Multiomics Analysis Of Bovine Muscle Development Related Genes And Functional Verification Of Polb Gene

Jiaxian Red cattle is considered to be an excellent material for the development of high quality beef due to its meat quality.However,due to the slow growth rate and low meat performance,Jiaxian Red cattle has a big gap in relative efficiency compared with international excellent beef cattle breeds.Improving its production performance is of great significance to improve economic benefits of Jiaxian Red cattle breeding.In this study,DNA polymerase beta(POLB),a candidate gene related to growth traits of Jiaxian Red cattle,was screened by multi-omics method and its function was verified on cell level.The main results of the study are as follows:(1)Screening of candidate genes related to growth traits in Jiaxian Red cattleIn order to screen out the major genes related to growth traits of Jiaxian Red cattle,this study used selective sweep method to obtain different genomic regions between Jiaxian Red cattle and Angus cattle.Genes in the regions were annotated.Comprehensive analysis of regional genes was conducted using Animal QTLdb Database,Animal Omics Database and longissimus dorsi muscle transcriptome data of Jiaxian Red cattle and Angus cattle.The results showed that 435 genomic regions were identified by selective clearance.36 overlapping QTL regions were obtained,and POLB gene was located in the QTL related to cattle daily gain(QTL 69373).POLB gene was highly expressed in bovine skeletal muscle(FPKM= 23.79)by Animal Omics Database.Transcriptome analysis showed that the expression of POLB gene in longissimus dorsi muscle of Jiaxian Red cattle was significantly higher than that in Angus cattle(P<0.01).In summary,POLB gene was screened as a candidate gene related to muscle development in cattle.(2)Detection of SNP in cattle POLB gene and correlation analysis on growth traits of Jiaxian Red cattleIn order to further explore the correlation between POLB gene and growth and development of Jiaxian Red cattle,this study conducted Single Nucleotide Polymorphism(SNP)scanning of the full length of POLB gene sequence using mixed pool sequencing.Correlation analysis was conducted between SNP and growth and body size phenotypic data of 100 Jiaxian Red cattle.The results showed that there was a 12 bp deletion site(c.-221＿-210del)at 210 bp upstream of the start codon of POLB gene,and a G>A mutation site(rs109209678)at 101 bp at 5’UTR.There was a C>T mutation site at the 16 th bp of exon 8(rs1092096781).The polymorphism of c-221＿-210 del mutation site was significantly correlated with body length Jiaxian Red cattle(P<0.05).In body weight,body height,body length and abdominal circumference,wild-type cattle with the mutation of c-221＿-210 del showed a higher trend than those with deletion,suggesting that POLB gene may be related to growth and development of Jiaxian Red cattle.(3)Effects of POLB gene overexpression on bovine primary muscle cellsIn order to explore the effects of POLB gene on the proliferation,apoptosis and cycle of primary bovine muscle cells,this study used POLB gene adenovirus overexpression vector(OE group)and negative control empty load(NC group)to infect primary bovine muscle cells.By Real-time PCR,Western blot,CCK-8 and flow cytometry,the differences of the effects of OE group and NC group on the proliferation,apoptosis and cycle of primary bovine muscle cells were analyzed.The results showed that the mRNA(P<0.05)and protein(P<0.0001)levels of Caspase 9 were significantly increased after POLB gene was overexpressed in primary bovine muscle cells.After48 h(P<0.01)and 72 h(P<0.05)infection with POLB gene adenovirus overexpression vector,cell proliferation was significantly inhibited.For 48 h,the apoptosis rate of primary bovine muscle cells infected with POLB gene adenovirus overexpression vector was significantly increased(P<0.05),and the cells were blocked in S phase(P<0.05).These results indicated that overexpression of POLB gene could inhibit the proliferation of primary bovine muscle cells,induce cell apoptosis and up-regulated apoptosis gene Caspase 9,and arrest the cells in S phase.In conclusion,POLB gene is related to growth and development of cattle,and the mutation site of c.-221＿-210 del of POLB gene can be used as a potential molecular marker to assist Jiaxian Red cattle breeding.POLB gene was overexpressed in primary bovine muscle cells,which inhibited the proliferation,induced apoptosis and affected the S-phase of muscle cells.This study lays a foundation for further exploring the biological function and regulation mechanism of POLB gene.