A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

Andrew Eamens; Chris L. Blanchard; Elizabeth S Dennis; Narayana M Upadhyaya

doi:10.1111/j.1467-7652.2004.00081.x

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A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

Journal article

Peer reviewed

A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)

Andrew Eamens, Chris L. Blanchard, Elizabeth S Dennis and Narayana M Upadhyaya

Plant Biotechnology Journal, Vol.2(5), pp.367-380

2004

DOI: https://doi.org/10.1111/j.1467-7652.2004.00081.x

PMID: 17168884

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Abstract

barnase

bidirectional T-DNA/Ds gene trap construct

flanking sequence tag (FST)

gus

insert-related sequence (IRS)

reporter gene expression

rice (Oryza sativa)

rice transformation

sgfpS65T

vector backbone (VB) sequence

A construct suitable for genome-wide transfer-DNA (T-DNA) and subsequent transposon-based (Ds) gene trapping has been developed for use in rice (Oryza sativa). This T-DNA/Ds construct contains: Ds terminal sequences immediately inside T-DNA borders for subsequent Ds mobilization; promoterless green fluorescent protein (sgfpS65T) and beta-glucuronidase (uidA) reporter genes, each fused to an intron (from Arabidopsis GPA1 gene) to enable bidirectional gene trapping by T-DNA or Ds; an ampicillin resistance gene (bla) and a bacterial origin of replication (ori) to serve as the plasmid rescue system; an intron-containing hygromycin phosphotransferase gene (hph) as a selectable marker or Ds tracer; and an intron-containing barnase gene in the binary vector backbone (VB) to select against transformants carrying unwanted VB sequences. More than a threefold increase over previously reported reporter gene-based gene trapping efficiencies was observed in primary T-DNA/Ds transformant rice lines, returning an overall reporter gene expression frequency of 23%. Of the plant organs tested, 3.3-7.4% expressed either reporter at varying degrees of organ or tissue specificity. Approximately 70% of the right border (RB) flanking sequence tags (FSTs) retained 1-6 bp of the RB repeat and 30% of the left border (LB) FSTs retained 5-23 bp of the LB repeat. The remaining FSTs carried deletions of 2-84 bp inside the RB or 1-97 bp inside the LB. Transposition of Ds from the original T-DNA was evident in T-DNA/Ds callus lines super-transformed with a transposase gene (Ac) construct, as indicated by gene trap reporter activity and rescue of new FSTs in the resulting double transformant lines.

Details

Title: A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.)
Authors: Andrew Eamens (Author) - CSIRO Plant Industry
Chris L. Blanchard (Author) - Charles Sturt University
Elizabeth S Dennis (Author) - CSIRO Plant Industry
Narayana M Upadhyaya (Author) - CSIRO Plant Industry
Publication details: Plant Biotechnology Journal, Vol.2(5), pp.367-380
Publisher: Wiley-Blackwell Publishing Ltd.
DOI: 10.1111/j.1467-7652.2004.00081.x
ISSN: 1467-7652
PMID: 17168884
Organisation Unit: School of Health and Behavioural Sciences - Legacy; University of the Sunshine Coast, Queensland; School of Health - Biomedicine
Language: English
Record Identifier: 99656589202621
Output Type: Journal article

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Collaboration types: Domestic collaboration
Web Of Science research areas: Biotechnology & Applied Microbiology; Plant Sciences

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