New Polymorphisms in Regulatory Region of CAPN3 Gene with no Effect on Gene Expression in Breast Muscle of Broiler Chickens

Piórkowska, Katarzyna; Nowak, Joanna; Połtowicz2, Katarzyna; Ropka-Molik, Katarzyna

doi:10.2478/aoas-2014-0032

Abstract

Calpains are enzymes that belong to calcium-dependent, non-lysosomal cysteine proteases. The CAPN3 gene encodes a major intracellular specific muscle protease with a high capacity for degradation of cytoskeletal and muscle fibre proteins. Therefore, they play an important role in fusion of myoblasts, proliferation, cell growth and migration. In chickens, the gene encoding for calpain 3 is localized on chromosome 5. Due to the function of encoded protein, the CAPN3 has beenchosen as a candidate gene for meat quality in chickens. Consequently, the aim of our study was to identify new polymorphisms in the regulatory region of CAPN3 gene and to investigate their impact on CAPN3 transcript abundance in breast muscles. The experiment used broilers of two genetic lines: fast- and slow-growing. The polymorphisms were identified by screening with High Resolution Melting (HRM) and sequencing based on the Sanger method. The CAPN3 gene expression was conducted by using succinate dehydrogenase complex, subunit A (SDHA) and 60S ribosomal protein L4 (RPL4) genes as endogenous controls. Four new polymorphisms were found: g.322176G>A in promoter region (GenBank: AADN03004661.1), c.176*C>T, c.144*G>C and c.137*_147*delCAGCCCTGCTT in 3`UTR sequence. The new polymorphisms were identified by using restriction enzymes ScrFI, BslI, AcuI, HpyAV, respectively. The frequency of polymorphisms found in 3`UTR region was similar in both lines. According to polymorphisms identified in 3`UTR region the alleles with deletion, C and C (c.137*_147*del, c.144*G>C and c.176*C>T) were rare. The polymorphism identified in the promoter region and 3`UTR regions changed a few binding sites for transcription factors, but did not alter any binding sites for the other important translation regulators such as miRNA. Analysis of the effect of new polymorphisms on CAPN3 gene expression showed that in fast-growing line the chickens with GG genotype according to polymorphism g.322176G>A inthe promoter region, were characterized by the highest CAPN3 transcript abundance. Other polymorphisms in 3>UTR region seem to have no effect on CAPN3 gene expression.

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New Polymorphisms in Regulatory Region of CAPN3 Gene with no Effect on Gene Expression in Breast Muscle of Broiler Chickens

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