RESUMO
Monitoring of the introduction of unapproved genetically modified (GM) events is required because the approval status of a GM event may differ from country to country. The on-site methods such as loop-mediated isothermal amplification (LAMP) offer a technological answer for the rapid GM detection beyond the laboratories. Real-time LAMP assays detecting one GM target were reported earlier. To increase the efficiency of the assay, a multiplex real-time LAMP simultaneously targeting Figwort Mosaic Virus promoter (P-FMV) that constructs region between the Cauliflower Mosaic Virus 35S promoter and cry1Ac gene (p35S-cry1Ac) and neomycin phosphotransferase II (nptII) marker gene was developed. The assay could detect as low as 0.1% for each GM target within 45 min. To the best of our knowledge, multiplexing in real-time LAMP using the Genie II system with applicability in GM detection has been reported herein for the first time. The developed method provides rapid, on-site, and real-time GM detection in seeds and food products.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Plantas Geneticamente Modificadas , Sementes , Técnicas de Amplificação de Ácido Nucleico/métodos , Plantas Geneticamente Modificadas/genética , Sementes/genética , Sementes/química , Caulimovirus/genética , Regiões Promotoras Genéticas , Técnicas de Diagnóstico MolecularRESUMO
In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.
Assuntos
Carica , DNA Polimerase Dirigida por DNA , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Carica/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Plantas Geneticamente Modificadas/genética , Alimentos Geneticamente Modificados , Caulimovirus/genética , Potyvirus/genética , Potyvirus/isolamento & purificaçãoRESUMO
Mucilage is a gelatinous and sticky hydrophilic polysaccharide released from epidermal cells of seed coat after the hydration of mature seeds and is composed primarily of unbranched rhamnogalacturonan I (RG-I). In this study, we produced a recombinant endo-RG-I hydrolase from Aspergillus aculeatus (AaRhgA) in the fission yeast Schizosaccharomyces pombe and examined its substrate preference for pyridylaminated (PA) RG-I with the various degrees of polymerization (DP). Recombinant AaRhgA requires PA-RG-I with a DP of 10 or higher for its hydrolase activity. We heterologously expressed the AarhgA gene under the strong constitutive promoter, cauliflower mosaic virus 35S promoter, in Arabidopsis thaliana. In a series of biochemical analyses of each mucilage fraction released from the water-imbibed seeds of the transgenic plants, we found the enhanced deposition of the transparent mucilage layer that existed in the peripheral regions of the adherent mucilage and was not stained with ruthenium red. This study demonstrated the feasibility of manipulating the mucilage organization by heterologous expression of the endo-RG-I hydrolase.
Assuntos
Arabidopsis , Aspergillus , Pectinas , Plantas Geneticamente Modificadas , Sementes , Arabidopsis/genética , Arabidopsis/metabolismo , Aspergillus/enzimologia , Aspergillus/genética , Aspergillus/metabolismo , Pectinas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/genética , Sementes/metabolismo , Mucilagem Vegetal/metabolismo , Mucilagem Vegetal/química , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Schizosaccharomyces/enzimologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Regiões Promotoras Genéticas , Caulimovirus/genética , Caulimovirus/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/química , Especificidade por SubstratoRESUMO
Genetically modified crops (GMCs) have been discussed due to unknown safety, and thus, it is imperative to develop an effective detection technology. CRISPR/Cas is deemed a burgeoning technology for nucleic acid detection. Herein, we developed a novel detection method for the first time, which combined thermostable Cas12b with loop-mediated isothermal amplification (LAMP), to detect genetically modified (GM) soybeans in a customized one-pot vessel. In our method, LAMP-specific primers were used to amplify the cauliflower mosaic virus 35S promoter (CaMV35S) of the GM soybean samples. The corresponding amplicons activated the trans-cleavage activity of Cas12b, which resulted in the change of fluorescence intensity. The proposed bioassay was capable of detecting synthetic plasmid DNA samples down to 10 copies/µL, and as few as 0.05% transgenic contents could be detected in less than 40 min. This work presented an original detection method for GMCs, which performed rapid, on-site, and deployable detection.
