RESUMO
Heavy metals (HMs) are among the most dangerous environmental variables for a variety of life forms, including crops. Accumulation of HMs in consumables and their subsequent transmission to the food web are serious concerns for scientific communities and policy makers. The function of essential plant cellular macromolecules is substantially hampered by HMs, which eventually have a detrimental effect on agricultural yield. Among these HMs, three were considered, i.e., arsenic, cadmium, and chromium, in this review, from agro-ecosystem perspective. Compared with conventional plant growth regulators, the use of nanoparticles (NPs) is a relatively recent, successful, and promising method among the many methods employed to address or alleviate the toxicity of HMs. The ability of NPs to reduce HM mobility in soil, reduce HM availability, enhance the ability of the apoplastic barrier to prevent HM translocation inside the plant, strengthen the plant's antioxidant system by significantly enhancing the activities of many enzymatic and nonenzymatic antioxidants, and increase the generation of specialized metabolites together support the effectiveness of NPs as stress relievers. In this review article, to assess the efficacy of various NP types in ameliorating HM toxicity in plants, we adopted a 'fusion approach', in which a machine learning-based analysis was used to systematically highlight current research trends based on which an extensive literature survey is planned. A holistic assessment of HMs and NMs was subsequently carried out to highlight the future course of action(s).
Assuntos
Metaloides , Metais Pesados , Nanotecnologia , Poluentes do Solo , Metais Pesados/toxicidade , Poluentes do Solo/análise , Poluentes do Solo/toxicidade , Agricultura/métodos , Ecossistema , Nanopartículas/química , Nanopartículas/toxicidade , Recuperação e Remediação Ambiental/métodos , Produtos AgrícolasRESUMO
Climate change has become increasingly intertwined with the occurrence and severity of droughts. As global temperatures rise due to greenhouse gas emissions, weather patterns are altered, leading to shifts in precipitation levels and distribution. These exacerbate the risk of drought in many regions, with potentially devastating consequences. A comprehensive transcriptome analysis was performed on Keteki Joha, an aromatic rice from North East India, with the aim of elucidating molecular responses to drought. Numerous genes linked to drought were activated, with both ABA-dependent and ABA-independent pathways playing crucial roles. Upregulated genes were enriched with gene ontology terms with response to abscisic acid and abscisic acid-activated signalling pathway, suggesting the existence of an ABA-dependent pathway for drought mitigation. The upregulated genes were also enriched with responses to stress, water, heat, jasmonic acid, and hydrogen peroxide, indicating the presence of an ABA-independent pathway alongside the ABA-dependent mechanism. Weighted Correlation Network Analysis (WGCNA) identified 267 genes that specifically govern drought mitigation in Keteki Joha. The late embryogenesis abundant (LEA) gene family emerges as the most overrepresented in both RNA sequencing data and WGCNA analysis, suggesting their dominant role in mitigating drought. Notably, 31 LEA genes were induced in seedlings and 32 in mature stages under drought stress. The LEA3-1, LEA14/WSI18, RAB16A, RAB16B, DHN1, DHN6, LEA1, LEA3, LEA17, and LEA33 exhibited and established co-expression with numerous other drought stress-related genes, indicating their inseparable role in alleviating drought. Consequently, LEA genes have been proposed to be primary and crucial responders to drought in Keteki Joha.
