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1.
Plant J ; 100(4): 851-862, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31169333

RESUMO

Molecular identification of mutant alleles responsible for certain phenotypic alterations is a central goal of genetic analyses. In this study we describe a rapid procedure suitable for the identification of induced recessive and dominant mutations applied to two Zea mays mutants expressing a dwarf and a pale green phenotype, respectively, which were obtained through pollen ethyl methanesulfonate (EMS) mutagenesis. First, without prior backcrossing, induced mutations (single nucleotide polymorphisms, SNPs) segregating in a (M2 ) family derived from a heterozygous (M1 ) parent were identified using whole-genome shotgun (WGS) sequencing of a small number of (M2 ) individuals with mutant and wild-type phenotypes. Second, the state of zygosity of the mutation causing the phenotype was determined for each sequenced individual by phenotypic segregation analysis of the self-pollinated (M3 ) offspring. Finally, we filtered for segregating EMS-induced SNPs whose state of zygosity matched the determined state of zygosity of the mutant locus in each sequenced (M2 ) individuals. Through this procedure, combining sequencing of individuals and Mendelian inheritance, three and four SNPs in linkage passed our zygosity filter for the homozygous dwarf and heterozygous pale green mutation, respectively. The dwarf mutation was found to be allelic to the an1 locus and caused by an insertion in the largest exon of the AN1 gene. The pale green mutation affected the nuclear W2 gene and was caused by a non-synonymous amino acid exchange in encoded chloroplast DNA polymerase with a predicted deleterious effect. This coincided with lower cpDNA levels in pale green plants.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Zea mays/genética , Análise Mutacional de DNA/métodos , Metanossulfonato de Etila/farmacologia , Genes Dominantes , Genes Recessivos , Genoma de Planta , Pólen/efeitos dos fármacos , Pólen/genética , Polimorfismo de Nucleotídeo Único , Fatores de Tempo , Zea mays/efeitos dos fármacos
2.
Nat Biotechnol ; 26(9): 1015-7, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18758448

RESUMO

Besides the well-documented integration of DNA flanked by the transfer DNA borders, occasional insertion of fragments from the tumor-inducing plasmid into plant genomes has also been reported during Agrobacterium tumefaciens-mediated transformation. We demonstrate that large (up to approximately 18 kb) gene-bearing fragments of Agrobacterium chromosomal DNA (AchrDNA) can be integrated into Arabidopsis thaliana genomic DNA during transformation. One in every 250 transgenic plants may carry AchrDNA fragments. This has implications for horizontal gene transfer and indicates a need for greater scrutiny of transgenic plants for undesired bacterial DNA.


Assuntos
Agrobacterium tumefaciens/genética , Biotecnologia/métodos , Cromossomos/metabolismo , Técnicas de Transferência de Genes , Plantas Geneticamente Modificadas/genética , Agrobacterium tumefaciens/metabolismo , DNA/genética , DNA/metabolismo , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Modelos Biológicos , Mutagênese , Plantas Geneticamente Modificadas/microbiologia , Plasmídeos/metabolismo
3.
Nucleic Acids Res ; 35(Database issue): D874-8, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17062622

RESUMO

Insertional mutagenesis approaches, especially by T-DNA, play important roles in gene function studies of the model plant Arabidopsis thaliana. GABI-Kat SimpleSearch (http://www.GABI-Kat.de) is a Flanking Sequence Tag (FST)-based database for T-DNA insertion mutants generated by the GABI-Kat project. Currently, the database contains >108,000 mapped FSTs from approximately 64,000 lines which cover 64% of all annotated A.thaliana protein-coding genes. The web interface allows searching for relevant insertions by gene code, keyword, line identifier, GenBank accession number of the FST, and also by BLAST. A graphic display of the genome region around the gene or the FST assists users to select insertion lines of their interests. About 3500 insertions were confirmed in the offspring of the plant from which the original FST was generated, and the seeds of these lines are available from the Nottingham Arabidopsis Stock Centre. The database now also contains additional information such as segregation data, gene-specific primers and confirmation sequences. This information not only helps users to evaluate the usefulness of the mutant lines, but also covers a big part of the molecular characterization of the insertion alleles.


Assuntos
Arabidopsis/genética , DNA Bacteriano/análise , Bases de Dados de Ácidos Nucleicos , Mutagênese Insercional , Sitios de Sequências Rotuladas , Internet , Interface Usuário-Computador
4.
Genomics ; 87(5): 645-52, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16488113

RESUMO

Large collections of sequence-indexed T-DNA insertion mutants are invaluable resources for plant functional genomics. Flanking sequence tag (FST) data from these collections indicated that T-DNA insertions are not randomly distributed in the Arabidopsis thaliana genome and that there are still a fairly high number of annotated genes without T-DNA insertions. We have analyzed FST data from the FLAGdb, GABI-Kat, and SIGnAL mutant populations. The lack of detectable transcriptional activity and the absence of suitable restriction sites were among the reasons genes are not covered by insertions. Additionally, a refined analysis of FSTs to genes with annotated noncoding regions showed that transcription initiation and polyadenylation site regions of genes are favored targets for T-DNA integration. These findings have implications for the use of T-DNA in saturation mutagenesis and for our chances to find a useful knockout allele for every gene.


