Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.497
Filtrar
1.
DNA Cell Biol ; 40(10): 1235-1250, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34558965

RESUMO

London plane tree is widely grown as a landscaping and street tree, but the release of its trichomes creates a serious air-borne pollution problem. Identifying the key genes that regulate the development of trichomes is, therefore, an important tool for the molecular breeding of Platanus acerifolia. In this study, a sequence homologous with the Arabidopsis Class II TCP subfamily was identified from London plane, and named PaTCP4. The expression of PaTCP4 was detected in various organs of London plane trees, significantly in the trichomes. Overexpression of PaTCP4 in Arabidopsis reduced the trichome density on the first pair of true leaves, and atypical 5-branched trichomes were also detected on those leaves. The expression of endogenous AtCPC and AtTCL2 was significantly increased in PaTCP4 transgenic lines, and was associated with a decrease in the expression of endogenous AtGL2. Furthermore, the expression of endogenous AtGL3 was significantly increased. In addition, the protein product of PaTCP4 was shown to directly activate AtCPC, AtTCL2, AtGL3, AtGIS, PaGIS, and PaGL3 in yeast one-hybrid assays and in the dual-luciferase reporter system. Taken together, these results identify a role for PaTCP4 in trichome initiation and branching in Arabidopsis. Thus, PaTCP4 represents a strong candidate gene for regulating the development of trichomes in London plane trees.


Assuntos
Melhoramento Vegetal , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Fatores de Transcrição/genética , Árvores/genética , Tricomas/genética , Arabidopsis/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Árvores/crescimento & desenvolvimento , Tricomas/crescimento & desenvolvimento
2.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575941

RESUMO

Drought is the main environmental factor that limits the yield and quality of apples (Malus × domestica) grown in arid and semi-arid regions. Nuclear factor Ys (NF-Ys) are important transcription factors involved in the regulation of plant growth, development, and various stress responses. However, the function of NF-Y genes is poorly understood in apples. Here, we identified 43 NF-Y genes in the genome of apples and conducted an initial functional characterization of the apple NF-Y. Expression analysis of NF-Y members in M. sieversii revealed that a large number of NF-Ys were highly expressed in the roots compared with the leaves, and a large proportion of NF-Y genes responded to drought treatment. Furthermore, heterologous expression of MsNF-YB21, which was significantly upregulated by drought, led to a longer root length and, thus, conferred improved osmotic and salt tolerance in Arabidopsis. Moreover, the physiological analysis of MsNF-YB21 overexpression revealed enhanced antioxidant systems, including antioxidant enzymes and compatible solutes. In addition, genes encoding catalase (AtCAT2, AtCAT3), superoxide dismutase (AtFSD1, AtFSD3, AtCSD1), and peroxidase (AtPER12, AtPER42, AtPER47, AtPER51) showed upregulated expression in the MsNF-YB21 overexpression lines. These results for the MsNF-Y gene family provide useful information for future studies on NF-Ys in apples, and the functional analysis of MsNF-YB21 supports it as a potential target in the improvement of apple drought tolerance via biotechnological strategies.


Assuntos
Arabidopsis/genética , Malus/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Arabidopsis/crescimento & desenvolvimento , Secas , Regulação da Expressão Gênica de Plantas/genética , Pressão Osmótica , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tolerância ao Sal/genética , Estresse Fisiológico/genética , Fatores de Transcrição/genética
3.
Int J Mol Sci ; 22(18)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34575962

