RESUMO
Vesicle trafficking plays a crucial role in the establishment of cell polarity in various cellular contexts, including axis-pattern formation in the developing egg chamber of Drosophila. The EGFR ligand, Gurken (Grk), is first localized at the posterior of young oocytes for anterior-posterior axis formation and later in the dorsal anterior region for induction of the dorsal-ventral (DV) axis, but regulation of Grk localization by membrane trafficking in the oocyte remains poorly understood. Here, we report that Syntaxin 1A (Syx1A) is required for efficient trafficking of Grk protein for DV patterning. We show that Syx1A is associated with the Golgi membrane and is required for the transportation of Grk-containing vesicles along the microtubules to their dorsal anterior destination in the oocyte. Our studies reveal that the Syx1A dependent trafficking of Grk protein is required for efficient EGFR signaling during DV patterning.
Assuntos
Padronização Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Receptores ErbB/metabolismo , Proteínas Qa-SNARE/metabolismo , Receptores de Peptídeos de Invertebrados/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador alfa/metabolismo , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Drosophila melanogaster/citologia , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Mutação/genética , Oócitos/citologia , Oócitos/metabolismo , Fenótipo , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
During the initial stages of carcinogenesis, transformation events occur in a single cell within an epithelial monolayer. However, it remains unknown what happens at the interface between normal and transformed epithelial cells during this process. In Drosophila, it has been recently shown that normal and transformed cells compete with each other for survival in an epithelial tissue; however the molecular mechanisms whereby "loser cells" undergo apoptosis are not clearly understood. Lgl (lethal giant larvae) is a tumor suppressor protein and plays a crucial role in oncogenesis in flies and mammals. Here we have examined the involvement of Lgl in cell competition and shown that a novel Lgl-binding protein is involved in Lgl-mediated cell competition. Using biochemical immunoprecipitation methods, we first identified Mahjong as a novel binding partner of Lgl in both flies and mammals. In Drosophila, Mahjong is an essential gene, but zygotic mahjong mutants (mahj(-/-)) do not have obvious patterning defects during embryonic or larval development. However, mahj(-/-) cells undergo apoptosis when surrounded by wild-type cells in the wing disc epithelium. Importantly, comparable phenomena also occur in Mahjong-knockdown mammalian cells; Mahjong-knockdown Madin-Darby canine kidney epithelial cells undergo apoptosis, only when surrounded by non-transformed cells. Similarly, apoptosis of lgl(-/-) cells is induced when they are surrounded by wild-type cells in Drosophila wing discs. Phosphorylation of the c-Jun N-terminal kinase (JNK) is increased in mahj(-/-) or lgl(-/-) mutant cells, and expression of Puckered (Puc), an inhibitor of the JNK pathway, suppresses apoptosis of these mutant cells surrounded by wild-type cells, suggesting that the JNK pathway is involved in mahj- or lgl-mediated cell competition. Finally, we have shown that overexpression of Mahj in lgl(-/-) cells strongly suppresses JNK activation and blocks apoptosis of lgl(-/-) cells in the wild-type wing disc epithelium. These data indicate that Mahjong interacts with Lgl biochemically and genetically and that Mahjong and Lgl function in the same pathway to regulate cellular competitiveness. As far as we are aware, this is the first report that cell competition can occur in a mammalian cell culture system.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Linhagem Celular , Células Clonais , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Epitélio/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases , Ubiquitina-Proteína Ligases , Asas de Animais/citologia , Asas de Animais/metabolismoRESUMO
NAC transcription factors play important roles in plant growth, development and stress responses. Previously, we identified multiple NAC genes in soybean (Glycine max). Here, we identify the roles of two genes, GmNAC11 and GmNAC20, in stress responses and other processes. The two genes were differentially induced by multiple abiotic stresses and plant hormones, and their transcripts were abundant in roots and cotyledons. Both genes encoded proteins that localized to the nucleus and bound to the core DNA sequence CGT[G/A]. In the protoplast assay system, GmNAC11 acts as a transcriptional activator, whereas GmNAC20 functions as a mild repressor; however, the C-terminal end of GmANC20 has transcriptional activation activity. Over-expression of GmNAC20 enhances salt and freezing tolerance in transgenic Arabidopsis plants; however, GmNAC11 over-expression only improves salt tolerance. Over-expression of GmNAC20 also promotes lateral root formation. GmNAC20 may regulate stress tolerance through activation of the DREB/CBF-COR pathway, and may control lateral root development by altering auxin signaling-related genes. GmNAC11 probably regulates DREB1A and other stress-related genes. The roles of the two GmNAC genes in stress tolerance were further analyzed in soybean transgenic hairy roots. These results provide a basis for genetic manipulation to improve the agronomic traits of important crops.
