Transcriptome Analysis Of Breast Muscle In Jinghai Yellow Chicken At Growth Stage And The Preliminary Functional Validation Of The Key Gene RAC2

Growth performance is very important for broiler production.In the growth process of broiler chickens,gene expression varies at different growth stages and the regulation mechanism of muscle growth and development is complex,but there are still few studies on the molecular mechanism of broiler growth.In this experiment,the RNA-seq for the breast muscles of Jinghai yellow chickens was studied,and the results of RNA-seq were verified by qPCR.RAC2,a key differentially expressed gene,was further selected to detect its regulation of downstream genes by lentivirus interference.The main results are as follows:1.Fold Change>2 and FDR:<0.05 were used as screening criteria for the differentially expressed genes(DEGs)in the study.A total of4608 DEGs were obtained from M4F,M8F and M12F groups by comparison in pairs.83,3445 and 3903 DEGs were separately obtained from M4FvsM8F,M4FvsM12F and M8FvsM12F.Further analysis of differentially expressed genes revealed that six common DEGs were found in the three groups,but only three of them were annotated,namely SNCG,MYH1A and ARHGDIB,suggesting that these genes may be closely related to growth and development at early stage of the Jinghai yellow chicken.2.GO enrichment analysis of 83,3445 and 3903 differentially expressed genes(Corrected p-value?0.05)was performed in M4FvsM8F?M4FvsM12F and M8FvsM12F groups and the results showed that 0,304 and 408 BP items were significantly enriched respectively.KEGG pathway enrichment analysis were also performed for the 4608 differentially expressed genes.1,2,4 and 4 signaling pathways were significantly enriched from M4FvsM8F5 M4FvsM12F,M8FvsM12F and all DEGs respectively according to the Corrected p-value ?0.05.They are Steroid biosynthesis,Carbon metabolism,Focal adhesion,Cytokine-cytokine receptor interaction,Biosynthesis of amino acids and Salmonella infection.The first five of these pathways were all closely related to chicken growth and development,and had important reference value in scientific research and production.3.Quantitative real-time PCR(qPCR)was used to verify the accuracy of RNA-seq.Pearson correlation coefficient was used as the criterion.Meanwhile,the significance test of r was carried out.The results showed that the correlation of the nine genes in the RNA-seq and qPCR at the three stages of weeks were all high(|r|?0.95)and the correlation all also reached the significant level(P<0.05),which further proved the reliability of the sequencing results.4.According to the protein interaction of DEGs and the significant enrichment pathway focal adhesion,combined with relvevant literatures,a differentially expressed gene(RAC2)were selecteded as the key gene.In order to preliminarily verify the function of RAC2,we constructed four lentiviral vectors of RAC2 and the results showed that the second one was an effective interfering vector,and the interference efficiency reached 60%.At the same time,the mRNA expression of the downstream genes PAK1,PAK3 and MAPK8 was detected,and they also had significant changes compared with the negative control group.The results might suggest that the RAC2 may regulate cell behaviors by regulating PAKs/MAPKs pathway and further affect growth and development of chickens.