Isolation of T-DNA flanking plant DNA from T-DNA insertional embryo-lethal mutants of Arabidopsis thaliana by plasmid rescue technique

Abstract

Three T-DNA insertional embryonic lethal mutants from NASC (The Nottingham Arabidopsis Stock Center) were first checked with their segregation ratio of abortive and normal seeds and the copy number of T-DNA insertion. The N4081 mutant has a segregation ratio of 1:3.04 in average and one T-DNA insertion site according to our assay. It was therefore chosen for further analysis. To isolate the joint fragment of T-DNA and plant DNA, the plasmid rescue technique was used. pEL-7, one of plasmids from left border of T-DNA, which contained pBR322 was selected from ampicillin plate. The T-DNA fragment of pEL-7 was checked by restriction enzyme analysis and Southern Blot. Restriction analysis confirmed the presence of known sites of EcoRI, PstI and PvuII on it. For confirming the presence of flanking plant DNA in this plasmid, pEL-7 DNA was labeled and hybridized with wild type and mutant plant DNA. The Southern Blot indicated the hybridization band in both of them. Furthermore, the junction of T-DNA/plant DNA was subcloned into bluescript SK+ and sequenced by Applied Biosystem 373A Sequencer. The results showed the 822 bp fragment contained a 274 bp sequence, which is 99.6% homolog (273bp/274 bp) to Ti plasmid pTi 15955 DNA. Ten bp of left 25 bp border repeat were also found in the juction of T-DNA and Plant DNA. Taken together, pEL-7 should contain a joint fragment of T-DNA and flanking plant DNA. This plasmid DNA could be used for the isolation of plant gene, which will be helpful to elucidate the relationship between gene function and plant embryo development.