Journal Article

RNAi-mediated Silencing of <i>OsGEN-L</i> (<i>OsGEN-like</i>), a New Member of the RAD2/XPG Nuclease Family, Causes Male Sterility by Defect of Microspore Development in Rice

Satoru Moritoh, Daisuke Miki, Masahiro Akiyama, Mihoko Kawahara, Takeshi Izawa, Hisaji Maki and Ko Shimamoto

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 46, issue 5, pages 699-715
Published in print May 2005 | ISSN: 0032-0781
Published online May 2005 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pci090
RNAi-mediated Silencing of OsGEN-L (OsGEN-like), a New Member of the RAD2/XPG Nuclease Family, Causes Male Sterility by Defect of Microspore Development in Rice

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We have cloned a new member of the RAD2/XPG nuclease family, OsGEN-L (OsGEN-like), from rice (Oryza sativa L.). OsGEN-L possesses two domains, the N- and I-regions, that are conserved in the RAD2/XPG nuclease family. Database searches and phylogenetic analyses revealed that OsGEN-L belongs to class 4 of the RAD2/XPG nuclease family, and OsGEN-L homologs were found in animals and higher plants. To elucidate the function of OsGEN-L, we generated rice OsGEN-L-RNAi transgenic plants in which OsGEN-L expression was silenced. Most of the OsGEN-L-RNAi plants displayed low fertility, and some of them were male-sterile. OsGEN-L-RNAi plants lacked mature pollen, resulting from a defect in early microspore development. A OsGEN-L–green fluorescent protein (GFP) fusion protein was localized in the nucleus, and the OsGEN-L promoter was specifically active in the anthers. Furthermore, a recombinant OsGEN-L protein possessed flap endonuclease activity and both single-stranded and double-stranded DNA-binding activities. Our results suggest that OsGEN-L plays an essential role in DNA metabolism required for early microspore development in rice.

Keywords: DNA-binding activity; Flap endonuclease activity; Male sterility; Microspore development; RAD2/XPG nuclease family; Rice (Oryza sativa L.); BAC, bacterial artificial chromosome; BER, base excision repair; DAPI, 4′,6-diamidino-2-phenylindole; DTT, dithiothreitol; EST, expressed sequence tag; GFP, green fluorescent protein; GUS, β-glucuronidase; HhH, helix–hairpin–helix; IPTG, isopropyl β-d-thiogalactopyranoside; MMS, methylmethane sulfonate; NER, nucleotide excision repair; RNAi, RNA interference; RT–PCR, reverse transcription–polymerase chain reaction; siRNA, short-interfering RNA

Journal Article.  11279 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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