The Promoter Region Methylation Status And MRNA Expression Of WFIKKN2 Gene In Pig

With the development of society,the demand for high quality pork is increasing.Although the foreign pigs grow fast,the pork tastes not so well.The local pigs grow slowly,but the taste of pork is excellent.However,the quality of pork can be improved rapidly by cross breeding,which could be sped up by studying the molecular mechanisms associated with skeletal muscle growth,further give full play to heterosis.Studies have shown that a variety of gene can affect the growth and development of skeletal muscle,while WFIKKN2 protein is able to regulate the growth and development of skeletal muscle by combining with myostatin.The WFIKKN2 gene,also known as growth differentiation factor-associated serum protein 1(GASP-1),belongs to the FS protein family.This study aimed at selecting the Duroc,Luchuan pig and their reciprocal F1 generation as research object to detecte the promoter activity and mRNA expression of WFIKKN2 gene in different pigs,meanwhile analyze the difference of mRNA expression in the view of DNA methylation.Also,bioinformatics analysis,tissue expression profiles and the different methylation level of CpG island in different tissues of WFIKKN2 gene was obtained.Furthermore,it may provide a theoretical basis for breeding work.The results of the study are as follows:1.The 1819bp promoter region upstream of the 5' end of pig WFIKKN2 gene published in NCBI was the object to be studied,in which the core promoter position,transcription factor binding site,transcription initiation site,TATA-box and some other structures were predicted by bioinformatics software.And then the target fragment in Duroc,Luchuan pig and their reciprocal F1 generation was obtained in this study and its promoter activity was detected by constructing promoter dual luciferase reporter gene vector.Here the results verified the promoter activity of WFIKKN2 gene in four kinds of pigs,and the promoter activity was lower significantly in the Duroc and Dulu pig than in the Luchuan pig and Ludu pig(P<0.05);2.The mRNA expression of WFIKKN2 gene in the longissimus dorsi of different pigs was detected by real-time PCR and it was lower significantly in the Duroc and Dulu pig than in the Luchuan and Ludu pig.Predicted on line,a CpG island containing 526 bp sequences existed in WFIKKN2 gene promoter.Next the Bisulfite sequencing PCR method was used to detect the CpG island methylation levels of WFIKKN2 gene in four different pigs.It revealed that the methylation level of CpG island was significantly higher both in Duroc and Dulu pig than in Luchuan pig and Ludu pig(P<0.05)and negatively correlated with mRNA expression.To investigate whether CpG island methylation level affects promoter activity,this experiment used the longissimus muscle DNA of Duroc and Luchuan pigs as a template.Further the activity of promoter deletion fragment containing the CpG island was detected by constructing luciferase reporter gene vector and it was found to be significantly lower in the Duroc than in the Luchuan pig(P<0.05).There were many binding sites for SP1 transcription factors existed in CpG islands,so it was concluded that the CpG island methylation level in WFIKKN2 gene may affect the SP1 transcription factor binding to promoter and result in the decrease of the promoter activity,thereby inhibit the mRNA expression of WFIKKN2 gene.3.The CDS region of WFIKKN2 gene in Luchuan pig and Duroc was cloned successfully which was 1743bp in length and encoded 580 amino acids showed by bioinformatics analysis.Comparing the sequences,there are 15 different bases between Duroc and Luchuan pigs,in addition seven of them were missense mutations.And WFIKKN2 gene was highly homologous in different mammals,also it was hydrophilic and belonged to the secreted protein.Moreover,multiple potential glycosylation and phosphorylation sites existed in the amino acid sequence,which may have regulatory effects on WFIKKN2 protein.The tissue expression profile of WFIKKN2 gene in Luchuan pig showed that it was expressed in different tissues.The methylation level of CpG island detected by BSP method was significantly lower in the heart than in the kidney(P<0.05),while the mRNA expression level of WFIKKN2 gene was significantly higher in the heart than in the kidney(P<0.05).It was speculated that the methylation of DNA also inhibited the expression of mRNA in different tissues.