猪肉嫩度性状评价体系研究及嫩度相关新基因的克隆、时空表达谱分析
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摘要
本研究采集18头六月龄DLY商品猪的背最长肌样品,通过24、72、144小时三种不同时间嫩化处理得到54份嫩度性状表现有差异的样品。利用Warner-Bratzler剪切力测定、质构剖面分析(TPA)和感官评定,分析了各样品的质构性状,并分析了仪器测定与感官测定数据间的线性关系。Warner-Bratzler剪切力测定中得到的最大剪切力、W-B硬度和平均剪切力与感官硬度、弹性、多汁性间均存在显著或极显著(P＜0.05或P＜0.01)的相关性,其中最强的相关关系表现在最大剪切力与感官硬度间(r=0.69385,P＜.0001);TPA分析数据与感官测定指标间的相关程度较弱,最强的相关关系表现在感官硬度与TPA硬度间(r=0.46075,P=0.0047)。进一步采用主成分-逐步回归法,以仪器测定指标为自变量,感官评定指标为依变量进行回归分析,分别得到具有统计意义的感官硬度、感官弹性和多汁性的预测方程,其中对感官硬度的预测效果较好,方程决定系数达到0.6146;而对感官弹性和多汁性的预测效果较差,方程决定系数为0.3977和0.4234。
采用RT-PCR结合克隆测序的方法,从猪肌肉组织总RNA中克隆出了猪组织蛋白酶B(CTSB)基因的cDNA序列,并推导出其编码的氨基酸序列。猪CTSB基因开放阅读框(ORF)全长1008bp,编码335个氨基酸。同源性分析结果表明,猪与人、小家鼠、牛的CTSB基因cDNA编码区(CDS)同源性分别为85%、81%、90%,推测的氨基酸序列同源性分别为81%、79%、91%。利用同源性结合序列特征预测表明该蛋白具有信号肽和前肽序列。蛋白质结构同源建模分析表明,该蛋白具有木瓜蛋白酶家族的典型空间结构,包括1个底物结合凹槽和3个相互靠近的活性位点。利用直接测序的方法,分别在第六和第七外显子内发现两处未造成编码氨基酸改变的T-C同义突变位点,另外还采用PCR-SSCP方法,在237头个体中分析了CTSB基因第6内含子内的SSCP位点多态性,检测到3个等位基因6种基因型。FF基因型个体的各项嫩度指标最高,其最大剪切力与硬度值分别为6.79 kg和24.06 kg.s,极显著地高于EE和EF基因型个体(P＜0.01),平均剪切力为5.23 kg,显著地高于EF基因型个体(P＜0.05)。另外发现CTSB基因的一种转录变异体CTSB2,在5'非编码区内比野生型CTSB mRNA多出一段140 bp的插入序列,推测可能是由外显子可变剪切造成的。Real-Time PCR结果表明CTSB基因mRNA表达量受到组织、发育阶段和品种因素影响。表达量最高的组织为肾脏和肝脏,而在心肌和骨骼肌中低丰度表达,并且在三处不同部位的骨骼肌间无显著表达量差异。CTSB基因mRNA表达量在0～5月龄长白猪和梅山猪背最长肌中表现出先升高后降低的变化趋势,梅山猪峰值出现较早,并在4月龄后再次出现急剧上升趋势。而在藏猪背最长肌中则表现出持续降低的变化趋势,且该基因表达量极显著地高于其他两个品种。
同样采用RT-PCR结合克隆测序的方法,从猪肌肉组织总RNA中克隆出了猪肉嫩度性状候选基因—半胱氨酸蛋白酶抑制素B(Cystatin B,CTSB)基因序列,并推导出其编码的氨基酸序列。猪Cystatin B基因ORF全长297bp,编码98个氨基酸。同源性分析结果表明,猪与人、鼠、牛的Cystatin B基因cDNA编码区(CDS)同源性分别为81%、85%、89%,推测的氨基酸序列同源性为83%、76%、85%。蛋白质结构同源建模分析表明,该蛋白与人、鼠Cystatin B类似,具有stefin类蛋白酶抑制剂的典型空间结构,包括5条平行的β-sheet和负责与被抑制酶结合的楔形边缘。另外还采用PCR-RFLP方法,分析了237头个体CSTB基因第二内含子内PvuⅡ酶切位点的多态性,检测到了AA、AB和BB三种基因型,关联性分析表明AA基因型个体的各项嫩度指标均极显著低于另两种基因型的个体(P＜0.01),最大剪切力为5.76kg、硬度值为20.15kg.s、平均剪切力为3.66kg。Real-Time PCR试验结果表明CSTB基因mRNA表达量受到组织、发育阶段和品种因素影响.表达量最高的组织为肾脏和肠系膜淋巴结,而在心肌和骨骼肌中低丰度表达,股四头肌组织表达量极显著高于眼肌。CSTB基因mRNA表达量在0～5月龄梅山和2～12月龄藏猪表现出生后初期较高,随后逐渐降低的表达模式:而外种猪长白猪的表达模式则为出生后表达量急剧升高,2月龄达到峰值,随后逐渐降低。CSTB和CTSB两基因的mRNA表达量表现出较高的正相关,但相关程度受到品种因素影响。
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.
