RESUMO
Anthonomus grandis Boheman is a key pest in cotton crops in the New World. Its larval stage develops within the flower bud using it as food and as protection against its predators. This behavior limits the effectiveness of its control using conventional insecticide applications and biocontrol techniques. In spite of its importance, little is known about its genome sequence and, more important, its specific expression in key organs like the midgut. Total mRNA isolated from larval midguts was used for pyrosequencing. Sequence reads were assembled and annotated to generate a unigene data set. In total, 400,000 reads from A. grandis midgut with an average length of 237 bp were assembled and combined into 20,915 contigs. The assembled reads fell into 6,621 genes models. BlastX search using the NCBI-NR database showed that 3,006 unigenes had significant matches to known sequences. Gene Ontology (GO) mapping analysis evidenced that A. grandis is able to transcripts coding for proteins involved in catalytic processing of macromolecules that allows its adaptation to very different feeding source scenarios. Furthermore, transcripts encoding for proteins involved in detoxification mechanisms such as p450 genes, glutathione-S-transferase, and carboxylesterases are also expressed. This is the first report of a transcriptomic study in A. grandis and the largest set of sequence data reported for this species. These data are valuable resources to expand the knowledge of this insect group and could be used in the design of new control strategies based in molecular information.
Assuntos
Transcriptoma , Gorgulhos/genética , Sequência de Aminoácidos , Animais , Simulação por Computador , Sistema Digestório/metabolismo , Larva/genética , Larva/metabolismo , Dados de Sequência Molecular , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Gorgulhos/crescimento & desenvolvimento , Gorgulhos/metabolismoRESUMO
BACKGROUND: Sclerotinia Head Rot (SHR) is one of the most damaging diseases of sunflower in Europe, Argentina, and USA, causing average yield reductions of 10 to 20 %, but leading to total production loss under favorable environmental conditions for the pathogen. Association Mapping (AM) is a promising choice for Quantitative Trait Locus (QTL) mapping, as it detects relationships between phenotypic variation and gene polymorphisms in existing germplasm without development of mapping populations. This article reports the identification of QTL for resistance to SHR based on candidate gene AM. RESULTS: A collection of 94 sunflower inbred lines were tested for SHR under field conditions using assisted inoculation with the fungal pathogen Sclerotinia sclerotiorum. Given that no biological mechanisms or biochemical pathways have been clearly identified for SHR, 43 candidate genes were selected based on previous transcript profiling studies in sunflower and Brassica napus infected with S. sclerotiorum. Associations among SHR incidence and haplotype polymorphisms in 16 candidate genes were tested using Mixed Linear Models (MLM) that account for population structure and kinship relationships. This approach allowed detection of a significant association between the candidate gene HaRIC_B and SHR incidence (P < 0.01), accounting for a SHR incidence reduction of about 20 %. CONCLUSIONS: These results suggest that AM will be useful in dissecting other complex traits in sunflower, thus providing a valuable tool to assist in crop breeding.
Assuntos
Ascomicetos/patogenicidade , Mapeamento Cromossômico/métodos , Resistência à Doença/genética , Helianthus/genética , Doenças das Plantas/imunologia , Locos de Características Quantitativas/genética , Sequência de Bases , Brassica napus/genética , Produtos Agrícolas , DNA de Plantas/genética , Genes de Plantas/genética , Genótipo , Helianthus/imunologia , Helianthus/microbiologia , Endogamia , Dados de Sequência Molecular , Fenótipo , Doenças das Plantas/microbiologia , Análise de Sequência de DNARESUMO
Cultivated cotton (Gossypium hirsutum) is heavily attacked by various species of insects worldwide and breeding of new varieties resistant to pests is still a hard battle to win. RNAi technology is an important reverse genetics tool to induce gene silencing in eukaryotic organisms and produce phenotypic modifications. In cotton, RNAi was applied to investigate gene function and enhance resistance to insects and pathogens. Different methods and techniques can be used to synthetize double stranded RNA (dsRNA) into plant cells. The Agrobacterium-mediated transformation is a common method to introduce RNAi binary plasmids into cotton genome and obtain stable transgenics plants. This methodology includes the coculture of cotton tissues with Agrobacterium cultures, selection of transgenic cells and induction of somatic embryogenesis to finally obtain transgenic plants after a relatively long period of time. The transient synthesis of dsRNA mediated by virus-induced gene silencing (VIGS) in cotton is an alternative to anticipate the silencing effect of a specific RNA sequence, prior to the development of a stable transgenic plant. VIGS vectors are incorporated into the plant by agroinfiltration technique. During VIGS replication inside plant cells, synthetized dsRNA allows the study on specific heterologous gene expression including the phenotypic effect on herbivorous target pests, thus facilitating a rapid evaluation of dsRNA expressed in cotton plants against individual insect target genes. Here we describe the complementation of these two techniques to evaluate RNAi-based cotton plant protection against insect pests.
