Porcine Troponin â…  Gene Family: Genomic Structure, Expression Profiling And Polymorphism Analysis

Troponin I（TnI） is one constituent protein of the troponin complex located on the thin filament of striated muscle that involves in the calcium-mediated regulation of striated muscle contraction.Three isoforms of the TnI are encoded by distinct genes that are expressed in a tissue-specific and developmentally regulated manner.The slow skeletal isoform（TNNI1） is expressed in both cardiac muscle and slow twitch skeletal muscle fibers during development,but is restricted to slow twitch skeletal muscle fibers in the adult.The fast skeletal isoform（TNNI2） is expressed in fast twitch skeletal muscle fibers.The cardiac isoform（TNNI3） is expressed exclusively in cardiac muscle.Their specificity of muscle fibers and the differential expressions are likely to exist in the differences of regulatory mechanism.So far,there are no reports about the genomic sequences and expression rules of the porcine TnI family.Here,we isolated and characterized the porcine TnI family.The results were as follows:1.The full-length cDNA of the TNNI1,TNNI2 and TNNI3 genes of TnI family were obtained and deposited under the GenBank accession number FJ719786,FJ719788 and FJ719790,respectively.They shared 54.4%- 58.3%similarity with each other in their predicted amino acid sequences.The phylogenetic tree showed a closer relationship between TNNI1 and TNNI3 proteins.2.The full-length genome sequences of the TnI genes were cloned and deposited into the GenBank under the accession number FJ719787（TNNI1）,FJ719789（TNNI2）,TNNI3 （TNNI3）.The three genes spanned over 9785 bp,2373 bp and 3648 bp genomic region,respectively.Comparison of the structure of the porcine TnI genes with that of human and mouse revealed that they had the same intron/exon organization.3.A PCR- Sma I-PFLP was developed to detect a mutation of Tâ†'C（g.1697 T＞C） in intron 3 of TNNI2 gene,and allele frequencies were determined among the ten different pig breeds.Association analysis was performed between polymorphisms and some important production traits in Yorkshire×Meishan F₂ offspring.There were significant associations between TNNI2- Sma I-RFLP genotypes and IFR（p＜0.05）, LMP（p＜0.05）,CL（p＜0.05）,LEH（p＜0.05）,LEA（p＜0.05）,BFT3（p＜0.05）,ABF （p＜0.05）,FP（p＜0.01） and BFT2（p＜0.01）.4.The promoter sequences of porcine TnI genes were obtained and the length is 1059bp, 499bp and 1075bp,respectively.The regulatory elements in the proximal promoter of TNNI2 and TNNI3 were conserved among human,mouse and pig.Nevertheless, regulatory elements of porcine TNNI1 promoter exhibited no homology with human and mouse.5.The expression of TnI genes in Yorkshire and Meishan breeds at six periods（fetal 65d, and after birth 3d,35d,60d,120d,180d） during three muscle fiber types（longissmus dorsi muscle,named LD;semitendinosus,ST;cardiac muscle,CM） were analyzed.At most of stages after birth,the expression of TNNI1 and TNNI2 were significantly higher in fast-twitch fibers dominated muscle（LD） than that in slow-twitch fibers dominated muscle（ST）.The relative expression of TNNI2 was constantly higher than that of TNNI1 in ST and LD,but on the contrary in the cardiac muscle.The TnI genes were widely expressed in the tissues studied,with the highest expression level of TNNI1 and TNNI2 in skeletal muscle,and TNNI3 in cardiac muscle.