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2.
J Vis Exp ; (168)2021 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-33720142

RESUMO

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22) or phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.


Assuntos
Agrobacterium/genética , Potexvirus/fisiologia , Potyvirus/fisiologia , Recombinação Genética/genética , Plântula/virologia , Zea mays/virologia , Células Clonais , DNA Bacteriano/genética , Fluorescência , Inativação Gênica , Vetores Genéticos/metabolismo , Genótipo , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/virologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas , Plasmídeos/genética , Plântula/genética , Zea mays/genética
4.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33443212

RESUMO

Agrobacterium spp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. Although Agrobacterium spp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis of Agrobacterium has been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of both Agrobacterium tumefaciens and Agrobacterium rhizogenes As an example, we generated EHA105 strains with loss-of-function mutations in recA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development by A. rhizogenes K599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of "engineering the engineer," leading to improved Agrobacterium strains for more efficient plant transformation and gene editing.


Assuntos
Agrobacterium/genética , Proteínas Associadas a CRISPR/genética , Edição de Genes/métodos , Agrobacterium tumefaciens/genética , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Sistemas CRISPR-Cas/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , DNA de Plantas/genética , Genes de Plantas/genética , Genoma de Planta/genética , Mutagênese/genética , Mutação/genética , Zea mays/genética
5.
Methods Mol Biol ; 2238: 3-17, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471321

RESUMO

Plant biotechnology provides a means for the rapid genetic improvement of crops including the enhancement of complex traits like yield and nutritional quality through the introduction and coordinated expression of multiple genes. GAANTRY (gene assembly in Agrobacterium by nucleic acid transfer using recombinase technology) is a flexible and effective system for stably stacking multiple genes within an Agrobacterium virulence plasmid transfer DNA (T-DNA) region. The system provides a simple and efficient method for assembling and stably maintaining large stacked constructs within the GAANTRY ArPORT1 Agrobacterium rhizogenes strain. The assembly process utilizes unidirectional site-specific recombinases in vivo and an alternating bacterial selection scheme to sequentially assemble multiple genes into a single transformation construct. A detailed description of the procedures used for bacterial transformation, selection, counter selection, and genomic PCR validation with the GAANTRY system are presented. The methods described facilitate the efficient assembly and validation of large GAANTRY T-DNA constructs. This powerful, yet simple to use, technology will be a convenient tool for transgene stacking and plant genetic engineering of rice and other crop plants.


Assuntos
Agrobacterium/genética , Produtos Agrícolas/genética , DNA Nucleotidiltransferases/metabolismo , Técnicas de Transferência de Genes , Engenharia Genética/métodos , Ácidos Nucleicos/genética , Plantas Geneticamente Modificadas/genética , Agrobacterium/patogenicidade , Produtos Agrícolas/microbiologia , DNA Nucleotidiltransferases/genética , Vetores Genéticos/administração & dosagem , Plantas Geneticamente Modificadas/microbiologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Recombinação Genética , Transgenes/fisiologia
6.
Methods Mol Biol ; 2238: 19-35, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471322

RESUMO

Efficient stacking of multiple genes is a critical element in metabolic engineering of complex pathways, synthetic biology, and genetic improvement of complex agronomic traits in plants. Here we present a high-efficiency multigene assembly and transformation vector system, TransGene Stacking II (TGS II), for these purposes. The operation process is described in detail, and the successful operation mainly depends on effective reagents, special Escherichia coli strains, and basic molecular biological means without other specific equipments.


Assuntos
Agrobacterium/genética , Produtos Agrícolas/metabolismo , Engenharia Metabólica/métodos , Plantas Geneticamente Modificadas/metabolismo , Biologia Sintética/métodos , Transformação Genética , Transgenes/fisiologia , Agrobacterium/patogenicidade , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Vetores Genéticos/administração & dosagem , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Recombinases/genética , Recombinases/metabolismo , Recombinação Genética
7.
Methods Mol Biol ; 2238: 241-257, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33471336

RESUMO

Gene targeting (GT) is a technique that alter the structure of the specific genes at their original loci in the genome by homologous recombination (HR). It plays an important role in functional genomics because it enables precise modification of the endogenous genes into desired forms such as knockout, knock-in, introduction of point mutations, as well as generation of fusion genes. Also, site-directed mutagenesis by GT can also be applied as an excellent technique for molecular breeding and gene therapy, because it can directly reflect the knowledge acquired from functional genomics. In this section, we introduce well-established GT procedure in rice in combination with positive-negative-selection (PNS) strategy.