Assuntos
Glycine max , Técnicas de Amplificação de Ácido Nucleico , Plantas Geneticamente Modificadas , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/química , Glycine max/genética , Glycine max/química , Bioensaio/métodos , Sistemas CRISPR-Cas , Caulimovirus/genética , Proteínas de Bactérias/genéticaRESUMO
Cauliflower mosaic virus (CaMV) has a double-stranded DNA genome and is globally distributed. The phylogeny tree of 121 CaMV isolates was categorized into two primary groups, with Iranian isolates showing the greatest genetic variations. Nucleotide A demonstrated the highest percentage (36.95%) in the CaMV genome and the dinucleotide odds ratio analysis revealed that TC dinucleotide (1.34 ≥ 1.23) and CG dinucleotide (0.63 ≤ 0.78) are overrepresented and underrepresented, respectively. Relative synonymous codon usage (RSCU) analysis confirmed codon usage bias in CaMV and its hosts. Brassica oleracea and Brassica rapa, among the susceptible hosts of CaMV, showed a codon adaptation index (CAI) value above 0.8. Additionally, relative codon deoptimization index (RCDI) results exhibited the highest degree of deoptimization in Raphanus sativus. These findings suggest that the genes of CaMV underwent codon adaptation with its hosts. Among the CaMV open reading frames (ORFs), genes that produce reverse transcriptase and virus coat proteins showed the highest CAI value of 0.83. These genes are crucial for the creation of new virion particles. The results confirm that CaMV co-evolved with its host to ensure the optimal expression of its genes in the hosts, allowing for easy infection and effective spread. To detect the force behind codon usage bias, an effective number of codons (ENC)-plot and neutrality plot were conducted. The results indicated that natural selection is the primary factor influencing CaMV codon usage bias.
Assuntos
Caulimovirus , Uso do Códon , Evolução Molecular , Genoma Viral , Filogenia , Doenças das Plantas , Genoma Viral/genética , Caulimovirus/genética , Doenças das Plantas/virologia , Fases de Leitura Aberta/genética , Códon/genética , Variação Genética , Brassica rapa/genética , Brassica rapa/virologia , Interações Hospedeiro-Patógeno/genética , Brassica/genética , Brassica/virologia , Raphanus/genética , Raphanus/virologia , Irã (Geográfico)RESUMO
BACKGROUND: Groundnut (Arachis hypogaea L.) is the 13th most important global crop grown throughout the tropical and subtropical regions of the world. One of the major constraints to groundnut production is viruses, which are also the most economically important and most abundant pathogens among cultivated legumes. Only a few studies have reported the characterization of RNA viruses in cultivated groundnuts in western Kenya, most of which deployed classical methods of detecting known viruses. METHODS: We sampled twenty-one symptomatic and three asymptomatic groundnut leaf samples from farmers' fields in western Kenya. Total RNA was extracted from the samples followed by First-strand cDNA synthesis and sequencing on the Illumina HiSeq 2500 platform. After removing host and rRNA sequences, high-quality viral RNA sequences were de novo assembled and viral genomes annotated using the publicly available NCBI virus database. Multiple sequence alignment and phylogenetic analysis were done using MEGA X. RESULTS: Bioinformatics analyses using as low as â¼3.5 million reads yielded complete and partial genomes for Cauliflower mosaic virus (CaMV), Cowpea polerovirus 2 (CPPV2), Groundnut rosette assistor virus (GRAV), Groundnut rosette virus (GRV), Groundnut rosette virus satellite RNA (satRNA) and Peanut mottle virus (PeMoV) falling within the species demarcation criteria. This is the first report of CaMV and the second report of CPPV2 on groundnut hosts in the world. Confirmation of the detected viruses was further verified through phylogenetic analyses alongside reported publicly available highly similar viruses. PeMoV was the only seed-borne virus reported. CONCLUSION: Our findings demonstrate the power of Next Generation Sequencing in the discovery and identification of novel viruses in groundnuts. The detection of the new viruses indicates the complexity of virus diseases in groundnuts and would require more focus in future studies to establish the effect of the viruses as sole or mixed infections on the crop. The detection of PeMoV with potential origin from Malawi indicates the importance of seed certification and cross-boundary seed health testing.
