The Cloning Of The Coding Region Of Dnmt2 And Dnmt3b Gene And The Study On Different Tissue Expression Pattern Of DNA Methyltransferese Genes In Pig

Here we cloned cDNA of Dnmt2 gene from total RNA in liver tissue of porcine by RT-PCR,and RACE technology.Sequence analysis indicated that the porcine Dnmt2 cDNA was 1227bp in length(Genbank accession number:FJ384441) and it encodes a deduced protein with 409 amino acids.Using amino acid sequence homology we found that there is a little differernces of Dnmt2 protein in human,cattle and mouse.The comparison anmino acids sequnence of porcine Dnmt3b with those of human,bos taurus,and mouse showed that the amino acids similarity were 84.76%,79.51%and 73.45%respectively.Cloning of the sequence and the electronic serial contig showed the similarity were 96.63%.Dnmt2 between different species of the amino acid homology between 87.25%～76.44%,whereas this study measured in the pig genes and different species Dnmt2 amino acid homology between the two intervals,so we can conclude that this study received Dnmt2 porcine gene sequence is the cDNA sequence.Through DNA sequence NJ tree,we can see that the clustering plan consistent with the biological evolution of Dnmt2,which further confirmed the sequence obtained in this study is Dnmt2 pig gene cDNA sequence.The molecular structure of pig Dnmt2 Obtained in this experiment embodies the characteristics of the family.They have 10 fixed with the characteristics of DNA methyl transferase motif,which showed 10 higher motif conservation,and in the eighth and ninth motif between a variable amino acid composition of the TRD.TRD only found Dnmt2 family.Porcine Dnmt3b cDNA was cloned from total RNA of liver in porcine by RT-PCR,and RACE.Sequence analysis indicated that the porcine Dnmt3b cDNA was 2559bp in length(Genbank accession number:FJ384442) and it encodes a deduced protein with 852 amino acids.The comparison of deduced anmino acids sequnence of porcine Dnmt3b with those of human,bos taurus,and mouse showed that the amino acids homology were 84.76%,79.51%and 73.45%.Between different species of nuclear Dnmt3b amino acid homology ranging from 87.75%～73.45%,whereas this study measured in the Dnmt3b gene in pig species with different amino acid homology between the two intervals,so we can conclude that the sequence obtained in this study is the Dnmt3b gene cDNA sequence of pig.Through DNA sequence NJ tree,we can see that the clustering plan consistent with the biological evolution of Dnmt2,which further confirmed the sequence obtained in this study is Dnmt2 pig gene cDNA sequence.Deduced Dnmt3b domain showed that Dnmt3b have the similar domain with human,bos taurus and mouse:a Cys-rich domain,a histone deacylation ATRX domain and a PWWP domain.Using real-time quantitative PCR,we detected gene Dnmt1,Dnmt2,Dnmt3a and Dnmt3b all expressed in a panel of porcine tissues(heart,liver,spleen and intestine).Dnmt1 expressed the highest level in intenstine,a little difference in liver and heart,low level in spleen.Dnmt2 expressed low level in intenstine,high level in liver.Dnmt3a expressed low level in intenstine that is similar to Dnmt2,second in spleen,a little difference in liver and heart.Dnmt3b also expressed low level in small intestine,second in liver and high level in spleen.