Assuntos
Agrobacterium/genética , Marcação de Genes/métodos , Recombinação Homóloga , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Seleção Genética , Vetores Genéticos/genética , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Transformação Genética
8.
Nucleic Acids Res ; 49(1): 529-546, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33313837

RESUMO

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.


Assuntos
Agrobacterium/metabolismo , Proteínas de Bactérias/fisiologia , Ácidos Cumáricos/metabolismo , Família Multigênica , Proteínas Repressoras/fisiologia , Agrobacterium/genética , Regulação Alostérica , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Genes Sintéticos , Modelos Moleculares , Regiões Promotoras Genéticas/genética , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Citrato de Sódio , Tetra-Hidrofolatos/fisiologia , Zinco/fisiologia
9.
Methods Mol Biol ; 2264: 219-244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33263914

RESUMO

For centuries, combining useful traits into a single tomato plant has been done by selective crossbreeding that resulted in hundreds of extant modern cultivars. However, crossbreeding is a labor-intensive process that requires between 5 and 7 years to develop a new variety. More recently, genome editing with the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has been established as an efficient method to accelerate the breeding process by introducing targeted modifications to plant genomes via generation of targeted double-strand breaks (DSBs). CRISPR/Cas9 has been used to generate a variety of specific changes ranging from gene knockouts to gene replacements, and can also be easily multiplexed to modify several targets simultaneously. Given that (1) generating knockout mutations only requires a DSB that is frequently repaired by the error-prone nonhomologous end joining (NHEJ) pathway resulting in gene function inactivation, and (2) the genetic basis of many useful agronomic traits consists of loss of gene function, multiple traits can be created in a plant in one generation by simultaneously introducing DSBs into multiple genes of interest. On the other hand, more precise modifications, such as allele replacement, can be achieved by gene targeting-a less efficient process in which an external template is used to repair the DSB by homologous recombination (HR). These technical breakthroughs allow the design and customization of plant traits to achieve the ideal plant type ("ideotype"). Here, we describe protocols to assemble CRISPR/Cas9 constructs for both single and multiplex gene knockouts as well as gene targeting and to generate and identify genome-edited tomato plants via Agrobacterium-mediated transformation in tissue culture.


Assuntos
Agrobacterium/genética , Sistemas CRISPR-Cas , Edição de Genes , Marcação de Genes , Genoma de Planta , Lycopersicon esculentum/genética , Transformação Genética , Vetores Genéticos , Lycopersicon esculentum/crescimento & desenvolvimento
10.
J Genet ; 992020.
Artigo em Inglês | MEDLINE | ID: mdl-32529984

RESUMO

CmFT homologous gene in muskmelon was obtained by homologous cloning, introducing CmFT gene by Agrobacterium mediated transformation. The results of subcellular localization showed that CmFT protein was expressed in cytoplasm and nucleus. qRTPCR results showed that the expression levels of AtLFY, AtFT, AtCO, AtFLC, AtSOC1 and AtAP1 were upregulated in the 35S::MeFT Arabidopsis line. The CmFT gene was introduced into wild-type Arabidopsis by Agrobacterium-mediated transformation, and the growth status of T2 transgenic Arabidopsis thaliana and wild-type A. thaliana was observed. The results showed that wild-type Arabidopsis began to bolt on the 25th day after sowing, we can initially confirm that the FT gene of melon can promote the early flowering of melon in the growth and development of melon.


Assuntos
Agrobacterium/genética , Arabidopsis/genética , Clonagem Molecular , Cucumis melo/genética , Genes de Plantas , Fenótipo , Plantas Geneticamente Modificadas , Transformação Genética
11.
J Biotechnol ; 318: 10-19, 2020 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-32387397