Assuntos
Regulação da Expressão Gênica de Plantas/genética , Glycine max/metabolismo , Proteínas de Plantas/metabolismo , Estresse Fisiológico/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Cotilédone/genética , Cotilédone/crescimento & desenvolvimento , Cotilédone/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Secas , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Congelamento , Proteínas de Fluorescência Verde , Ácidos Indolacéticos/metabolismo , Motivos de Nucleotídeos/genética , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Caules de Planta/genética , Caules de Planta/crescimento & desenvolvimento , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Protoplastos , Tolerância ao Sal , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Cloreto de Sódio/farmacologia , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
WRKY-type transcription factors are involved in multiple aspects of plant growth, development and stress response. WRKY genes have been found to be responsive to abiotic stresses; however, their roles in abiotic stress tolerance are largely unknown especially in crops. Here, we identified stress-responsive WRKY genes from wheat (Triticum aestivum L.) and studied their functions in stress tolerance. Forty-three putative TaWRKY genes were identified and two multiple stress-induced genes, TaWRKY2 and TaWRKY19, were further characterized. TaWRKY2 and TaWRKY19 are nuclear proteins, and displayed specific binding to typical cis-element W box. Transgenic Arabidopsis plants overexpressing TaWRKY2 exhibited salt and drought tolerance compared with controls. Overexpression of TaWRKY19 conferred tolerance to salt, drought and freezing stresses in transgenic plants. TaWRKY2 enhanced expressions of STZ and RD29B, and bound to their promoters. TaWRKY19 activated expressions of DREB2A, RD29A, RD29B and Cor6.6, and bound to DREB2A and Cor6.6 promoters. The two TaWRKY proteins may regulate the downstream genes through direct binding to the gene promoter or via indirect mechanism. Manipulation of TaWRKY2 and TaWRKY19 in wheat or other crops should improve their performance under various abiotic stress conditions.
Assuntos
Arabidopsis/fisiologia , Proteínas de Plantas/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Triticum/genética , Arabidopsis/genética , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Secas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Pressão Osmótica , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Regiões Promotoras Genéticas , Cloreto de Sódio/farmacologia , Fatores de Transcrição/genéticaRESUMO
The formation of an anterior-posterior (AP) gradient of microtubules in Drosophila oocytes is essential for specification of the AP axis. Proper microtubule organization in the oocyte requires the function of serine/threonine kinase Par-1. The N1S isoform of Par-1 is enriched at the posterior cortex of the oocyte from stage 7 of oogenesis. Here we report that posterior restriction of Par-1 (N1S) kinase activity is critical for microtubule AP gradient formation. Egg chambers with excessive and ectopic Par-1 (N1S) kinase activity in the germline cells display phenotypes similar to those of egg chambers treated with the microtubule-depolymerizing drug colcemid: depolymerization of microtubules in the oocyte and disruption of oocyte nucleus localization. A phosphorylation target of Par-1, the microtubule-associated protein Tau, is also involved in oocyte polarity formation, and overexpression of Tau alleviates the phenotypes caused by ectopic Par-1 (N1S) kinase activity, suggesting that Par-1 regulates oocyte polarity at least partly through Tau. Our findings reveal that maintaining proper levels of Par-1 at correct position in the oocyte is key to oocyte polarity formation and that the conserved role of Par-1 and Tau is crucial for the establishment of an AP gradient of microtubules and for AP axis specification.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Microtúbulos/metabolismo , Oócitos/citologia , Oogênese/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas tau/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Quinase 3 da Glicogênio Sintase , Oócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas tau/genéticaRESUMO
WRKY-type transcription factors have multiple roles in the plant defence response and developmental processes. Their roles in the abiotic stress response remain obscure. In this study, 64 GmWRKY genes from soybean were identified, and were found to be differentially expressed under abiotic stresses. Nine GmWRKY proteins were tested for their transcription activation in the yeast assay system, and five showed such ability. In a DNA-binding assay, three proteins (GmWRKY13, GmWRKY27 and GmWRKY54) with a conserved WRKYGQK sequence in their DNA-binding domain could bind to the W-box (TTGAC). However, GmWRKY6 and GmWRKY21, with an altered sequence WRKYGKK, lost the ability to bind to the W-box. The function of three stress-induced genes, GmWRKY13, GmWRKY21 and GmWRKY54, was further investigated using a transgenic approach. GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, possibly through the regulation of DREB2A and STZ/Zat10. Transgenic plants over-expressing GmWRKY13 showed increased sensitivity to salt and mannitol stress, but decreased sensitivity to abscisic acid, when compared with wild-type plants. In addition, GmWRKY13-transgenic plants showed an increase in lateral roots. These results indicate that the three GmWRKY genes play differential roles in abiotic stress tolerance, and that GmWRKY13 may function in both lateral root development and the abiotic stress response.
