T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants,and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype.In this study,we screen...T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants,and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype.In this study,we screened 4416 rice TI tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents,ZH 11 and ZH 15.We found many lines showed obvious morphological mutations,including two types-fake-homozygous mutation and separating mutation.The mutation phenotype was related to 14 kinds of trait such as plant height,heading date,leaf shape,leaf color,tiller number,panicle shape,spikelet number,grain shape,disease-like mutation,male sterility,awn,and so on.Among them,plant height,heading date,leaf color and male sterility had a comparatively high mutation frequency(over 1%).The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents,but the frequency of head-ing date and male sterility varied greatly,suggesting that environment had a great effect on the expression of latter two traits.By conducting continuously co-segregating analyses in TI and T2 generation,we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets,which would provide a base for cloning correlative functional genes.At the same time,we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR,of which,26 were vector backbone sequence,14 had good identity to rice genome sequence.The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.展开更多
Objective To investigate the 23 bp and 12 bp insertion/deletion(indel)mutations within the bovine prion protein(PRNP)gene in Chinese dairy cows,and to detect the associations of two indel mutations with BSE susceptibi...Objective To investigate the 23 bp and 12 bp insertion/deletion(indel)mutations within the bovine prion protein(PRNP)gene in Chinese dairy cows,and to detect the associations of two indel mutations with BSE susceptibility and milk performance.Methods Based on bovine PRNP gene sequence,two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed.The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations.Moreover,based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study,the corrections between the two indel mutations and BSE susceptibility were tested,as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses.Results In the analyzed Chinese Holstein population,the frequencies of two"del"alleles in 23 bp and 12 bp indel muations were more frequent.The frequency of haplotype of 23del-12del was higher than those of 23del-12ins and 23ins-12del.From the estimated r2and D’values,two indel polymorphisms were linked strongly in the Holstein population(D’=57.5%,r2=0.257).Compared with the BSE-affected cattle populations from the reported data,the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations(P<0.05 or P<0.01).Similarly,there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle(P<0.05 or P<0.01).Moreover,association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population,but no significant differences were found(P>0.05).Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits,which layed the foundation for future selection of resistant animals,and for improving health conditions for dairy breeding against BSE in China.展开更多
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
[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
A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was develope...A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was developed to systemi- cally study the rice seed quality control. Genome-wide analysis of the FST distribution showed that T-DNA insertions were positively correlated with expressed genes, but negatively with transposable elements and small RNAs. In addition, the recovered T-DNAs were preferentially located at the untranslated region of the expressed genes. More than 11 000 putative homozygous lines were obtained through multi-generations of planting and resistance screening, and measurement of seed quality of around half of them, including the contents of starch, amylose, protein and fat, with a nondestructive near-infrared spectroscopy method, identified 551 mutants with unique or multiple altered param- eters of seed quality. Analysis of the corresponding FSTs showed that genes participating in diverse functions, including metabolic processes and transcriptional regulation, were involved, indicating that seed quality is regulated by a complex network.展开更多
Agrobacterium tumefaciens-mediated DNA transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-re...Agrobacterium tumefaciens-mediated DNA transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-resistant gene cassette between the right and left borders of T-DNA, Optimal co-cultivation of N.sylviforrne with A. tumefaciens containing pBI121-43 led to 110- 130 hygromycin-resistant transformants per＂ million eonidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N. sylviforme fusant HDF-68, and the development of such svstem lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.展开更多
Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F....Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.展开更多
