RESUMO
KEY MESSAGE: We found metabolites, enzyme activities and enzyme transcript abundances vary significantly across the maize lifecycle, but weak correlation exists between the three groups. We identified putative genes regulating nitrate assimilation. Progress in improving nitrogen (N) use efficiency (NUE) of crop plants has been hampered by the complexity of the N uptake and utilisation systems. To understand this complexity we measured the activities of seven enzymes and ten metabolites related to N metabolism in the leaf and root tissues of Gaspe Flint maize plants grown in 0.5 or 2.5 mM NO3 (-) throughout the lifecycle. The amino acids had remarkably similar profiles across the lifecycle except for transient responses, which only appeared in the leaves for aspartate or in the roots for asparagine, serine and glycine. The activities of the enzymes for N assimilation were also coordinated to a certain degree, most noticeably with a peak in root activity late in the lifecycle, but with wide variation in the activity levels over the course of development. We analysed the transcriptional data for gene sets encoding the measured enzymes and found that, unlike the enzyme activities, transcript levels of the corresponding genes did not exhibit the same coordination across the lifecycle and were only weakly correlated with the levels of various amino acids or individual enzyme activities. We identified gene sets which were correlated with the enzyme activity profiles, including seven genes located within previously known quantitative trait loci for enzyme activities and hypothesise that these genes are important for the regulation of enzyme activities. This work provides insights into the complexity of the N assimilation system throughout development and identifies candidate regulatory genes, which warrant further investigation in efforts to improve NUE in crop plants.
Assuntos
Regulação da Expressão Gênica de Plantas , Nitrogênio/metabolismo , Zea mays/genética , Zea mays/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Locos de Características Quantitativas/genética , Zea mays/enzimologia , Zea mays/crescimento & desenvolvimentoRESUMO
An understanding of nitrate (NO3-) uptake throughout the lifecycle of plants, and how this process responds to nitrogen (N) availability, is an important step towards the development of plants with improved nitrogen use efficiency (NUE). NO3- uptake capacity and transcript levels of putative high- and low-affinity NO3- transporters (NRTs) were profiled across the lifecycle of dwarf maize (Zea mays) plants grown at reduced and adequate NO3-. Plants showed major changes in high-affinity NO3- uptake capacity across the lifecycle, which varied with changing relative growth rates of roots and shoots. Transcript abundances of putative high-affinity NRTs (predominantly ZmNRT2.1 and ZmNRT2.2) were correlated with two distinct peaks in high-affinity root NO3- uptake capacity and also N availability. The reduction in NO3- supply during the lifecycle led to a dramatic increase in NO3- uptake capacity, which preceded changes in transcript levels of NRTs, suggesting a model with short-term post-translational regulation and longer term transcriptional regulation of NO3- uptake capacity. These observations offer new insight into the control of NO3- uptake by both plant developmental processes and N availability, and identify key control points that may be targeted by future plant improvement programmes to enhance N uptake relative to availability and/or demand.
Assuntos
Nitratos/metabolismo , Nitrogênio/farmacologia , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo , Aminoácidos/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Biomassa , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Transportadores de Nitrato , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Zea mays/efeitos dos fármacos , Zea mays/genéticaRESUMO
Ectopic gene expression, or the gain-of-function approach, has the advantage that once the function of a gene is known the gene can be transferred to many different plants by transformation. We previously reported a method, called FOX hunting, that involves ectopic expression of Arabidopsis full-length cDNAs in Arabidopsis to systematically generate gain-of-function mutants. This technology is most beneficial for generating a heterologous gene resource for analysis of useful plant gene functions. As an initial model we generated more than 23,000 independent Arabidopsis transgenic lines that expressed rice fl-cDNAs (Rice FOX Arabidopsis lines). The short generation time and rapid and efficient transformation frequency of Arabidopsis enabled the functions of the rice genes to be analyzed rapidly. We screened rice FOX Arabidopsis lines for alterations in morphology, photosynthesis, element accumulation, pigment accumulation, hormone profiles, secondary metabolites, pathogen resistance, salt tolerance, UV signaling, high light tolerance, and heat stress tolerance. Some of the mutant phenotypes displayed by rice FOX Arabidopsis lines resulted from the expression of rice genes that had no homologs in Arabidopsis. This result demonstrated that rice fl-cDNAs could be used to introduce new gene functions in Arabidopsis. Furthermore, these findings showed that rice gene function could be analyzed by employing Arabidopsis as a heterologous host. This technology provides a framework for the analysis of plant gene function in a heterologous host and of plant improvement by using heterologous gene resources.
