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

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.