Fatemeh Ravanrouy1, Ali Niazi2, Ali Moghadam3, Seyed Mohsen Taghavi4. 1. Institute of Biotechnology, Shiraz University, Shiraz, Iran. 2. Institute of Biotechnology, Shiraz University, Shiraz, Iran. niazi@shirazu.ac.ir. 3. Institute of Biotechnology, Shiraz University, Shiraz, Iran. ali.moghadam@shirazu.ac.ir. 4. Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz, Iran.
Abstract
BACKGROUND: DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation. METHODS AND RESULTS: In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6 days and the concentrations of 3 and 10 μM Azacytidine for 10 and 3 days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies. CONCLUSIONS: Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.
BACKGROUND: DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation. METHODS AND RESULTS: In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6 days and the concentrations of 3 and 10 μM Azacytidine for 10 and 3 days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies. CONCLUSIONS: Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.
Authors: Miloslava Fojtová; Annick Bleys; Jana Bedrichová; Helena Van Houdt; Katerina Krízová; Anna Depicker; Ales Kovarík Journal: Nucleic Acids Res Date: 2006-05-02 Impact factor: 16.971