Molecular cloning,sequence analysis and expression patterns of porcine CCT5 gene | 猪CCT5基因的cDNA克隆、序列分析及时空表达

中国人. 基于2-DE和MS的蛋白质组学方法分析梅山猪骨骼肌发育过程中蛋白质动态表达变化,发现CCT5差异表达。为进一步获得猪CCT5基因的序列特征、时空表达及生物学功能等相关信息,克隆猪CCT5基因,分析不同物种序列进化关系,检测CCT5在猪不同组织和骨骼肌不同发育阶段中的表达。序列分析结果表明:在获得的2 286 bp猪CCT5 cDNA序列中包括1 626 bp的完整开放阅读框(ORF),编码541个氨基酸,其氨基酸序列与人的相似性为97%,表明该蛋白在不同物种间高度保守。荧光定量PCR结果显示,CCT5基因在猪肌肉和脂肪中表达量最高,心、肝等组织中表达量很低,没有肌纤维特异性;不同发育阶段骨骼肌中,该基因在出生后3 d的表达量最高,胚胎期表达量最低;在不同品种间,胚胎期及出生后3、30和180 d的表达量大白猪显著高于梅山猪(P0.05),而60 d却显著低于梅山猪(P0.05)。结果提示:CCT5基因在猪骨骼肌发育过程中起着重要作用。

英语. The 2-DE and MS based proteomics approach was used to analyse the dynamic expression of proteins in Meishan pigs skeletal muscle development,and CCT5(chaperonin-containing t-complex peptide-1 subunit epsilon) was identified as one of differential expression proteins.The aim of this research was to obtain the information about the sequence structure,spatio-temporal expression and biology function of porcine CCT5 gene.In the present study,CCT5 was cloned by real-time PCR,the sequence was analysed by bioinformatics method,and the mRNA expression was detected by quantitative real-time PCR.Sequence analysis showed that the obtained 2 286 bp of porcine CCT5 cDNA contained the 1 626 bp opening reading-frame(ORF),encoding 541 acids,and the high sequence similarity of CCT5 between the pig and human was 97%.The tissue analysis showed that the mRNA expression was the highest in pig muscle and adipose tissue and very low in heart and liver tissue,but there was no specific muscle fibers.The result of different developmental stages expression analysis indicated that CCT5 mRNA had the highest expression in 3 days after birth and lowest in embryonic.Furthermore,comparing the different breeds,the mRNA abundance of CCT5 in Large White pigs was higher than in Meishan pigs at embryonic,3,30 and 180 day-old(P0.05),whereas at 60 d,the abundance in Meishan was higher than in Large White pigs(P0.05).These results suggest that CCT5 is probably playing a key role in porcine skeletal muscle development.