Molecular Characterization And Expression Pattern Of Dnmt2 Gene And DNA Methylation Detection In Crustacean

So far, three DNA methyltransferase families (Dnmt1, Dnmt2 and Dnmt3) have been identified in eukaryote. These enzymes are characterized by a set of ten motifs in a typical order. In vivo, they use S-adenosyl-L-methionine (AdoMet) as the donor of an activated methyl group and catalyze the transfer of a methyl group to cytosine in DNA, so that control the genes expression.Among these three families, the biological function of Dnmt1 and Dnmt3 are comparative understandable. They are required genes during the mammal development. The Dnmt1 and Dnmt3 knock-out animals show a lethal phenotype. However, because Dnmt2 mutants caused no readily observable phenotypes in organism, its biological function is still unknown. Then analysis of Dnmt2 gene expression is a useful method to understand its biological function.In this research, we used two crustaceans Artemia franciscana and Macrobrachium rosenbergii as research material, isolated each Dnmt2 gene, detected each the DNA methylation level and characterized Dnmt2 gene expression patterns during the each development. The result indicated that Dnmt2 gene involved in the embryo development, but the main substracte is not DNA. This research provides the basic information about Dnmt2 research in Crustacean.The main results include the following four parts:1. By using CODEHOP degenerated primer strategy, Dnmt2 genes were isolated from the Artemia franciscana and Macrobrachium rosenbergii. They were named as AfDnmt2 and MarDnmt2 respectively. The length of AfDnmt2 cDNA we identified was 1054-bp. It contained an 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids. The whole MarDnmt2 cDNA was 1472-bp in length. It contained an 1191 -bp open reading frame that encoded a putative Dnmt2 protein of 396 amino acids. Compared with other known Dnmt2 homologs, AfDnmt2 and MarDnmt2 are both exhibiting approximately 30%-40% identities, ten DNA methyltransferase characteristic motifs and a target recognized domain. This is the first report of Dnmt2 gene from crustacean, and is the first report of DNA methyltransferase gene from crustacean as well. Using PCR and Southern blot to analyze the structure of AfDnmt2 in the genome. The result indicated that there was no intron of AfDnmt2 and multiple copies of genes in Artemia franciscana.2. Because Northern blot analysis could not detect AfDnmt2 mRNA expression, semi-quantitative RT-PCR was performed to analyze AfDnmt2 mRNA expression. The result showed that AfDnmt2 mRNA expression rose steadily in the activated cysts within hatch medium and reached the maximum at the stage of 10 hours incubation beyond which an obvious decrease was observed at the next nauplius stage. During the maturation of adult females, an obvious decreasing in AfDnmt2 mRNA expression from larval stage L7 to Larval stage L12. Afterwards, with further development, AfDnmt2 mRNA expression showed no obvious difference. Additionally, AfDnmt2 mRNA expression did not present great difference between adult females and males. Meanwhile, we detected the DNA methylation of Artemia franciscana by the method of C~5-methylcytosine antibody. The results showed about 0.02% cytosine residues were found to be methylated in the DNA of Artemia franciscana and the expression of AfDnmt2 was not involved in the DNA methylation.3. By real-time PCR, we characterized the MarDnmt2 expression in various tissue and embryo development in Macrobrachium rosenbergii. The result showed that especially high mRNA level of Mar-Dnmt2 was found in the ovary, approximately 5～16 times higher than other tissues. Moreover, MarDnmt2 mRNA presented obvious downtrend during the embryo development. At the late embryo stage of zoea, the level of MarDnmt2 mRNA was approximately one fifth of the maximum stage. We also detected the DNA methylation of Macrobrachium rosenbergii by the method of C~5-methylcytosine antibody. The results showed about 0.05% cytosine residues were found to be methylated in the DNA of Artemia franciscana and the expression of MarDnmt2 was also not involved in the DNA methylation.