Dissociation (Ds) constructs, mapped Ds launch pads and a transiently-expressed transposase system suitable for localized insertional mutagenesis in rice.
We have developed a transiently-expressed transposase (TET)-mediated Dissociation (Ds) insertional mutagenesis system for generating stable insertion lines in rice which will allow localized mutagenesis of a chromosomal region. In this system, a Ds containing T-DNA construct was used to produce Ds launch pad lines. Callus tissues, from single-copy Ds/T-DNA lines, were then transiently infected with Agrobacterium harbouring an immobile Ac (iAc) construct, also containing a green fluorescent protein gene (sgfpS65T) as the visual marker. We have regenerated stable Ds insertion lines at a frequency of 9-13% using selection for Ds excision and GFP counter selection against iAc and nearly half of them were unique insertion lines. Double transformants (iAc/Ds) were also obtained and their progeny yielded approximately 10% stable insertion lines following excision and visual marker screening with 50% redundancy. In general, more than 50% of the Ds reinsertions were within 1 cM of the launch pad. We have produced a large number of single-copy Ds/T-DNA launch pads distributed over the rice chromosomes and have further refined the Ds/T-DNA construct to enrich for "clean" single-copy T-DNA insertions. The availability of single copy "clean" Ds/T-DNA launch pads will facilitate chromosomal region-directed insertion mutagenesis. This system provides an opportunity for distribution of gene tagging tasks among collaborating laboratories on the basis of chromosomal locations.
Upadhyaya
Narayana M
NM
CSIRO Plant Industry, GPO Box 1600, 2601, Canberra, ACT, Australia. Narayana.upadhyaya@csiro.au
Zhu
Qian-Hao
QH
Zhou
Xue-Rong
XR
Eamens
Andrew L
AL
Hoque
Mohammad S
MS
Ramm
Kerrie
K
Shivakkumar
Ramannee
R
Smith
Kathryn F
KF
Pan
Shu-Ting
ST
Li
Suzhi
S
Peng
Kefan
K
Kim
Song J
SJ
Dennis
Elizabeth S
ES
D002874:Chromosome Mapping; D032461:Chromosomes, Plant; D018983:DNA Footprinting; D018744:DNA, Plant; D016208:Databases, Factual; D018628:Gene Dosage; D017343:Genes, Plant; D017930:Genes, Reporter; D018745:Genome, Plant; D016254:Mutagenesis, Insertional; D012275:Oryza / Q000235:genetics*; D014170:Transformation, Genetic; D019076:Transgenes; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
D018744:DNA, Plant; D019895:Transposases