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. ...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 1:3.04in 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 plan DNA, the plasmid rescue technique waJs 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.展开更多
The expression of FOAl （F-box overexpressed/oppressed ABA signaling） in different organs of Arabidopsis, and in response to ABA and NaCI, was analyzed. The expression level of FOAl is higher in the root and is lower...The expression of FOAl （F-box overexpressed/oppressed ABA signaling） in different organs of Arabidopsis, and in response to ABA and NaCI, was analyzed. The expression level of FOAl is higher in the root and is lower in the stem, and is induced rapidly by ABA and NaC1. The phenotypes of T-DNA insertion mutant foal and FOAl overexpression lines FOAloxl and FOAlox2 were analyzed. The foal mutant exhibited a lower germination rate, shorter root length, more stomatal opening, in- creased proline accumulation and hypersensitivity to ABA compared with the wild type. In contrast, the overexpression lines showed lower sensitivity to ABA than the wild type. The expression levels of several ABA and stress-responsive transcription factors and genes were altered in the foal mutant in response to ABA. Compared with the wild type, the expression levels of ABA-responsive transcription factors were higher, but ABA and stress-responsive genes were lower in foal mutant. This study demonstrates that FOAl is an ABA signaling-related gene, and may play a negative role in ABA signaling.展开更多
文摘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 1:3.04in 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 plan DNA, the plasmid rescue technique waJs 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.
基金supported by the National Natural Science Foundation of China(Grant No.31171176)Research Fund for the Doctoral Program of Higher Education of China(Grant No.755228001)+1 种基金Natural Science Foundation of Hunan Province(Grant No.11JJA002)the National Key Laboratory of Plant Molecular Genetics(Grant No.Y109Z711T1)
文摘The expression of FOAl （F-box overexpressed/oppressed ABA signaling） in different organs of Arabidopsis, and in response to ABA and NaCI, was analyzed. The expression level of FOAl is higher in the root and is lower in the stem, and is induced rapidly by ABA and NaC1. The phenotypes of T-DNA insertion mutant foal and FOAl overexpression lines FOAloxl and FOAlox2 were analyzed. The foal mutant exhibited a lower germination rate, shorter root length, more stomatal opening, in- creased proline accumulation and hypersensitivity to ABA compared with the wild type. In contrast, the overexpression lines showed lower sensitivity to ABA than the wild type. The expression levels of several ABA and stress-responsive transcription factors and genes were altered in the foal mutant in response to ABA. Compared with the wild type, the expression levels of ABA-responsive transcription factors were higher, but ABA and stress-responsive genes were lower in foal mutant. This study demonstrates that FOAl is an ABA signaling-related gene, and may play a negative role in ABA signaling.
