Identification,promoter Activity And Genetic Variationof LncRNA LncFAM200B Brelated To Muscle And Fat In Cattle

Muscle and fat are the main components of beef,thus,Uncovering the function genesassociated with muscle and adipocyte development is meaningful for the improvement of the beef quality and growth speed.Until now,some genes have been identified to regulate the development of muscle and adipocyte,such as Myo D and My HC associated with muscle development,C/EBPα and PPARγlinked to adipocyte generation,and Sirt 1 and Klf5 which are associated both the muscle and adipocyte development.In recent years,the functions of lnc RNA were uncovered gradually,but the studies about the bovinemuscle and adipocyte development only focused on uncovering the candidate genes.And the studies about the identification of the function of key genes are limited.Therefore,this study identified the candidate gene from the RNA-Seq results and investigated the function of candidate gene on muscle and adipocyte development by using RACE,bioinformatics analysis,prokaryotic expression,dual-luciferase reporter assay system and q RT-PCR methods.The aims of this study were to lay the foundation for the function research of lnc RNA and to provide scientific data for the improvement of beef quality and the development.1.The full length cloning,identificaton and expression pattern of bovine lnc FAM200BBased on the Ribo-ZeroRNA-Seq data from our lab,the new lncRNA lncFAM200 B was differently expressed in the muscle of fetal,calf and adult Qinchuan cattle.This study used RACE technology identified the full length of lnc FAM200B（472 bp）.The prokaryotic expression analysis showed that lnc FAM200 B was a non-coding RNA;Transcription factor binding site prediction showed that the 3000 bp promoter region containedseveral potential binding sites,e.g.30 C/EBPα,7 C/EBPβ and 43 SP1 easily binding to Myo D.The expression level analysis results showed that the expression level of lnc FAM200 B in calf Qinchuan cattle muscle was significantly lower than those in fetal and adult muscle.Meanwhile,the expression level in fat was higher than those in muscle under the same stage.These findingssuggested that lnc FAM200 B might influence the development of muscle and adipocyte.2.Promoter activity identification of bovine lnc FAM200 B geneConsidering the potential roles of lncFAM200 Binmuscle and adipocyte development,this study constructed p GL3-Basic recombinant vectors containing different fragments of lnc FAM200 B promoter,and transfected the recombinant vectors into 293 T,C2C12 and 3T3-L1 cell lines.Thenthe dual-luciferase activity were detected and the activity region of lnc FAM200 B promoter region were analyzed.As a result,the same variation trend of luciferase activity were found in these three cell lines.When the p GL3-pro4 was shortened to p GL3-pro5,the luciferase activity reduced significantly（P<0.01）.This result demonstrated that the promoter activity region is between the-403 to-139 bp from the transcriptional start site.Moreover,the luciferase activity in the three cell lines are 293 T < C2C12 <3T3-L1（P<0.01）.These results demonstrated that the promoter activity was highest in 3T3-L1 cell,followed by C2C12 cell,which was consistent with its tissue expression profile.Further predicted thetranscription factor binding site in the activity region,the result showed the 264 bp activity regioncontained6 SP1 and 2 C/EBPα binding sites.This results suggested that lnc FAM200 B might have function on the regulation of the development ofmuscle and adipocyte.3.The genetic variation and genetic effect of cattle lnc FAM200 B geneThe development of muscle and fat is one of the main influence factors of individual growth and development.Thus this study detected the association between the SNP of lnc FAM200 B geneand the growth related traits in Nanyang cattle.One intronic mutation AC₀00163:g.2472 T>C（Taq I）was found in cattle lnc FAM200 B gene.Then thegenotypes of 145 Nanyang cattle were identified by using PCR-RFLP method.The statistic analysis showed that the frequency of TC was highest（0.57）,and CC was lowest（0.02）.The association analysis demonstrated that the mutation of this SNP locus was significantly associated with the body weight,body height,body length and daily gain of six months old Nanyang cattle,as well as the body height and body length of twelve months old Nanyang cattle（P<0.05）.These findings not only would lay the foundation for the further research of lnc FAM200 B on growth and development,but also would provide basic data for the marker assisted selection（MAS）breeding of beef quality and development improvement.