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Plant Physiol. 2011 Jun;156(2):466-73. doi: 10.1104/pp.111.172981. Epub 2011 Apr 4.

Targeted mutagenesis of duplicated genes in soybean with zinc-finger nucleases.

Author information

1
Department of Agronomy and Plant Genetics, University of Minnesota, St Paul, Minnesota 55108, USA.

Abstract

We performed targeted mutagenesis of a transgene and nine endogenous soybean (Glycine max) genes using zinc-finger nucleases (ZFNs). A suite of ZFNs were engineered by the recently described context-dependent assembly platform--a rapid, open-source method for generating zinc-finger arrays. Specific ZFNs targeting dicer-like (DCL) genes and other genes involved in RNA silencing were cloned into a vector under an estrogen-inducible promoter. A hairy-root transformation system was employed to investigate the efficiency of ZFN mutagenesis at each target locus. Transgenic roots exhibited somatic mutations localized at the ZFN target sites for seven out of nine targeted genes. We next introduced a ZFN into soybean via whole-plant transformation and generated independent mutations in the paralogous genes DCL4a and DCL4b. The dcl4b mutation showed efficient heritable transmission of the ZFN-induced mutation in the subsequent generation. These findings indicate that ZFN-based mutagenesis provides an efficient method for making mutations in duplicate genes that are otherwise difficult to study due to redundancy. We also developed a publicly accessible Web-based tool to identify sites suitable for engineering context-dependent assembly ZFNs in the soybean genome.

PMID:
21464476
PMCID:
PMC3177250
DOI:
10.1104/pp.111.172981
[Indexed for MEDLINE]
Free PMC Article

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