African Journal of Biotechnology Vol. 5 (15), pp. 1375-1382, 3 August 2006   

ISSN 1684–5315 © 2006 Academic Journals        

Full Length Research Paper

Effect of short day photoperiod on DNA methylation and expression of a gene in rice KDML105

Theerachai Thanananta^1,2*, Pradit Pongtongkam², Amara Thongpan², Lilly

Kaveeta³, Surin Peyachoknagul²

¹Department of Biotechnology, Faculty of Science and Technology, Thammasat

University, Pathum Thani 12121, Thailand.

²Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10903, Thailand.

³Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10903, Thailand.

*Corresponding authors E-mail: thanananta@yahoo.com. Tel: 662-564-4444 ext. 2833. Fax: 662-564-4500.

Accepted 23 June, 2006

Methylation-sensitive amplified polymorphism (MSAP) technique was adapted to screen a photoperiod-responsive gene of rice KDML105. Six out of thirty-two primer combinations gave twelve EcoRI/HpaII-generated MSAP markers from the genomic DNA of KDML105 after exposure to short-day (SD) photoperiod. These MSAP fragments were cloned and used as probes to hybridize to MSAP fingerprints. Positive fragments generating the signals correlated to the MSAP fingerprints were sequenced and aligned to the database. Ten out of twelve MSAP markers showed 93-100% homology to database sequences and the best homology fragment (F1) was chosen for complete gene cloning, sequencing, and alignment. This candidate gene fragment showed 98% homology to the sequences of rice genomic DNA gi|23237916 and rice cDNA gi|32986083. It also has an open reading frame (ORF) of 402 bp and deduced a polypeptide of 134 amino acid residues, while those of gi|23237916 and gi|32986083 have a deduced polypeptide of 137 amino acid residues resulting in the overall homology of 97%. In addition, RT-PCR products and the Southern blot hybridization of the genomic DNA of KDML105 digested with HpaII or MspI indicated that the expression of this candidate gene depends on DNA demethylation, which is induced by 10 days of SD photoperiod. It is speculated that this gene might belong to another class of regulatory gene for the flowering time or heading-time loci in rice or it is a downstream product of flower initiation.     

Key words: DNA methylation, photoperiod, rice, Oryza sativa L., MSAP, methylation sensitive AFLP.