Research On The Regulation Function Of Transcription Factor AP-2? Targeting On The GHR Gene Promoter Of Guangxi Bama Mini-pig

It's well known to that the unique and abundant miniature pigs resources exit in China.And Guangxi Bama mini-pig shared high genomic homology of 83%with human,meanwhile it is similar to humans in terms of physiology,disease mechanism and anatomy etc.Therefore,miniature pigs was the ideal experimental animal model to study human physiology,pathological mechanisms and xenotransplantation[1].Guangxi Bama mini-pig is one of the representative miniature pigs used in the experiment,and its characteristics was short stature,early sexual maturity and slower growth compared with large lean pigs.There are numerous and complex factors that can affect the growth and development of animals,such as genetic,nutritional,environmental,hormones and some other regulators,of which regulating method was the neuroendocrine growth axis(GH-GHR-insulin-like growth factor).Studies have shown that the concentrations of GHR and IGF-I in the serum of Guangxi Bama mini-pigs were significantly lower than that of Duroc,Yorkshire and Landrace pigs,but there was no significant difference in serum GH concentrations among the 4 varieties.The gene transcription was a very crucial stage in the gene expression regulation mechanism,and it played the role as "bridge" in the process of genetic information transmission.But the onset time,initiation site and frequency of transcription were regulated by important element the promoter which mainly played a regulatory role through combining to transcription factors.To explore the relationship between promoter SNP loci of GHR gene and GHR expression in swine,the GHR gene promoter sequences of two native pig breeds in Guangxi and three foreign pig breeds were cloned in this experiment to screen the different SNP sites between local pig breeds and foreign pig breeds,further to study the effect of differential SNP loci on GHR expression,the main findings are as follows:1.The length of GHR was about 1300 bp amplificated by PCR in Guangxi Bama mini-pigs(n=48),Luchuan pigs(n=28),Landrace pigs(n=42),Duroc pigs(n=45)and Yorkshire pigs(n=40).After sequencing,the alignment results found that the Guangxi Bama mini-pigs and the Luchuan pigs were all 'A',while Landrace pigs,Duroc pigs,and Yorkshire pigs were all 'G' at position-758 in the upstream region of GHR promoter.Besides,this site was the binding site of AP-2a transcription factor according to the prediction of the online software transcription predicting factor binding site.2.The mutant(-758 A?G)and wild-type(-758 A)dual luciferase reporter vector of the core region in GHR gene promoter successfully constructed in Guangxi Bama mini-pig and transfected PK15 and C2C12 cells.The results showed that the promoter activity of mutant gene vector was significantly higher than that of the wild-type vector in Guangxi Bama mini-pig(p<0.05).3.The overexpression vector pcDNA3.1-AP-2? of transcription factor AP-2? gene was successfully constructed.After transfection of PK15 cells,the relative expression of AP-2? and GHR genes was detected by qPCR.The results showed that after overexpressing AP-2? gene,the relative expression of AP-2?gene increased significantly(p<0.01),and the relative expression of GHR gene increased significantly too(p<0.01).4.After co-transforming PK15 cells with transcription factor overexpression vector pcDNA3.1-AP-2? together with the mutant(-758A? G)and wild-type(-758A)dual-luciferase reporter gene vector respectively in Guangxi Bama mini-pig,the promoter activity of mutant vector and pcDNA3.1-AP-2? co-transformation group(experimental group)was significantly higher than that of mutation vector and pcDNA3.1 co-transformation group(control group)(p<0.05),the promoter activity of wild-type vector pcDNA3.1-AP-2? co-transformation group(experimental group)was also significantly higher than that of mutation vector and pcDNA3.1 co-transformation group(control group)(p<0.05).Thus,the activity of mutant(-75 8A? G)promoter was significantly higher than that of wild-type up-regulated by overexpressing AP-2? gene(p<0.01).5.Five shRNA vectors were designed to interfere with the expression of endogenous AP-2? gene in porcine kidney cell PK15.The expression of AP-2?gene and GHR gene in the cells was quantitatively detected after shRNA vector transfection into PK15.The results showed that after the interference of AP-2?gene,the expression of AP-2a gene was significantly decreased(p<0.01),and the expression of GHR gene was also significantly decreased(p<0.01).6.The specific biotin-labeled probes,cold-competitive probes and mutant probes were designed according to the DNA base sequence of the transcription factor AP-2a binding site in the GHR gene promoter of Guangxi Bama mini-pig,and then carried out the EMSA experiment.The results of EMSA experiments showed that the transcription factor AP-2a can bind to the designed probe,furthermore it can be proved that the transcription factor AP-2a could target its corresponding binding site on GHR gene promoter.