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
采用静水方法研究甲醛、高锰酸钾和消毒粉(主要成分三氯异氰脲酸,TCCA)对锦绣龙虾胚胎的急性毒性效应。结果表明:甲醛对锦绣龙虾胚胎24、48、72、96 h的半致死浓度(LC_(50))分别是14.86、11.20、10.87、9.80 m L/m^3,安全浓度是0.98 m L...采用静水方法研究甲醛、高锰酸钾和消毒粉(主要成分三氯异氰脲酸,TCCA)对锦绣龙虾胚胎的急性毒性效应。结果表明:甲醛对锦绣龙虾胚胎24、48、72、96 h的半致死浓度(LC_(50))分别是14.86、11.20、10.87、9.80 m L/m^3,安全浓度是0.98 m L/m^3。高锰酸钾对锦绣龙虾胚胎24、48、72、96 h的半致死浓度(LC_(50))分别是1.88、1.52、1.26、1.06 mg/L,安全浓度为0.11 mg/L。消毒粉对锦绣龙虾胚胎24、48、72、96 h的半致死浓度(LC_(50))分别为19.91、16.76、16.38、16.38 mg/L,安全浓度为1.64 mg/L。展开更多
In the absence of photosynthesis, ATP is imported into chloroplasts and non-green plastids by ATP/ADP transporters or formed during glycolysis, the latter requiring continuous regeneration of NAD＋, supplied by the pl...In the absence of photosynthesis, ATP is imported into chloroplasts and non-green plastids by ATP/ADP transporters or formed during glycolysis, the latter requiring continuous regeneration of NAD＋, supplied by the plastidial isoform of NAD-MDH. During screening for T-DNA insertion mutants in the plNAD-MDH gene of Arabidopsis, only heterozygous plants could be isolated and homozygous knockout mutants grew only after complementation. These heterozygous plants show higher transcript levels of an alternative NAD＋-regenerating enzyme, NADH-GOGAT, and, remarkably, improved growth when ammonium is the sole N-source. In situ hybridization and GUS-histochemical stain- ing revealed that plNAD-MDH was particularly abundant in male and female gametophytes. Knockout plNAD-MDH pollen exhibit impaired tube growth in vitro, which can be overcome by adding the substrates of NADH-GOGAT. In vivo, knockout pollen is able to fertilize the egg cell. Young siliques of selfed heterozygous plants contain both green and white seeds corresponding to wild-type/heterozygous （green） and homozygous knockout mutants （white） in a （1：2）：1 ratio. Embryos of the homozygous knockout seeds only reached the globular stage, did not green, and developed to tiny wrinkled seeds. Complementation with the gene under the native promoter rescued this defect, and all seeds developed as wild-type. This suggests that a blocked major physiological process in plNAD-MDH mutants stops both embryo and endosperm development, thus avoiding assimilate investment in compromised offspring.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.
文摘In the absence of photosynthesis, ATP is imported into chloroplasts and non-green plastids by ATP/ADP transporters or formed during glycolysis, the latter requiring continuous regeneration of NAD＋, supplied by the plastidial isoform of NAD-MDH. During screening for T-DNA insertion mutants in the plNAD-MDH gene of Arabidopsis, only heterozygous plants could be isolated and homozygous knockout mutants grew only after complementation. These heterozygous plants show higher transcript levels of an alternative NAD＋-regenerating enzyme, NADH-GOGAT, and, remarkably, improved growth when ammonium is the sole N-source. In situ hybridization and GUS-histochemical stain- ing revealed that plNAD-MDH was particularly abundant in male and female gametophytes. Knockout plNAD-MDH pollen exhibit impaired tube growth in vitro, which can be overcome by adding the substrates of NADH-GOGAT. In vivo, knockout pollen is able to fertilize the egg cell. Young siliques of selfed heterozygous plants contain both green and white seeds corresponding to wild-type/heterozygous （green） and homozygous knockout mutants （white） in a （1：2）：1 ratio. Embryos of the homozygous knockout seeds only reached the globular stage, did not green, and developed to tiny wrinkled seeds. Complementation with the gene under the native promoter rescued this defect, and all seeds developed as wild-type. This suggests that a blocked major physiological process in plNAD-MDH mutants stops both embryo and endosperm development, thus avoiding assimilate investment in compromised offspring.
