Transcriptome Analysis Of Leg Muscle In Haiyang Yellow Chicken And The Effects Of Alox5 Gene On Myoblast Proliferation And Differentiation

The growth and development of skeletal muscle has undergone a series of changes in the expression of regulatory genes from embryonic stage to adulthood,and the expression of these genes changes in the process of muscle growth.The regulation of muscle and development is very complex.To better understand the regulation of muscle growth and development of broiler chickens,in this study,Haiyang yellow chicken was used as the test material,RNA-seq sequencing technology was used to screen differentially expressed gene that affect muscle growth and development.And study the effects of key genes Alox5 on myoblast proliferation and differentiation.The main results were as follows:1.The transcriptome level of leg muscles at different growth stages(E12,E16,1d and 10W,three males and three females at each growth stages)were analyzed,the results showed that there were 5743 and 6150 differentially expression genes in male and female chickens,respectively(Fold Change?2 and FDR?0.05).Among these,AHSG,ACACB,PHGDH,MYH1C and NTRK2 genes belong to the common differentially expressed gene in male and female chickens.This indicates that these genes are closely related to growth and development at different growth stages in male and female chickens.2.The results of GO function annotation showed that the main enriched GO terms in male and female chickens were basically same.And there were some GO terms significantly enriched and related to muscle growth and development,such as cell proliferation,growth and developmental process.Including some known genes that affect muscle growth and development of broiler chickens,such as MYOCD,MUSTN1,MYOG,MYOD1,FGF8,FGF9 and IGF-I;KEGG pathway enrichment analysis showed there three signaling pathways related to muscle growth and development(P<0.05),such as ECM-receptor interaction,Focal adhesion and MAPK signaling pathway.They have played an important role in regulating muscle growth and development.3.Using RT-qPCT to detect the spatiotemporal expression of Alox5 gene in chicken myoblast differentiation.The results showed that the expression of Alox5 gene gradually increased with the prolongation of differentiation time.This indicates that Alox5 gene plays an important role in the differentiation of myoblasts.4.In order to study the effect of Alox5 gene on the proliferation and differentiation of myoblast cells,we designed interference sequences and constructed overexpression vectors of Alox5 gene.And transfected them into myoblasts,then detecting the changes of myoblast proliferation and differentiation.The results showed that Alox5 gene could promote the proliferation and differentiation of myoblasts.5.To compare the different expression miRNA between Alox5-G and Alox5-N groups after the overexpression of Alox5 gene.The result showed that the expression of miRNA in chicken myoblast is relatively low,the number of TPM rang 1-10 accounts for more than 87%of total miRNA,while the number greater than 10000 accounts for only 0.8%of total miRNA.6.A total of 16532 target genes were obtained from 2599 different expression miRNAs,and the corresponding target genes of 188 significantly different expressed miRNAs were 13991.Screening the target genes,we found many genes related to muscle growth and development,such as EGFR,FGFR3,FGF13,FGF6,MYLK3,FGF10,MYO1F,FGF9,FGF1,MYH9,FGR,MYOD1,MYOG and IGFBP4.This suggesting that the regulation miRNAs of these genes may play a key role in myoblast proliferation and differentiation.7.To analyze the GO functional annotation of target genes,some significantly enriched GO terms related to muscle growth and development were found,such as cell proliferation,myeloid cell differentiation,growth,regulation of cell growth and tissue development.The target genes enriched in these terms may be involved in myoblast proliferation and differentiation,thus affecting muscle growth and development.KEGG pathway analysis showed that there were three significantly enriched pathways affecting muscle growth and development(P<0.05),Cytokine-cytokine receptor interaction and FoxO signaling pathway,which are played an important role in the regulatory of chicken growth development.