Assuntos
Adaptação Fisiológica/genética , Arabidopsis/genética , Congelamento , Genes de Plantas , Glycine max/genética , Cloreto de Sódio/farmacologia , Fatores de Transcrição/genética , Adaptação Fisiológica/efeitos dos fármacos , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , DNA de Plantas/metabolismo , Dimerização , Desastres , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Fenótipo , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Glycine max/efeitos dos fármacos , Fatores de Transcrição/química , Ativação Transcricional/efeitos dos fármacosRESUMO
BACKGROUND: Trihelix transcription factors play important roles in light-regulated responses and other developmental processes. However, their functions in abiotic stress response are largely unclear. In this study, we identified two trihelix transcription factor genes GmGT-2A and GmGT-2B from soybean and further characterized their roles in abiotic stress tolerance. FINDINGS: Both genes can be induced by various abiotic stresses, and the encoded proteins were localized in nuclear region. In yeast assay, GmGT-2B but not GmGT-2A exhibits ability of transcriptional activation and dimerization. The N-terminal peptide of 153 residues in GmGT-2B was the minimal activation domain and the middle region between the two trihelices mediated the dimerization of the GmGT-2B. Transactivation activity of the GmGT-2B was also confirmed in plant cells. DNA binding analysis using yeast one-hybrid assay revealed that GmGT-2A could bind to GT-1bx, GT-2bx, mGT-2bx-2 and D1 whereas GmGT-2B could bind to the latter three elements. Overexpression of the GmGT-2A and GmGT-2B improved plant tolerance to salt, freezing and drought stress in transgenic Arabidopsis plants. Moreover, GmGT-2B-transgenic plants had more green seedlings compared to Col-0 under ABA treatment. Many stress-responsive genes were altered in GmGT-2A- and GmGT-2B-transgenic plants. CONCLUSION: These results indicate that GmGT-2A and GmGT-2B confer stress tolerance through regulation of a common set of genes and specific sets of genes. GmGT-2B also affects ABA sensitivity.
Assuntos
Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Glycine max/metabolismo , Plantas Geneticamente Modificadas , Fatores de Transcrição/genética , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Dimerização , Secas , Etiquetas de Sequências Expressas , Modelos Genéticos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Ativação Transcricional , Técnicas do Sistema de Duplo-HíbridoRESUMO
Specification of the anteroposterior (AP) axis in Drosophila oocytes requires proper organization of the microtubule and actin cytoskeleton. The establishment and regulation of cytoskeletal polarity remain poorly understood, however. Here, we show important roles for the tumor suppressor Lethal (2) giant larvae (Lgl) and atypical protein kinase C (aPKC) in regulating microtubule polarity and setting up the AP axis of the oocyte. Lgl in the germline cells regulates the localization of axis-specifying morphogens. aPKC phosphorylation of Lgl restricts Lgl activity to the oocyte posterior, thereby dividing the cortex into different domains along the AP axis. Active Lgl promotes the formation of actin-rich projections at the oocyte cortex and the posterior enrichment of the serine/threonine kinase Par-1, a key step for oocyte polarization. Our studies suggest that Lgl and its phosphorylation by aPKC may form a conserved regulatory circuitry in polarization of various cell types.