Assuntos
Arabidopsis/genética , DNA Bacteriano/genética , Códon de Terminação , Elementos de DNA Transponíveis , Perfilação da Expressão Gênica , Genes de Plantas , Genoma de Planta , Mutagênese Insercional , Poli A , Biossíntese de Proteínas , Pseudogenes , Mapeamento por Restrição , Sitios de Sequências Rotuladas , Sítio de Iniciação de Transcrição
5.
Plant Physiol ; 139(3): 1155-62, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16244137

RESUMO

Urease is a nickel-containing urea hydrolase involved in nitrogen recycling from ureide, purine, and arginine catabolism in plants. The process of urease activation by incorporation of nickel into the active site is a prime example of chaperone-mediated metal transfer to an enzyme. Four urease accessory proteins are required for activation in Klebsiella aerogenes. In plants urease accessory proteins have so far been only partially defined. Using reverse genetic tools we identified four genes that are necessary for urease activity in Arabidopsis (Arabidopsis thaliana; ecotypes Columbia and Nössen). Plants bearing T-DNA or Ds element insertions in either the structural gene for urease or in any of the three putative urease accessory genes AtureD, AtureF, and AtureG lacked the corresponding mRNAs and were defective in urease activity. In contrast to wild-type plants, the mutant lines were not able to support growth with urea as the sole nitrogen source. To investigate whether the identified accessory proteins would be sufficient to support eukaryotic urease activation, the corresponding cDNAs were introduced into urease-negative Escherichia coli. In these bacteria, urease activity was observed only when all three plant accessory genes were coexpressed together with the plant urease gene. Remarkably, plant urease activation occurred as well in cell-free E. coli extracts, but only in extracts from cells that had expressed all three accessory proteins. The future molecular dissection of the plant urease activation process may therefore be performed in vitro, providing a powerful tool to further our understanding of the biochemistry of chaperone-mediated metal transfer processes in plants.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Urease/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Dados de Sequência Molecular , Mutação/genética , Nitratos/farmacologia , Compostos de Amônio Quaternário/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ureia/farmacologia
6.
Plant Mol Biol ; 58(1): 41-51, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-16028115

RESUMO

Plants exposed to photoinhibitory conditions respond by accumulation of the early light-induced proteins (ELIPs) with a potential photoprotective function. In Arabidopsis thaliana two genes (Elip1 and Elip2) encode for two ELIP proteins: evidence exists that the two genes are differentially regulated but their precise function is unclear. Mutants null for one or the other Elip gene can help in elucidating ELIPs role and here we describe the expression profile of ELIP1 and ELIP2, and the phenotype of such null mutants. Both ELIPs accumulate during greening of etiolated seedlings and in mature plants the transcripts fluctuate diurnally without protein accumulation. Steady-state transcript level of both genes increases in response to high light with transcription of Elip1 much more sensitive than that of Elip2 to increasing irradiation at 22 degrees C. At 4 degrees C instead Elip2 is strongly transcribed even at growing light. Furthermore, only ELIP1 accumulates under high light at 22 degrees C while both proteins accumulate at 4 degrees C. These results indicate the existence of a differential regulation of ELIPs expression in response to light or chilling stress with mechanisms active either at transcriptional and post-transcriptional level. Phenotypically, the mutants behave as the wild type as far as sensitivity to light- or light and cold-induced short-term photoinhibition, while both ELIPs are necessary to ensure a high rate of chlorophyll accumulation during deetiolation in continuous high light.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Perfilação da Expressão Gênica , Mutação , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/metabolismo , Western Blotting , Clorofila/metabolismo , Ritmo Circadiano , Temperatura Baixa , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Cinética , Fenótipo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Plântula/genética , Plântula/metabolismo , Plântula/efeitos da radiação , Fatores de Tempo
7.
Development ; 132(7): 1555-65, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15728668

RESUMO

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Flores/crescimento & desenvolvimento , Morfogênese/fisiologia , Oxirredutases/genética , Proteína AGAMOUS de Arabidopsis/biossíntese , Proteína AGAMOUS de Arabidopsis/genética , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis , Sequência de Bases , Cisteína/genética , Cisteína/metabolismo , Flores/genética , Flores/metabolismo , Glutarredoxinas , Dados de Sequência Molecular , Morfogênese/genética , Família Multigênica , Oxirredutases/metabolismo
8.
Plant Cell ; 17(3): 760-75, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15722468