RESUMO

This study was to obtain stable transformed roots of Salvia bulleyana using A. rhizogenes strain A4 and then evaluate their phytochemical profile and selected the most productive clone. Our results indicated that the type of explant and medium used for bacterium and explant incubation had an influence on the frequency of hairy root formation. The best response was obtained on leaves infected with bacteria cultivated on YMB medium supplemented with acetosyringone. Of the four selected transformed root clones, after five-week cultivation in Woody Plant (WP) medium, the highest growth indexes were demonstrated for line C1: i.e. 13 for fresh and 15 for dry weight (81.4 and 8.2 g/l fresh and dry weight, respectively). The qualitative analysis of hydromethanolic extracts of hairy roots of S. bulleyana using UPLC-PDA-ESI-MS/MS method showed the presence of 10 polyphenolic compounds including predominant rosmarinic acid (RA), its derivatives (hexoside and methyl rosmarinate), caffeic acid, its derivatives and several salvianolic acids: K, E and F. Their production varied among the four root clones studied; the highest RA (39.6 mg/g dry weight) and total polyphenol (48.9 mg/g dry weight) level were found in the roots of C4 clone. These values were significantly higher than those of the roots of plants grown for several years under field conditions. The transformation of the obtained root cultures was confirmed by polymerase chain reaction using aux1, aux2, rolB, rolC and rolD primers.


Assuntos
Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Polifenóis/biossíntese , Salvia , Agrobacterium/genética , Técnicas de Cultura de Células , Extratos Vegetais/química , Raízes de Plantas/química , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Polifenóis/química , Transformação Genética
12.
Sheng Wu Gong Cheng Xue Bao ; 36(4): 700-706, 2020 Apr 25.
Artigo em Chinês | MEDLINE | ID: mdl-32347064

RESUMO

The responsibility of root is absorbing water and nutrients, it is an important plant tissue, but easily to be affected by biotic and abiotic stresses, affecting crop growth and yield. The design of a synthetic root-specific promoter provides candidate promoters for the functional analysis and efficient expression of stress-related genes in crop roots. In this study, a synthetic root-specific module (pro-SRS) was designed using tandem four-copies of root specific cis-acting elements (OSE1ROOTNODULE, OSE2ROOTNODULE, SP8BFIBSP8AIB, and ROOTMOTIFAPOX1), and fused with minimal promoter from the CaMV 35S promoter to synthesize an artificially synthetic SRSP promoter. The SRSP promoter was cloned in pCAMBIA2300.1 by replacing CaMV 35S promoter so as to drive GUS expression. The constructs with SRSP promoter were transformed in tobacco by Agrobacterium-mediated method. SRSP promoter conferred root-specific expression in transgenic tobacco plants through Real-time PCR (RT-PCR) analysis and GUS histochemical staining analysis. It is indicated that the repeated arrangement of cis-acting elements can realize the expected function of the promoter. This study laid a theoretical foundation for the rational design of tissue-specific promoters.


Assuntos
Regulação da Expressão Gênica de Plantas , Raízes de Plantas , Regiões Promotoras Genéticas , Tabaco , Agrobacterium/genética , Clonagem Molecular , Raízes de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Estresse Fisiológico , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Transformação Genética
13.
Sci Rep ; 10(1): 2836, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071340

RESUMO

Peach is an economically import fruit crop worldwide, and serves as a model species of the Rosaceae family as well. However, peach functional genomics studies are severely hampered due to its recalcitrance to regeneration and stable transformation. Here, we report a fast and efficient Agrobacterium rhizogenes-mediated transformation system in peach. Various explants, including leaf, hypocotyl and shoot, were all able to induce transgenic hairy roots, with a transformation efficiency of over 50% for hypocotyl. Composite plants were generated by infecting shoots with A. rhizogenes to induce transgenic adventitious hairy roots. The composite plant system was successfully used to validate function of an anthocyanin-related regulatory gene PpMYB10.1 in transgenic hairy roots, and two downstream genes, PpUFGT and PpGST, were strongly activated. Our stable and reproductive A. rhizogenes-mediated transformation system provides an avenue for gene function assay, genetic engineering, and investigation of root-rhizosphere microorganism interaction in peach.