Mutator transposable element （Mu） has been used as an effective tool to clone maize （Zea mays L.） genes. One opaque endosperm mutant （miol6） was identified in a pool of Mu inserted mutants. A modified method, te...Mutator transposable element （Mu） has been used as an effective tool to clone maize （Zea mays L.） genes. One opaque endosperm mutant （miol6） was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments （DSAIFF）, was employed to isolate the Mu flanking fragments （MFFs） of miol6. The target site duplications （TSDs） isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and miol6 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of miol6 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of miol6 is alternated by Mu insertion.展开更多
Genetic algorithms(GAs)are very good metaheuristic algorithms that are suitable for solving NP-hard combinatorial optimization problems.AsimpleGAbeginswith a set of solutions represented by a population of chromosomes...Genetic algorithms(GAs)are very good metaheuristic algorithms that are suitable for solving NP-hard combinatorial optimization problems.AsimpleGAbeginswith a set of solutions represented by a population of chromosomes and then uses the idea of survival of the fittest in the selection process to select some fitter chromosomes.It uses a crossover operator to create better offspring chromosomes and thus,converges the population.Also,it uses a mutation operator to explore the unexplored areas by the crossover operator,and thus,diversifies the GA search space.A combination of crossover and mutation operators makes the GA search strong enough to reach the optimal solution.However,appropriate selection and combination of crossover operator and mutation operator can lead to a very good GA for solving an optimization problem.In this present paper,we aim to study the benchmark traveling salesman problem(TSP).We developed several genetic algorithms using seven crossover operators and six mutation operators for the TSP and then compared them to some benchmark TSPLIB instances.The experimental studies show the effectiveness of the combination of a comprehensive sequential constructive crossover operator and insertion mutation operator for the problem.The GA using the comprehensive sequential constructive crossover with insertion mutation could find average solutions whose average percentage of excesses from the best-known solutions are between 0.22 and 14.94 for our experimented problem instances.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： Although the kinase insert domain-containing receptor （KDR） gene play an very important role in the metastasis of cancer and is also as one of the molecular targets used in cancer therapy, mutation in th...Objective： Although the kinase insert domain-containing receptor （KDR） gene play an very important role in the metastasis of cancer and is also as one of the molecular targets used in cancer therapy, mutation in the tyrosine kinase （TK） domain of the KDR gene has not been reported. Here we detected the mutations and polymorphisms in the TK domain of KDR gene in human lung cancer patients and to give the basic evidence and clue for cancer prevention and target therapy. Methods： The entire sequence of exons 21, 22, 23 and 27 （which contain the coding sequence of tyrosine phosphorylation） in the TK domain of KDR gene in the patients with lung cancer and control healthy individuals were assayed by PCR and DNA sequencing. We also analyzed one non-coding single nucleotide polymorphisms （SNPs） in the KDR gene. Results： No mutations were found in exon 22, 23 and 27. One heterozygous mutation of c.＋2837 in exon 21 was found at a frequency of 2.08% （2/96） in the patients with lung cancer and none were detected in the healthy control individuals. The mutation was from a G to a A resulting in substitution of arginine with histidine residue. Conclusion： Our data suggested that we should focus on the mutation or SNP in the other regions or the expression levels of KDR gene, and the function of c.＋2837 mutation of KDR .qene may be needed further study in the future.展开更多
[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobac...[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobacterium mediated method. [ Result] The best infection effect appeared under the OD600 value of Agrobacterium within 0.6 -0.8, the addition of 250 mg/L AS could increase the transformation efficiency. The optimal concentration of G418 for screening is 50 mg/L. Some putative transgenic plants of Lilium longiflorum with resistance to G418 showed positive in PCR, preliminarily proving that T-DNA gene had integrated into the genome of lily. [ Conclusion] The study may lay a foundation for breeding excellent lily varieties through TDNA integration technique.展开更多
转座子是一类可以在基因组中不同遗传位点间移动的DNA序列,在其转移过程中有时会伴随自身拷贝数的增加。作为基因组的重要组成部分,转座子可以通过多种方式影响宿主基因及基因组的结构与功能,进而在宿主的演化过程中扮演重要角色。目前...转座子是一类可以在基因组中不同遗传位点间移动的DNA序列,在其转移过程中有时会伴随自身拷贝数的增加。作为基因组的重要组成部分,转座子可以通过多种方式影响宿主基因及基因组的结构与功能,进而在宿主的演化过程中扮演重要角色。目前依据转座过程中间体类型的不同可以将其分为I类转座子和II类转座子。Mutator超家族转座子是20世纪70年代在玉米(Zea may L.)中发现的一类特殊的转座子,其属于II类转座子,广泛存在于真核生物基因组中,包含遗传特征明晰可分的众多转座子家族。此外,该超家族转座子转座频率高,倾向于插入基因富含区及低拷贝序列区,可快速产生大量新的突变体,目前已被广泛应用于正向及反向遗传学研究。本文结合近年来相关研究结果,围绕Mutator超家族转座子的分类组成、结构特征、转座机制、插入偏好、靶位点重复序列以及玉米自主性MULEs元件展开综述,并对转座子研究面临的问题及未来研究方向进行了探讨,旨在与研究领域内的同行探讨相关研究的可能突破点、未来发展方向及可能产生的重大影响。展开更多
基金This work was supported by a Grant from the National Special Key Project on Functional Genomics and Biochip of China.
