Molecular Cloning,Characterization And Tissue Expression Analysis Of The TNNI2 And TNNI3 From Small-tailed Han Sheep

Troponin I(Troponin I,TnI)is essential to exercise the functions of normal muscle contraction,it can also affect many aspects of muscle quality.Troponin I family had three members,Troponin I1(TNNI1),Troponin I2(TNNI2)and Troponin I3(TNNI3).This experiment has been based on Small-tailed Han sheep and Dorper sheep transcriptome resequencing data,selecting differentially expressed genes TNNI2 and TNNI3.The full-length cDNA of the TNNI2 and TNNI3 genes were cloned from Small-tailed Han sheep by rapid amplification of cDNA ends-PCR.The Structure and characteristics of cDNA and protein were analyzed by bioinformatics software.m RNA expressing was analyzed by qRT-PCR.The results are as follows:(1)The full length was cloned from Small-tailed Han Sheep by methods of reverse transcription PCR and rapid amplification of c DNA ends.The obtained TNNI2 full-length cDNA of 714 bp which contained a open reading frame of 549 bp,a 88 bp 3 ‘end untranslated region(not including the polyA tail),a 5 ‘end untranslated region of 77 bp.The gene encodes 182 amino acids.The full-length TNNI3 cDNA is 847bp(not including the polyA tail).It have an open reading frame of 639 bp,3 ‘end untranslated region of 65 bp and 5’ end untranslated region of 143 bp.It encodes 212 amino acids.The GenBank accession numbers of the full-length TNNI2 and TNNI3 cDNA sequences are KX110054 and KX110055,respectively.(2)Bioinformatics analysis showed that TNNI2 amino acid sequences are highly similar to those of other vertebrates.The predicted molecular weight is 21.4kDa and the isoelectric point is 8.8,it is a good hydrophilic basic protein;The secondary structure containing more than 70% α-helix and a small amount of random coil;there is no predicted signal peptide sites;it has an O-glycosylation sites and nine phosphorylation sites.Sheep is highly homologous with Cattle from the point of TNNI3 protein.The predicted molecular weight of TNNI3 protein is 24.1kDa and the isoelectric point is 9.8,it is a good hydrophilic basic protein;its secondary structure have a high similarity with TNNI2,the proportion of α-helix of 70.75%;It is predicted has no signal peptide site;the protein has 14 phosphorylation sites,but no glycosylation sites.(3)The expression of two genes has a big difference in mRNA levels.TNNI2 was mainly detected in intercostal muscles,longissimus and biceps femoris.These data indicates that TNNI2 gene has higher specificity in expression.TNNI2 was expressed to a higher level in the Intercostal muscles of Dorper sheep than in Small-tailed Han sheep(P <0.01);TNNI2 was expressed to a higher level in the biceps femoris and lung of Dorper sheep than in Small-tailed Han sheep(P <0.05);the difference in longissimus muscle,liver,stomach and heart tissue was not notable.TNNI3 was mainly detected in myocardial.TNNI3 was expressed to a higher level in the of myocardial Dorper sheep than in Small-tailed Han sheep(P <0.01).Other organizations do not express the gene.(4)TNNI2 protein has a different expressionin in the blood vessels,longissimus,intercostal muscle,biceps femoris,heart,liver and lung.TNNI2 specifically expressed in muscle tissue.TNNI3 specifically expressed in the myocardium.In summary,this study successfully cloned TNNI2 gene and TNNI3 gene,analyzed the structural characteristics of genes and they deduced potein and the expression abundance at transcription levels in different tissues.The whole results may provide some basis for subsequent research in explaining the function of TNNI2 and TNNI3.