Assuntos
Ácido Abscísico , Secas , Regulação da Expressão Gênica de Plantas , Redes Reguladoras de Genes , Oryza , Oryza/genética , Oryza/fisiologia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Perfilação da Expressão Gênica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estresse Fisiológico/genética , Genes de Plantas , Transcriptoma/genéticaRESUMO
Pea (Pisum sativum L.), a legume, has a high nutritional content, but arsenic (As) in the agro-ecosystem poses a significant bottleneck to its yield, especially in South East Asia, by severely hampering ontogeny. The present study proposes a rhizospheric nano-remediation strategy to evade As-genotoxicity and improve crop yield using biogenic zinc-oxide nanoparticles (ZnONPs). Similar to any other source of environmental stress, As-toxicity caused rapid oxidative bursts with deterioration in morpho-physiological attributes (germination rate, shoot length, and root length decreased by 62 %, 16 %, and 14.9 % respectively in the negative control, over normal control). Reactive oxygen species (ROS) accumulation (12.8 and 9-fold increase in leaves and roots) overburdened antioxidative defense, and loss of cellular homeostasis resulted in membrane damage (82.75 % increase) and electrolyte-leakage (2.6-fold increase) in negative control. The study also reveals a significant increase in nuclear area, nuclear fragmentation, and micronuclei formation in root tip cells under As-stress, indicating severe genomic instability and increased programmed cell death (3.3-fold increase in early apoptotic cells) due to leaky plasma membrane and unrepaired DNA damage. Application of ZnONPs significantly reduced As-toxicity in peas due to its adsorption in the rhizosphere, causing diminished As-uptake and better antioxidant response. Improved phytochelatin synthesis enhanced vacuolar sequestration of arsenic, which reduced As-interference. Comparatively better flowering time (7.74-19.36 % reduction in flowering delay) with greater transcript abundance of GIGANTIA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) genes; better photosynthetic activity (1.3-1.9-fold increased chlorophyll autofluorescence); increased pollen viability; lesser genotoxicity (decreased tail DNA in comet assay) was noticed. A maximum increase of 37.5 % in pod number and seed zinc content (1.67-fold) was observed while seed arsenic content decreased under ZnONPs treatment. However, the highest dose of ZnONPs (400 mg L-1) induced NP-toxicity in pea plants under our experimental conditions, while optimum stress-alleviation was observed up to 300 mg L-1.
Assuntos
Arsênio , Nanopartículas , Óxido de Zinco , Pisum sativum , Óxido de Zinco/toxicidade , Arsênio/toxicidade , Ecossistema , Estresse Oxidativo , Antioxidantes/metabolismo , Nanopartículas/toxicidade , ZincoRESUMO
Nicotinamide adenine dinucleotide (NAD) is an essential cofactor of numerous enzymatic reactions found in all living cells. Pyridine nucleotides (NAD + and NADH) are also key players in signaling through reactive oxygen species (ROS), being crucial in the regulation of both ROS-producing and ROS-consuming systems in plants. NAD content is a powerful modulator of metabolic integration, protein de-acetylation, and DNA repair. The balance between NAD oxidized and reduced forms, i.e ., the NADH/NAD + ratio, indicates the redox state of a cell, and it is a measurement that reflects the metabolic health of cells. Here we present an easy method to estimate the NAD + and NADH content enzymatically, using alcohol dehydrogenase (ADH), an oxido-reductase enzyme, and with MTT (3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) as the substrate and 1-methoxy PMS (1-Methoxy-5-methylphenazinium methyl sulfate) as the electron carrier. MTT is reduced to a purple formazan, which is then detected. We used Arabidopsis leaf samples exposed to aluminum toxicity and under untreated control conditions. NADH/NAD + connects many aspects of metabolism and plays vital roles in plant developmental processes and stress responses. Therefore, it is fundamental to determine the status of NADH/NAD + under stress.
RESUMO
Iron (Fe) toxicity is one of the major abiotic stresses which limits the yield of lowland rice. This study aims to investigate the physiological, biochemical, and molecular aspects of two contrasting aromatic Joha rice, viz., Keteki and Kola Joha of Assam. Oxidative damage caused due to Fe2+ toxicity was quantitatively determined. Fe2+ toxicity in the growth medium increases the level of ROS and anti-oxidative enzyme activity. Along with the aforementioned damage caused due to Fe2+ toxicity, chlorophyll content decreases in both the rice varieties. Detection of Fe3+ and Fe2+ was also conducted by Perls' Prussian and Turnbull blue method, respectively. In addition, spectrophotometric quantification of Fe2+ was determined by 2, 2'-Bipyridyl (Bpy). Above 2.5 mM, Fe2+ toxicity was found to be lethal in rice seedlings affecting their total growth and biomass. Gene expression analysis of iron-regulated transporter 1 (OsIRT1), Yellow Stripe-Like 15 (OsYSL15), and ferritin 1 (OsFer1) revealed the differential gene expression over a time period of Fe2+ toxicity. Our study suggested that the different parameters which are considered here can be helpful for the better understanding of how aromatic Joha rice performed under Fe2+ toxicity which can also help to reveal broader aspects that how gene players are involved in the iron homeostasis mechanism in Joha rice in coming future.
