Genetische Veränderung von Tabak mittels Zielsequenz-spezifizierter Endonukleasen

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VII Jornadas de Expania

Sección: Noticias. Noticias externasLos días 27 y 28 de mayo se celebraron en Santiago de Compostela las VII Jornadas de Expania, la Asociación de Usuarios de Ex Libris en España.N

True-Breeding Targeted Gene Knock-Out in Barley Using Designer TALE-Nuclease in Haploid Cells

<div><p>Transcription activator-like effector nucleases (TALENs) are customizable fusion proteins able to cleave virtually any genomic DNA sequence of choice, and thereby to generate site-directed genetic modifications in a wide range of cells and organisms. In the present study, we expressed TALENs in pollen-derived, regenerable cells to establish the generation of instantly true-breeding mutant plants. A <i>gfp-</i>specific TALEN pair was expressed via <i>Agrobacterium</i>-mediated transformation in embryogenic pollen of transgenic barley harboring a functional copy of <i>gfp</i>. Thanks to the haploid nature of the target cells, knock-out mutations were readily detected, and homozygous primary mutant plants obtained following genome duplication. In all, 22% of the TALEN transgenics proved knocked out with respect to <i>gfp,</i> and the loss of function could be ascribed to the deletions of between four and 36 nucleotides in length. The altered <i>gfp</i> alleles were transmitted normally through meiosis, and the knock-out phenotype was consistently shown by the offspring of two independent mutants. Thus, here we describe the efficient production of TALEN-mediated gene knock-outs in barley that are instantaneously homozygous and non-chimeric in regard to the site-directed mutations induced. This TALEN approach has broad applicability for both elucidating gene function and tailoring the phenotype of barley and other crop species.</p></div

Root tips of reporter and mutated lines visualized.

<p>A root tip of a reporter line (a) under white light and (b) when excited with far blue light. A root tip of a TALEN <i>gfp</i> knock-out individual (24/2-1) visualized (c) under white light and (d) when excited by far blue light.</p

Characterization of primary <i>gfp</i> knock-out plants.

1<p>according to DNA gel blot analysis of <i>Hin</i>dIII-digested DNA.</p>2<p>according to DNA gel blot analysis of <i>Ssp</i>I-digested DNA.</p>3<p>haploid plantlets treated with colchicine to induce chromosome doubling.</p

Modifications of the <i>gfp</i> sequence induced by TALENs.

<p>(a) Alignment of the <i>gfp</i> sequences of the various mutants and the donor line. The underlined sequence in red represents the left and that in blue the right TALEN binding site; the sequence shown in green is a duplication of the underlined motif. The number of nucleotides deleted (dashes) or inserted (upper case letters in green) is shown to the right of each sequence. (b) Intact <i>gfp</i> (WT) and <i>gfp</i> knock-out peptide sequences. Stop codons indicated by asterisks.</p

Various <i>gfp</i> alleles (A–F) present in T₁ individuals derived from the primary transformant 32/2-2.

<p>The underlined sequence in red represents the left and that in blue the right TALEN binding site. Note that allele 32/2-2_B (not shown in this figure) that was identified in the primary mutant could not be detected in the progeny.</p

Schematic representation of the T-DNA TALEN constructs.

<p>The right-TALEN unit is shown above the left one (not drawn to scale). Primer binding sites are indicated by arrows and the <i>Fok</i>I and <i>BAR</i> probe regions used for DNA gel blot analysis by bars. LB: T-DNA left border, UBIP: maize <i>UBIQUITIN-1</i> promoter, NLS: SV40 nuclear localization signal, HA: hemagglutinin tag, TALEN-Right: customized DNA binding domain of right TALEN unit, TALEN-Left: customized DNA binding domain of left TALEN unit, FokI: DNA cleavage domain of <i>Fok</i>I endonuclease, NOST: <i>A. tumefaciens NOPALINE SYNTHASE</i> terminator, 35ST: <i>CaMV 35S</i> terminator, BAR: bialaphos resistance gene, d35SP: <i>CaMV 35S</i> promoter harbouring a doubled enhancer sequence, RB: T-DNA right border, <i>Hin</i>dIII: restriction site targeted in the DNA gel blot analysis.</p

Summary of <i>gfp</i>-specific TALEN transformation outputs.

<p>Summary of <i>gfp</i>-specific TALEN transformation outputs.</p

The inheritance of mutated <i>gfp</i> in the T₁ generation bred from plants 24/2-1 and 24/2-2 all of which carried a knock-out copy of <i>gfp</i>.

<p>Scale bar: 0.2 mm. Root tips imaged (a–c): under bright field, (d–f): after excitation with far blue light. (a, d) <i>gfp</i> donor, (b, e) 24/2-1, (c, f) 24/2-2. Some auto-fluorescence was detectable in the root hairs of the mutants (see e, f) and also in those of non-transformed plants (not shown).</p