Studies On SNPs In5’ Regulatory Region Of GPR54Gene And Their Promoter Efficiency In Cattle

In this study, GPR54gene was filtered as a candidate gene in the local cattle, Simmental, Holstein and Wan Bei breeding groups which were gathered as the experimental groups for prematurity character. This study sought to identify the polymorphism, promoter efficiency, and theirs Association with cattle prematurity by means of the analysis on the5’regulatory region of GPR54gene in cattle. After genomic DNA was extracted from ear and blood tissues of cattles,973bp sequence in5’ regulatory region of GPR54gene was cloned using PCR(GPR973). The functions of GPR973were determined by using sequencing, structure prediction, SNP typing, bioinformatics analysis, dual-luciferase reporter gene validation, and analysis of variance. The results showed that:1. The homologies of GPR973were93%and43%with goat and human, respectively. Two potential promoters were found which one was between the site-110bp and-160bp, of which a TATA box existed, and the other was between the site-744bp and-794bp by online software.2. The analysis of transcription factor binding sites showed that Sox-5, SRY, Mat1-M, Dfd, GATA-2, c-Myb, NIT2, STRE, MZF1, MyoD, MATa1, cap, Dof2, GCR1, PolyA, GATA-1, AP-4, STATx, ADR1and other potential regulatory elements existed in the GPR973sequence.3. Two SNP sites were detected, which were the Tâ†'C mutation at the site of754bp (T754C) and the Câ†'T mutation at the site of816bp(C816T). The PCR products were digested into three genotypes C816C(157bp and184bp), T816T(341bp) and C816T for C816T by Maeâ…¡restriction enzyme The PCR products were also digested into three genotypes, T754T(218bp and123bp), C754C(341bp) and T754C for T754C by Nlaâ…£ restriction enzyme For the4genotypes of two SNP sites, C816CT754T, C816TT754C, T816TC754C and C816CT754C, were found in the Wanbei breeding group and the gene frequencies were0.5431,0.4310,0.0172and0.0086, respectively. Only C816CT754T genotype was found in the simmental cattle and Holstein cattle, and T816TC754C in Anhui local cattle.4.Dual luciferase reporter gene assay showed that the ratio of fluorescence intensity of firefly luciferase to sea-kidney for GPR973-816CT754promoter was significantly higher than that for GPR973-816TC754promoter, and the promoter efficiency increased79.31%(p <0.01). 5. Association analysis showed that, the first oestrus age of the cattle of816CC754TT genotype was earlier than the cattle of816CC754TT by1.28months, so the variation in GPR54gene had some cattle association with early maturity.