Assuntos
Fabaceae , Luteoviridae , Vírus de Plantas , Vírus de RNA , Tombusviridae , Caulimovirus/genética , Quênia , Filogenia , Vírus de Plantas/genética , Vírus de RNA/genética , Fabaceae/genética , Luteoviridae/genética , Perfilação da Expressão GênicaRESUMO
Dahlia (Dahlia variabilis) is a widely cultivated ornamental and medicinal plant in China. Recently, dahlia plants with symptoms of leaf mottling and distortion were collected in Hohhot, Inner Mongolia, China. The presence of dahlia common mosaic virus (DCMV), an unassigned species in the genus Caulimovirus, was confirmed by high-throughput sequencing. Three fragments of DCMV Inner Mongolia isolate (DCMV-IN) were PCR-amplified with specific primers, sequenced and assembled into the complete genome sequence with a GenBank accession number of OR494328. The double-stranded circular DNA genome of DCMV-IN consists of 7949 bp and contains six open reading frames (ORFs). Sequence analysis showed that DCMV-IN shared high sequence identities with other DCMV isolates available in the GenBank database. Phylogenetic analysis of DCMV isolates and other representative caulimoviruses based on genome sequence clustered four DCMV isolates to a single branch which was closest to dahlia mosaic virus (DMV). No recombination event was detected among the four DCMV isolates.
Assuntos
Caulimovirus , Dahlia , Caulimovirus/genética , Dahlia/genética , Filogenia , Genoma Viral , Reação em Cadeia da PolimeraseRESUMO
Viruses are intimately linked with their hosts and especially dependent on gene-for-gene interactions to establish successful infections. On the host side, defence mechanisms such as tolerance and resistance can occur within the same species, leading to differing virus accumulation in relation to symptomology and plant fitness. The identification of novel resistance genes against viruses and susceptibility factors is an important part of understanding viral patho-genesis and securing food production. The model plant Arabidopsis thaliana displays a wide symptom spectrum in response to RNA virus infections, and unbiased genome-wide association studies have proven a powerful tool to identify novel disease-genes. In this study we infected natural accessions of A. thaliana with the pararetrovirus cauliflower mosaic virus (CaMV) to study the phenotypic variations between accessions and their correlation with virus accumulation. Through genome-wide association mapping of viral accumulation differences, we identified several susceptibility factors for CaMV, the strongest of which was the abscisic acid synthesis gene NCED9. Further experiments confirmed the importance of abscisic acid homeostasis and its disruption for CaMV disease.
Assuntos
Arabidopsis , Ácido Abscísico , Arabidopsis/genética , Caulimovirus/genética , Estudo de Associação Genômica Ampla , Doenças das Plantas/genéticaRESUMO
Biomolecular condensation is a multipurpose cellular process that viruses use ubiquitously during their multiplication. Cauliflower mosaic virus replication complexes are condensates that differ from those of most viruses, as they are nonmembranous assemblies that consist of RNA and protein, mainly the viral protein P6. Although these viral factories (VFs) were described half a century ago, with many observations that followed since, functional details of the condensation process and the properties and relevance of VFs have remained enigmatic. Here, we studied these issues in Arabidopsis thaliana and Nicotiana benthamiana. We observed a large dynamic mobility range of host proteins within VFs, while the viral matrix protein P6 is immobile, as it represents the central node of these condensates. We identified the stress granule (SG) nucleating factors G3BP7 and UBP1 family members as components of VFs. Similarly, as SG components localize to VFs during infection, ectopic P6 localizes to SGs and reduces their assembly after stress. Intriguingly, it appears that soluble rather than condensed P6 suppresses SG formation and mediates other essential P6 functions, suggesting that the increased condensation over the infection time-course may accompany a progressive shift in selected P6 functions. Together, this study highlights VFs as dynamic condensates and P6 as a complex modulator of SG responses.