文摘T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants,and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype.In this study,we screened 4416 rice TI tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents,ZH 11 and ZH 15.We found many lines showed obvious morphological mutations,including two types-fake-homozygous mutation and separating mutation.The mutation phenotype was related to 14 kinds of trait such as plant height,heading date,leaf shape,leaf color,tiller number,panicle shape,spikelet number,grain shape,disease-like mutation,male sterility,awn,and so on.Among them,plant height,heading date,leaf color and male sterility had a comparatively high mutation frequency(over 1%).The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents,but the frequency of head-ing date and male sterility varied greatly,suggesting that environment had a great effect on the expression of latter two traits.By conducting continuously co-segregating analyses in TI and T2 generation,we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets,which would provide a base for cloning correlative functional genes.At the same time,we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR,of which,26 were vector backbone sequence,14 had good identity to rice genome sequence.The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.
基金supported by the National Natural Science Foundation of China (Grant No. 31272408 30972080)+2 种基金the National 863 Program of China (Grant No. 2013AA102505)the Program of National Beef Cattle and yak Industrial Technology System (CARS-38)the Agricultural Science and Technology Innovation Projects of Shanxi Province (No. 2012NKC01-13).
文摘Objective To investigate the 23 bp and 12 bp insertion/deletion(indel)mutations within the bovine prion protein(PRNP)gene in Chinese dairy cows,and to detect the associations of two indel mutations with BSE susceptibility and milk performance.Methods Based on bovine PRNP gene sequence,two pairs of primers for testing the 23 bp and 12 bp indel mutations were designed.The PCR amplification and agarose electrophoresis were carried out to distinguish the different genotypes within the mutations.Moreover,based on previous data from other cattle breeds and present genotypic and allelic frequencies of two indels mutations in this study,the corrections between the two indel mutations and BSE susceptibility were tested,as well as the relationships between the mutations and milk performance traits were analyzed in this study based on the statistical analyses.Results In the analyzed Chinese Holstein population,the frequencies of two"del"alleles in 23 bp and 12 bp indel muations were more frequent.The frequency of haplotype of 23del-12del was higher than those of 23del-12ins and 23ins-12del.From the estimated r2and D’values,two indel polymorphisms were linked strongly in the Holstein population(D’=57.5%,r2=0.257).Compared with the BSE-affected cattle populations from the reported data,the significant differences of genotypic and allelic frequencies were found among present Holstein and some BSE-affected populations(P<0.05 or P<0.01).Similarly,there were significant frequency distribution differences of genotypes and alleles among Chinese Holstein and several previous reported healthy dairy cattle(P<0.05 or P<0.01).Moreover,association of genotype and combined genotypes of two indel polymorphisms with milk performance and resistant mastitis traits were analyzed in Holstein population,but no significant differences were found(P>0.05).Conclusions These observations revealed that the influence of two indel mutations within the bovine PRNP gene on BSE depended on the breed and they did not affect the milk production traits,which layed the foundation for future selection of resistant animals,and for improving health conditions for dairy breeding against BSE in China.
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
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.
文摘A rice （Oryza sativa） T-DNA insertion population, which included more than 63 000 independent transgenic lines and 8 840 identified flanking sequence tags （FSTs） that were mapped onto the rice genome, was developed to systemi- cally study the rice seed quality control. Genome-wide analysis of the FST distribution showed that T-DNA insertions were positively correlated with expressed genes, but negatively with transposable elements and small RNAs. In addition, the recovered T-DNAs were preferentially located at the untranslated region of the expressed genes. More than 11 000 putative homozygous lines were obtained through multi-generations of planting and resistance screening, and measurement of seed quality of around half of them, including the contents of starch, amylose, protein and fat, with a nondestructive near-infrared spectroscopy method, identified 551 mutants with unique or multiple altered param- eters of seed quality. Analysis of the corresponding FSTs showed that genes participating in diverse functions, including metabolic processes and transcriptional regulation, were involved, indicating that seed quality is regulated by a complex network.