Assuntos
Genoma de Planta/genética , Genômica , Plantas Geneticamente Modificadas/genética , Prunus persica/genética , Agrobacterium/genética , Antocianinas/genética , Antocianinas/metabolismo , Engenharia Genética/tendências , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Prunus persica/crescimento & desenvolvimento
14.
Appl Microbiol Biotechnol ; 104(6): 2435-2451, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32002599

RESUMO

Compact plant growth is an economically important trait for many crops. In practice, compactness is frequently obtained by applying chemical plant growth regulators. In view of sustainable and environmental-friendly plant production, the search for viable alternatives is a priority for breeders. Co-cultivation and natural transformation using rhizogenic agrobacteria result in morphological alterations which together compose the Ri phenotype. This phenotype is known to exhibit a more compact plant habit, besides other features. In this review, we highlight the use of rhizogenic agrobacteria and the Ri phenotype with regard to sustainable plant production and plant breeding. An overview of described Ri lines and current breeding applications is presented. The potential of Ri lines as pre-breeding material is discussed from both a practical and legal point of view.


Assuntos
Agrobacterium/genética , Melhoramento Vegetal/legislação & jurisprudência , Melhoramento Vegetal/métodos , Plantas/genética , Produtos Agrícolas/genética , Produtos Agrícolas/microbiologia , Fenótipo , Desenvolvimento Vegetal , Raízes de Plantas/microbiologia , Plantas/microbiologia , Rhizobium
16.
J Virol ; 94(8)2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-31996436

RESUMO

Viral capsids are dynamic assemblies that undergo controlled conformational transitions to perform various biological functions. The replication-derived four-molecule RNA progeny of Brome mosaic virus (BMV) is packaged by a single capsid protein (CP) into three types of morphologically indistinguishable icosahedral virions with T=3 quasisymmetry. Type 1 (B1V) and type 2 (B2V) virions package genomic RNA1 and RNA2, respectively, while type 3 (B3+4V) virions copackage genomic RNA3 (B3) and its subgenomic RNA4 (sgB4). In this study, the application of a robust Agrobacterium-mediated transient expression system allowed us to assemble each virion type separately in planta Experimental approaches analyzing the morphology, size, and electrophoretic mobility failed to distinguish between the virion types. Thermal denaturation analysis and protease-based peptide mass mapping experiments were used to analyze stability and the conformational dynamics of the individual virions, respectively. The crystallographic structure of the BMV capsid shows four trypsin cleavage sites (K65, R103, K111, and K165 on the CP subunits) exposed on the exterior of the capsid. Irrespective of the digestion time, while retaining their capsid structural integrity, B1V and B2V released a single peptide encompassing amino acids 2 to 8 of the N-proximal arginine-rich RNA binding motif. In contrast, B3+4V capsids were unstable with trypsin, releasing several peptides in addition to the peptides encompassing four predicted sites exposed on the capsid exterior. These results, demonstrating qualitatively different dynamics for the three types of BMV virions, suggest that the different RNA genes they contain may have different translational timing and efficiency and may even impart different structures to their capsids.IMPORTANCE The majority of viruses contain RNA genomes protected by a shell of capsid proteins. Although crystallographic studies show that viral capsids are static structures, accumulating evidence suggests that, in solution, virions are highly dynamic assemblies. The three genomic RNAs (RNA1, -2, and -3) and a single subgenomic RNA (RNA4) of Brome mosaic virus (BMV), an RNA virus pathogenic to plants, are distributed among three physically homogeneous virions. This study examines the thermal stability by differential scanning fluorimetry (DSF) and capsid dynamics by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analyses following trypsin digestion of the three virions assembled separately in vivo using the Agrobacterium-mediated transient expression approach. The results provide compelling evidence that virions packaging genomic RNA1 and -2 are distinct from those copackaging RNA3 and -4 in their stability and dynamics, suggesting that RNA-dependent capsid dynamics play an important biological role in the viral life cycle.


Assuntos
Bromovirus/metabolismo , Proteínas do Capsídeo/metabolismo , Capsídeo/metabolismo , Vírion/metabolismo , Montagem de Vírus/fisiologia , Agrobacterium/genética , Bromovirus/genética , Proteínas do Capsídeo/genética , Genoma Viral , Mapeamento de Peptídeos , RNA Bacteriano , RNA Viral/genética , Vírion/genética , Montagem de Vírus/genética , Replicação Viral
17.
Curr Pharm Biotechnol ; 21(6): 516-527, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31775597

