Volume 6 Issue 2, December 1996: 125-136
ORIGINAL ARTICLES
Isolation of T-DNA flanking plant DNA from T-DNA insertional embryo-lethal mutants of Arabidopsis thaliana by plasmid resced technique
YAO XIAO LI,JIAN GE SUN,ZHI PING ZHU
Chinese National Laboratory of Plant Molecular Genetics
Shanghai Institute of plant Physiology , Chinese Academy of Sciences, 320 yueyang Road, Shanghai 200031, China
Correspondence: ZHI PING ZHU
Three T-DNA insertional embryonic lethal mutants from NASC( The Nottingham Arabidopsis Stock Center) were first checked with their segreation ration of abortive and normal seeds and the copy number of T-DNA insertion . The N4081 mutant has a segreation ration of 1:3:04 in average and one T-DNA insertion for further analysis. To isolate the joint fragment of T-DNA ,which contained pBR322 was selected 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 hypridized 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 Plasmid rescue and cloning of T-DNA /Plant DNA junction
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