RESUMO

The SHORT VEGETATIVE PHASE (SVP) gene is a transcription factor that integrates flowering signals and plays an important role in the regulation of flowering time in many plants. In this study, two full-length cDNA sequences of SVP homologous genes-MiSVP1 and MiSVP2-were obtained from 'SiJiMi' mango. Sequence analysis showed that the MiSVPs had typical MADS-box domains and were highly conserved between each other. The analysis of expression patterns showed that the MiSVPs were expressed during flower development and highly expressed in vegetative tissues, with low expression in flowers/buds. The MiSVPs could responded to low temperature, NaCl, and PEG treatment. Subcellular localization revealed that MiSVP1 and MiSVP2 were localized in the nucleus. Transformation of Arabidopsis revealed that overexpression of MiSVP1 delayed flowering time, overexpression of MiSVP2 accelerated flowering time, and neither MiSVP1 nor MiSVP2 had an effect on the number of rosette leaves. Overexpression of MiSVP1 increased the expression of AtFLC and decreased the expression of AtFT and AtSOC1, and overexpression of MiSVP2 increased the expression levels of AtSOC1 and AtFT and decreased the expression levels of AtFLC. Point-to-point and bimolecular fluorescence complementation (BiFC) assays showed that MiSVP1 and MiSVP2 could interact with SEP1-1, SOC1D, and AP1-2. These results suggest that MiSVP1 and MiSVP2 may play a significant roles in the flowering process of mango.


Assuntos
Proteínas de Arabidopsis/genética , Mangifera/genética , Estado Vegetativo Persistente/genética , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Mangifera/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
4.
Int J Mol Sci ; 22(18)2021 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-34576057

RESUMO

Stomatal regulation is crucial to reduce water consumption under drought conditions. Extracellular ATP (eATP) serves as a signaling agent in stomatal regulation; however, it is less known whether the eATP mediation of stomatal aperture is linked to apyrases (APYs), the principal enzymes that control the concentration of eATP. To clarify the role of APYs in stomatal control, PeAPY1 and PeAPY2 were isolated from Populus euphratica and transferred into Arabidopsis. Compared with the wild-type Arabidopsis and loss-of-function mutants (Atapy1 and Atapy2), PeAPY1- and PeAPY2-transgenic plants decreased stomatal aperture under mannitol treatment (200 mM, 2 h) and reduced water loss during air exposure (90 min). The role of apyrase in stomatal regulation resulted from its control in eATP-regulated stomatal movements and increased stomatal sensitivity to ABA. The bi-phasic dose-responses to applied nucleotides, i.e., the low ATP (0.3-1.0 mM)-promoted opening and high ATP (>2.0 mM)-promoted closure, were both restricted by P. euphratica apyrases. It is noteworthy that eATP at a low concentration (0.3 mM) counteracted ABA action in the regulation of stomatal aperture, while overexpression of PeAPY1 or PeAPY2 effectively diminished eATP promotion in opening, and consequently enhanced ABA action in closure. We postulate a speculative model of apyrase signaling in eATP- and ABA-regulated stomatal movements under drought.


Assuntos
Apirase/genética , Arabidopsis/genética , Plantas Geneticamente Modificadas/genética , Populus/enzimologia , Arabidopsis/crescimento & desenvolvimento , Secas , Regulação da Expressão Gênica de Plantas/genética , Estômatos de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Populus/genética , Estresse Fisiológico/genética
5.
Nutrients ; 13(8)2021 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-34445042

RESUMO

The consumption of plant sprouts as part of human day-to-day diets is gradually increasing, and their health benefit is attracting interest across multiple disciplines. The purpose of this review was to (a) critically evaluate the phytochemicals in selected sprouts (alfalfa, buckwheat, broccoli, and red cabbage), (b) describe the health benefits of sprouts, (c) assess the recent advances in sprout production, (d) rigorously evaluate their safety, and (e) suggest directions that merit special consideration for further novel research on sprouts. Young shoots are characterized by high levels of health-benefitting phytochemicals. Their utility as functional ingredients have been extensively described. Tremendous advances in the production and safety of sprouts have been made over the recent past and numerous reports have appeared in mainstream scientific journals describing their nutritional and medicinal properties. However, subjects such as application of sprouted seed flours in processed products, utilizing sprouts as leads in the synthesis of nanoparticles, and assessing the dynamics of a relationship between sprouts and gut health require special attention for future clinical exploration. Sprouting is an effective strategy allowing manipulation of phytochemicals in seeds to improve their health benefits.