RESUMO

BACKGROUND: Tropane Alkaloids (TAs) are important drugs for curing many diseases in the medical industry. METHODS: To sustainably exploit TA resources in endangered traditional Tibetan herbs, the hairy root (HR) systems of Przewalskia tangutica Maxim. and Anisodus tanguticus Maxim. were compared under the same culture conditions. RESULTS: The results indicated that both the Agrobacterium rhizogenes strains and explants affected the HR induction frequency, MSU440, A4 and LBA9402 strains could induce hairy roots following infection of cotyledon and hypocotyl of A. tanguticus while LBA9402 could not induce HR on either explants of P. tangutica. The efficiency of LBA9402 was higher than A4 and MSU440 on A. tanguticus and A4 was better strain than MSU440 on P. tangutica. The hypocotyl explant was more suitable for P.tangutica and cotyledon explant was better for A.tangutica with a transformation frequency of 33.3% (P. tangutica) and 82.5% (A. tanguticus), respectively. In a flask reactor system, both the growth curves of HR for two species both appeared to be "S" curve; however, the HR of P. tangutica grew more rapidly than that of A. tanguticus, and the latter accumulated more biomass than the former. As the culture volume increased, the HR proliferation coefficient of both the species increased. HPLC analysis results showed that the content of TAs in the HR of P. tangutica was 257.24mg/100g·DW, which was more than that of A. tanguticus HR (251.08mg/100g·DW), and the anisodamine in the Pt- HR was significantly higher than that in At-HR. Moreover, tropane alkaloids in the HR of the two species were all significantly higher than that of the roots of aseptic seedlings. CONCLUSION: Our results suggest that HR of P. tangutica and A. tanguticus both could provide a useful platform for sustainable utilization of two Tibetan medicinal plants in the Qinghai-Tibetan Plateau in the future.


Assuntos
Raízes de Plantas/química , Plantas Medicinais/química , Solanaceae/química , Tropanos/análise , Agrobacterium/genética , Agrobacterium/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Genes Bacterianos , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/microbiologia , Plantas Medicinais/crescimento & desenvolvimento , Plantas Medicinais/microbiologia , Solanaceae/crescimento & desenvolvimento , Solanaceae/microbiologia , Alcaloides de Solanáceas/análise , Tibet
18.
Mol Biotechnol ; 62(2): 79-90, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31758488

RESUMO

Vaccines are biological preparations that improve immunity to particular diseases and form an important innovation of 19th century research. It contains a protein that resembles a disease-causing microorganism and is often made from weak or killed forms of the microbe. Vaccines are agents that stimulate the body's immune system to recognize the antigen. Now, a new form of vaccine was introduced which will have the power to mask the risk side of conventional vaccines. This type of vaccine was produced from plants which are genetically modified. In the production of edible vaccines, the gene-encoding bacterial or viral disease-causing agent can be incorporated in plants without losing its immunogenic property. The main mechanism of action of edible vaccines is to activate the systemic and mucosal immunity responses against a foreign disease-causing organism. Edible vaccines can be produced by incorporating transgene in to the selected plant cell. At present edible vaccine are developed for veterinary and human use. But the main challenge faced by edible vaccine is its acceptance by the population so that it is necessary to make aware the society about its use and benefits. When compared to other traditional vaccines, edible vaccines are cost effective, efficient and safe. It promises a better prevention option from diseases.


Assuntos
Produtos Biológicos/imunologia , Imunidade nas Mucosas/efeitos dos fármacos , Organismos Geneticamente Modificados/imunologia , Plantas Geneticamente Modificadas/imunologia , Vacinas de Plantas Comestíveis/imunologia , Administração Oral , Agrobacterium/genética , Agrobacterium/imunologia , Animais , Biolística/métodos , Clorófitas/genética , Clorófitas/imunologia , Técnicas de Transferência de Genes , Humanos , Insetos/genética , Insetos/imunologia , Lactobacillales/genética , Lactobacillales/imunologia , Agricultura Molecular , Vírus de Plantas/genética , Vírus de Plantas/imunologia , Leveduras/genética , Leveduras/imunologia
19.
J Plant Physiol ; 245: 153095, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31877472