基金the National Natural Science Foundation of China(No.30570025)the Education Department of Heilongjiang Province(No.10551238)
文摘Agrobacterium tumefaciens-mediated DNA transiormation method was applied to transform Noclulisporium sylviforme fusant HDF-68, a taxol-produeing fungus. We constructed a binary vector pBI121-43 canting a hygromycin-resistant gene cassette between the right and left borders of T-DNA, Optimal co-cultivation of N.sylviforrne with A. tumefaciens containing pBI121-43 led to 110- 130 hygromycin-resistant transformants per＂ million eonidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N. sylviforme fusant HDF-68, and the development of such svstem lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.
基金supported by grants from the National Natural Science Foundation of China(31901835)the Science and Technology Planning Project of Henan Province of China(212102110145)the International(Regional)Cooperation and Exchange Program of the National Natural Science Foundation of China(31961143018).
文摘Fusarium pseudograminearum is a devastating pathogen that causes Fusarium crown rot(FCR)in wheat and poses a significant threat to wheat production in terms of grain yield and quality.However,the mechanism by which F.pseudograminearum infects wheat remains unclear.In this study,we aimed to elucidate these mechanisms by constructing a T-DNA insertion mutant library for the highly virulent strain WZ-8A of F.pseudograminearum.By screening this mutant library,we identified nine independent mutants that displayed impaired pathogenesis in barley leaves.Among these mutants,one possessed a disruption in the gene FpRCO1 that is an ortholog of Saccharomyces cerevisiae RCO1,encoding essential component of the Rpd3S histone deacetylase complex in F.pseudograminearum.To further investigate the role of FpRCO1 in F.pseudograminearum,we employed a split-marker approach to knock out FpRCO1 in F.pseudograminearum WZ-8A.FpRCO1 deletion mutants exhibit reduced vegetative growth,conidium production,and virulence in wheat coleoptiles and barley leaves,whereas the complementary strain restores these phenotypes.Moreover,under stress conditions,the FpRCO1 deletion mutants exhibited increased sensitivity to NaCl,sorbitol,and SDS,but possessed reduced sensitivity to H_(2)O_(2)compared to these characteristics in the wild-type strain.RNA-seq analysis revealed that deletion of FpRCO1 affected gene expression(particularly the downregulation of TRI gene expression),thus resulting in significantly reduced deoxynivalenol(DON)production.In summary,our findings highlight the pivotal role of FpRCO1 in regulating vegetative growth and development,asexual reproduction,DON production,and pathogenicity of F.pseudograminearum.This study provides valuable insights into the molecular mechanisms underlying F.pseudograminearum infection in wheat and may pave the way for the development of novel strategies to combat this devastating disease.
基金supported by the High-Tech R&D Program of China(2006AA10A106)the open funds of the National Key Laboratory of Crop Genetic Improvement and China National Fundamental Fund of Personnel Training (J0730649)
文摘Mutator transposable element （Mu） has been used as an effective tool to clone maize （Zea mays L.） genes. One opaque endosperm mutant （miol6） was identified in a pool of Mu inserted mutants. A modified method, termed the double selected amplification of insertion flanking fragments （DSAIFF）, was employed to isolate the Mu flanking fragments （MFFs） of miol6. The target site duplications （TSDs） isolated from the Msp I and Mse I digested MFFs had a same 9-bp sequence and were confirmed to be the flanking sequence of one identically inserted gene. Co-segregation analysis suggested that the MFFs were associated with the mutant opaque endosperm, and miol6 was mapped in silico onto the physical position ranged from 229 965 021 to 229 965 409 bp of the maize chromosome 4.09 bin. The full-length cDNA of the wild-type gene was obtained by an RT-PCR primer-scanning technique, and Mio16 was found to putatively encode a homolog of the Arabidopsis MAP3K delta-1 protein kinase. RT-PCR result the mRNA expression of miol6 region anchored by primers Mu20 and af276 was not interrupted by Mu insertion. Further researches will be done to elucidate how the expression of miol6 is alternated by Mu insertion.
基金the Deanship of Scientific Research at Imam Mohammad Ibn Saud Islamic University(IMSIU)(Grant Number IMSIU-RP23030).