Assuntos
Dieta Saudável/tendências , Valor Nutritivo , Compostos Fitoquímicos/análise , Plantas Comestíveis/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plântula/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Animais , Qualidade de Produtos para o Consumidor , Manipulação de Alimentos , Germinação , Humanos , Plantas Comestíveis/efeitos adversos , Plantas Comestíveis/genética , Plantas Comestíveis/metabolismo , Plantas Geneticamente Modificadas/efeitos adversos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plântula/efeitos adversos , Plântula/genética , Plântula/metabolismo , Sementes/efeitos adversos , Sementes/genética , Sementes/metabolismo
6.
Virus Genes ; 57(5): 469-473, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34379307

RESUMO

Citrus yellow mosaic badnavirus (CMBV) causes mosaic disease in all economically important citrus cultivars of India, with losses reaching up to 70%. CMBV belongs to the genus Badnavirus, family Caulimoviridae, possessing a circular double-stranded (ds) DNA genome with six open reading frames (ORFs I to VI), whose functions are yet to be deciphered. The RNA-silencing suppressor (RSS) activity has not been assigned to any CMBV ORF as yet. In the present study, it was found that ORFI exhibited RSS activity among all the six CMBV ORFs tested. Studies were done by employing the well-established Agrobacterium-mediated transient assay based on the transgenic Nicotiana benthamiana 16c plant line expressing the green fluorescent protein (GFP). The RSS activity of ORFI was confirmed by the analysis of the GFP visual expression in the agroinfiltrated leaves, further supported by quantification of GFP expression by RT-PCR. Based on the GFP visual expression, the CMBV ORFI was a weak RSS when compared to the p19 protein of tomato bushy stunt virus. In contrast, the ORFII, ORFIV, ORFV, ORFVI, and CP gene did not exhibit any RSS activity. Hence, ORFI is the first ORF of CMBV to be identified with RNA-silencing suppression activity.


Assuntos
Badnavirus/isolamento & purificação , Citrus/genética , Doenças das Plantas/virologia , Vírus de Plantas/genética , Badnavirus/genética , Badnavirus/patogenicidade , Citrus/crescimento & desenvolvimento , Citrus/virologia , Proteínas de Fluorescência Verde/genética , Índia , Fases de Leitura Aberta/genética , Doenças das Plantas/genética , Vírus de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/virologia , RNA/genética , Interferência de RNA , Tabaco/virologia , Tombusvirus/genética
7.
Int J Mol Sci ; 22(15)2021 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-34360645

RESUMO

Environmental adversities, particularly drought and nutrient limitation, are among the major causes of crop losses worldwide. Due to the rapid increase of the world's population, there is an urgent need to combine knowledge of plant science with innovative applications in agriculture to protect plant growth and thus enhance crop yield. In recent decades, engineering strategies have been successfully developed with the aim to improve growth and stress tolerance in plants. Most strategies applied so far have relied on transgenic approaches and/or chemical treatments. However, to cope with rapid climate change and the need to secure sustainable agriculture and biomass production, innovative approaches need to be developed to effectively meet these challenges and demands. In this review, we summarize recent and advanced strategies that involve the use of plant-related cyanobacterial proteins, macro- and micronutrient management, nutrient-coated nanoparticles, and phytopathogenic organisms, all of which offer promise as protective resources to shield plants from climate challenges and to boost stress tolerance in crops.


Assuntos
Adaptação Fisiológica , Mudança Climática , Produtos Agrícolas/crescimento & desenvolvimento , Secas , Desenvolvimento Vegetal , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Produtos Agrícolas/genética , Engenharia Genética , Plantas Geneticamente Modificadas/genética
8.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299210

RESUMO

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of ß-glucosidases. Evaluation of previously characterized recombinant rice ß-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher ß-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The ß-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular ß-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.


