Cloning And Expression Of Common Carp Calpain Gene Family And Correlation With Textural Propertiesof Its Muscle

Calpain family is an extensively studied of proteolytic enzymes, which has been proven related to meat tenderizer. Calpain family is a complex regulatory system. As research continues, calpain and calpastatin genes have been used as the candidate genes that influence meat tenderness; and have been widely used to evaluate meat quality in animal breeding and farming. Currently, there is no report on the relationship between the common carp calpain system and muscle tenderness. Therefore, the homologous cloning method was used for cloning major genes in common carp calpain system and bioinformatic analysis was applied to understand the basic characteristics of capn genes family in this study; while real-time PCR was used to identify the expression profiles of capn genes family in tissues, in order to find out the gene expression patterns in common carp tissue system during the process of development. The genetic markers on the gene system can be developed, which can provide theoretical support for the good varieties breeding, and to indicate the direction of the genetic regulatory.(1) The completely c DNA of common carp capn1, capn2 a, capn2 b, capn3 and cast gene with Genbank accession number KP878555, KF669387, KF669386 and KF669385, respectively, are obtained in the study. However we dose not find any fragments on common carp capn1 b gene, suggesting that the common carp may have no capn1 b genes. The homologies of common carp capn2 a and capn2 b with those in zebrafish capn2 were 90 % and 85 %, respectively. The homology of capn3 between common carp and zebrafish gene can reach 89 %; while fish homology between these two branches was 69 %. The homology of common carp and grass carp cast gene can be 88 %, followed by 83 % between the zebrafish and Atlantic salmon. But the homology of common carp cast gene can not reach to 50 % with rainbow trout. The phylogenetic kinship tree of capn gene family shows genetic relationship consistent with the traditional classification.(2) The CAPN protein family of common carp is hydrophilic protein. There may be a transmembrane region, so CAPN protein could be a potential transmembrane protein family. This protein family does not contain any signal peptide sequence. CAPN1 protein subcellular was located in mitochondria, CAPN2 and CAPN3 protein subcellular were located in the cytoplasm, CASN protein subcellular is located in the nucleus. It has been found that CAPN protein family contains phosphorylation sites, amongwhich P52 site of CAPN1 protein and D727 site of CPNA3 protein are unusual phosphorylation site. N-linked glycosylation sites have been detected; while C-linked glycosylation sites have not been found in CAPN protein. One N-linked glycosylation site and 36 O-linked glycosylation sites have been found in CAST protein. Since we did not find the signal peptide, the 36 potential O-glycosylation sites can not be glycosylated.(3) There are three protein secondary structures, including alpha-helix, random coil and extended strand in CAPN protein family of common carp. Tertiary structure of CAPN protein in common carp is highly similar with tertiary structure through diffraction of CAPN2 protein in human, but there is difference on the structure of domain I. The similarity of tertiary structure of CAST protein between common carp and human is 31.46 %. There are four typical domains with CAPN protein found in common carp. However, CAPN3 protein dose not contain the three typical insertion regions. CAST protein contains four repeat conserved domains; the homology among which canreach 44.02 %.(4) Capn genes family can expressed in eight tissues of common carp. The expression levels of these tissues are remarkably different, with the relatively high expression in muscle fibers within the organization. The expression profiles of common carp at different stages(blastocyst stage, somites stage, embryo of pharynx, feeding stage) are significantly different. A negative correlation has been found between common carp embryonic capn3 and cast. Capn gene family expression in the muscle had a correlation. In two common carp varieties, cast gene expression levels are higher, but the capn1 gene expression is lower, suggesting that cast gene can inhibit capn1 genes, but has less effect on capn2 and capn3. It is found that a positive relationship existed between capn1 gene common carp and body weight, width and water content. And there are significant negative correlations between capn2 a and body length and fat content, with other indicators are positively correlated. Capn3 gene and ash are positively correlated. Cast gene is positively correlated with protein and fat content, and has negative relationships with other indicators. The measure of CAPN protein family by extracting is almost the same change trend with relative expression of this protein family by fluorescence quantitative PCR detection. And CAPN3 and CAST protein expression is significantly higher than CAPN1 and CAPN2 proteins.(5) Frozen can significantly delay muscle quality changes of a fish, and the lower temperature, the slower quality declines. The texture of-80 ℃ frozen common carp muscle is slightly better than-20 ℃ sample. Significantly positive correlations have been found in following parameter pairs: water-adhesion, weight-cohesiveness, gumminess-chewiness, body width-chewiness, muscle protein-resilience. Capn2 b gene is only positively correlated with protein and ash content; and negatively correlated with other indicators. Capn genes family are correlated with muscle texture, except a significant negative correlation between the cohesiveness and capn2 b, capn1 and adhesion, capn2 a and recovery, capn2 b and hardness, capn3 and chewiness and cast and cohesiveness are positively correlated. Therefore, common carp capn genes are candidate indicators of meat tenderness traits in molecular nutrition regulation and use to select breeding new varieties of common carp with excellent tenderness.