Assuntos
Gossypium , Agrobacterium/genética , Animais , Gossypium/genética , Insetos , Melhoramento Vegetal , Plantas Geneticamente Modificadas/genética , Interferência de RNA , RNA de Cadeia Dupla/genéticaRESUMO
Weeds are one of the main causes of the decrease in crop yields, with Johnsongrass (Sorghum halepense L.) being one of the most significant. Weeds can be controlled by herbicides, but some have developed resistance. Quantitative PCR is the technique of choice for studying gene expression related to herbicide resistance because of its high sensitivity and specificity, although its quantitative accuracy is highly dependent on the stability of the reference genes. Thus, in this study we evaluated the stability of different reference genes of glyphosate-resistant S. halepense. Nine genes frequently used as reference genes were selected: MDH, ADP, PP2A, EIF4α, ACT, ARI8, DnaJ, Hsp70, and ALS1, and their expression analyzed in susceptible and resistant biotypes at 0, 24 and 72 h post-application of glyphosate. The stability was analyzed with the geNorm, NormFinder, and BestKeeper software programs and using the ΔCt method. RefFinder was used to generate a comprehensive stability ranking. The results showed that PP2A and ARI8 were the most stable genes under the test conditions. EPSPS expression was also verified against the best two and the worst two reference genes. This study provides useful information for gene expression analysis under glyphosate stress and will facilitate resistance mechanism studies in this weed species.
RESUMO
BACKGROUND: In a large cropping area of northern Argentina, Sorghum halepense (Johnsongrass) has evolved towards glyphosate resistance. This study aimed to determine the molecular and biochemical basis conferring glyphosate resistance in this species. Experiments were conducted to assess target EPSPS gene sequences and (14)C-glyphosate leaf absorption and translocation to meristematic tissues. RESULTS: Individuals of all resistant (R) accessions exhibited significantly less glyphosate translocation to root (11% versus 29%) and stem (9% versus 26%) meristems when compared with susceptible (S) plants. A notably higher proportion of the applied glyphosate remained in the treated leaves of R plants (63%) than in the treated leaves of S plants (27%). In addition, individuals of S. halepense accession R(2) consistently showed lower glyphosate absorption rates in both adaxial (10-20%) and abaxial (20-25%) leaf surfaces compared with S plants. No glyphosate resistance endowing mutations in the EPSPS gene at Pro-101-106 residues were found in any of the evaluated R accessions. CONCLUSION: The results of the present investigation indicate that reduced glyphosate translocation to meristems is the primary mechanism endowing glyphosate resistance in S. halepense from cropping fields in Argentina. To a lesser extent, reduced glyphosate leaf uptake has also been shown to be involved in glyphosate-resistant S. halepense.
Assuntos
Glicina/análogos & derivados , Resistência a Herbicidas , Herbicidas/metabolismo , Sorghum/efeitos dos fármacos , Transporte Biológico , Glicina/metabolismo , Glicina/farmacologia , Herbicidas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sorghum/genética , Sorghum/metabolismo , GlifosatoRESUMO
Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.
Assuntos
Helianthus/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologiaRESUMO
Mal de Río Cuarto virus (MRCV) is a plant virus of the genus Fijivirus within the family Reoviridae that infects several monocotyledonous species and is transmitted by planthoppers in a persistent and propagative manner. Other members of the family replicate in viral inclusion bodies (VIBs) termed viroplasms that are formed in the cytoplasm of infected plant and insect cells. In this study, the protein coded by the first ORF of MRCV segment S9 (P9-1) was shown to establish cytoplasmic inclusion bodies resembling viroplasms after transfection of Spodoptera frugiperda insect cells. In accordance, MRCV P9-1 self-associates giving rise to high molecular weight complexes when expressed in bacteria. Strong self-interaction was also evidenced by yeast two-hybrid assays. Furthermore, biochemical characterization showed that MRCV P9-1 bound single stranded RNA and had ATPase activity. Finally, the MRCV P9-1 region required for the formation of VIB-like structures was mapped to the protein carboxy-terminal half. This extensive functional and biochemical characterization of MRCV P9-1 revealed further similarities between plant and animal reovirus viroplasm proteins.