Assuntos
Ácido Abscísico/farmacologia , Ésteres/química , Glucose/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , beta-Glucosidase/metabolismo , Ácido Abscísico/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Secas , Giberelinas/farmacologia , Hidrólise , Família Multigênica , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Tabaco/efeitos dos fármacos , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo , beta-Glucosidase/genética
9.
Biomolecules ; 11(5)2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063698

RESUMO

Cyclic AMP (cAMP) is a pivotal signaling molecule existing in almost all living organisms. However, the mechanism of cAMP signaling in plants remains very poorly understood. Here, we employ the engineered activity of soluble adenylate cyclase to induce cellular cAMP elevation in Arabidopsis thaliana plants and identify 427 cAMP-responsive genes (CRGs) through RNA-seq analysis. Induction of cellular cAMP elevation inhibits seed germination, disturbs phytohormone contents, promotes leaf senescence, impairs ethylene response, and compromises salt stress tolerance and pathogen resistance. A set of 62 transcription factors are among the CRGs, supporting a prominent role of cAMP in transcriptional regulation. The CRGs are significantly overrepresented in the pathways of plant hormone signal transduction, MAPK signaling, and diterpenoid biosynthesis, but they are also implicated in lipid, sugar, K+, nitrate signaling, and beyond. Our results provide a basic framework of cAMP signaling for the community to explore. The regulatory roles of cAMP signaling in plant plasticity are discussed.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Perfilação da Expressão Gênica/métodos , Canais de Potássio/genética , Fatores de Transcrição/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Canais de Potássio/metabolismo , Proteínas Recombinantes/metabolismo , Análise de Sequência de RNA , Transdução de Sinais
10.
Elife ; 102021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34059197

RESUMO

Genetically identical plants growing in the same conditions can display heterogeneous phenotypes. Here we use Arabidopsis seed germination time as a model system to examine phenotypic variability and its underlying mechanisms. We show extensive variation in seed germination time variability between Arabidopsis accessions and use a multiparent recombinant inbred population to identify two genetic loci involved in this trait. Both loci include genes implicated in modulating abscisic acid (ABA) sensitivity. Mutually antagonistic regulation between ABA, which represses germination, and gibberellic acid (GA), which promotes germination, underlies the decision to germinate and can act as a bistable switch. A simple stochastic model of the ABA-GA network shows that modulating ABA sensitivity can generate the range of germination time distributions we observe experimentally. We validate the model by testing its predictions on the effects of exogenous hormone addition. Our work provides a foundation for understanding the mechanism and functional role of phenotypic variability in germination time.


Assuntos
Ácido Abscísico/farmacologia , Arabidopsis/efeitos dos fármacos , Germinação/efeitos dos fármacos , Giberelinas/farmacologia , Sementes/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Modelos Biológicos , Fenótipo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , Transdução de Sinais , Processos Estocásticos , Fatores de Tempo
11.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064885

RESUMO

Genetically modified (GM) crops possess some superior characteristics, such as high yield and insect resistance, but their biosafety has aroused broad public concern. Some genetic engineering technologies have recently been proposed to remove exogenous genes from GM crops. Few approaches have been applied to maintain advantageous traits, but excising exogenous genes in seeds or fruits from these hybrid crops has led to the generation of harvested food without exogenous genes. In a previous study, split-Cre mediated by split intein could recombine its structure and restore recombination activity in hybrid plants. In the current study, the recombination efficiency of split-Cre under the control of ovule-specific or pollen-specific promoters was validated by hybridization of transgenic Arabidopsis containing the improved expression vectors. In these vectors, all exogenous genes were flanked by two loxP sites, including promoters, resistance genes, reporter genes, and split-Cre genes linked to the reporter genes via LP4/2A. A gene deletion system was designed in which NCre was driven by proDD45, and CCre was driven by proACA9 and proDLL. Transgenic lines containing NCre were used as paternal lines to hybridize with transgenic lines containing CCre. Because this hybridization method results in no co-expression of the NCre and CCre genes controlled by reproduction-specific promoters in the F1 progeny, the desirable characteristics could be retained. After self-crossing in F1 progeny, the expression level and protein activity of reporter genes were detected, and confirmed that recombination of split-Cre had occurred and the exogenous genes were partially deleted. The gene deletion efficiency represented by the quantitative measurements of GUS enzyme activity was over 59%, with the highest efficiency of 73% among variable hybrid combinations. Thus, in the present study a novel dual reproductive cell-specific promoter-mediated gene deletion system was developed that has the potential to take advantage of the merits of GM crops while alleviating biosafety concerns.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Deleção de Genes , Integrases/metabolismo , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Transgenes , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Engenharia Genética , Vetores Genéticos , Integrases/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Recombinação Genética , Reprodução
12.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063649

