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Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus

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Abstract

Two genomic clones (OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice (Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56–75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.

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Abbreviations

BAH:

bromo adjacent homology (domain)

GFP:

green fluorescent protein

GUS:

β-glucuronidase

MTase:

methyltransferase

RACE:

rapid amplification of cDNA ends

RT:

reverse transcription

35S:

cauliflower mosaic virus 35S promoter

uidA :

β-glucuronidase gene from Escherichia coli

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Acknowledgements

This work was funded by the Texas Advanced Technology Program (010366-0092-1999) and NSF (MCB 0110477). M.B.C. was supported by NSF grant MCB 9974706. P.T. and J.N. were funded in part by The Royal Golden Jubilee Ph.D. Program (PHD/00075/2541), The Thailand Research Fund. We thank Guojun Yang, Xiangyu Shi, Lakshminarayan Iyer and Amaret Bhumiratana for valuable discussions.

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  1. Institute of Developmental and Molecular Biology, and Department of Biology, Texas A&M University, College Station, TX 77843–3155, USA

    Prapapan Teerawanichpan, Mahesh B. Chandrasekharan, Yiming Jiang & Timothy C. Hall

  2. Department of Biotechnology, Faculty of Science, Mahidol University, Rama 6 Road, 10400, Bangkok, Thailand

    Prapapan Teerawanichpan & Jarunya Narangajavana

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  1. Prapapan Teerawanichpan
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  2. Mahesh B. Chandrasekharan
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Correspondence to Timothy C. Hall.

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Teerawanichpan, P., Chandrasekharan, M.B., Jiang, Y. et al. Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus. Planta 218, 337–349 (2004). https://doi.org/10.1007/s00425-003-1112-6

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