Preliminary Study Of The Biological Function Of Rice DNA Methyltransferase By Saccharomyces Cerevisiae And Pichia Pastoris

DNA methylation, as an important epigenetic modification, exists in most creatures.Since the DNA methyltransferase system plays an important role in controlling geneexpression and maintaining genomic stability in animals and plants, more attention has beenpaid to its biological significance and functional mechanism. We chose Saccharomycescerevisiae AH109, which is thoroughly free of DNA cytosine methylation, as the host of therecombinant plasmids. By using bioinformatics method, we got the putative rice (Oryza sativaL.) DNA methyltransferase-coing gene (OsDMT) and constructed thepBridge_OsDMT_EGFP fusion gene eukaryotic expression vector. At the same time, we usedthe mammalian de novo methyltransferase DMNT3A and DNMT3B2 as the positive controls.Since all of the exogenous OsDMT, positive and negative controls were transferred into theyeast AH109 seperately, we used the gradient dotting assay to investigate the yeast growthinhibition and check the exogenous gene expression level by measuring GFP (greenfluorescent protein) fluorescence. We obtained the correct reconstructed plasmids includingpBridge_OsDMT_EGFP, pBridge_OsDMT, pBridge_DNMT3A, pBridge_DNMT3B2 andpBridge_EGFP, which were verified by PCR amplification, double-enzymes digestion andsequencing. All of them were successfully transformed into the AH109. Gradient DottingAssay showed that DNMT3A and DNMT3B2 inhibited the growth of AH109, while theinhibition effect of OsDMT was not obvious. By using fluorescence microscope, the greenfluorescence were observed in both of the yeasts with and without transformed EGFP, makingit uncertain as to whether the exogenous genes were expressed,or autofluorescence of hoststrain AH109 may explain why all the yeast are emission under Fluorescence microscope. Themethylation-sensitive molecular marker, MSAP, was used to test yeast genomic DNAmethylation level of randomly chosed transformants of hDNMT3A, DNMT3B2 and OsDMT.Very few demethylation events were observed while de novo methylation happened, whichhower might be due to sequence variations and need to be verified by further studies.We also performed Pichia pastoris expression experiments, but to this end onlytranscriptional expression were detected. The exogenous proteins may be expressed too low tobe detected.