Assuntos
Corpos de Inclusão Viral/metabolismo , Reoviridae/metabolismo , Spodoptera/virologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Corpos de Inclusão Viral/química , Corpos de Inclusão Viral/genética , Fases de Leitura Aberta , Reoviridae/química , Reoviridae/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
As a step towards developing a safe and effective edible vaccine against Newcastle disease virus (NDV), we have explored the use of plants genetically engineered to express viral proteins. We report the construction of transgenic potato plants expressing the genes coding for immunogenic proteins of NDV under the regulation of CaMV 35S promoter and its immunogenicity in mice. All mice receiving transgenic plant extracts in incomplete Freund adjuvant produced specific anti-NDV antibodies. Animals fed with transgenic leaves showed a specific response against NDV. Detection of IgA released from in vitro-cultured intestinal tissue fragments indicated the presence of IgA-secreting cells in the gut.
Assuntos
Doença de Newcastle/imunologia , Vírus da Doença de Newcastle/genética , Plantas Geneticamente Modificadas , Vacinas Virais/genética , Animais , Anticorpos Antivirais/biossíntese , Formação de Anticorpos , Imunidade nas Mucosas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Vacinas Virais/administração & dosagem , Vacinas Virais/imunologiaRESUMO
The plant vp1 gene encodes a transcription factor originally identified in maize that participates in the control of the transition from embryogenesis to seed germination. Different lines of evidence suggest that vp1 participates in preharvest sprouting (PHS) resistance in cereals, but the genetic relationship is not yet established. Thus, the aim of this study was to investigate if there is a connection between vp1 and the formerly documented quantitative trait loci for PHS in Sorghum bicolor. This gene was mapped in sorghum using two well-studied inbred lines displaying contrasting PHS phenotypes. Linkage analysis revealed that the S. bicolor vp1 (Sbvp1) locus is linked to markers located on chromosomes 3 and 8 in maize. Analysis indicated that this gene is not correlated with PHS at a statistically significant level. Structural analysis of the Sbvp1 gene from both inbred lines showed a high degree of overall conservation (low polymorphism), even in the promoter region. These findings, taken together with the previously observed correlation between the pattern of expression of this gene and PHS performance, suggest that Sbvp1 might act downstream in the signalling pathway that leads to seed germination, or even be differentially regulated as a consequence rather than as a cause of the phenotypic behaviour.
Assuntos
Mapeamento Cromossômico , Genes de Plantas , Germinação/genética , Locos de Características Quantitativas/genética , Sorghum/genética , Cromossomos de Plantas , Ligação Genética , Marcadores Genéticos , Genoma de Planta , Germinação/fisiologia , Polimorfismo de Fragmento de Restrição , Sementes/genética , Sementes/fisiologia , Análise de Sequência de DNA , Sorghum/fisiologiaRESUMO
Oxalis tuberosa is an octoploid Andean tuber crop called oca that belongs to the worldwide distributed genus Oxalis. The genus is very heterogeneous and its systematics is still problematic. It has been proposed that O. tuberosa evolved by polyploidization of a still not defined ancestor that belongs to an alliance of species sharing the same basic chromosome number (x = 8). Nuclear rDNA units of O. tuberosa and a selected group of four related diploid species were characterised by RFLP using different restriction endonucleases and southern hybridization probes to produce a restriction map for EcoRI and BamHI. The major rDNA unit length in O. tuberosa was estimated at 10.7 kbp. As expected, restriction site variation was observed mainly in the intergenic spacer (IGS), but was also detected in coding regions. Restriction site mapping organization of the transcribed rDNA unit of O. tuberosa is very similar to O. oblongiformis. Nucleotide sequencing of a region of O. peduncularis IGS generated a complex organization pattern of repeats and subrepeats. Diploid species O. peduncularis, O. tabaconasensis and O. aff. villosula exhibited a ladder pattern that is a consequence of a 170 bp subrepeat unit indicating that these species share organization similarity and sequence homology. The variation pattern provided information to compare among diploid species, although it did not help to clarify taxonomic relationships between O. tuberosa and the putative diploid ancestors analysed in this study. Nonetheless, the RFLP pattern exhibited by O. tuberosa for the IGS region was quite unique and will be a useful tool to prospect in other related species.