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An Ac/Ds-mediated gene trap system for functional genomics in barley.

ABSTRACT: BACKGROUND: Gene trapping is a powerful tool for gene discovery and functional genomics in both animals and plants. Upon insertion of the gene trap construct into an expressed gene, splice donor and acceptor sites facilitate the generation of transcriptional fusions between the flanking sequence and the reporter. Consequently, detection of reporter gene expression allows the identification of genes based on their expression pattern. Up to now rice is the only cereal crop for which gene trap approaches exist. In this study we describe a gene trap system in barley (Hordeum vulgare L.) based on the maize transposable elements Ac/Ds. RESULTS: We generated gene trap barley lines by crossing Ac transposase expressing plants with multiple independent transformants carrying the Ds based gene trap construct GTDsB. Upstream of the beta-Glucuronidase start codon GTDsB carries splice donor and acceptor sites optimized for monocotyledonous plants. DNA blot analysis revealed GTDsB transposition frequencies of 11% and 26% in the F1 and F2 generation of gene trap lines and perpetuation of transposition activity in later generations. Furthermore, analysis of sequences flanking transposed GTDsB elements evidenced preferential insertion into expressed regions of the barley genome. We screened leaves, nodes, immature florets, pollinated florets, immature grains and seedlings of F2 plants and detected GUS expression in 51% (72/141) of the plants. Thus, reporter gene expression was found in 24 of the 28 F1 lines tested and in progeny of all GTDsB parental lines. CONCLUSION: Due to the frequent transposition of GTDsB and the efficient expression of the GUS reporter gene, we conclude that this Ac/Ds-based gene trap system is an applicable approach for gene discovery in barley. The successful introduction of a gene trap construct optimized for monocots in barley contributes a novel functional genomics tool for this cereal crop.

SUBMITTER: Lazarow K

PROVIDER: S-EPMC2647555 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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An Ac/Ds-mediated gene trap system for functional genomics in barley.

Lazarow Katina K Lütticke Stephanie S

BMC genomics 20090129

<h4>Background</h4>Gene trapping is a powerful tool for gene discovery and functional genomics in both animals and plants. Upon insertion of the gene trap construct into an expressed gene, splice donor and acceptor sites facilitate the generation of transcriptional fusions between the flanking sequence and the reporter. Consequently, detection of reporter gene expression allows the identification of genes based on their expression pattern. Up to now rice is the only cereal crop for which gene tr ...[more]

PMID: 19178688

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Project description:BackgroundRapid improvements in the development of new sequencing technologies have led to the availability of genome sequences of more than 300 organisms today. Thanks to bioinformatic analyses, prediction of gene models and protein-coding transcripts has become feasible. Various reverse and forward genetics strategies have been followed to determine the functions of these gene models and regulatory sequences. Using T-DNA or transposons as tags, significant progress has been made by using "Knock-in" approaches ("gain-of-function" or "activation tagging") in different plant species but not in perennial plants species, e.g. long-lived trees. Here, large scale gene tagging resources are still lacking.ResultsWe describe the first application of an inducible transposon-based activation tagging system for a perennial plant species, as example a poplar hybrid (P. tremula L. × P. tremuloides Michx.). Four activation-tagged populations comprising a total of 12,083 individuals derived from 23 independent "Activation Tagging Ds" (ATDs) transgenic lines were produced and phenotyped. To date, 29 putative variants have been isolated and new ATDs genomic positions were successfully determined for 24 of those. Sequences obtained were blasted against the publicly available genome sequence of P. trichocarpa v2.0 (Phytozome v7.0; http://www.phytozome.net/poplar) revealing possible transcripts for 17 variants.In a second approach, 300 randomly selected individuals without any obvious phenotypic alterations were screened for ATDs excision. For one third of those transposition of ATDs was confirmed and in about 5% of these cases genes were tagged.ConclusionsThe novel strategy of first genotyping and then phenotyping a tagging population as proposed here is, in particular, applicable for long-lived, difficult to transform plant species. We could demonstrate the power of the ATDs transposon approach and the simplicity to induce ATDs transposition in vitro. Since a transposon is able to pass chromosomal boundaries, only very few primary transposon-carrying transgenic lines are required for the establishment of large transposon tagging populations. In contrast to T-DNA-based activation tagging, which is plagued by a lack of transformation efficiency and its time consuming nature, this for the first time, makes it feasible one day to tag (similarly to Arabidopsis) every gene within a perennial plant genome.

Project description:BACKGROUND: The nonautonomous maize Ds transposons can only move in the presence of the autonomous element Ac. They comprise a heterogeneous group that share 11-bp terminal inverted repeats (TIRs) and some subterminal repeats, but vary greatly in size and composition. Three classes of Ds elements can cause mutations: Ds-del, internal deletions of the 4.6-kb Ac element; Ds1, ~400-bp in size and sharing little homology with Ac, and Ds2, variably-sized elements containing about 0.5 kb from the Ac termini and unrelated internal sequences. Here, we analyze the entire complement of Ds-related sequences in the genome of the inbred B73 and ask whether additional classes of Ds-like (Ds-l) elements, not uncovered genetically, are mobilized by Ac. We also compare the makeup of Ds-related sequences in two maize inbreds of different origin. RESULTS: We found 903 elements with 11-bp Ac/Ds TIRs flanked by 8-bp target site duplications. Three resemble Ac, but carry small rearrangements. The others are much shorter, once extraneous insertions are removed. There are 331 Ds1 and 39 Ds2 elements, many of which are likely mobilized by Ac, and two novel classes of Ds-l elements. Ds-l3 elements lack subterminal homology with Ac, but carry transposase gene fragments, and represent decaying Ac elements. There are 44 such elements in B73. Ds-l4 elements share little similarity with Ac outside of the 11-bp TIR, have a modal length of ~1 kb, and carry filler DNA which, in a few cases, could be matched to gene fragments. Most Ds-related elements in B73 (486/903) fall in this class. None of the Ds-l elements tested responded to Ac. Only half of Ds insertion sites examined are shared between the inbreds B73 and W22. CONCLUSIONS: The majority of Ds-related sequences in maize correspond to Ds-l elements that do not transpose in the presence of Ac. Unlike actively transposing elements, many Ds-l elements are inserted in repetitive DNA, where they probably become methylated and begin to decay. The filler DNA present in most elements is occasionally captured from genes, a rare feature in transposons of the hAT superfamily to which Ds belongs. Maize inbreds of different origin are highly polymorphic in their DNA transposon makeup.

Dataset Information

An Ac/Ds-mediated gene trap system for functional genomics in barley.

Publications

An Ac/Ds-mediated gene trap system for functional genomics in barley.

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