Assuntos
Polaridade Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Oócitos/citologia , Oócitos/enzimologia , Proteína Quinase C/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Actinas/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Células Clonais , Quinase 3 da Glicogênio Sintase , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transporte ProteicoRESUMO
From soybean plant, 131 bZIP genes were identified and named as GmbZIPs. The GmbZIPs can be classified into ten groups and more than one third of these GmbZIPs are responsive to at least one of the four treatments including ABA, salt, drought and cold stresses. Previous studies have shown that group A bZIP proteins are involved in ABA and stress signaling. We now chose four non-group A genes to study their features. The four proteins GmbZIP44, GmbZIP46, GmbZIP62 and GmbZIP78 belong to the group S, I, C and G, respectively, and can bind to GLM (GTGAGTCAT), ABRE (CCACGTGG) and PB-like (TGAAAA) elements with differential affinity in both the yeast one-hybrid assay and in vitro gel-shift analysis. GmbZIP46 can form homodimer or heterodimer with GmbZIP62 or GmMYB76. Transgenic Arabidopsis plants overexpressing the GmbZIP44, GmbZIP62 or GmbZIP78 showed reduced ABA sensitivity. However, all the transgenic plants were more tolerant to salt and freezing stresses when compared with the Col plants. The GmbZIP44, GmbZIP62 and GmbZIP78 may function in ABA signaling through upregulation of ABI1 and ABI2 and play roles in stress tolerance through regulation of various stress-responsive genes. These results indicate that GmbZIP44, GmbZIP62 and GmbZIP78 are negative regulators of ABA signaling and function in salt and freezing tolerance.
Assuntos
Ácido Abscísico/metabolismo , Arabidopsis/fisiologia , Fatores de Transcrição de Zíper de Leucina Básica/genética , Glycine max/genética , Arabidopsis/genética , Clonagem Molecular , Temperatura Baixa , Dimerização , Expressão Gênica , Família Multigênica , Filogenia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Salinidade , Transdução de Sinais , Especificidade por Substrato , Ativação Transcricional , Água/metabolismoRESUMO
Soybean is one of the most important leguminous seed crops among the oil crops. Although the pathways for lipid biosynthesis have been identified, the factors that regulate the biosynthetic pathways at the transcriptional level are largely unknown. Here, we report our findings on the involvement of soybean Dof-type transcription factor genes in the regulation of the lipid content in soybean seeds. We identified 28 Dof-type transcription factor genes in soybean plants, and these genes displayed diverse patterns of expression in various organs. Seven flower/pod-specific genes and one constitutively expressed gene were further investigated. The proteins encoded by these seven genes were localized in the nucleus, and exhibited different abilities for transcriptional activation and DNA binding. Two genes, GmDof4 and GmDof11, were found to increase the content of total fatty acids and lipids in GmDof4 and GmDof11 transgenic Arabidopsis seeds. We also found that the 1000-seed weight was increased in the GmDof4 and GmDof11 transgenic plants. Using microarray and DNA binding analysis, we found that the two Dof-like proteins, GmDof4 and GmDof11, activated the acetyl CoA carboxylase gene and long-chain-acyl CoA synthetase gene, respectively, by direct binding to the cis-DNA elements in their promoter regions. In addition, both proteins downregulated the storage protein gene, CRA1, through direct binding. These results suggest that the two GmDof genes may augment the lipid content of soybean seeds by upregulating genes that are associated with the biosynthesis of fatty acids.
Assuntos
Arabidopsis/metabolismo , Lipídeos/análise , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Sementes/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sementes/genética , Glycine max/genética , Fatores de Transcrição/genéticaRESUMO
Three DREB homologue genes, GmDREBa, GmDREBb, and GmDREBc, were isolated from soybean, Glycine max (L.) Merr. Each of the deduced proteins contains an AP2 domain of 64 amino acids. Yeast one-hybrid assay revealed that all of the three dehydration-responsive, element-binding proteins specifically bound to the dehydration-responsive element. Analysis of transcriptional activation abilities of these proteins in yeast indicated that GmDREBa and GmDREBb could activate the expression of a reporter gene, whereas GmDREBc could not. The transcriptions of GmDREBa and GmDREBb were induced by salt, drought, and cold stresses in leaves of soybean seedlings. The expression of GmDREBc was not significantly affected in leaves but apparently induced in roots by salt, drought, and abscisic acid treatments. These results suggest that these three genes function specifically in response to abiotic stresses in soybean.
