EPIGENETIC ANALYSIS ON WATER STRESS-ASSOCIATED GENE IN TOMATO

GONZALEZ RODRIGO; RICARDI MARTINIANO; IUSEM NORBERTO

Hotel Catalinas Park, San Miguel del Tucumán, Argentina.

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Our goal is to investigate the possible connection between epigenetics and water stress in plants, by analyzing the tomato Asr1 gene, associated with water stress, as a model example. To test occurrence of cytosine methylation covering regulatory or coding regions, we used the following restriction enzymes recognizing the site 5’CCGG3’: HpaII, sensitive to CG methylation, and MspI, sensitive to CNG methylation. Plant growth: plants were grown in pots with soil for 90 days, when watering was stopped. From that time on, samples were collected after 1, 2, 4, 7, 9 and 11 days DNA restriction: 100 ng DNA from each treatment was digested overnight by Hpa II or Msp I. Monitoring cutting effiency: by means of Real-time PCR, we analyzed a CCGG site 1700 bp 5’ to the transcription start point and another one in exon 1, using either treated or untreated DNA as template. As a control, we used a primer pair whose amplicon does not contain such a site. For comparisons, Ct values were previously normalized to those from this non-relevant amplicon. Conclusion: while the site in the regulatory region was constitutively methylated at both cytosines, remaining unchanged without irrigation, the cytosines studied in the coding region behave differentially: no methylation occurs in a CNG context but it does occur in a CG context, where the internal cytosine is demethylated as a consequence of water stress.