采用RT-PCR结合克隆测序的方法,从猪肌肉组织总RNA中克隆出了猪组织蛋白酶B(CTSB)基因的cDNA序列,并推导出其编码的氨基酸序列。猪CTSB基因开放阅读框(ORF)全长1008bp,编码335个氨基酸。同源性分析结果表明,猪与人、小家鼠、牛的CTSB基因cDNA编码区(CDS)同源性分别为85%、81%、90%,推测的氨基酸序列同源性分别为81%、79%、91%。利用同源性结合序列特征预测表明该蛋白具有信号肽和前肽序列。蛋白质结构同源建模分析表明,该蛋白具有木瓜蛋白酶家族的典型空间结构,包括1个底物结合凹槽和3个相互靠近的活性位点。利用直接测序的方法,分别在第六和第七外显子内发现两处未造成编码氨基酸改变的T-C同义突变位点,另外还采用PCR-SSCP方法,在237头个体中分析了CTSB基因第6内含子内的SSCP位点多态性,检测到3个等位基因6种基因型。FF基因型个体的各项嫩度指标最高,其最大剪切力与硬度值分别为6.79 kg和24.06 kg.s,极显著地高于EE和EF基因型个体(P＜0.01),平均剪切力为5.23 kg,显著地高于EF基因型个体(P＜0.05)。另外发现CTSB基因的一种转录变异体CTSB2,在5'非编码区内比野生型CTSB mRNA多出一段140 bp的插入序列,推测可能是由外显子可变剪切造成的。Real-Time PCR结果表明CTSB基因mRNA表达量受到组织、发育阶段和品种因素影响。表达量最高的组织为肾脏和肝脏,而在心肌和骨骼肌中低丰度表达,并且在三处不同部位的骨骼肌间无显著表达量差异。CTSB基因mRNA表达量在0～5月龄长白猪和梅山猪背最长肌中表现出先升高后降低的变化趋势,梅山猪峰值出现较早,并在4月龄后再次出现急剧上升趋势。而在藏猪背最长肌中则表现出持续降低的变化趋势,且该基因表达量极显著地高于其他两个品种。
同样采用RT-PCR结合克隆测序的方法,从猪肌肉组织总RNA中克隆出了猪肉嫩度性状候选基因—半胱氨酸蛋白酶抑制素B(Cystatin B,CTSB)基因序列,并推导出其编码的氨基酸序列。猪Cystatin B基因ORF全长297bp,编码98个氨基酸。同源性分析结果表明,猪与人、鼠、牛的Cystatin B基因cDNA编码区(CDS)同源性分别为81%、85%、89%,推测的氨基酸序列同源性为83%、76%、85%。蛋白质结构同源建模分析表明,该蛋白与人、鼠Cystatin B类似,具有stefin类蛋白酶抑制剂的典型空间结构,包括5条平行的β-sheet和负责与被抑制酶结合的楔形边缘。另外还采用PCR-RFLP方法,分析了237头个体CSTB基因第二内含子内PvuⅡ酶切位点的多态性,检测到了AA、AB和BB三种基因型,关联性分析表明AA基因型个体的各项嫩度指标均极显著低于另两种基因型的个体(P＜0.01),最大剪切力为5.76kg、硬度值为20.15kg.s、平均剪切力为3.66kg。Real-Time PCR试验结果表明CSTB基因mRNA表达量受到组织、发育阶段和品种因素影响.表达量最高的组织为肾脏和肠系膜淋巴结,而在心肌和骨骼肌中低丰度表达,股四头肌组织表达量极显著高于眼肌。CSTB基因mRNA表达量在0～5月龄梅山和2～12月龄藏猪表现出生后初期较高,随后逐渐降低的表达模式:而外种猪长白猪的表达模式则为出生后表达量急剧升高,2月龄达到峰值,随后逐渐降低。CSTB和CTSB两基因的mRNA表达量表现出较高的正相关,但相关程度受到品种因素影响。
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.
引文
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