RESUMO

Previous work showed in tomato plants harbouring the Agrobacterium rhizogenes rolB gene overexpression of genes involved in chloroplast function and stress response, significant increase in non-photochemical quenching and chlorophyll a and b content, and reduced chlorophyll a/b ratio. The latter condition being typical of plant shade where far-red is dominant, suggested a role for rolB in improving photosynthesis in such condition. To gain a better insight into these results, the photosynthetic performance of transgenic and control plants was compared by means of variable fluorescence kinetics with a WATER-PAM chlorophyll fluorometer, after 6 days-exposure to white light and to a far-red-enriched light source. Photosynthetic parameters analysed were quantum yield of photosystem II photochemistry Y(II); qL, corresponding to the fraction of open PSII reaction centers in a "lake" model of photosystem II; non-photochemical quenching and Y(NO), describing, respectively, regulated and non-regulated pathways for dissipation of excess energy. Chlorophyll a and b content was also analysed by HPLC. Finally, real-time PCR was performed to quantify the expression level of some of the chloroplast-related genes already shown to be overexpressed in transgenic plants. Quantum yield of photosystem II photochemistry decreased with increasing light intensity, showing no significant differences in both plant genotypes and light regimen. qL, on the other hand, was significantly higher at low PAR intensities, in particular in FR-treated transgenic plants. Fate of remaining light energy, channelled into regulated or non-regulated dissipation pathways, was different in transgenic and control plants, indicating a higher capability for protection from photodamage in rolB plants, particularly after exposure to far-red-enriched light. Chlorophyll a/b ratio was also decreased in transgenic plants under far-red-enriched light with respect to white light. Finally, qPCR showed that the expression of genes encoding small heat shock protein, chlorophyll a/b binding protein and carbonic anhydrase was significantly induced by far-red-enriched condition. Taken together, these data suggest the involvement of rolB in photosynthesis modulation under far-red-rich light in tomato.


Assuntos
Agrobacterium/genética , Proteínas de Bactérias/metabolismo , Clorofila A/metabolismo , Clorofila/metabolismo , Lycopersicon esculentum/metabolismo , Fotossíntese/efeitos da radiação , Plantas Geneticamente Modificadas/metabolismo , beta-Glucosidase/metabolismo , Proteínas de Bactérias/genética , Clorofila/análise , Clorofila A/análise , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Proteínas de Choque Térmico Pequenas/genética , Proteínas de Choque Térmico Pequenas/metabolismo , Luz , Lycopersicon esculentum/genética , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , beta-Glucosidase/genética
20.
J Appl Microbiol ; 128(3): 828-839, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31755153

RESUMO

AIMS: Crown gall, a phytobacteriosis characterized by the formation of tumours on plant roots was observed in recently planted vineyards of the Meknes region (Morocco). The objective of this research was to analyse the diversity of pathogenic agrobacteria isolated from grapevine in Morocco. METHODS AND RESULTS: Eighty-two isolates from 11 affected vineyards were characterized by recA sequencing and were found to belong to Agrobacterium tumefaciens genomospecies G1, G4 or G7, Rhizobium rhizogenes, and to Allorhizobium vitis. Only the All. vitis isolates appeared to be pathogenic on tomato and multilocus sequence analysis phylogenetic analyses revealed a weak genetic diversity, with the definition of only four genomic groups. Definition of the All. vitis genomic groups correlated with specific pathogenic traits: indeed, genomic groups differed with respect to the severity of hypersensitive response symptoms on tobacco leaves, the intensity of necrotic response on grapevine explants and opine profiles. Both vitopine and octopine were detected by UHPLC in tumours induced by isolates of three genomic groups, an opine signature scarcely ever reported. CONCLUSIONS: Allorhizobium vitis is the only causative agent of crown gall on grape in Morocco, pathogenic isolates can be separated into four genomic groups. SIGNIFICANCE AND IMPACT OF THE STUDY: This study of recently crown-gall-infested vineyards demonstrated that All. vitis is the only causative agent and revealed the presence of nonpathogenic Agrobacterium strain within tumours. Moreover, as the genetic diversity of the All. vitis isolates is relatively narrow, this study lays the basis for further analyses on the evolution of the disease, on the dissemination of the pTi and more globally on the fate of the different genomic groups in this newly colonized environment.


Assuntos
Agrobacterium/classificação , Agrobacterium/fisiologia , Filogenia , Vitis/microbiologia , Agrobacterium/genética , Agrobacterium/patogenicidade , Arginina/análogos & derivados , Arginina/metabolismo , Proteínas de Bactérias/genética , Variação Genética , Genoma Bacteriano/genética , Glutamina/análogos & derivados , Glutamina/metabolismo , Marrocos , Tumores de Planta/microbiologia
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