Assuntos
Desidratação/metabolismo , Regulação da Expressão Gênica de Plantas , Glycine max/genética , Folhas de Planta/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Análise por Conglomerados , Primers do DNA , Desidratação/genética , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Estrutura Terciária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , LevedurasRESUMO
Cation transport is thought to be an important process for ion homeostasis in plant cells. Here, we report that a soybean putative cation/proton antiporter GmCAX1 may be a mediator of this process. GmCAX1 is expressed in all tissues of the soybean plants but at a lower level in roots. Its expression was induced by PEG, ABA, Ca(2+), Na(+) and Li(+) treatments. The GmCAX1-GFP fusion protein was mainly localized in plasma membrane of the transgenic Arabidopsis plant cells and onion epidermal cells. Transgenic Arabidopsis plants overexpressing GmCAX1 accumulated less Na(+), K(+), and Li(+), and were more tolerant to elevated Li(+) and Na(+) levels during germination when compared with the controls. These results suggest that GmCAX1 may function as an antiporter for Na(+), K(+) and Li(+). Modulation of this antiporter may be beneficial for regulation of ion homeostasis and thus plant salt tolerance.
Assuntos
Antiporters/genética , Antiporters/metabolismo , Arabidopsis/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Glycine max/genética , Lítio/farmacologia , Cloreto de Sódio/farmacologia , Sequência de Aminoácidos , Antiporters/química , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/química , Proteínas de Transporte de Cátions/química , Regulação da Expressão Gênica de Plantas , Lítio/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Cloreto de Sódio/metabolismoRESUMO
By using cDNA-AFLP, we analyzed a recombinant inbred line population of soybean that was derived from a soybean mosaic virus (SMV) resistant cultivar Kefeng No.1 and a susceptible cultivar Nannong 1138-2. One hundred and eight fragments showing polymorphism between SMV resistant and susceptible pools were identified. One fragment w27 was 96 bp in length and showed homology to homeobox ggth with a coding region of 738 bp, encoding a protein of 245 amino acids. The genomic sequence analysis defined an intron of 521 bp in the coding region. GmHZ1 was characterized by the presence of a homeodomain (HD) with a closely linked leucine zipper motif (Zip). Southern blot analysis indicated that there was a single copy of GmHZ1 in the soybean genome. When inoculated with SMV strain N3, resistant and susceptible varieties showed reduced and increased expression of the GmHZ1, respectively. The fusion protein of GmHZ1 with GFP was targeted only in nucleus. Yeast two hybrid studies revealed that the GmHZ1 had transcriptional activation activity and can form homodimer. GmHZ1 can bind two 9-bp pseudopalindromic elements (CAAT(A/T)ATTG and CAAT(C/G)ATTG) with different affinity. Using GUS as a reporter gene, GmHZ1 was proved to be a transcriptional activator and enhanced GUS expression by binding with the two elements in plant cells. These results indicate that the GmHZ1 may have a transcriptional activator function in plant response to SMV infection.
Assuntos
Glycine max/genética , Proteínas de Homeodomínio/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/metabolismo , Zíper de Leucina , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Glycine max/metabolismo , Transcrição Gênica , Ativação TranscricionalRESUMO
A full-length gene GmPti1 was identified from soybean in an EST sequencing project by its homology to tomato Pti1. It encoded a protein of 366 amino acids. RT-PCR analysis showed that the GmPti1 expression was induced by salicylic acid and wounding. The deduced amino acid sequence had a Ser/Thr/Tyr kinase domain. GmPti1 protein was expressed in E. coli as an MBP fusion, purified by amylose resin and examined for its autophosphorylation ability. The phosphorylation assay in vitro showed that GmPti1 had kinase activity in the presence of Mn2+. These results demonstrated that GmPti1 represented a new Pti1-like gene, unlike the two published genes sPti1a and sPti1b, which encoding proteins had no autophosphorylation ability.
