Cloning,Bioinformatics Analysis and Spatiotemporal Expression of CKMT2 Gene in Jianzhou Big-eared Goats
[Objective]This study was aimed to clone the creatine kinase mitochondrial 2(CKMT2)gene and explore its biological characteristics,and clarify its expression characteristics in different tissues and myoblasts of different differentiation stages of Jianzhou Big-eared goats.[Method]The samples of heart,liver,spleen,kidney,longissimus dorsi muscle and triceps brachii muscle in Jianzhou Big-eared goats were collected.The CKMT2 gene was amplified by PCR method,cloned and sequenced,and bioinformatic analysis was conducted by online software.Real-time quantitative PCR was used to detect the expression of CKMT2 gene in different tissues and myoblasts of different differentiation stages in Jianzhou Big-eared goats.[Result]The total length of CKMT2 gene in Jianzhou Big-eared goats was 1 314 bp,including 1 259 bp in CDS region,encoding 419 amino acids,which was closely related to Ovis aries and Oryx dammah.CKMT2 protein was an alkaline hydrophophilic stable protein without signal peptide and transmembrane domain,mainly located in mitochondria,peroxasome and cytoplasm,which contained 32 phosphorylation sites,1 N-glycosylation site and 4 O-glycosylation sites.The secondary structure of the protein mainly consisted of alpha helix(39.14％),random coil(36.28％),extended chain(16.23％)and beta turn(8.35％),and the tertiary structure was basically consistent with the predicted secondary structure.The results of protein interaction analysis showed that CKMT2 protein was associated with cysteine and glycine-rich protein 3(CSRP3),creatine kinase M-type(CKM)and phosphoglycerate mutase 2(PGAM2)and other proteins.The results of tissue expression profile showed that the expression of CKMT2 gene was the highest in heart of Jianzhou Big-eared goats,which was significantly higher than that in other tissues(P＜0.05).The expression of longissimus dorsi muscle was higher than that of spleen and triceps brachii muscle(P＜0.05).The results of time series expression profile showed that the expression of CKMT2 gene in Jianzhou Big-eared goats reached a peak at 4 days after differentiation,which was significantly higher than 0 and 6 days(P＜0.05).[Conclusion]CKMT2 gene sequence was successfully cloned and its molecular characteristics were clarified,and its expression was higher in heart,longissimus dorsi muscle and myoblast at 4 days of differentiation.The results laid the foundation for further elucidating the molecular mechanism of CKMT2 gene regulating the proliferation and differentiation of myoblasts in Jianzhou Big-eared goats.
万方数据
维普