RESUMO

Plastids of nongreen tissues can import carbon in the form of glucose 6-phosphate via the glucose 6-phosphate/phosphate translocator (GPT). The Arabidopsis thaliana genome contains two homologous GPT genes, AtGPT1 and AtGPT2. Both proteins show glucose 6-phosphate translocator activity after reconstitution in liposomes, and each of them can rescue the low-starch leaf phenotype of the pgi1 mutant (which lacks plastid phosphoglucoisomerase), indicating that the two proteins are also functional in planta. AtGPT1 transcripts are ubiquitously expressed during plant development, with highest expression in stamens, whereas AtGPT2 expression is restricted to a few tissues, including senescing leaves. Disruption of GPT2 has no obvious effect on growth and development under greenhouse conditions, whereas the mutations gpt1-1 and gpt1-2 are lethal. In both gpt1 lines, distorted segregation ratios, reduced efficiency of transmission in males and females, and inability to complete pollen and ovule development were observed, indicating profound defects in gametogenesis. Embryo sac development is arrested in the gpt1 mutants at a stage before the fusion of the polar nuclei. Mutant pollen development is associated with reduced formation of lipid bodies and small vesicles and the disappearance of dispersed vacuoles, which results in disintegration of the pollen structure. Taken together, our results indicate that GPT1-mediated import of glucose 6-phosphate into nongreen plastids is crucial for gametophyte development. We suggest that loss of GPT1 function results in disruption of the oxidative pentose phosphate cycle, which in turn affects fatty acid biosynthesis.


Assuntos
Antiporters/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Proteínas de Plantas/metabolismo , Alelos , Antiporters/genética , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , DNA de Plantas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Microscopia Eletrônica , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Pólen/crescimento & desenvolvimento , Pólen/ultraestrutura
9.
Plant Physiol ; 134(1): 286-95, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14671019

RESUMO

Pectins are major components of primary plant cell walls and the seed mucilage of Arabidopsis. Despite progress in the structural elucidation of pectins, only very few enzymes participating in or regulating their synthesis have been identified. A first candidate gene involved in the synthesis of pectinaceous rhamnogalacturonan I is RHM2, a putative plant ortholog to NDP-rhamnose biosynthetic enzymes in bacteria. Expression studies with a promoter beta-glucuronidase construct and reverse transcription PCR data show that RHM2 is expressed ubiquitously. Rhm2 T-DNA insertion mutant lines were identified using a reverse genetics approach. Analysis of the rhm2 seeds by various staining methods and chemical analysis of the mucilage revealed a strong reduction of rhamnogalacturonan I in the mucilage and a decrease of its molecular weight. In addition, scanning electron microscopy of the seed surface indicated a distorted testa morphology, illustrating not only a structural but also a developmental role for RGI or rhamnose metabolism in proper testa formation.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Pectinas/biossíntese , Adesivos/metabolismo , Sequência de Aminoácidos , Arabidopsis/embriologia , Sequência de Bases , DNA de Plantas/genética , Genes de Plantas , Dados de Sequência Molecular , Mutação , Pectinas/química , Fenótipo , Plantas Geneticamente Modificadas , Sementes/metabolismo , Homologia de Sequência de Aminoácidos
10.
Biotechniques ; 35(6): 1164-8, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-14682050

RESUMO

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.


Assuntos
Arabidopsis/genética , DNA Bacteriano/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/métodos , Robótica/métodos , Análise de Sequência de DNA/métodos , Genoma de Planta , Mutagênese Sítio-Dirigida/genética , Plantas Geneticamente Modificadas/genética , Sequências Repetidas Terminais/genética
11.
Bioinformatics ; 19(11): 1441-2, 2003 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-12874060

RESUMO

SUMMARY: GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY: http://www.mpiz-koeln.mpg.de/GABI-Kat/


Assuntos
Arabidopsis/genética , Elementos de DNA Transponíveis/genética , DNA Bacteriano/genética , Sistemas de Gerenciamento de Base de Dados , Bases de Dados de Ácidos Nucleicos , Etiquetas de Sequências Expressas , Armazenamento e Recuperação da Informação/métodos , Mutagênese Insercional/métodos , Perfilação da Expressão Gênica/métodos , Internet , Plantas Geneticamente Modificadas/genética , Análise de Sequência de DNA/métodos , Sequências Repetidas Terminais/genética , Interface Usuário-Computador
12.
Plant Mol Biol ; 53(1-2): 247-59, 2003 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-14756321

RESUMO

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the population's functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.


Assuntos
Arabidopsis/genética , DNA Bacteriano/genética , DNA de Plantas/genética , Mutagênese Insercional , Proteína AGAMOUS de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Sítios de Ligação/genética , DNA de Plantas/química , Genoma de Planta , Dados de Sequência Molecular , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Análise de Sequência de DNA , Fatores de Transcrição/genética
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