Assuntos
Clorofila/química , Ferro/metabolismo , Oryza/química , Plântula/química , ÍndiaRESUMO
Expression pattern of aluminum (Al) tolerance genes is one of the major determinants of Al avoidance/tolerance within plant cultivars. We have performed transcriptome analysis of two contrasting (Al-tolerant, Disang; Al-sensitive, Joymati) cultivars of India's North Eastern region, an indica rice diversity hotspot, on exposure to excess Al3+ treatment in acidic condition. Co-expression analysis and SNPs enrichment analysis proposed the role of both trans-acting and cis-acting polymorphisms in Al signaling in the Al-tolerant cultivar. We proposed ten major genes, including arginine decarboxylase, phytase, and beta-glucosidase aggregating factor as candidates responsible for Al tolerance based on transcriptome analysis. Al3+ stress led to changes in the alternative splicing profile of the Al-tolerant cultivar. These studies demonstrated the transcriptional variations affiliated to Al avoidance/tolerance in contrasting indica rice of North East India and provided us with several candidate genes responsible for Al tolerance.
Assuntos
Alumínio/química , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/química , Proteínas de Plantas/química , Raízes de Plantas/química , Análise de Sequência de RNA/métodos , ÍndiaRESUMO
Aluminum (Al) toxicity is a serious problem for rice crop productivity in acidic soils worldwide. The present work was conducted to look out for the alteration in ROS homeostasis; metabolic fingerprint; and morphology in two contrasting Indica rice cultivars of North East India (NE India) to Al toxicity. Al stress led to excess accumulation of ROS (H2O2 and O2-), and this in turn induced ROS mediated cellular damage, as indicated by lipid peroxidation both qualitatively as well as quantitatively. This excessive ROS production also led to significant reduction in chlorophyll content and stomatal conductance. This was followed by the loss of photosynthetic efficiency as detected by chlorophyll fluorescence. This excessive damage due to ROS prompted us to check the anti-oxidative machinery. Antioxidants, especially enzymes (SOD, APX, POX, GR, CAT, DHAR, MDHAR) are very important players in maintenance of ROS homeostasis. In tolerant variety Disang, higher activity of these enzymes and vice versa in sensitive variety, was observed in response to Al treatment. The non-enzymatic antioxidants (proline, ascorbate and glutathione) also showed similar trend. Though the tolerant variety showed strong anti-oxidative machinery, it was unable to completely nullify the stress experienced by the seedlings. Organic acids are also important players in detoxification of Al stress through efflux in the rhizosphere. In tolerant genotype, citrate exudate was found to be more when compared to sensitive genotypes on exposure to high dose of Al. This is supported by higher abundance of FRDL4, a citrate transporter. Not only FRDL4, other stakeholders for Al stress response like ART1 and ALS1 depicted prominent transcript abundance in the tolerant variety. In conclusion, through this study detailed physiological and metabolic characterisation of two contrasting Indica rice varieties Disang and Joymati, native to NE India for Al tolerance was performed for the very first time.