Assuntos
Perfilação da Expressão Gênica/métodos , Genes de Plantas , Oryza/genética , Arabidopsis/metabolismo , Composição de Bases , DNA Complementar/genética , DNA de Plantas/genética , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Oryza/metabolismo , Fenótipo , Plantas Geneticamente Modificadas/metabolismoRESUMO
BACKGROUND: Wheat is an allopolyploid plant that harbors a huge, complex genome. Therefore, accumulation of expressed sequence tags (ESTs) for wheat is becoming particularly important for functional genomics and molecular breeding. We prepared a comprehensive collection of ESTs from the various tissues that develop during the wheat life cycle and from tissues subjected to stress. We also examined their expression profiles in silico. As full-length cDNAs are indispensable to certify the collected ESTs and annotate the genes in the wheat genome, we performed a systematic survey and sequencing of the full-length cDNA clones. This sequence information is a valuable genetic resource for functional genomics and will enable carrying out comparative genomics in cereals. RESULTS: As part of the functional genomics and development of genomic wheat resources, we have generated a collection of full-length cDNAs from common wheat. By grouping the ESTs of recombinant clones randomly selected from the full-length cDNA library, we were able to sequence 6,162 independent clones with high accuracy. About 10% of the clones were wheat-unique genes, without any counterparts within the DNA database. Wheat clones that showed high homology to those of rice were selected in order to investigate their expression patterns in various tissues throughout the wheat life cycle and in response to abiotic-stress treatments. To assess the variability of genes that have evolved differently in wheat and rice, we calculated the substitution rate (Ka/Ks) of the counterparts in wheat and rice. Genes that were preferentially expressed in certain tissues or treatments had higher Ka/Ks values than those in other tissues and treatments, which suggests that the genes with the higher variability expressed in these tissues is under adaptive selection. CONCLUSION: We have generated a high-quality full-length cDNA resource for common wheat, which is essential for continuation of the ongoing curation and annotation of the wheat genome. The data for each clone's expression in various tissues and stress treatments and its variability in wheat and rice as a result of their diversification are valuable tools for functional genomics in wheat and for comparative genomics in cereals.
Assuntos
Adaptação Biológica/genética , Evolução Molecular , Oryza/genética , Plantas Tolerantes a Sal/genética , Triticum/genética , DNA Complementar/genética , DNA de Plantas/genética , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Genes de Plantas , Genômica , Análise de Sequência de DNA , Estresse FisiológicoRESUMO
Enhancing nitrogen use efficiency (NUE) of wheat is a major focus for wheat breeding programs. NUE may be improved by identifying genotypes that are competitive for nitrogen (N) uptake in early vegetative stages of growth and are able to invest that N in grain. Breeders tend to select high yielding genotypes under conditions of medium to high N supply, but it is not known whether this influences the selection of root plasticity traits or whether, over time, breeders have selected genotypes with higher N uptake efficiency. To address this, genotypes were selected from CIMMYT (1966-1985) and Australian (1999-2007) breeding programs. Genotypes from both programs responded to low N supply by expanding their root surface area through increased total root number and/or length of lateral roots. Australian genotypes were N responsive (accumulated more N under high N than under low N) whereas CIMMYT genotypes were not very N responsive. This could not be explained by differences in N uptake capacity as shown by 15N flux analysis of two representative genotypes with contrasting N accumulation. Expression analysis of nitrate transporter genes revealed that the high-affinity transport system accounted for the majority of root nitrate uptake in wheat seedlings under both low and high N conditions.
RESUMO
When challenged with the crucifer pathogen Colletotrichum higginsianum, Arabidopsis thaliana ecotype Columbia (Col-0) was colonized by the fungus within 2 to 3 days, developing brown necrotic lesions surrounded by a yellow halo. Lesions spread from the inoculation site within 3 to 4 days, and subsequently continued to expand until they covered the entire leaf. Electron microscopy confirmed that C. higginsianum is a hemibiotroph on Arabidopsis, feeding initially on living cells as a biotroph before switching to a necrotrophic mode of growth. A collection of 37 ecotypes of Arabidopsis varied in their responses to infection by C. higginsianum. The ecotype Eil-0 was highly resistant, with symptoms limited to necrotic flecking and with only very limited fungal colonization. Analyses suggested that the hypersensitive response and reactive oxygen species may be important in this defense response. Expression analyses with cDNA microarrays indicated that the defense reaction depends primarily on the jasmonic acid- and ethylene-dependent signaling pathways and, to a lesser extent, on the salicylate-dependent pathway. Crosses between the Eil-0 and Col-0 ecotypes suggested that the resistance in Eil-0 was dominant and was conferred by a single locus, which we named RCH1. RCH1 is the first resistance locus to be identified from Arabidopsis against the hemibiotrophic fungus genus Colletotrichum.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Colletotrichum/crescimento & desenvolvimento , Folhas de Planta/genética , Arabidopsis/microbiologia , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/metabolismo , Ciclopentanos/farmacologia , Etilenos/farmacologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Indóis/metabolismo , Microscopia Eletrônica , Análise de Sequência com Séries de Oligonucleotídeos , Oxilipinas , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Ácido Salicílico/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tiazóis/metabolismoRESUMO
A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39,936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5' and 3' ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22,674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5'- and 3'-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.