Assuntos
Arabidopsis , Caulimovirus , Caulimovirus/genética , Caulimovirus/metabolismo , Grânulos de Estresse , Proteínas Virais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Arabidopsis/metabolismoRESUMO
BACKGROUND: Reliable and efficient methods for detecting genetically modified organisms (GMOs) in unprocessed and processed food will be essential for establishing an effective system for traceability all along the supply chain. It is important to understand the detection of GMOs following microwave treatment, which is a common processing method used in various food products such as flours. Therefore, this study aimed to detect the presence of Cauliflower mosaic virus (CaMV) 35S promoter (P-35S), Figwort mosaic virus (FMV) promoter (P-FMV), and T-NOS (nopaline synthase terminator) genetic elements in DNA samples from untreated and microwave-treated genetically modified (GM) cereal flour samples using the qualitative polymerase chain reaction (PCR) based screening method. METHODS AND RESULTS: DNA was extracted from all samples, and the efficiency of the qualitative PCR screening technique was tested by the verification studies. We performed an inhibition study with plant-specific actin (ACT) gene to the effectiveness of confirming the DNA extraction method. Then, we made the confirming of the qualitative PCR system by method performance testing criteria. The high quality and quantity of the DNA extracts from untreated and microwave-treated flour samples indicated the applicability of qualitative PCR screening assays. The results showed that microwave radiation does not significantly impact the genetic element screening in flour materials. CONCLUSION: Untreated and microwave-treated flour samples had amplifiable DNA for the simultaneous screening of three genetic elements. The qualitative screening tests conducted in this study produced dependable outcomes, thus, can be successfully used for monitoring in control laboratories.
Assuntos
Caulimovirus , Grão Comestível , Caulimovirus/genética , Plantas Geneticamente Modificadas/genética , Grão Comestível/genética , Micro-Ondas , Farinha , DNARESUMO
Cauliflower mosaic virus (CaMV) was the first discovered plant virus with genomic DNA that uses reverse transcriptase for replication. The CaMV 35S promoter is a constitutive promoter and thus, an attractive driver of gene expression in plant biotechnology. It is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. In the last century, producing food for the world's population while preserving the environment and human health is the main topic of agriculture. The damage caused by viral diseases has a significant negative economic impact on agriculture, and disease control is based on two strategies: immunization and prevention to contain virus spread, so correct identification of plant viruses is important for disease management. Here, we discuss CaMV from different aspects: taxonomy, structure and genome, host plants and symptoms, transmission and pathogenicity, prevention, control and application in biotechnology as well as in medicine. Also, we calculated the CAI index for three ORFs IV, V, and VI of the CaMV virus in host plants, the results of which can be used in the discussion of gene transfer or antibody production to identify the CaMV.
Assuntos
Caulimovirus , Interações entre Hospedeiro e Microrganismos , Humanos , Caulimovirus/genética , Plantas , Regiões Promotoras Genéticas , BiotecnologiaRESUMO
There is growing evidence that plant viruses manipulate their hosts and vectors in ways that increase transmission. However, to date only few viral components underlying these phenomena have been identified. Here we show that cauliflower mosaic virus (CaMV) protein P2 modifies the feeding behavior of its aphid vector. P2 is necessary for CaMV transmission because it mediates binding of virus particles to the aphid mouthparts. We compared aphid feeding behavior on plants infected with the wild-type CaMV strain Cabb B-JI or with a deletion mutant strain, Cabb B-JIΔP2, which does not produce P2. Only aphids probing Cabb B-JI infected plants doubled the number of test punctures during the first contact with the plant, indicating a role of P2. Membrane feeding assays with purified P2 and virus particles confirmed that these viral products alone are sufficient to cause the changes in aphid probing. The behavior modifications were not observed on plants infected with a CaMV mutant expressing P2Rev5, unable to bind to the mouthparts. These results are in favor of a virus manipulation, where attachment of P2 to a specific region in the aphid stylets-the acrostyle-exercises a direct effect on vector behavior at a crucial moment, the first vector contact with the infected plant, which is essential for virus acquisition.