Assuntos
Glycine max/enzimologia , Glycine max/genética , Proteínas de Plantas , Proteínas Serina-Treonina Quinases/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/isolamento & purificação , Proteínas Serina-Treonina Quinases/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Using mixed resistance gene analogs as probes, a putative resistance gene (KR1) was isolated from soybean and characterized further. The KR1 protein consists of a Toll/interleukin receptor (TIR) domain, a nucleotide binding site (NBS) domain, an imperfect leucine-rich repeat (LRR) domain and two C-terminal transmembrane segments. Due to these features, KR1 represents a distinct member in the TIR-NBS-LRR class of resistance genes. Southern-blot analysis indicated that there were several KR1-related sequences within the soybean genome, and two polymorphic loci were mapped onto linkage group L. KR1 was induced by SA treatment and soybean mosaic virus (SMV) infection in the resistant line (Kefeng 1). An orthologue (NR1) and a homologue (NR2) of the KR1 gene were also identified in the SMV susceptible-line Nannong1138-2. Sequencing analysis revealed that NR2 was highly homologous to KR1 and NR1, but had a 21-bp deletion. Moreover, the NR1, NR2 transcription and the ratio of NR1/ NR2 was up-regulated by viral infection in Nannong1138-2. These results indicated the complexity of the regulatory mechanism in the plant responses to SMV infection.
Assuntos
Glycine max/genética , Vírus de Plantas/fisiologia , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Sequência de Bases , Southern Blotting , Mapeamento Cromossômico , Ligação Genética , Interações Hospedeiro-Parasita/efeitos dos fármacos , Interações Hospedeiro-Parasita/genética , Dados de Sequência Molecular , Filogenia , Vírus de Plantas/efeitos dos fármacos , Ácido Salicílico/farmacologia , Análise de Sequência de DNA , Glycine max/efeitos dos fármacos , Glycine max/virologiaRESUMO
A full-length gene GmSAMDC1, encoding the S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in polyamine biosynthesis, was identified from soybean expressed sequence tags and was characterized. GmSAMDC1 encoded a peptide of 355 amino acids. When compared with other plant SAMDCs, the GmSAMDC1 protein had several highly conserved regions including a putative pro-enzyme cleavage site and a PEST sequence. The 5' leader sequence of the the GmSAMDC1 mRNA contained two additional open reading frames (ORFs), which may regulate the translational process. The genomic sequence of the GmSAMDC1 gene contained three introns in the 5' leader sequence, but no intron in the 3'-UTR or the main pro-enzyme ORF. A simple sequence repeat (SSR) was found in intron 2, and the GmSAMDC1 gene was mapped to linkage group D1 using this SSR. The genomic organization of the GmSAMDC1 gene in the subgenus Glycine and the subgenus Soja was found to be different by Southern-blot and PCR analysis. A pseudogene, GmSAMDC2, was also identified. This gene contained no intron and lost its two uORFs. Northern-blot analysis showed that the GmSAMDC1 gene expression was induced by salt, drought and cold, but not induced by wounding; suggesting that the gene was implicated in response to multiple-stress conditions.
Assuntos
Adenosilmetionina Descarboxilase/genética , Genoma de Planta , Glycine max/genética , Regiões 3' não Traduzidas , Adenosilmetionina Descarboxilase/química , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Southern Blotting , Primers do DNA , DNA Complementar , Genes de Plantas , Íntrons , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Pseudogenes , RNA Mensageiro/genética , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
We analyzed 314,254 soybean expressed sequence tags (ESTs), including 29,540 from our laboratory and 284,714 from GenBank. These ESTs were assembled into 56,147 unigenes. About 76.92% of the unigenes were homologous to genes from Arabidopsis thaliana ( Arabidopsis). The putative products of these unigenes were annotated according to their homology with the categorized proteins of Arabidopsis. Genes corresponding to cell growth and/or maintenance, enzymes and cell communication belonged to the slow-evolving class, whereas genes related to transcription regulation, cell, binding and death appeared to be fast-evolving. Soybean unigenes with no match to genes within the Arabidopsis genome were identified as soybean-specific genes. These genes were mainly involved in nodule development and the synthesis of seed storage proteins. In addition, we also identified 61 genes regulated by salicylic acid, 1,322 transcription factor genes and 326 disease resistance-like genes from soybean unigenes. SSR analysis showed that the soybean genome was more complex than the Arabidopsis and the Medicago truncatula genomes. GC content in soybean unigene sequences is similar to that in Arabidopsis and M. truncatula. Furthermore, the combined analysis of the EST database and the BAC-contig sequences revealed that the total gene number in the soybean genome is about 63,501.