Molecular Cloning,Characterization And Tissue Expression Analysis Of Three Slow Skeletal Muscle Troponin Genes In Small-tailed Han Sheep

Troponin C type 1(TNNC1), troponin I type 1(TNNI1) and troponin T type 1(TNNT1) are slow skeletal muscle troponin genes. Early study showed that the genes are not only related to skeletal muscle contraction but are also associated with slow skeletal muscle growth, muscle fiber characteristics and cardiomyopathy. The full-length cDNA sequences of these troponin genes in the sheep have not been reported. In the present work, the complete cDNA sequence of the genes from the Small-tailed Han sheep was cloned using reverse transcription-polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(RACE). The gene structures and characteristics of the predicted amino acids sequences were analyzed using bioinformatics analysis software. Gene expression analyses were performed using quantitative reverse transcription PCR(qRT-PCR). All of the works are to explore the main structure and the expression characteristics of the genes of Small-tailed Han sheep from the molecular level. The main results were showed as follows:(1)The cDNA sequences of the TNNC1, TNNI1 and TNNT1 genes from Small-tailed Han sheep were cloned and sequenced. Especially, the TNNI1 gene contains two different 5' end sequences which were named as TNNI1 a and TNNI1 b. The full-length cDNA sequences of the TNNC1, TNNI1 a and TNNT1 genes and the partial cDNA sequence of the TNNI1 b gene were submitted to Gen Bank under accession numbers KR153938, KT218688, KT218689 and KT218690, and encoded 161, 187, 194, 263 amino acids, respectively.(2)The cTnC protein was weakly acidic, stable in structure and had good hydrophilicity and high mobility. The two isoforms of ssTnI protein were both slightly alkaline, stable in structure and had good hydrophilicity and high mobility. The ssTnT protein was weakly acidic, unstable in structure and had good hydrophilicity and high mobility. The proteins were predicted to be non-secretory proteins with several phosphorylation sites and without any transmembrane helices and N-glycosylation sites. The secondary structural analysis predicted that the proteins were mainly composed of alpha helices and random coils.(3)Multiple alignments demonstrated that the cTnC protein was highly conserved between the investigated species. ssTn I and ssTnT were highly similar to the analogous proteins in other mammals and showed little differences when compared to the analogous proteins in non-mammalian species. Phylogenetic trees showed that for cTnC protein, Ovis shared the closest evolutionary relationship with S. scrofa, B.taurus and C. hircus. For ssTn I protein, the closest evolutionary relationships were observed with C. hircus and B.taurus, and for ssTnT protein, the closest relationships were with S. scrofa and B. taurus. Multiple alignments and phylogenetic tree analyses showed that the proteins were relatively conserved in mammals.(4)The expression levels of TNNC1, TNNI1 and TNNT1 differed among the eight tissues of the two sheep species; however, the pattern of mRNA expression was consistent. All of these genes showed higher expression levels in the intercostal muscles(IM), biceps femoris(BF) and longissimus dorsi(LD), and the expression level is IM>BF>LD. The expression level of TNNI1 a in Small-tailed Han sheep and Dorper sheep in each tissue was higher than that of TNNI1 b in various tissues. Additionally, differences were observed between the two sheep breeds. TNNI1 a was expressed at a significantly higher levels in the intercostal muscles of Small-tailed Han sheep compared with Dorper sheep(p<0.05). The cTnC, ssTn I and ssTnT proteins existed expression in longissimus dorsi, intercostal muscles, biceps femoris muscle. In addition, the cTnC protein existed expression in the heart, but ssTnI and ssTnT proteins in the heart not exsited expression.In summary, this study successfully cloned TNNC1, TNNI1 and TNNT1 in Small-tailed Han sheep and studied in a systematic way. Exploring the characteristics and functions of these genes at the molecular level would provide basic theoretical foundation for the subsequent study of the gene function and molecular breeding.