Assuntos
Afídeos , Vírus de Plantas , Animais , Caulimovirus/genética , Comportamento Alimentar , Plantas , Doenças das PlantasRESUMO
MAIN CONCLUSION: We characterized an efficient chimeric sub-genomic transcript promoter from Horseradish Latent Virus, FHS4, active in both dicot and monocot plants, and it could be a potential tool for plant biotechnology. Plant pararetroviruses are a rich source of novel plant promoters widely used for biotechnological applications. Here, we comprehensively characterized a unique sub-genomic transcript (Sgt) promoter of Horseradish Latent Virus (HRLV) and identified a fragment (HS4; - 340 to + 10; 351 bp) that showed the highest expression of reporter genes in both transient and transgenic assays as evidenced by biochemical, histochemical GUS reporter assay and transcript analysis of uidA gene by qRT-PCR. Phylogenetic analysis showed that the HSgt promoter was closely related to the sub-genomic promoter of the Cauliflower Mosaic Virus (CaMV19S). We found that the as-1 element and W-box played an important role in the transcriptional activity of the HS4 promoter. Furthermore, the HS4 promoter was also induced by salicylic acid. Alongside, we enhanced the activity of the HS4 promoter by coupling the enhancer region from Figwort Mosaic Virus (FMV) promoter to the upstream region of it. This hybrid promoter FHS4 was around 1.1 times stronger than the most commonly used promoter, 35S (Cauliflower Mosaic Virus full-length transcript promoter), and was efficient in driving reporter genes in both dicot and monocot plants. Subsequently, transgenic tobacco plants expressing an anti-microbial peptide BrLTP2.1 (Brassica rapa lipid transport protein 2.1), under the control of the FHS4 promoter, were developed. The in vitro anti-fungal assay revealed that the plant-derived BrLTP2.1 protein driven by an FHS4 promoter manifested increased resistance against an important plant fungal pathogen, Alternaria alternata. Finally, we concluded that the FHS4 promoter can be used as an alternative to the 35S promoter and has a high potential to become an efficient tool in plant biotechnology.
Assuntos
Armoracia , Caulimovirus , Caulimovirus/genética , Armoracia/genética , Armoracia/metabolismo , Filogenia , Regiões Promotoras Genéticas/genética , Plantas Geneticamente Modificadas/genética , Genômica , Nicotiana/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismoRESUMO
Viral infections impose extraordinary RNA stress, triggering cellular RNA surveillance pathways such as RNA decapping, nonsense-mediated decay, and RNA silencing. Viruses need to maneuver among these pathways to establish infection and succeed in producing high amounts of viral proteins. Processing bodies (PBs) are integral to RNA triage in eukaryotic cells, with several distinct RNA quality control pathways converging for selective RNA regulation. In this study, we investigated the role of Arabidopsis thaliana PBs during Cauliflower mosaic virus (CaMV) infection. We found that several PB components are co-opted into viral factories that support virus multiplication. This pro-viral role was not associated with RNA decay pathways but instead, we established that PB components are helpers in viral RNA translation. While CaMV is normally resilient to RNA silencing, dysfunctions in PB components expose the virus to this pathway, which is similar to previous observations for transgenes. Transgenes, however, undergo RNA quality control-dependent RNA degradation and transcriptional silencing, whereas CaMV RNA remains stable but becomes translationally repressed through decreased ribosome association, revealing a unique dependence among PBs, RNA silencing, and translational repression. Together, our study shows that PB components are co-opted by the virus to maintain efficient translation, a mechanism not associated with canonical PB functions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Caulimovirus/genética , Caulimovirus/metabolismo , Proteínas Correpressoras/metabolismo , Corpos de Processamento , RNA Viral/genéticaRESUMO
