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Plant J. 2013 Dec;76(5):888-99. doi: 10.1111/tpj.12335. Epub 2013 Nov 5.

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

Author information

1
DuPont/Pioneer Agricultural Biotechnology, 8305 N.W. 62nd Avenue, Johnston, IA, 50131, USA.

Abstract

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 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 MS26 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-CreI-based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.

KEYWORDS:

Zea mays L; double-strand break; homing endonuclease; maize; mutagenesis; technical advance

PMID:
24112765
DOI:
10.1111/tpj.12335
[Indexed for MEDLINE]
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