RESUMO

The waxy (Wx) gene, encoding the granule-bound starch synthase (GBSS), is responsible for amylose biosynthesis and plays a crucial role in defining eating and cooking quality. The waxy locus controls both the non-waxy and waxy rice phenotypes. Rice starch can be altered into various forms by either reducing or increasing the amylose content, depending on consumer preference and region. Low-amylose rice is preferred by consumers because of its softness and sticky appearance. A better way of improving crops other than downregulation and overexpression of a gene or genes may be achieved through the posttranslational modification of sites or regulatory enzymes that regulate them because of their significance. The impact of posttranslational GBSSI modifications on extra-long unit chains (ELCs) remains largely unknown. Numerous studies have been reported on different crops, such as wheat, maize, and barley, but the rice starch granule proteome remains largely unknown. There is a need to improve the yield of low-amylose rice by employing posttranslational modification of Wx, since the market demand is increasing every day in order to meet the market demand for low-amylose rice in the regional area that prefers low-amylose rice, particularly in China. In this review, we have conducted an in-depth review of waxy rice, starch properties, starch biosynthesis, and posttranslational modification of waxy protein to genetically improve starch quality in rice grains.


Assuntos
Amilose/genética , Oryza/genética , Proteínas de Plantas/genética , Sintase do Amido/genética , Grão Comestível/genética , Grão Comestível/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Processamento de Proteína Pós-Traducional/genética , Amido/genética
13.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068763

RESUMO

Heat shock transcription factors (HSFs) play critical roles in several types of environmental stresses. However, the detailed regulatory mechanisms in response to salt stress are still largely unknown. In this study, we examined the salt-induced transcriptional responses of ThHSFA1-ThWRKY4 in Tamarix hispida and their functions and regulatory mechanisms in salt tolerance. ThHSFA1 protein acts as an upstream regulator that can directly activate ThWRKY4 expression by binding to the heat shock element (HSE) of the ThWRKY4 promoter using yeast one-hybrid (Y1H), chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. ThHSFA1 and ThWRKY4 expression was significantly induced by salt stress and abscisic acid (ABA) treatment in the roots and leaves of T. hispida. ThHSFA1 is a nuclear-localized protein with transactivation activity at the C-terminus. Compared to nontransgenic plants, transgenic plants overexpressing ThHSFA1 displayed enhanced salt tolerance and exhibited reduced reactive oxygen species (ROS) levels and increased antioxidant enzyme activity levels under salt stress. Therefore, we further concluded that ThHSFA1 mediated the regulation of ThWRKY4 in response to salt stress in T. hispida.


Assuntos
Proteínas de Arabidopsis/genética , Fatores de Transcrição de Choque Térmico/genética , Estresse Salino/genética , Tamaricaceae/genética , Fatores de Transcrição/genética , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino/efeitos dos fármacos , Tolerância ao Sal/efeitos dos fármacos , Tolerância ao Sal/genética , Sais/toxicidade , Tamaricaceae/efeitos dos fármacos , Tamaricaceae/crescimento & desenvolvimento
14.
Int J Biol Macromol ; 184: 955-966, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34153360

RESUMO

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.


Assuntos
Hemoglobina Fetal/genética , Oxigênio/metabolismo , Tabaco/genética , Hemoglobina Fetal/isolamento & purificação , Hemoglobina Fetal/metabolismo , Humanos , Ligação de Hidrogênio , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo
15.
Methods Mol Biol ; 2317: 77-94, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028763

RESUMO

Expression of transgenes from the plastid genome offers a number of attractions to biotechnologists, with the potential to attain very high protein accumulation levels arguably being the most attractive one. High-level transgene expression is of particular importance in resistance engineering (e.g., for expression of insecticidal proteins) and molecular farming (e.g., for expression of pharmaceutical proteins and industrial enzymes). Over the past decades, the production of many commercially valuable proteins in chloroplast-transgenic (transplastomic) plants has been attempted, including pharmaceutical proteins (e.g., subunit vaccines and protein antibiotics) and industrial enzymes. Although in some cases, spectacularly high foreign protein accumulation levels have been obtained, expression levels were disappointingly poor in other cases. In this review, I summarize our current knowledge about the factors influencing the efficiency of plastid transgene expression, and highlight possible optimization strategies to alleviate problems with poor expression levels. I also discuss available techniques for inducible expression of chloroplast transgenes.


