenderness Evaluation System And Cloning, Spatiotemporal Expression Profiles Analysis Of Novel Genes Related To Tenderness

Two methods for assessing tenderness characteristics of pork,Warner-Bratzler(W-B) and texture profile analysis(TPA),were tested in 54 samples of in.longissimus dorsi muscle of 18 DLY commercial pigs,aged 24,72 and 144 hours post-mortem.A trained panelists sensory analysis was also performed on 54 samples.The Warner-Bratzler measures of Shear Force, Toughness,and Mean Shear Force were moderately correlated(P＜0.05 or P＜0.01) with trained panel sensory characteristics of Hardness,Springiness,and Juiciness.Sensory characteristics of Hardness had the highest correlations values with Shear Force(r=0.69385,P＜.0001).The TPA measures were weakly correlated with sensory texture characteristics.Sensory characteristics of Hardness had the highest correlations values with TPA Hardness(r=0.46075,P=0.0047).Principal component-Stepwise regression analysis generated prediction equations that included the TPA measures and Warner-Bratzler measures,which accounted for 61.46,39.77 and 42.34%of the variation in sensory Hardness,Springiness,and Juiciness,respectively.Instrumental measures explained more of the variation in subjective sensory measures of hardness than of Springiness and Juiciness.The porcine cathepsin B(CTSB) nucleotide and amino acid sequence were obtained by RT-PCR and sequencing.The open reading frames(ORFs) were 1008 bp long(encoding for 335 amino acids).The nucleotide sequence identities of porcine cathepsin B ORFs were 85%,81%and 90% compare to that of the human,rat,and bovine cathepsin B ORFs respectively.The deduced amino acid sequence identities of the porcine cathepsin B protein were 81%,79%,and 91% compare to that of the rat,human,and bovine cathepsin B proteins respectively.The structure of cathepsin B protein was predicted by homologous modelling which was similar to other papains. The substrate-binding cleft and the catalytic triad were found in the structure model.A splice variant CTSB2 were cloned from skeletal muscle and liver,which had a 140bp long insertion sequence in 5'UTR.Two synonymous mutation loci were detected in exon6 and exon7.The polymorphisms of porcine cathepsin B gene was detected in 237 pigs by PCR-SSCP.There were six genotypes be detected on the SSCP locus.The genotype FF on CTSB gene SSCP locus had the highest tenderness traits,it had significantly higher Shear force(6.76 kg) and Toughness(24.06 kg.s) than genotype EE and EF(P＜0.01),and it had significantly higher Mean shear force(5.23kg) than genotype EF(P＜0.05).The tissue- and developmental-specific expression patterns of CTSB mRNA were revealed by Real-time PCR analyses.In adult pigs,CTSB mRNA expression was widely detected in many tissue.The highest levels of expression occurred in kidney and the lowest levels of expression were in myocardium and skeletal muscle,respectively.There were no significant differences in the expression of CTSB among muscles from three different location. The expression of CTSB mRNA in m.longissimus dorsi muscle was elevated at first then depressed between 0 and 5 month old of Landrace and Taihu pigs.The peak of expression was occur earlier in Taihu than in Landrace.A continuously decreased expression pattern were detected between 2 and 12 month old of Tibeten pigs.Utilizing technique of RT-PCR and sequencing,the nucleotide and deduced amino acid sequence of the porcine cystatin B(CSTB) gene were obtained in this study.The open reading frames(ORFs) of the CSTB gene were 297 bp long(encoding for 98 amino acids).The nucleotide sequence identities of porcine CSTB Complete Coding Sequence(CDS) were 81%,85%and 89% compare to that of the human,rat,and bovine CSTB CDS respectively.The deduced amino acid sequence identities of the porcine CSTB protein were 83%,76%,and 85%compare to that of the rat,human,and bovine CSTB proteins,respectively.The structure of CSTB protein was predicted by homologous modelling which was similar to the results observed previously in humans and mouse.The five-stranded antiparallelβ-sheet and the wedge shaped edge were found in the structure model.The polymorphisms of porcine CSTB gene was detected in 237 pigs by PCR-RFLP.Base on the polymorphism detection,the influence of CSTB genotype on tenderness traits were analyzed.The genotype AA in CSTB gene PvuⅡlocus had significantly lower Shear force(5.76 kg),Toughness(20.15 kg.s) and Mean Shear force(3.66 kg) than genotype AB and BB (P＜0.01).In adult pigs,CSTB mRNA expression was widely detected in many tissue.The highest levels of expression occurred in kidney and the lowest levels of expression were in myocardium and skeletal muscle,respectively.There were no significant differences in the expression of CSTB among muscles from three different location.Continuously decreased expression patterns were detected among different month in m.longissimus dorsi muscle of native Taihu and Tibeten pigs. And in Landrace pig the expression was soar at 2 month old,then depressed.The expression of CSTB mRNA were highly correlated with CTSB mRNA,and the coefficients of correlation depended on the breed.