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GrainGenes Reference Report: PMB-46-215

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Reference
PMB-46-215
Title
Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis.
Journal
Plant Molecular Biology
Year
2001
Volume
46
Pages
215-227
Author
Greco R
[ Show all 9 ]
Abstract
Summary: A GFP excision assay was developed to monitor the excision of Ac introduced into rice by Agrobacterium-mediated transformation. The presence of a strong double enhancer element of the CaMV 35S promoter adjacent to the Ac promoter induced very early excision, directly after transformation into the plant cell, exemplified by the absence of Ac in the T-DNA loci. Excision fingerprint analysis and characterization of transposition events from related regenerants revealed an inverse correlation between the number of excision events and transposed Ac copies, with single early excisions after transformation generating Ac amplification. New transpositions were generated at a frequency of 15-50% in different lines, yielding genotypes bearing multiple insertions, many of which were inherited in the progeny. The sequence of DNA flanking Ac in three representative lines provided a database of insertion tagged sites suitable for the identification of mutants of sequenced genes that can be examined for phenotypes in a reverse genetics strategy to elucidate gene function. Remarkably, two-thirds of Ac tagged sites showing homology to sequences in public databases were in predicted genes. A clear preference of transposon insertions in genes that are either predicted by protein coding capacity or by similarity to ESTs suggests that the efficiency of recovering knockout mutants of genes could be about three times higher than random. Linked Ac transposition, suitable for targeted tagging, was documented by segregation analysis of a crippled Ac element and by recovery of a set of six insertions in a contiguous sequence of 70 kb from chromosome 6 of rice
Keyword
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35s promoter
ac
ac transposition
agrobacterium tumefaciens
agrobacterium-mediated transformation
amplification
animal proteins
assay
camv 35s
camv 35s promoter
capacity
cauliflower mosaic caulimovirus
cell
chromosome
copies
correlation
database
dna
element
enhancer
enhancer element
est
ests
events
excision
fingerprint
fingerprint analysis
frequencies
function
gene
genetic transformation
genotype
gfp
homologies
homology
insertion
insertional mutagenesis
jellyfish
loci
multiple
mutagenesis
mutant
mutation
nucleotide sequence
oryza sativa
p
phenotype
preference
progeny
promoter
promoters
protein
regenerants
reporter gene
reverse genetics
rice
segregation
segregation analysis
sequence
similarities
site
strategies
strategy
t-dna
transformation
transposable elements
transposition
transposon
zea mays

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