Biological Effects Of VGLL2 Gene On Proliferation And Differentiation Of Chicken Myoblasts

In China,chicken has become the second higher consumption of meat products,and meat performance is one of the most economically significant traits of chicken.The proliferation and differentiation of myoblast are critical steps of muscle development.Therefore,the researches of the mechanisms of proliferation and differentiation of myoblasts are hot issue in animal husbandry science.It is reported that VGLL2(vestigial like family member 2)belongs to VGLL family.Its sequence is relatively conservative among mammals,reptiles and amphibians,and its expression increases with the differentiation of myoblast.Therefore,VGLL2 gene plays a very important role in the growth and development of animal skeletal muscle.However,its function in the development of chicken skeletal muscle is not clear.Given this,in this study,using bioinformatics analysis,the differences of VGLL2 expression among different species and tissues were analyzed,the temporal and spatial expression of VGLL2 gene were evaluated by qRT-PCR,and the expression pattern of VGLL2 gene of muscle tissues at different embryonic stages was also detected.The chicken primary myoblast was isolated and induced into differentiation in vitro.The expression of VGLL2 gene in the process of myoblast proliferation and differentiation was detected,the effect and molecular mochenisms of VGLL2 gene underlying chichen myoblast proliferation and differentiation were investigated by adenovirus overexpression and RNA interference()In addition,after injection of purified adenovirus into chicken muscle,the effects of VGLL2 on daily gain and fiber characteristics were analyzed to explore the molecular mechanisms of VGLL2 in muscle growth and development of chicken.The main results of the study are as follows:1.By bioinformatics analysis,VGLL2 specificly expresses in muscle tissue among different species of mammal,reptile,and amphibian,and using qRT-PCR,it was found that VGLL2 gene was specific and highly expressed in muscle of chicken.By detecting the expression profile of VGLL2 gene in chicken muscle at different embryo stages and after hatching,it found that VGLL2 showed higher expression level at embryo stage than that after hatching,and the value peak at E16.Because chicken embryo E12-E16 key period of muscle fiber formation,it supposed that VGLL2 might be involved in the process of chicken muscle fiber formation.2.The primary chicken myoblasts were isolated by enzyme digestion and cultured in vitro.After purification by differential adhension methodand immunofluorescence identification,myoblasts that meet the requirements were collected for further researches.The chicken VGLL2 recombinant adenovirus overexpression vector was constructed and packaged,siRNA that specificly targeting on VGLL2 gene were designed and synthyzed,and their interference efficiency were detected by qRT-PCR.After the infection of adenovirus,and the cell viability was detected by CCK8.It was found that overexpression of VGLL2 significantly improves the viability of myoblasts(P<0.05).EdU and flow cytometry showed that overexpression of VGLL2 inhibits the proliferation of myoblasts and increases the proportion of blocking myoblasts entering S phase(P<0.05).On the contrary,interfere with VGLL2 could significantly inhibits the viability of myoblasts(P<0.05).and improves the proliferation of myoblasts and increases the proportion of blocking myoblasts entering S phase(P<0.05).These results suggest that VGLL2 inhibits the proliferation of chicken myoblast.3.The proliferative cells with different confluence and differently induced differentiated myoblasts were collected to detect the expression of VGLL2 gene.The results showed that the expression of VGLL2 increased during the differentiation of chicken myoblasts,which indicated that VGLL2 might play an important role in skeletal muscle development.qRT-PCR was used to detected the expression of myoblast differentiation marker genes MyoD,MyoG and Myomaker,and they all increased after overexpression of VGLL2,and immunofluorescence showed that overexpression of VGLL2 promotes the fusion of myoblasts.On the contrary,interfere with VGLL2 could significantly reduce the expression of marker genes,and immunofluorescence showed that interfere with VGLL2 inhibits the fusion of myoblasts.These results showed that VGLL2 could promote differentiation of chicken myoblast.4.The results showed that VGLL2 gene expression and daily gain in muscle tissue of treatment group(overexpressing VGLL2)were significantly higher than those of control group(P<0.05).The muscle fiber characteristics of the control group and the treatment group were detected by tissue section technique.the results showed that the muscle fiber diameter of the treatment group was significantly higher than that of the control group(P<0.05).These results showed that VGLL2 could promote the muscle growth and development of chicks by increasing daily gain and muscle fiber diameter.Collectively,chicken VGLL2 gene inhibits the proliferation of myoblasts,promotes the differentiation of chicken myoblasts into myotubes,and increases the diameter of individual muscle fibers.and then promotes chicken growth and development.These results will provide a theoretical basis for supplementary of regulatory network underlying muscle development in chicken and improvement of chicken breeding...