Effects Of GPR54 Gene Polymorphism On Precociousness Of Cattle And Their Molecular Mechanisms

Precociousness,an important character for assessment and utilization of cattle strains,which refers to sexual and economic characters maturation,has been used as a principle indicator of many economic characters such as the age of puberty,body weight and reproduction.precociousness has usually been used to choose parents and evaluate breeding during the husbandry of cattle.The process of puberty initiation vary as much as its breeding resources,the regulation network is complicated and little has been known about the genetic mechanism of puberty,leading the precociousness a main issue in beef cattle breeding field.Currently,some studies have found that GPR54 gene is a key candidate gene for regulating puberty initiation.It’s not clear about the regulation and function of cattle GPR54 gene,nor the role of mutation of GPR54 gene on precociousness.It’s of great importance to investigate the variation,expression,transcriptional regulation of GPR54 gene in different populations of cattle in puberty and its influence on precociousness for elucidating the molecular mechanism of precociousness and genetic manipulation of cattle breeding.In this work,we tested the polymorphisms in 5’ proximal regulatory region(PRR)of GPR54 gene of 6 cattle breeds(Wannan,WN,Dabieshan,BS,Wanbei,WB,Wandong,WD,Simmental,ST and Holstein,HT),1 Jianghuai buffalo(JH),and 1 hybrid strain(WS)in Anhui Province,analyzed the distribution of PRR polymorphisms.Besides,the relation between first estrus age and PRR polymorphisms was tested and analyzed.ASPAR CORE and PROTTER were used to predict the mechanism of PRR polymorphism,and investigated the relation among GPR54 expression level,PRR polymorphism and transcription factors by reporter genetic techniques.Moreover,the impact of the PRR polymorphisms on related gene transcription level and signaling pathway was performed in genome level via RNAseq.GPR54 Interaction Network based on Whole-genome and its association of the Interaction Network-related genes with precociousness were investigated.The summary of the research was followed:1)PRR polymorphism is closely related to precocity.In 8 cattle strains,we found there were three SNP loci,-794A/C,-663T/C and-601C/T,which combined to form the haplotypes of ATC,CCT and CTC.Haplotype was significantly correlated with the first estrus age and relative abundance of GPR54 m RNA(P < 0.01),in which ATC was the dominant haplotype in WB,WN and BS,CCT in WD,ST,HT and WS,and CTC in JH.2)The prediction shows that,changes in protein structure and expression level were two important ways for GPR54 gene affecting the precocity of cattle.Compared with cattle,the N-terminal amino acid substitution of GPR54 in buffalo resulted in the second ligand binding site narrowing the distance between the N-terminal and the third extracellular ring(from 0.10 ? to 0 ?),which changes the level of signal transmission and amplification.On the other hand,GPR54 gene selectively binds transcription factors through promoter polymorphism,leading to the changes of GPR54 transcription level,affecting cattle precociousness.3)By dual luciferase reporter gene assay,the results showed that PRR promoter polymorphism affected the activity of transcription factors,resulting in different expression of GPR54 gene.CCT promoter was the most efficient promoter,CTC was the less efficient and ATC was the least efficient one among them.Different PRR promoters selectively recruited specific transcription factor to participate initiation and regulation of GPR54 gene expression level,leading to the promoter polymorphism regulation mechanism.4)The results of transcriptome analysis showed that gene expression differences were mainly in 4 pathways,immune system(6 vs 11),infectious diseases(14 vs 15),inherited signaling(7 vs 7)and environment signaling(10 vs 23).The Ca M,BCR and Tp12/Cot gene transcription level was upregulated in CCCCTT strain,influencing the expression and secretion of Gonadotropin.While Ca M,PLD,MKP,RGS2 and MMP9 genes were down-regulated in AATTCC strain to reduce the expression and secretion of Gonadotropin.Furthermore,Ferritin gene was upregulated in CCCCTT and Ca BP9 K and DMT1 were down-regulated in AATTCC strain,which caused the difference in transplant of embryo.5)The interaction network of GPR54 was analyzed by open database and STRING.The protein partners of GPR54 and their expression levels in WD and BS were investigated.The results showed that GPR54 interacted with Gn RH signaling pathway and other signaling pathways,and co-expressed with UTS2 R,FOXB1 and NEUROG2.The expression levels of TAC3,POMC and transcription factors(FOXB1,NEUROG2,ZFPM1 and ZSCAN25)were related to different promoter genotypes of the GPR54 gene.