Electrochemical polymerase chain reaction (PCR) represents a valid alternative to the optical-based PCR due to reduced costs of signaling labels, use of simpler instrumentation, and possibility of miniaturization and portability of the systems, which can facilitate decentralized detection. The high intrinsic electroactivity and strong linear relationship between the material concentration and its redox signal suggest a possible use of oxidized nanocarbon materials as electroactive tags for PCR. Herein, we compared three different nanographene oxide materials namely nGO-1, nGO-2 and nGO-3 as signaling tags for the detection of genetically modified organisms (GMO) by electrochemical PCR. The three materials differ in size, chemical composition as well as type and amount of oxygen functionalities verified by extensive characterization with X-ray photoelectron spectroscopy (XPS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), transmission electron microscopy (TEM) and electrochemical methods. A sense primer sequence belonging to the Cauliflower Mosaic Virus 35S promoter (a common genetic marker for GMO screening) was used to conjugate to the nanocarbon materials by carbodiimide chemistry before PCR amplification with a biotinylated antisense strand. Finally, the amplified electroactive PCR product was detected, where the reduction signal derived from the electrochemically reducible oxygenated functionalities on the nanocarbon material surface was directly correlated to the presence of GMO. Overall, we were able to correlate the different material characteristics with their performance as electroactive labels and identify the nanocarbon material that exhibits the highest potential to be used as innovative electroactive label for PCR in the amplification and detection of the selected target sequence.
Assuntos
Caulimovirus , Técnicas Eletroquímicas , Caulimovirus/genética , Oxirredução , Óxidos/química , Reação em Cadeia da Polimerase/métodosRESUMO
The adoption of Arabidopsis thaliana in the 1980s as a universal plant model finally enabled researchers to adopt and take full advantage of the molecular biology tools and methods developed in the bacterial and animal fields since the early 1970s. It further brought the plant sciences up to speed with other research fields, which had been employing widely accepted model organisms for decades. In parallel with this major development, the concurrent establishment of the plant transformation methodology and the description of the cauliflower mosaic virus (CaMV) 35S promoter enabled scientists to create robust transgenic plant lines for the first time, thereby providing a valuable tool for studying gene function. The ability to create transgenic plants launched the plant biotechnology sector, with Monsanto and Plant Genetic Systems developing the first herbicide- and pest-tolerant plants, initiating a revolution in the agricultural industry. Here I review the major developments over a less than 10-year span and demonstrate how they complemented each other to trigger a revolution in plant molecular biology and launch an era of unprecedented progress for the whole plant field. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.
Assuntos
Arabidopsis , Regulação da Expressão Gênica de Plantas , Arabidopsis/genética , Caulimovirus/genética , Biologia Molecular , Plantas Geneticamente Modificadas/genéticaRESUMO
BACKGROUND: Promoters are important factors affecting gene expression in cells. The driven activities of viral promoters were generally assessed to screen available promoters for transgenic and research and biotech industries. In this study, we cloned a full-length promoter from a Chinese isolate of strawberry vein banding virus (SVBV) and produced several deletion mutants for evaluation of applications in production of reporter proteins in stable transgenic plants. METHODS: The full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction. The effects of SVBV SP1, SP2, SP3, and SP4 on gene expression were evaluated using ß-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes. RESULTS: Transient expression assays showed that the SVBV SP1 promoter and its three deletion mutants all expressed the reporter genes, albeit at very different levels. Interestingly, transcriptional activity driven by the SP1 promoter was much higher than that of the cauliflower mosaic virus (CaMV) 35S promoter. After stable transformation of the GUS gene into Nicotiana tabacum plants, SVBV SP1-driven transgene expression was approximately 2.6-fold higher than CaMV 35S promoter-driven transgene expression. In addition, GUS gene expression levels were enhanced by co-inoculation of the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing SVBV open reading frame (ORF) V or ORF VI. CONCLUSIONS: The SVBV SP1 promoter from the Chinese isolate evaluated in this study could successfully drive transient and stable expression in plants, it was a stronger promoter than the CaMV 35S and FLt-US promoters and may be more useful for the production of stable transgenic plants.
