Cloning And Expression Of CAPN3 Coding Region In Goose And Its Role In Muscle Growth And Development

Calpain(CAPN),as a candidate gene,plays an important role in muscle fiber growth and degradation.Calpain3(CAPN3),a member of this family,is expressed exclusively in skeletal muscle.Existing research results show that CAPN3 activity and muscle fiber type,such as the contraction of muscle fibers are closely related,therefore,we hypothesized that CAPN3 gene may be affected by regulating the growth and development of muscle fiber and meat quality,but at the moment about CAPN3 expression pattern has not been reported in the goose skeletal muscle at the same time,CAPN3 in goose regulation mechanism in the process of development of muscle fiber is not yet clear,all these problems need to be researched.Therefore,Sichuan white goose and Gang goose were used as the experimental materials in this study.Firstly,the CDS region sequence of CAPN3 gene of goose was obtained through gene cloning,and the corresponding protein sequence was predicted.Then,the diameters and densities of muscle fibers of breast and leg muscles of geese of different ages were measured,and the expressions of CAPN3,PPP3 CA,PPP3CB and PPP3R1 genes in muscle tissues at different growth stages were detected by qPCR.Finally,at the cellular level,through interfering and overexpressing CAPN3 gene in skeletal muscle satellite cells,mRNA changes of Cn-NFAT signaling pathway related genes in the muscle growth and development of geese were detected,and the role of CAPN3 gene in the growth and development of skeletal muscle of geese was preliminarily discussed.The main research results are as follows:(1)The CDS sequence of CAPN3 gene was obtained for the first time,2316 bp,which encodes 771 amino acids with a molecular mass of 89,019 kDa.The protein was predicted to have no signal peptide,but several N-glycosylation,O-glycosylation,and phosphorylation sites.The secondary structure of CAPN3 was predicted to be 38.65%α-helical.Sequence alignment showed that CAPN3 of Sichuan white goose shared more than 90% amino acid sequence similarity with those of Japanese quail,turkey,helmeted guineafowl,duck,pigeon,and chicken.Phylogenetic tree analysis showed that goose CAPN3 has a close genetic relationship and small evolutionary distance with those of the birds.(2)The pectoral muscle fibers and leg muscle fibers of the two varieties of goose had different changes with the increase of age,and there were significant differences.At different ages,the diameter and density of the muscle fibers of the same breed were significantly different(P<0.01).At the same age,there was no significant difference in thefiber diameter and density of the chest muscle and leg muscle between different breeds(P>0.05).(3)The expression of CAPN3 mRNA in goose and its muscle fiber characters were linearly correlated.In the chest muscle,the expression of CAPN3 mRNA in both Sichuan white goose and Gang goose showed significant or extremely significant positive and negative correlation with the muscle fiber density(P<0.05,P<0.01).In the leg muscle,the expression of CAPN3 mRNA in Sichuan white goose was negatively correlated with the muscle fiber density and reached a significant level(P<0.05),while the expression of CAPN3 mRNA in Gang goose was negatively correlated with the muscle fiber diameter(P<0.01).(4)In Sichuan white goose and Gang goose,the expression of PPP3 CA showed a trend of "ascending first and descending later" in the pecs muscle,but showed different expression patterns in the leg muscle.It was found that at E15,E22,49 d and 70 d,the expression of Gang goose in the leg muscle was significantly higher than that of Sichuan white goose(P<0.05).The expression of PPP3 CB gene in chest muscle of Sichuan white goose and Gang goose showed a trend of "down-up-down-up-up-up-down",while the expression trend in leg muscle was consistent.It was found that the expression of PPP3 CB gene in leg muscle of Gang goose was significantly higher than that of Sichuan white goose at different embryo age(day age)points(P<0.05).The expression of PPP3R1 gene in both chest and leg muscles was significantly higher in Gang geese than in Sichuan white geese(P<0.05).(5)The relative expression levels of PPP3 CA,PPP3CB and PPP3R1 genes in CAPN3 siRNA interfered skeletal muscle satellite cells all changed significantly.After 24 h and 72 h of interference,the relative expression levels of PPP3 CA and PPP3 CB genes in the interference group were extremely significant and significantly lower than those in the blank control group and the negative control group(P<0.01,P<0.05).The relative expression level of PPP3R1 gene was lower in the treatment group than in the other two control groups at all three time points,and reached extremely significant level(P<0.01).(6)The expression levels of CAPN3 mRNA in different muscle tissues of the two geese were significantly positively correlated with the expression levels of PPP3 CA,PPP3CB and PPP3R1 genes(P<0.01).(7)In the satellite cells treated with CAPN3 gene overexpression,the relative expression levels of PPP3 CA and PPP3 CB genes in the over-expression group showed significant or extremely significant difference compared with the blank control group and the no-load control group(P<0.05,P<0.01)at 24 h,48h and 72 h after transfection.There was no significant difference in the expression of PPP3R1 gene between the two control groups.(8)In satellite cells treated with CAPN3 gene overexpression,the expression level of PPP3 CA gene was significantly different from that of blank control group and blank vector control group(P<0.05).At 24 h,48h and 72 h after transfection,the relative expression level of PPP3 CB gene in the over-expression group was significantly higher than that in the blank control group and the blank vector control group(P<0.01),and the gene expression level remained at a high level at 72 h.At 24 h,48h and 72 h after transfection,the expression level of PPP3R1 gene was not significantly different from that of the other two control groups.All this being said,we obtained the full-length cDNA of CAPN3 gene of goose for the first time,2316 bp.By homology comparison,it was found that the amino acid sequence of CAPN3 protein of goose was highly similar to that of other species.The tissue expression patterns of CAPN3 gene at different developmental stages indicate that CAPN3 plays an important role in the development of skeletal muscle fibers in geese.There was a linear correlation between CAPN3,PPP3 CA,PPP3CB and PPP3R1 genes and the characteristics of goose muscle fibers.At the cellular level,CAPN3 has a positive regulatory effect on the calcineurin gene in the Cn-NFAT signaling pathway during the growth and development of skeletal muscle in geese.Therefore,we concluded that CAPN3 gene may regulate muscle growth and development through the Cn-NFAT signaling pathway.