Characterization Of CAPN3Splicing Variants From The Skeletal Muscles And Association Between CAPN3Polymorphisms And Meat Quality Traits In Cattle

CAPN3is a member of the calpain family of Ca2+-regulated cysteine proteases. In cattle, itplays an important role in sarcomere remodelling and mitochondrial protein turnover, thusregulating beef tenderness. At present, multiple transcripts produced by the CAPN3gene （CAPN3）have been detected in human, monkey, rat and rabbit, however, it remains unknown in cattle.Here, we identified three CAPN3transcripts from the skeletal muscle in the certain cattleindividuals by RT-PCR. While one transcript corresponds to the known full-length which wasnamed CAPN3a, one transcript absents from exon2to exon19as well as a single nucleotideinsert in the penultimate of exon1compared to CAPN3a, named CAPN3b, and another transcriptinserts intron16, named CAPN3c. CAPN3a could be detected in all individuals, but CAPN3b onlyexist in an individual of Changbaishan Black cattle and CAPN3c present in the certain individualsof each species of cattle. This explained that the CAPN3mRNA splicing is not exactly the same indifferent varieties of cattle, the splicing in different individuals of the same species are alsodifferences. To determine the transcription levels of three CAPN3transcripts, Gray-scale analysisor real-time RT-PCR was carried out. The results showed that the expression level of CAPN3a wasabout48-fold compared to CAPN3b and12-fold compared to CAPN3c. The amino acid sequencesof CAPN3splice variants were predicted. Translation of the CAPN3a leads to a94kDa protein of822amino acids. However, the predicted CAPN3b protein only had the N-terminal103aminoacids of the CAPN3a protein and an additional19amino acids, but lacked all of other domains.The IS1and IS2regions and the cysteine, histidine and asparagine residues of the active site. TheCAPN3c protein lacked domains IV, in which consist of five EF hand structures, and the IS2regions was destruct. The alteration of amino acids structures to be bound to affect the expressionof protein function, and needs to be further studied.CAPN3gene polymorphism was detected by PCR-SSCP and sequencing for detected SNPsin Chinese Red Steppes, Simmental, Limousin and Changbaishan Black Cattle. Correlativitybetween CAPN3genetic polymorphism and meat quality traits was analysed with SPSS software.The result indicated that a SNP （G→T） was found in intron12（NC₀07308）in Chinese RedSteppes and Changbaishan Black Cattle. Moreover, the individual of different genotype hadsignificant deviation on marbling, water lose rate, drip loss and shearing force.In summary, we described the CAPN3mRNA splicing variants and gene expression in skeletal muscle, in addition, anaylzed the CAPN3gene polymorphism and correlation with meatquality traits. These results of the study provided a theoretical basis for analyze the geneticcharacters of CAPN3gene and genetic markers of productive traits in cattle.