Single-nucleotide Polymorphisms And Activity Analysis Of The Promoter And Enhancer Of The Pig Lactase Gene

ObjectiveTo detect the polymorphisms site in promoter and enhancer sequences oflactase gene of pigs in domestic and abroad,to analyze the activities of differentgenotypes of promoter and enhancer of pig lactase gene,to build a theoreticalbasis for the mechanism of the relationship between polymorphisms of piglactase gene with lactose intolerance in pigs.MethodCollect ear tissue of5domestic and foreign pig breeds(16heads of largewhite,14heads of Landrace,16heads of Duroc,18heads of Hebao pig and14heads of min pigs), extract DNA from pig ear tissue using traditional method byphenol-chloroform,and saved in-20℃.Polymerase chain reaction(PCR),TAclone and sequence comparative analysis were used to analyze thepolymorphisms of5’UTR of pig lactase gene,get the different genotypes ofpromoter and enhancer of pig lactase gene by PCR amplification,6groups ofplasmids were constructed and confirmed by sequencing, which we named:ProCG-pGL3.0Basic,ProGA-pGL3.0Basic,EnhA-ProCG-pGL3.0Basic,EnhA-ProGA-pGL3.0Basic,EnhG-ProCG-pGL3.0Basic,EnhG-ProGA-pGL3.0Basic,then transfected Caco-2cells with constructions of recombinant plasmids DNA,Dual-Glo Luciferase Assay System was also used to test Fluorescence signalvalue. Result1.Obtain the5’UTR,promoter and enhancer sequences of pig LCT gene bygene synthesis technology.2.One SNP in enhancer region:-797*G/A and two SNPs located promoterregion:-308*G/C and-301*A/G were found in our study, Moreinterestingly,-308*G is corresponding to-301*A,and-308*C is corresponding to-301*G. Namely,when-308is G genotype,-301is A genotype,while-308is Cgenotype,-301is G genotype.3.Successfully constructed six sets of luciferase reporter gene vectors.4.The fluorescence signal value shows that the value of the Pro-CG is3.3times of the Pro-GA,Enh-A can increase the activity of the Pro-GA by2.3times,but decrease the activity of the Pro-CG by2.6times,Enh-G decrease theactivity of Pro-GA and Pro-CG by1.6times and2.6times respectively.Conclusion1.we found polymorphisms in the promoter and enhancer of pig LCT gene.2.The effect order of different genotypes of enhancer and promoter on piglactase gene,from high to low, as follows: Enh-A﹢Pro-GA>Enh-A/G+Pro-CG>Enh-G﹢Pro-GA.