Assuntos
Caulimovirus , Caulimovirus/genética , Genes Reporter , Plantas Geneticamente Modificadas/genética , Regiões Promotoras GenéticasRESUMO
Paper mulberry vein banding virus (PMVBV), a member of the genus Badnavirus in the family Caulimoviridae, infects paper mulberry (Broussonetia papyrifera), a dicotyledonous plant. Putative promoter regions in the PMVBV genome were tested using recombinant plant expression vectors, revealing that the promoter activity of three genome fragments was about 1.5-fold higher than that of the 35S promoter of cauliflower mosaic virus in Nicotiana benthamiana. In transformed transgenic Arabidopsis thaliana plants, these promoter constructs showed constitutive expression. Based on the activity and gene expression patterns of these three promoter constructs, a fragment of 384 bp (named PmVP) was deduced to contain the full-length promoter of the PMVBV genome. The results suggest that the PMVBV-derived promoter can be used for the constitutive expression of transgenes in dicotyledonous plants.
Assuntos
Badnavirus , Morus , Badnavirus/genética , Caulimovirus/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Nicotiana/genéticaRESUMO
The Arabidopsis (Arabidopsis thaliana) BTB-TAZ DOMAIN PROTEIN 2 (BT2) contains an N-terminal BTB domain, a central TAZ zinc-finger protein-protein interaction domain, and a C-terminal calmodulin-binding domain. We previously demonstrated that BT2 regulates telomerase activity and mediates multiple responses to nutrients, hormones, and abiotic stresses in Arabidopsis. Here, we describe the essential role of BT2 in activation of genes by multimerized Cauliflower mosaic virus 35S (35S) enhancers. Loss of BT2 function in several well-characterized 35S enhancer activation-tagged lines resulted in suppression of the activation phenotypes. Suppression of the phenotypes was associated with decreased transcript abundance of the tagged genes. Nuclear run-on assays, mRNA decay studies, and bisulfite sequencing revealed that BT2 is required to maintain the transcriptionally active state of the multimerized 35S enhancers, and lack of BT2 leads to hypermethylation of the 35S enhancers. The TAZ domain and the Ca++/calmodulin-binding domain of BT2 are critical for its function and 35S enhancer activity. We further demonstrate that BT2 requires CULLIN3 and two bromodomain-containing Global Transcription factor group E proteins (GTE9 and GTE11), to regulate 35S enhancer activity. We propose that the BT2-CULLIN3 ubiquitin ligase, through interactions with GTE9 and GTE11, regulates 35S enhancer activity in Arabidopsis.
Assuntos
Arabidopsis/genética , Caulimovirus/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ativação Transcricional/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Plantas Geneticamente ModificadasRESUMO
Endogenous viral elements (EVEs) have been for the most part described in animals and to a less extent in plants. The endogenization was proposed to contribute toward evolution of living organisms via horizontal gene transfer of novel genetic material and resultant genetic diversity. During the last two decades, several full-length and fragmented EVEs of pararetroviral and non-retroviral nature have been identified in different plant genomes, both monocots and eudicots. Prior to this work, no EVEs have been reported in alfalfa (Medicago sativa L.), the most cultivated forage legume in the world. In this study, taking advantage of the most recent developments in the field of alfalfa research, we have assessed alfalfa genome on the presence of viral-related sequences. Our analysis revealed segmented EVEs resembling two dsDNA reverse-transcribing virus species: Soybean chlorotic mottle virus (family Caulimoviridae, genus Soymovirus) and Figwort mosaic virus (family Caulimoviridae, genus Caulimovirus). The EVEs appear to be stable constituents of the host genome and in that capacity could potentially acquire functional roles in alfalfa's development and response to environmental stresses.