Assuntos
Adaptação Fisiológica/efeitos dos fármacos , Alumínio/toxicidade , Oryza/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Adaptação Fisiológica/genética , Antioxidantes/metabolismo , Clorofila/metabolismo , Metabolismo Energético/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genótipo , Índia , Peroxidação de Lipídeos/efeitos dos fármacos , Oryza/classificação , Oryza/genética , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fotossíntese/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/metabolismo , Especificidade da EspécieRESUMO
Plant NAC (NAM, ATAF, and CUC) transcription factors (TF) have important roles to play in abiotic stress responses through activation of a battery of functional genes/transcriptional regulators responsible for stress tolerance. Here we report the cloning of a novel Solanum lycopersicum L., NAC2 TF having 960 nucleotides long CDS (GenBank: KT740994.1). Phylogenetic analysis depicted the similarity of SlNAC2 to other orthologs. SlNAC2 was overexpressed in Arabidopsis thaliana to assess and characterize its role in plant abiotic stress responses. The transgenic events were first confirmed by genomic DNA PCR and qRT PCR; then the T3 generation plants were used for stress assays. Soil stress assay depicted better survivability of the transgenic plants under both salt (NaCl) and drought (PEG) stress. The transgenic plants showed enhanced endurance; with better antioxidative response, reduced accumulation of reactive oxygen species (ROS) molecules and better retention of water in tissue. This study for the very first time analyzed the different stakeholders of the glutathione metabolism in SlNAC2 overexpressing transgenic lines on exposure to both salinity and PEG stress. The expression of the two genes (ɤ-ECS, GS) responsible for glutathione biosynthesis increased with SlNAC2 overexpression. Further glutathione reductase responsible for reduction of glutathione disulfide (GSSG) to glutathione (GSH) also increased significantly which suggested the regulation of glutathione metabolism as a mechanism for the osmotic stress tolerance conferred to plants upon NAC overexpression.
Assuntos
Arabidopsis/genética , Glutationa/metabolismo , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Estresse Fisiológico/genética , Antioxidantes/metabolismo , Arabidopsis/fisiologia , Clorofila/metabolismo , Clonagem Molecular , Secas , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Glutationa/genética , Malondialdeído/metabolismo , Estresse Oxidativo , Filogenia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Salino/genética , Estresse Fisiológico/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Lipid peroxidation is a physiological indicator of both biotic and abiotic stress responses, hence is often used as a biomarker to assess stress-induced cell damage or death. Here we demonstrate an easy, quick and cheap staining method to assess lipid peroxidation in plant tissues. In this methodology, Schiff's reagent, is used to assay for membrane degradation. Histochemical detection of lipid peroxidation is performed in this protocol. In brief, Schiff's reagent detects aldehydes that originate from lipid peroxides in stressful condition. Schiff's reagent is prepared and applied to plants tissue. After the reaction, plant tissue samples are rinsed with a sulfite solution to retain the staining color. From this analysis, qualitative visualization of lipid peroxidation in plant tissue is observed in the form of magenta coloration. This reagent is useful for visualization of stress induced lipid peroxidation in plants. In this protocol, Indica rice root, Assam tea root and Indian mustard seedlings are used for demonstration.
RESUMO
Salinity is the second most prevalent abiotic stress faced by plants, and rice is not an exception. Through this study, it has been tried upon, to study the relative salinity tolerance of eight local varieties of North East India. Preliminary screening was based on their dose- and time-dependent physiological responses to salinity stress. Among the cultivars, Tampha was found to be relatively more tolerant, whereas MSE9 the most sensitive. To further ascertain their tolerance capacity, MDA and H2O2 content was determined, which also confirmed the tolerance level of the two cultivars. Histochemical assays for root plasma membrane integrity and leaf and root H2O2 and O2- content also showed more damage in Tampha in comparison to MSE9. Finally, gene expression analysis for Na+/K+ co-transporters, OsHKT2;1, OsHKT2;3 and OsHKT2;4, was performed to observe how the expression level of these transporters varies with the tolerance capacity of these two cultivars in leaves and roots under different time frames. The study reveals Tampha to be the most tolerant and MSE9 the most sensitive when compared to the other six screened cultivars for salinity stress.
