CDNA Cloning And Tissue Expression Of CAPN1 Gene And CAST Transcript â…¡ In Goat

The calpain system was closely related with meat quality in livestock, it mainly comprised calpain and calpastatin. The calpain system was involved in many physiological procedure, such as cell signal transduction, cell proliferation and muscle growth. especially in the renovating of the myofibril and postmortem tenderization of muscle. Calpains (CAPN) are widely distributed and are intracellular cysteine proteases activated in a Ca2+-dependent manner, Calpains play a key role in muscle protein degradation in postmortem tissue and were responsible for proteolysis of those myofibrillar proteins. Calpastatin (CAST) is a specific, endogenous inhibitor of the calpains activated by Ca2+, it can inhibit the activity of calpain and reduce the hydrolyzation of protein after slaughter. Researches have showed that calpain system was associated with meat tenderness, meanwhile, CAPN and CAST can be regarded as important candidate genes for meat quality character.Based on bovine and ovine mRNA sequences, T-A and RACE technique were used to clone CAPN1 and CASTⅡgene of goat, respectively. We obtained cDNA sequences of CAPN1, which was 2267 bp in length with an open reading frame (ORF) 2151 bp long and encoded 716 amino acids, cDNA sequences of CASTⅡwas 2474 bp in length with an open reading frame (ORF) 1695 bp long and encoded 564 amino acids, and there were four conserved domains and one conserved seven-peptide domain in amino acids sequences. both of them were submitted to NCBI and two Genbank ID were obtained(CAPN1:HQ718593, CASTⅡ:HM053645), this was the first report of gene sequence of CAPN1 and CASTⅡin goat. Bioinformatics analysis showed that the identity of CAPN 1 amino acids in goat was high when compared with other species, especially with mammals, but the identity of CASTⅡin goat was low when compared with other species, except sheep and cattle; both CAPN1 and CASTⅡprotein are hydrophilic and non-transmembrane; phosphorylation sites prediction showed that amino acids sequence of CAPN1 contained 40 phosphorylation sites totally,24 Ser phosphorylation sites,8 Thr phosphorylation sites,8 Tyr phosphorylation sites, CASTⅡcontained 35 phosphorylation sites totally,20 Ser phosphorylation sites,14 Thr phosphorylation sites,1 Tyr phosphorylation site; secondary structure prediction showed that amino acids sequence of CAPN1 contained 42.04%α-helix,43.16% random coil,14.80% extended fragment, CASTⅡcontained 25.89%α-helix,71.45% random coil,2.66% extended fragment.The expression of CAPN1 and CASTⅡin patial selected tissues in goat were analysed by real-time fluorescence quantitative PCR. Results showed that CAPN] and CASTⅡwere all expressed in seven selected tissues(heart, liver, spleen, lung, kidney, longissimus, leg muscle). Meanwhile, the expression of CAPN1 and CAST II gene were different in different tissues, on the whole, the expression in muscle tissues were higher than that in internal organs, when the goat was of 6-month age, the highest expression was observed in leg muscle, which was significantly higher than that of longissimus and liver (P<0.01), the expression in longissimus and liver was significantly higher than that of other tissues (P<0.01). Meanwhile, the expression of CAPN1 increased first and then decreased with the rise of the age, it was the highest when the goat was at one-year age. When the goat was of 6-month age, the highest expression of CASTⅡgene was observed in longissimusdorsi, which was significantly higher than that of crureus (P<0.05) and other internal organ tissues (P<0.01). Furthermore, the expression of CASTⅡincreased with the rise of the age and became the highest when the goat was at three-year age.