Assuntos
Clonagem Molecular , DNA Complementar , Biblioteca Gênica , Glycine max , Análise de Sequência de DNA , Animais , Etiquetas de Sequências Expressas/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Dados de Sequência Molecular , Nematoides/fisiologia , Doenças das Plantas/parasitologia , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Glycine max/parasitologia , Glycine max/fisiologiaRESUMO
The latest report has estimated the number of rice genes to be approximately 32,000. To elucidate the functions of a large population of rice genes and to search efficiently for agriculturally useful genes, we have been taking advantage of the Full-length cDNA Over-eXpresser (FOX) gene-hunting system. This system is very useful for analyzing various gain-of-function phenotypes from large populations of transgenic plants overexpressing cDNAs of interest and others with unknown or important functions. We collected the plasmid DNAs of 13,980 independent full-length cDNA (FL-cDNA) clones to produce a FOX library by placing individual cDNAs under the control of the maize Ubiquitin-1 promoter. The FOX library was transformed into rice by Agrobacterium-mediated high-speed transformation. So far, we have generated approximately 12,000 FOX-rice lines. Genomic PCR analysis indicated that the average number of FL-cDNAs introduced into individual lines was 1.04. Sequencing analysis of the PCR fragments carrying FL-cDNAs from 8615 FOX-rice lines identified FL-cDNAs in 8225 lines, and a database search classified the cDNAs into 5462 independent ones. Approximately 16.6% of FOX-rice lines examined showed altered growth or morphological characteristics. Three super-dwarf mutants overexpressed a novel gibberellin 2-oxidase gene,confirming the importance of this system. We also show here the other morphological alterations caused by individual FL-cDNA expression. These dominant phenotypes should be valuable indicators for gene discovery and functional analysis.
Assuntos
Perfilação da Expressão Gênica/métodos , Genoma de Planta , Oryza/genética , Sequência de Bases , Primers do DNA , DNA Complementar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhizobium/genéticaRESUMO
From Arabidopsis full-length cDNA libraries, we collected ca. 7000 (7K) independent full-length cDNAs to prepare a cDNA microarray. The 7K cDNA collection contains 49 cytochrome P450 genes. In this study, expression patterns of these cytochrome P450 genes were analyzed by a full-length cDNA microarray under various treatments, such as hormones (salicylic acid, jasmonic acid, ethylene, abscisic acid), pathogen-inoculation ( Alternaria brassicicola , Alternaria alternata ), paraquat, rose bengal, UV stress (UV-C), heavy metal stress (CuSO4), mechanical wounding, drought, high salinity and low temperature. Expression of 29 cytochrome P450 genes among them was induced by various treatments. Inoculation with A. brassicicola and A. alternata as biotic stresses increased transcript levels of 12 and 5 genes in Arabidopsis plants, respectively. In addition, some of the genes were also expressed by abiotic stresses. This suggests crosstalk between abiotic and biotic stresses. The promoter sequences and cis -acting elements of each gene were studied on the basis of full-length cDNA sequences. Most cytochrome P450 genes induced by both abiotic and biotic stresses contained the recognition sites of MYB and MYC, ACGT-core sequence, TGA-box and W-box for WRKY transcription factors in their promoters. These cis -acting elements are known to participate in the regulation of plant defense. The response of each gene to multiple stresses is strictly regulated.