Assuntos
Oryza/química , Estresse Fisiológico/fisiologia , Índia , SalinidadeRESUMO
The accessibility of particle-coupled antibodies is important for many analytical applications, but comprehensive data on parameters controlling the accessibility are scarce. Here we report on the site-specific accessibility of monoclonal antibodies, immobilized on magnetic nanoparticles (500 nm) by the widely used covalent EDC coupling method, with the variation of four key coupling parameters (surface activation and immobilization pH, crosslinker and antibody concentration ratios). By developing quantitative radio-labelled assays, the number of immobilized antibodies, the Fab domain accessibility (in a sandwich immunoassay), and the Fc domain accessibility (in a Protein G assay) were determined. For sub-monolayer surface coverage, the observed numbers of accessible Fab and Fc domains are equal and scale linearly with the antibody density. For above monolayer coverage, the fractions of accessible Fab and Fc domains decrease, in an unequal manner. The results show that the antibody accessibility is primarily determined by the antibody surface density, rather than by chemical reactivity or the charge state, and that crowded conditions affect Fab and Fc accessibility in an unequal manner.
Assuntos
Anticorpos Imobilizados/química , Anticorpos Monoclonais/química , Nanopartículas/química , Imunoensaio , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/química , MagnetismoRESUMO
Aluminum (Al) is the third most abundant metal in earth crust, whose chemical form is mainly dependent on soil pH. The most toxic form of Al with respect to plants is Al3+, which exists in soil pH <5. Acidic soil significantly limits crop production mainly due to Al3+ toxicity worldwide, impacting approximately 50% of the world's arable land (in North-Eastern India 80% soil are acidic). Al3+ toxicity in plants ensues root growth inhibition leading to less nutrient and water uptake impacting crop productivity as a whole. Rice is one of the chief grains which constitutes the staple food of two-third of the world population including India and is not untouched by Al3+ toxicity. Al contamination is a critical constraint to plant production in agricultural soils of North East India. 24 indigenous Indica rice varieties (including Badshahbhog as tolerant check and Mashuri as sensitive check) were screened for Al stress tolerance in hydroponic plant growth system. Results show marked difference in growth parameters (relative growth rate, Root tolerance index, fresh and dry weight of root) of rice seedlings due to Al (100 µM) toxicity. Al3+ uptake and lipid peroxidation level also increased concomitantly under Al treatment. Histochemical assay were also performed to elucidate uptake of aluminum, loss of membrane integrity and lipid peroxidation, which were found to be more in sensitive genotypes at higher Al concentration. This study revealed that aluminum toxicity is a serious harmful problem for rice crop productivity in acid soil. Based on various parameters studied it's concluded that Disang is a comparatively tolerant variety whereas Joymati a sensitive variety. Western blot hybridization further strengthened the claim, as it demonstrated more accumulation of Glutathione reductase (GR) protein in Disang rice variety than Joymati under stressed condition. This study also observed that the emergence of lethal toxic symptoms occurs only after 48h irrespective of the dose used in the study.
Assuntos
Alumínio/toxicidade , Oryza/efeitos dos fármacos , Oryza/fisiologia , Alumínio/metabolismo , Transporte Biológico , Biomassa , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genótipo , Hemina/metabolismo , Peróxido de Hidrogênio/metabolismo , Índia , Peroxidação de Lipídeos/efeitos dos fármacos , Oryza/genética , Oryza/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Superóxidos/metabolismo , Água/metabolismoRESUMO
Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.
Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência , DNA/análise , Proteínas Luminescentes/química , FluorescênciaRESUMO
Alternative oxidase (AOX) is one of the terminal oxidases of the plant mitochondrial electron transport chain. AOX acts as a means to relax the highly coupled and tensed electron transport process in mitochondria thus providing and maintaining the much needed metabolic homeostasis by directly reducing oxygen to water. In the process AOX also act as facilitator for signaling molecules conveying the metabolic status of mitochondria to the nucleus and thus able to influence nuclear gene expression. Since AOX indirectly, is able to control the synthesis of important signaling molecules like hydrogen peroxide, superoxide, nitric oxide, thus it is also helping in stress signaling. AOX mediated signaling and metabolic activities are very much important for plant stress response. This include both biotic (fungal, bacterial, viral, etc.) and abiotic (drought, salinity, cold, heavy metal, etc.) stresses. The review provides a gist of regulation and functioning of AOX.
