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Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P 450‐like gene ( MS 26 ) using a re‐designed I– C re I homing endonuclease
Author(s) -
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
Publication year - 2013
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.12335
Subject(s) - homing endonuclease , biology , genetics , mutagenesis , mutant , homologous recombination , endonuclease , gene , microbiology and biotechnology , insertional mutagenesis , gene targeting , dna
Summary The I– Cre I 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 MS 26 , a maize fertility gene. Three versions of a single‐chain endonuclease, called E ms26, E ms26+ and E ms26++, 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 B lack M exican 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 T 0 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 T 0 plants carried two mutated alleles of the MS 26 gene. As expected, the bi‐allelic mutant T 0 plant and the T 1 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– C re I–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.