Details

Autor(en) / Beteiligte

• Djukanovic, Vesna
• Smith, Jeff
• Lowe, Keith
• Yang, Meizhu
• Gao, Huirong
• Jones, Spencer
• Nicholson, Michael G
• West, Ande
• Lape, Janel
• Bidney, Dennis
• Carl Falco, Saverio
• Jantz, Derek
• Alexander Lyznik, Leszek

Titel

Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease

Ist Teil von

• The Plant journal : for cell and molecular biology, 2013-12, Vol.76 (5), p.888-899

Ort / Verlag

England: Blackwell Publishing Ltd

Erscheinungsjahr

2013

Quelle

Wiley Blackwell Single Titles

Beschreibungen/Notizen

• The I–CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double‐strand breaks and enhancing DNA recombination reactions in maize cells. The DNA‐binding properties of this protein were re‐designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single‐chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium‐mediated transformation, the cleavage resulted in mutations at a co‐delivered extra‐chromosomal ms26‐site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26‐site in 5.8% of transgenic T₀ plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double‐strand break repair generated by non‐homologous end joining. One of 21 mutagenized T₀ plants carried two mutated alleles of the MS26 gene. As expected, the bi‐allelic mutant T₀ plant and the T₁ progeny homozygous for the ms26 mutant alleles were male‐sterile. This paper described the second maize chromosomal locus (liguless‐1 being the first one) mutagenized by a re‐designed I–CreI–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.