Assuntos
Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Secas , Metais Pesados/toxicidade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Plantas/genética , Plantas/efeitos dos fármacos , Plantas/metabolismo , Plantas/microbiologia , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Cloreto de Sódio/farmacologiaRESUMO
The biological activity of the immobilized enzyme is crucial for the performance of different nanoparticle mediated enzymatic assays, where enzymatic conversion can be used for label-free analyte detection. In this article we have addressed two significant aspects of enzyme-nanoparticle interactions. First, we have developed copper sulfide (CuS) nanoparticles with an average diameter of 25 nm as a potential enzyme-interface using trypsin protease as a model enzyme. CuS nanoparticles showed high trypsin immobilization capacity of about 14.0 mg m(-2) with the significant retention of native enzymatic activity (75-98%) at room temperature, even beyond the calculated tightly packed monolayer coverage (which is around 4.1 mg m(-2)). Second, we report a quantitative correlation between the structure-functional relationship and the density of immobilized trypsin on a nanoparticle surface. The in situ conformation of immobilized trypsin could be efficiently analyzed by fluorescence, circular dichroism and FT-IR spectroscopic measurements because of the small size of the nanoparticles. Trypsin molecules appear to retain their close-native tertiary and secondary structural features (with a small loss of 1-2% of helical content) in the entire surface density range (2.0-14.0 mg m(-2)) on the CuS nanoparticles. However, interestingly, at a low surface coverage (2.0 mg m(-2)), immobilized trypsin retains almost 98% of its native enzymatic activity, leading to a highly functional bio-nanocomposite. However, at higher surface coverages, the enzyme activity decreases to 77%, indicating the influence of steric crowding. Furthermore, the high functionality of the immobilized trypsin at low surface density on CuS nanoparticle was also confirmed by determining the kinetic parameters of enzymatic activity.
Assuntos
Enzimas Imobilizadas/metabolismo , Nanopartículas Metálicas/química , Nanocompostos/química , Tripsina/metabolismo , Dicroísmo Circular , Cobre/química , Ensaios Enzimáticos , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacocinética , Conformação Molecular , Ligação Proteica , Conformação Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , Sulfetos/química , Tripsina/química , Tripsina/farmacocinéticaRESUMO
Agrobacterium tumefaciens-mediated transformation is an efficient method for incorporating genes and recovering stable transgenic plants in cowpea because this method offers several advantages such as the defined integration of transgenes, potentially low copy number, and preferential integration into transcriptional active regions of the chromosome. Cotyledonary node explants of cowpea present an attractive target for T-DNA delivery followed by regeneration of shoots via axillary proliferation without involvement of a de novo regeneration pathway. In this chapter, we describe a detailed protocol for Agrobacterium-mediated transformation of the cowpea variety Pusa Komal. The seedling cotyledonary node explants are used for cocultivation with an Agrobacterium strain EHA105 harboring standard binary vector, pCAMBIA2301 or pNOV2819, and putative transformed plants are selected using aminoglycoside antibiotic or mannose as sole carbon source, respectively. The entire process includes explant infection to transgenic seed generation in greenhouse.
Assuntos
Fabaceae/genética , Técnicas Genéticas , Plantas Geneticamente Modificadas , Agricultura/métodos , Agrobacterium tumefaciens/genética , Cotilédone/genética , DNA Bacteriano , Fabaceae/crescimento & desenvolvimento , Vetores Genéticos , Germinação , Raízes de Plantas/genética , Sementes/genética , Seleção Genética , Esterilização/métodos , Transformação BacterianaRESUMO
The antigen-capturing activity of antibody-coated nanoparticles is very important for affinity-based bioanalytical tools. In this paper, a comprehensive study is reported of the antigen-capturing activity of antibodies that are nondirectionally immobilized on a nanoparticle surface. Superparamagnetic nanoparticles (500 nm) were covalently functionalized with different quantities of monoclonal antibodies against cardiac troponin I (cTnI). At a low antibody surface coverage, up to 4% of the immobilized antibodies could capture antigen molecules from solution. At high antibody coverage (≥50 × 10(2) antibodies per nanoparticle, i.e., ≥ 64 × 10(2) antibodies per µm(2)), the fraction of antigen-capturing antibodies drops well below 4% and the number of active antibodies saturates at about 120 per nanoparticle. The fraction of active antibodies is small, yet surprisingly their dissociation constants (Kd) are low, between 10 and 200 pM. In addition, the surface-binding activity of the antibody-coated nanoparticles was analyzed in an optomagnetic sandwich immunoassay biosensor, measuring cTnI in undiluted blood plasma. The data show that the immunoassay response scales with the number of active antibodies, increasing initially and saturating at higher antibody densities. The observations are summarized in a molecular sketch of the attachment, ordering, and functionality of antibodies on the nanoparticle surface.