文摘Genetic algorithms(GAs)are very good metaheuristic algorithms that are suitable for solving NP-hard combinatorial optimization problems.AsimpleGAbeginswith a set of solutions represented by a population of chromosomes and then uses the idea of survival of the fittest in the selection process to select some fitter chromosomes.It uses a crossover operator to create better offspring chromosomes and thus,converges the population.Also,it uses a mutation operator to explore the unexplored areas by the crossover operator,and thus,diversifies the GA search space.A combination of crossover and mutation operators makes the GA search strong enough to reach the optimal solution.However,appropriate selection and combination of crossover operator and mutation operator can lead to a very good GA for solving an optimization problem.In this present paper,we aim to study the benchmark traveling salesman problem(TSP).We developed several genetic algorithms using seven crossover operators and six mutation operators for the TSP and then compared them to some benchmark TSPLIB instances.The experimental studies show the effectiveness of the combination of a comprehensive sequential constructive crossover operator and insertion mutation operator for the problem.The GA using the comprehensive sequential constructive crossover with insertion mutation could find average solutions whose average percentage of excesses from the best-known solutions are between 0.22 and 14.94 for our experimented problem instances.
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.
基金Supported by the grants from the National Natural Science Foundation of China (No. 30772531)Guangdong Provincial Medical Science and Technology Research Foundation (No. B2006001)China Postdoctoral Science Foundation (No. 20060400212)
文摘Objective： Although the kinase insert domain-containing receptor （KDR） gene play an very important role in the metastasis of cancer and is also as one of the molecular targets used in cancer therapy, mutation in the tyrosine kinase （TK） domain of the KDR gene has not been reported. Here we detected the mutations and polymorphisms in the TK domain of KDR gene in human lung cancer patients and to give the basic evidence and clue for cancer prevention and target therapy. Methods： The entire sequence of exons 21, 22, 23 and 27 （which contain the coding sequence of tyrosine phosphorylation） in the TK domain of KDR gene in the patients with lung cancer and control healthy individuals were assayed by PCR and DNA sequencing. We also analyzed one non-coding single nucleotide polymorphisms （SNPs） in the KDR gene. Results： No mutations were found in exon 22, 23 and 27. One heterozygous mutation of c.＋2837 in exon 21 was found at a frequency of 2.08% （2/96） in the patients with lung cancer and none were detected in the healthy control individuals. The mutation was from a G to a A resulting in substitution of arginine with histidine residue. Conclusion： Our data suggested that we should focus on the mutation or SNP in the other regions or the expression levels of KDR gene, and the function of c.＋2837 mutation of KDR .qene may be needed further study in the future.
基金the Fund of Basis Scientific Research Operation of Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciencesthe Grant of Scientific Fund of Chinese Academy of Tropical Agricultural Sciences (NoRky0529)~~
文摘[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobacterium mediated method. [ Result] The best infection effect appeared under the OD600 value of Agrobacterium within 0.6 -0.8, the addition of 250 mg/L AS could increase the transformation efficiency. The optimal concentration of G418 for screening is 50 mg/L. Some putative transgenic plants of Lilium longiflorum with resistance to G418 showed positive in PCR, preliminarily proving that T-DNA gene had integrated into the genome of lily. [ Conclusion] The study may lay a foundation for breeding excellent lily varieties through TDNA integration technique.
文摘转座子是一类可以在基因组中不同遗传位点间移动的DNA序列,在其转移过程中有时会伴随自身拷贝数的增加。作为基因组的重要组成部分,转座子可以通过多种方式影响宿主基因及基因组的结构与功能,进而在宿主的演化过程中扮演重要角色。目前依据转座过程中间体类型的不同可以将其分为I类转座子和II类转座子。Mutator超家族转座子是20世纪70年代在玉米(Zea may L.)中发现的一类特殊的转座子,其属于II类转座子,广泛存在于真核生物基因组中,包含遗传特征明晰可分的众多转座子家族。此外,该超家族转座子转座频率高,倾向于插入基因富含区及低拷贝序列区,可快速产生大量新的突变体,目前已被广泛应用于正向及反向遗传学研究。本文结合近年来相关研究结果,围绕Mutator超家族转座子的分类组成、结构特征、转座机制、插入偏好、靶位点重复序列以及玉米自主性MULEs元件展开综述,并对转座子研究面临的问题及未来研究方向进行了探讨,旨在与研究领域内的同行探讨相关研究的可能突破点、未来发展方向及可能产生的重大影响。
