AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （...AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （ABA） and ethylene stimulus ATSYR1 gene encodes a syntaxin localizing at the plasma membrane in Arabidopsis, which can be induced by abiotic stress. To identify mutation lines for gene functional analysis, real-time PCR was employed to detect the expression level of AtERF4 and ATSYR1 in homozygous T-DNA insertion mutant line, respectively. Real-time PCR is a powerful tool which can be used to detect steady-state mRNA levels specifically, sensitively and reproducibly. Comparing to other forms of quantitative RT-PCR, the amount of amplified products can be detected by real-time PCR instantly and thus is a preferable alternative. In this study, RNA with T-DNA inserting into exon could be detected in AtERF4 knock-out mutation line. The results indicated that AtERF4 had been trucked in transcription level. On the other hand, T-DNA inserting into the promoter of gene ATSYR1 had no effect on reducing the expression level ofATSYR1 gene. Further molecular and phenotype studies now are ongoing to clarify the potential consequences of AtERF4 and ATSYR1 deficiency in Arabidopsis展开更多
Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-st...Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.展开更多
The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Th...The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Through sequencing PCR-amplifiedfragments adjacent to the inserted elements,wecan construct the T-DNA flanking database,which would be useful for cloning the genestagged by T-DNA.展开更多
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
bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mut...bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.展开更多
[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-...[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-DNA tag lines, the progeny of homozygous plants carrying T-DNA insertion were screened for mutants with mutated phenotypes. The genetic analysis of the mutant and test for the linkage between the mutated phenotype and the T-DNA insertion were carried out to determine its genetic characteristics. [Result] In the present study, a grain shape mutant induced by T-DNA insertion in rice was identified, which showed small grain. Genetic analysis of the mutant showed that the two types of phenotype, normal and small grain in the segregating populations derived from the T-DNA heterozygotes, fit the ratio of 3∶1. Test for Basta resistance showed that all the mutants were resistant while the normal plants segregated for resistant and susceptible by the ratio of 2∶1. The results indicated that the mutant phenotype cosegregated with Bar gene. The small grain mutant caused by T-DNA insertion was confirmed by PCR amplification aiming at T-DNA. [Conclusion] The grain shape mutant is useful for isolation of the tagged gene and genetic improvement in rice.展开更多
基金Supported by National High Technology Program (2008ZX08004-002, 2009ZX08009-032B)Key Research Plan of Heilongjiang Province (GA06B103)Education Department Plan of Heilongjiang Province(11521021, 1152024)
文摘AtERF4 （ethylene response factor） is a negative regulator in jasmonic acid mediated signal transduction pathway and ethylene mediated signal transduction pathway of Arabidopsis. It could respond to abscisic acid （ABA） and ethylene stimulus ATSYR1 gene encodes a syntaxin localizing at the plasma membrane in Arabidopsis, which can be induced by abiotic stress. To identify mutation lines for gene functional analysis, real-time PCR was employed to detect the expression level of AtERF4 and ATSYR1 in homozygous T-DNA insertion mutant line, respectively. Real-time PCR is a powerful tool which can be used to detect steady-state mRNA levels specifically, sensitively and reproducibly. Comparing to other forms of quantitative RT-PCR, the amount of amplified products can be detected by real-time PCR instantly and thus is a preferable alternative. In this study, RNA with T-DNA inserting into exon could be detected in AtERF4 knock-out mutation line. The results indicated that AtERF4 had been trucked in transcription level. On the other hand, T-DNA inserting into the promoter of gene ATSYR1 had no effect on reducing the expression level ofATSYR1 gene. Further molecular and phenotype studies now are ongoing to clarify the potential consequences of AtERF4 and ATSYR1 deficiency in Arabidopsis
基金the National Key R&D Program of China(Nos.2018YFD0901506,2018YFD0900305)the Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology(Qingdao)(No.2018 SDKJ0406-3)。
文摘Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.
文摘The use of transfer DNA(T-DNA)as amutagen has been developed for tagging genes inmany crops,and results showed that T-DNAinsertion is a random event,and that theinserted genes are stable through multiplegenerations.Through sequencing PCR-amplifiedfragments adjacent to the inserted elements,wecan construct the T-DNA flanking database,which would be useful for cloning the genestagged by T-DNA.
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 National Natural Science Foundation of China (30570990)National Major Project for Cultivation of Transgenic Crops (20082x08004)+1 种基金Key Research Plan of Heilongjiang Province (GA06B103)Innovation Research Group of NEAU (CXT004)
文摘bZIP transcription factor family is one of the largest groups of the plant transcription factor families and plays an important role in plant growth and adaption to the abiotic stresses. In this study, two AtbZIP1 mutant Arabidopsis （bzipl） were used with T-DNA inserted into two different sites, designated as SALK-556773 and SALK-660942, in order to identify different effects on AtbZIP1 gene expression by different T-DNA insertion sites. PCR and RT-PCR results revealed that T-DNA insertion in CDS region could effectively inhibit AtbZIP1 gene expression, while T-DNA insertion in 3＇-UTR couldn＇t. The phenotype analysis further confirmed the differences and showed that T-DNA insertion in CDS region decreased plants＇ drought resistance, while in 3＇-UTR couldn＇t. The phenotype assays also suggested that AtbZIP1 held pivotal roles in plant response to drought stress.
文摘[Objective] The aim of this study is to understand the genetic characteristics of a grain shape mutant and its possible role in genetic improvement of grain yield in rice. [Method] On the basis of the collection of T-DNA tag lines, the progeny of homozygous plants carrying T-DNA insertion were screened for mutants with mutated phenotypes. The genetic analysis of the mutant and test for the linkage between the mutated phenotype and the T-DNA insertion were carried out to determine its genetic characteristics. [Result] In the present study, a grain shape mutant induced by T-DNA insertion in rice was identified, which showed small grain. Genetic analysis of the mutant showed that the two types of phenotype, normal and small grain in the segregating populations derived from the T-DNA heterozygotes, fit the ratio of 3∶1. Test for Basta resistance showed that all the mutants were resistant while the normal plants segregated for resistant and susceptible by the ratio of 2∶1. The results indicated that the mutant phenotype cosegregated with Bar gene. The small grain mutant caused by T-DNA insertion was confirmed by PCR amplification aiming at T-DNA. [Conclusion] The grain shape mutant is useful for isolation of the tagged gene and genetic improvement in rice.