Assuntos
Anticorpos Imobilizados/química , Anticorpos Monoclonais/química , Nanopartículas/química , Troponina I/sangue , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Técnicas Biossensoriais/métodos , Humanos , Imunoensaio/métodos , Cinética , Troponina I/imunologiaRESUMO
The work we have undertaken is to investigate the adsorption of two different proteins (BSA and BLG) having near same IEP and differing in their conformational flexibility, onto the surface of ZnS nanoparticles (ZnS NPs). BSA and BLG both have an IEP value around pH~5. BSA is more prone to conformational deformation and considered "soft" while BLG holds the conformational rigidity and considered as "hard" protein. To ascertain the differences in surface coverage and conformation of the protein onto ZnS surface (PZC ~ 3.7), we have evaluated the adsorption profile at pH 7, where the entire surface behaves negatively. An integrated approach was taken by incorporating zeta (ζ) potential, fluorescence and CD for analyzing the adsorption process. In both systems, an increase in protein surface coverage was observed with the increase in free protein concentration in the solution and ζ values approaching that of native protein at high surface coverage. An alteration in the tertiary structure was observed for both BSA and BLG. The CD spectra analysis reveals that the secondary structure of the BSA was more deviated from the native protein structure, accommodating the increased adsorption value. For BLG no such prominent structural alteration was observed. These findings help us to understand better, how adjustment of the protein adsorption amount can be achieved onto the surface of nanoparticles having like charges.
Assuntos
Lactoglobulinas/química , Soroalbumina Bovina/química , Adsorção , Animais , Bovinos , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Nanopartículas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Eletricidade Estática , Sulfetos/química , Propriedades de Superfície , Compostos de Zinco/químicaRESUMO
A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T0 transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.
Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Fabaceae/crescimento & desenvolvimento , Fabaceae/genética , Técnicas de Transferência de Genes , Genes Bacterianos/genética , Manose-6-Fosfato Isomerase/genética , Bioensaio , Segregação de Cromossomos/efeitos dos fármacos , Segregação de Cromossomos/genética , Cotilédone/efeitos dos fármacos , Cotilédone/crescimento & desenvolvimento , Cruzamentos Genéticos , Fabaceae/efeitos dos fármacos , Marcadores Genéticos , Manose/farmacologia , Fenolsulfonaftaleína/análogos & derivados , Fenolsulfonaftaleína/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas , Plasmídeos/genética , Regeneração/efeitos dos fármacos , Sacarose/farmacologia , Transformação Genética/efeitos dos fármacosRESUMO
Malachite nanoparticles of 100-150 nm have been efficiently and for the first time used as an adsorbent for the removal of toxic arsenate and chromate. We report a high adsorption capacity for chromate and arsenate on malachite nanoparticle from both individual and mixed solution in pH â¼4-5. However, the adsorption efficiency decreases with the increase of solution pH. Batch studies revealed that initial pH, temperature, malachite nanoparticles dose and initial concentration of chromate and arsenate were important parameters for the adsorption process. Thermodynamic analysis showed that adsorption of chromate and arsenate on malachite nanoparticles is endothermic and spontaneous. The adsorption of these anions has also been investigated quantitatively with the help of adsorption kinetics, isotherm, and selectivity coefficient (K) analysis. The adsorption data for both chromate and arsenate were fitted well in Langmuir isotherm and preferentially followed the second order kinetics. The binding affinity of chromate is found to be slightly higher than arsenate in a competitive adsorption process which leads to the comparatively higher adsorption of chromate on malachite nanoparticles surface.