Study On Promoter Activity And Transcription Regulation And Polymorphism Of ANK1 Gene In Porcine

At present,China is the largest pork production and consumption country in the world.The increasing demands of pork with higher quality as the rapid development of China's economy will lead to the improvement of pork meat quality become an important task in breeding programs.ANK1(Ankyrin1)gene belongs to the family of Ankyrins,and its encoded protein Ankrin(Ankyrin-R)is composed of membrane-binding domain,spectrin-binding domain and regulatory domain.It had been found that the polymorphism of ANK1 gene is related to the meat quality of pigs and cattle.Studies indicated that the ANK1 gene belongs to the susceptibility gene of type 2 diabetes and is associated with hereditary spherocytosis,coronary heart disease and Alzheimer's disease.Gene transcription regulation is a very important process in the process of gene expression regulation,which leads to the transmission of genetic information.The transcription process of the gene is regulated by the promoter which contains key elements where bounded with the RNA polymerase and transcriptional binding factors.In order to further understand the transcriptional regulation mechanism of pig ANK1 gene,we cloned the promoter region of ANK1 gene from Guangxi Bama mini-pig and Landrace pig and screened its core region and SNP.The results are as follows:1.The sequence of the promoter region of ANK1 gene was successfully cloned from Landrace and Guangxi Bama mini-pig.The online software PromoterScan,NNPP,Promoter2.0,Gene-regulation and MethPrimer were used to analyze the cloned 2074bp sequence,and it was found that a lots of regulating elements such as TATAbox,GATA-1,SP1,myogenin,AP-2alpha,CEP-bind were detected.Two CpG islands and two core promoter regions were also been found.2.Eights double luciferase reporter gene vectors containing different deletions of ANK1 promoter were constructed and transfected into C2C12 cells respectively.The luciferase activity of each vector was measured.The results showed that fragment PGL3-P3 from Landrace and the fragment PGL3-P2 from Bama mini-pig showed the highest luciferase activity.It was imply that the promoter core region may be located within upstream-1080?-1991 of intervening fragment.3.Sixteen single nucleotide polymorphism(SNP)have been found in Guangxi Bama mini-pig and Landrace ANK1 gene promoter core region,which were located on-19,-133,-147,-187,-228,-246,-322,-360,-369,-468,-549,-550,-615,-696,-725,-798 loci.The base on-798,-468 and-369 locus were C?C and G respectively in Guangxi Bama mini-pig and T?G and A respectively in Landrace.The base on-468 locus was C in Guangxi Bama mini-pig?Debao pig and Luchuan pig,but it was G in Landrace.4.Analysis of the ANK1 gene core promoter revealed the presence of SP1?GCN4?C/EBP alph transcription factor binding sites at SNP of-468.Double-luciferase reporter gene vectors containing the promoter with wild-type or mutation of-468 SNP were constructed by point mutation experiments.The activity of luciferase with significantly different between the double-luciferase reporter gene vectors containing the promoter with wild-type and mutation of-468 SNP(P>0.01).When the eukaryotic expression vector of SP1 was constructed and co-transfected with the double-luciferase reporter gene vectors containing the wild-type or mutation of-468 SNP,it was found that the activity of the luciferase was significantly decreased(P<0.05).5.The different expression of ANK1 gene in 11 tissues from Guangxi Bama mini-pig was detected by fluorescence quantitative PCR,and it was found the highest expression was in pancreas and the lowest expression in adipose tissue.The eukaryotic expression vector of ANK1 gene CDS was successfully constructed and expressed in C2C12 cells..