Assuntos
Biotecnologia/métodos , Cloroplastos/genética , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Transformação Genética , Transgenes , Animais , Cloroplastos/metabolismo , Humanos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo
16.
Methods Mol Biol ; 2317: 95-107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028764

RESUMO

Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.


Assuntos
DNA de Plantas/genética , Marcadores Genéticos , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Recombinação Genética , Transformação Genética , Transgenes , DNA Nucleotidiltransferases , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sequências Repetitivas de Ácido Nucleico
17.
Methods Mol Biol ; 2317: 135-153, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028766

RESUMO

The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.


Assuntos
Genoma de Cloroplastos , Genomas de Plastídeos , Resistência a Canamicina/genética , Plastídeos/genética , Tabaco/genética , Transformação Genética , Transgenes , Marcadores Genéticos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Espectinomicina/farmacologia , Tabaco/crescimento & desenvolvimento
18.
Methods Mol Biol ; 2317: 155-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028767

RESUMO

Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (106 viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed plastids are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.


Assuntos
DNA de Plantas/genética , Resistência a Medicamentos , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Polietilenoglicóis/farmacologia , Tabaco/genética , Transformação Genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tabaco/efeitos dos fármacos , Tabaco/crescimento & desenvolvimento
19.
Methods Mol Biol ; 2317: 167-175, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028768

RESUMO

Chloroplast transformation has been extremely valuable for the study of plastid biology and gene expression, but the tissue culture methodology involved can be laborious and it can take several months to obtain homoplasmic regenerated plants useful for molecular or physiological studies. In contrast, transformation of tobacco suspension cell plastids provides an easy and efficient system to rapidly evaluate the efficacy of multiple constructs prior to plant regeneration. Suspension cell cultures can be initiated from many cell types, and once established, can be maintained by subculture for more than a year with no loss of transformation efficiency. Using antibiotic selection, homoplasmy is readily achieved in uniform cell colonies useful for comparative gene expression analyses, with the added flexibility to subsequently regenerate plants for in planta studies. Plastids from suspension cells grown in the dark are similar in size and cellular morphology to those in embryogenic culture systems of monocot species, thus providing a useful model for understanding the steps leading to plastid transformation in those recalcitrant species.


Assuntos
DNA de Plantas/genética , Genomas de Plastídeos , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Tabaco/genética , Transformação Genética , Células Cultivadas , Vetores Genéticos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
20.
Methods Mol Biol ; 2317: 283-290, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028776

RESUMO

Chloroplast biotechnology has assumed great importance in the past 20 years and, thanks to the numerous advantages as compared to conventional transgenic technologies, has been applied in an increasing number of plant species but still very much limited. Hence, it is of outmost importance to extend the range of species in which plastid transformation can be applied. Sugar beet (Beta vulgaris L.) is an important industrial crop of the temperate zone in which chloroplast DNA is not transmitted trough pollen. Transformation of the sugar beet genome is performed in several research laboratories, conversely sugar beet plastome genetic transformation is far away from being considered a routine technique. We describe here a method to obtain transplastomic sugar beet plants trough biolistic transformation. The availability of sugar beet transplastomic plants should avoid the risk of gene flow between these cultivated genetic modified sugar beet plants and the wild-type plants or relative wild species.


Assuntos
Beta vulgaris/genética , DNA de Cloroplastos/genética , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Transformação Genética , Beta vulgaris/crescimento & desenvolvimento , Produtos Agrícolas , Plantas Geneticamente Modificadas/crescimento & desenvolvimento
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...