| Muscovy duck has the characteristics of fast growth and high lean meat rate,which is a very excellent breeding material for duck.However,compared with the other ducks,the muscle of Muscovy duck is very rough.Therefore,the urgent problem in Muscovy duck breeding is to improve muscle tenderness.Calpain-1(CAPN1)can degrade myofibrils and affect muscle tenderness.The research on CAPN1 gene has been reported in pigs,cattle,sheep,chickens and other animals,but the research on CAPN1 gene of Muscovy duck is less at home and abroad.In order to improve the muscle tenderness of Muscovy duck and promote the breeding of new muscovy duck,CAPN1 gene was cloned by homologous recombination and bioinformatics analysis was carried out;The qPCR was used to analyze the expression of m RNA in different tissues,genders and developmental periods.The myoblasts of the leg muscle of Muscovy duck were isolated successfully by enzyme digestion.According to the different principles of adhesion speed of fibroblasts and myoblasts,myoblasts were purified.The myoblasts were then identified by immunofluorescence staining.Finally,overexpression and interference of CAPN1 gene were carried out to study the expression of genes related to muscle development.The results of our study are as follows:(1)In this experiment,the coding region of CAPN1 gene of Muscovy duck was cloned with a length of 2148 bp and 715 amino acids.The sequence has been submitted to Gen Bank with a accession number of MT419750.1.The results of homology analysis showed that CAPN1 gene was highly conserved.The results of phylogenetic analysis were consistent with those of traditional taxonomy.The analysis of physical and chemical properties showed that the relative molecular weight of CAPN1 protein was 81 368.72 Da;the theoretical isoelectric point was 5.82;leucine was the most frequently used and histidine was the least frequently used.The protein is stable,fat-soluble and hydrophilic.There are two transmembrane regions,but there is no signal peptide,which is mainly distributed in the cytoplasm.There were 59 potential phosphorylation sites,9 O-glycosylation sites and 3 N-glycosylation sites.In the secondary structure,the ratio of α-helix is the highest,and that of β-angle is the lowest.The number of conservative domains is 3.Finally,a tertiary structure model of CAPN1 was established.(2)CAPN1 gene can be expressed in chest,leg,heart,liver,lung,spleen,kidney,stomach,small intestine of black muscovy duck,without obvious tissue specificity;however,the expression of CAPN1 gene in female duck was higher than that in male duck.In addition,there were similar expression patterns in pectoral muscle and leg muscle,similar expression patterns in heart and liver,similar expression patterns in lung,spleen and small intestine,and similar expression patterns in kidney and stomach.(3)The myoblasts from the leg muscles of black Muscovy ducks were successfully isolated and purified.The cells distributed evenly and grew well,which accords with the morphological characteristics of myoblasts.The Desmin protein,a special marker protein of myoblasts,was detected by immunofluorescence staining.It was confirmed that the isolated cells were indeed myoblasts of Muscovy duck.(4)When the expression of CAPN1 gene was up-regulated,the expression of MyoD and MyoG gene were up regulated,and the expression of Mrf5 gene was down regulated.But when the expression of CAPN1 gene is down regulated,the expression of these genes is the opposite;the up regulated or down regulated expression of CAPN1 gene has no effect on MSTN gene.This study preliminarily confirmed that CAPN1 gene is involved in the regulation of muscle development,which provides a basis for further study on the function of CAPN1 gene in Muscovy duck. |
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