Assuntos
Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ácido Abscísico/farmacologia , Alternaria/crescimento & desenvolvimento , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Northern Blotting , Botrytis/crescimento & desenvolvimento , Análise por Conglomerados , Ciclopentanos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Etilenos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Família Multigênica/genética , Oxilipinas , Paraquat/farmacologia , Filogenia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Elementos de Resposta/genética , Rosa Bengala/farmacologia , Ácido Salicílico/farmacologia , Cloreto de Sódio/farmacologia , Temperatura , Raios UltravioletaRESUMO
Transcriptional regulation in response to hyperosmotic, high-salinity and oxidative stress, and abscisic acid (ABA) treatment in Arabidopsis suspension-cultured cell line T87 was investigated with a cDNA microarray containing 7000 independent full-length Arabidopsis cDNAs. The transcripts of 102, 11, 84 and 73 genes were increased more than 5-fold within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. On the other hand, the transcripts of 44, 57, 25 and 34 genes were down-regulated to less than one-third within 5h after treatment with 0.5M mannitol, 0.1M NaCl, 50 microM ABA and 10mM H2O2, respectively. Venn diagram analysis revealed 11 genes were induced significantly by mannitol, NaCl, and ABA, indicating crosstalk among these signaling pathways. Comparison of the genes induced by each stress revealed that 32%, 17% and 33% of mannitol-, NaCl- and ABA-inducible genes were also induced by H2O2, indicating the crosstalk between the signaling pathways for osmotic stress and oxidative stress. Although the expression profiles revealed that the T87 cells had most of the regulatory systems seen in Arabidopsis seedlings, the T87 cells did not have one of ABA-dependent signaling pathways.
Assuntos
Ácido Abscísico/farmacologia , Arabidopsis/genética , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Cloreto de Sódio/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Northern Blotting , Células Cultivadas , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Soluções Hipertônicas , Manitol/farmacologia , Pressão Osmótica , Estresse Oxidativo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/efeitos dos fármacos , Plântula/genética , Análise de Sequência de DNA , Fatores de Tempo , Transcrição Gênica/efeitos dos fármacosRESUMO
Full-length cDNAs are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones were isolated. The 3'-end expressed sequence tags (ESTs) of all 155,144 RAFL cDNAs were clustered into 14,668 non-redundant cDNA groups, about 60% of predicted genes. The sequence database of the RAFL cDNAs is useful for promoter analysis and the correct annotation of predicted transcription units and gene products. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. RAFL cDNA microarrays were prepared, containing independent full-length cDNA groups for analysing the expression profiles of genes under various stress- and hormone-treatment conditions and in various mutants and transgenic plants. In this review, recent progress on transcriptome analysis using the RAFL cDNA microarray is highlighted.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Perfilação da Expressão Gênica , DNA Complementar/genética , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas , Análise de Sequência com Séries de OligonucleotídeosRESUMO
We examined the transcripts that showed changes among the ca.7,000 Arabidopsis full-length cDNAs under biotic and abiotic stresses. Expression of Arabidopsis phospholipase A IIA (AtPLA IIA) gene was induced by various treatments such as pathogen inoculation (Alternaria alternata, Alternaria brassicicola and Colletotrichum higginsianum), cold, high-salinity, abscisic acid, salicylic acid, methyl jasmonate, ethephon, paraquat, rose bengal, UV-C and CuSO(4)-treatments. The regulation of AtPLA IIA gene expression under biotic and abiotic stresses was analyzed with AtPLA IIA promoter region (from +95 to -1,405) fused to the GUS reporter gene. In conclusion, the promoter activity is induced under these stresses.
Assuntos
Arabidopsis/enzimologia , Perfilação da Expressão Gênica , Fosfolipases A/genética , Arabidopsis/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacosRESUMO
Full-length cDNAs are essential for functional analysis of plant genes. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. Microarray technology is a powerful tool for identifying genes induced by environmental stimuli or stress and for analyzing their expression profiles in response to environmental signals. We prepared an Arabidopsis full-length cDNA microarray containing around 7,000 independent full-length cDNA groups and analyzed the expression profiles of genes. The transcripts of 245, 299, 54 and 213 genes increased after abscisic acid (ABA), drought-, cold-, and salt-stress treatments, respectively, with inducibilities more than fivefold compared with those of control genes [corrected]. The cDNA microarray analysis showed that many ABA-inducible genes were induced after drought- and high-salinity-stress treatments, and that there is more crosstalk between drought and ABA responses than between ABA and cold responses. Among the ABA-inducible genes identified, we identified 22 transcription factor genes, suggesting that many transcriptional regulatory mechanisms exist in the ABA signal transduction pathways.
Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Temperatura Baixa , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Pressão Osmótica , Regiões Promotoras GenéticasRESUMO
The hypersensitive response (HR) was induced in a wild-type Arabidopsis thaliana plant (Columbia) (Col-wt) by inoculation with Alternaria brassicicola that causes the development of small brown necrotic lesions on the leaves. By contrast, pad3-1 mutants challenged with A. brassicicola produced spreading lesions. The cell death in pad3-1 mutants could not inhibit the pathogen growth and development, although both production of H(2)O(2) and localized cell death were similar in Col-wt and pad3-1 plants after the inoculation. The difference between Col-wt and pad3-1 plants is defense responses after the occurrence of cell death. In other words, PAD3 is necessary for defense response to A. brassicicola. Therefore, we examined the changes in the expression patterns of ca. 7,000 genes by cDNA microarray analysis after inoculation with A. brassicicola. The cDNA microarrays were also done to analyze Arabidopsis responses after treatment with signal molecules, reactive oxygen species (ROS)-inducing compounds and UV-C. The results suggested that the pad3-1 mutation altered not only the accumulation of camalexin but also the timing of expression of many defense-related genes in response to the challenge with A. brassicicola. Furthermore, the plants integrate two or more signals that act together for promoting the induction of multiple defense pathways.
Assuntos
Alternaria/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Perfilação da Expressão Gênica/métodos , Oxigenases de Função Mista/genética , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Northern Blotting , Sistema Enzimático do Citocromo P-450/metabolismo , DNA Complementar/química , DNA Complementar/genética , Imunidade Inata/genética , Oxigenases de Função Mista/metabolismo , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de DNARESUMO
Full-length complementary DNAs (cDNAs) are essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We isolated 155,144 RIKEN Arabidopsis full-length (RAFL) cDNA clones. The 3'-end expressed sequence tags (ESTs) of 155,144 RAFL cDNAs were clustered into 14,668 nonredundant cDNA groups, about 60% of predicted genes. We also obtained 5' ESTs from 14,034 nonredundant cDNA groups and constructed a promoter database. The sequence database of the RAFL cDNAs is useful for promoter analysis and correct annotation of predicted transcription units and gene products. Furthermore, the full-length cDNAs are useful resources for analyses of the expression profiles, functions, and structures of plant proteins.
Assuntos
Arabidopsis/genética , DNA Complementar , Etiquetas de Sequências Expressas , Genes de Plantas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , Biologia Computacional , DNA Complementar/isolamento & purificação , DNA de Plantas/genética , Bases de Dados de Ácidos Nucleicos , Expressão Gênica , Biblioteca Gênica , Genoma de Planta , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Análise de Sequência de DNARESUMO
Full-length cDNAs are essential for functional analysis of plant genes in the post-sequencing era of the Arabidopsis genome. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. We have prepared a full-length cDNA microarray containing approximately 7000 independent, full-length cDNA groups to analyse the expression profiles of genes under drought, cold (low temperature) and high-salinity stress conditions over time. The transcripts of 53, 277 and 194 genes increased after cold, drought and high-salinity treatments, respectively, more than fivefold compared with the control genes. We also identified many highly drought-, cold- or high-salinity- stress-inducible genes. However, we observed strong relationships in the expression of these stress-responsive genes based on Venn diagram analysis, and found 22 stress-inducible genes that responded to all three stresses. Several gene groups showing different expression profiles were identified by analysis of their expression patterns during stress-responsive gene induction. The cold-inducible genes were classified into at least two gene groups from their expression profiles. DREB1A was included in a group whose expression peaked at 2 h after cold treatment. Among the drought, cold or high-salinity stress-inducible genes identified, we found 40 transcription factor genes (corresponding to approximately 11% of all stress-inducible genes identified), suggesting that various transcriptional regulatory mechanisms function in the drought, cold or high-salinity stress signal transduction pathways.
Assuntos
Arabidopsis/genética , Temperatura Baixa , Dessecação , Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Doenças das Plantas/genética , Cloreto de Sódio/farmacologia , Arabidopsis/efeitos dos fármacos , Desastres , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Doenças das Plantas/induzido quimicamente , Regiões Promotoras Genéticas/genética , RNA de Plantas/análise , RNA de Plantas/genética , Fatores de Tempo , Ativação TranscricionalRESUMO
Functional analysis of a genome requires accurate gene structure information and a complete gene inventory. A dual experimental strategy was used to verify and correct the initial genome sequence annotation of the reference plant Arabidopsis. Sequencing full-length cDNAs and hybridizations using RNA populations from various tissues to a set of high-density oligonucleotide arrays spanning the entire genome allowed the accurate annotation of thousands of gene structures. We identified 5817 novel transcription units, including a substantial amount of antisense gene transcription, and 40 genes within the genetically defined centromeres. This approach resulted in completion of approximately 30% of the Arabidopsis ORFeome as a resource for global functional experimentation of the plant proteome.