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1.
Food Chem ; 327: 127048, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32454285

RESUMO

In our previous study, a novel LMW-GS designated as LMW-N13 with a unique molecular structure was identified from Aegilops uniaristata. LMW-N13 has been characterized as the largest LMW-GS, so far, and possesses an extra cysteine residue compared with typical LMW-GS. In order to analyze the contribution of LMW-N13 to dough quality, in this work, three transgenic wheat lines overexpressing LMW-N13 were generated. Compared with non-transformation (NT) lines, transgenic (TG) lines demonstrated superior dough properties. These superior dough properties were accompanied by the higher contents of glutenin macropolymer (GMP) and total protein. The microstructure of the dough was further investigated by scanning electron microscopy; starch granules in NT lines were smaller than those in transgenic lines. The protein matrix in NT lines was relatively loose and discontinuous. Conversely, the protein matrix in transgenic lines was more continuous and tight. The application of LMW-N13 in wheat breeding is also discussed.


Assuntos
Farinha/análise , Glutens/química , Plantas Geneticamente Modificadas/química , Triticum/química , Aegilops/genética , Dissulfetos/química , Glutens/genética , Glutens/metabolismo , Microscopia Eletrônica de Varredura , Peso Molecular , Plantas Geneticamente Modificadas/metabolismo , Amido/química , Triticum/metabolismo , Água/química
2.
Mol Biotechnol ; 62(6-7): 344-354, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32246385

RESUMO

Newcastle disease (ND) is considered as one of the most devastating infectious diseases targeting domestic birds and has considerable threat to the commercial poultry production. Two surface glycoproteins, hemagglutinin-neuraminidase (HN) and fusion (F), act as antigens in the virus structure and also play important roles in infecting host cells. In the current study, the expression of the chimeric HN-F protein in canola seeds and its immunogenicity in chickens were investigated. The HN-F gene was cloned downstream of the fatty acid elongase 1 (FAE1) promoter in the binary expression vector, pBI1400-HN-F, and introduced into rapeseed (Brassica napus L.) using Agrobacterium-mediated transformation. The amount of the HN-F glycoprotein was estimated up to 0.18% and 0.11% of the total soluble protein (TSP) in transgenic seeds and leaves of canola, respectively. Confirmatory analyses of 36 transgenic lines revealed that the HN-F gene was integrated into the genome. Subsequently, HN-F protein could be expressed and accumulated in the seed tissue. Specific pathogen-free (SPF) chickens immunized orally with recombinant HN-F showed a significant rise in specific and hemagglutination inhibition (HI) antibodies 35 to 42 days post the first administration. The results implied the potential of transgenic canola seed-based expression for oral delivery of NDV immunogenic glycoproteins.


Assuntos
Brassica napus/química , Proteína HN/imunologia , Vírus da Doença de Newcastle/imunologia , Óleos Vegetais/química , Plantas Geneticamente Modificadas/química , Sementes/química , Animais , Galinhas , Elongases de Ácidos Graxos/genética , Elongases de Ácidos Graxos/metabolismo , Folhas de Planta/química
3.
Food Chem Toxicol ; 137: 111129, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31935424

RESUMO

A subchronic toxicity study were conducted in Wistar Han RCC rats to evaluate the potential health effects of genetically modified (GM), drought-tolerant wheat MGX11-10. Rats were fed a rodent diet formulated with MGX11-10 and were compared with rats fed a diet formulated with its corresponding non-transgenic control Jimai22 and rats fed a basal diet. MGX11-10 and Jimai22 were ground into flour and formulated into diets at concentrations of 16.25, 32.5, or 65%, w/w% and fed to rats (10/sex/group) for 13 weeks. Compared with rats fed Jimai22 and the basal-diet group, no biologically relevant differences were observed in rats fed the GM diet with respect to body weight/gain, food consumption/efficiency, clinical signs, mortality, ophthalmology, clinical pathology (hematology, prothrombin time, urinalysis, clinical chemistry), organ weights, and gross and microscopic pathology. Under the conditions of this study, the MGX11-10 diets did not cause any treatment-related effects in rats following at least 90 days of dietary administration as compared with rats fed diets with the corresponding non-transgenic control diet and the basal-diet group. The MGX11-10 diets are considered equivalent to the diets prepared from conventional comparators. The results demonstrated that MGX11-10 wheat is as safe and wholesome as the corresponding non-transgenic control wheat.


Assuntos
Triticum/genética , Triticum/metabolismo , Ração Animal/análise , Animais , Secas , Feminino , Farinha/efeitos adversos , Farinha/análise , Alimentos Geneticamente Modificados/efeitos adversos , Masculino , Valor Nutritivo , Plantas Geneticamente Modificadas/efeitos adversos , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ratos , Ratos Wistar , Triticum/efeitos adversos , Triticum/química
4.
Plant Biol (Stuttg) ; 22(1): 70-80, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31283085

RESUMO

The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid-deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild-type cv. Xanthi plants, where salicylic acid (SA) contents are normal. Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R-2-oxothiazolidine-4-carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation. SA-deficient NahG tobacco was hyper-susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA-deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild-type plants (i.e. enhanced basal resistance). However, expression of the SA-mediated pathogenesis-related gene (NtPR-1a) did not increase significantly in GSH or OTC-pretreated and powdery mildew-inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH. Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA-deficient tobacco.


Assuntos
Ascomicetos , Resistência à Doença , Glutationa , Ácido Salicílico , Tabaco , Ascomicetos/efeitos dos fármacos , Ascomicetos/fisiologia , Resistência à Doença/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Glutationa/metabolismo , Glutationa/farmacologia , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/microbiologia , Tabaco/química , Tabaco/microbiologia
5.
J Chem Ecol ; 46(2): 217-231, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31879865

RESUMO

Despite active research, antiherbivore activity of specific plant phenolics remains largely unresolved. We constructed silver birch (Betula pendula) lines with modified phenolic metabolism to study the effects of foliar flavonoids and condensed tannins on consumption and growth of larvae of a generalist herbivore, the autumnal moth (Epirrita autumnata). We conducted a feeding experiment using birch lines in which expression of dihydroflavonol reductase (DFR), anthocyanidin synthase (ANS) or anthocyanidin reductase (ANR) had been decreased by RNA interference. Modification-specific effects on plant phenolics, nutrients and phenotype, and on larval consumption and growth were analyzed using uni- and multivariate methods. Inhibiting DFR expression increased the concentration of flavonoids at the expense of condensed tannins, and silencing DFR and ANR decreased leaf and plant size. E. autumnata larvae consumed on average 82% less of DFRi plants than of unmodified controls, suggesting that flavonoids or glandular trichomes deter larval feeding. However, larval growth efficiency was highest on low-tannin DFRi plants, indicating that condensed tannins (or their monomers) are physiologically more harmful than non-tannin flavonoids for E. autumnata larvae. Our results show that genetic manipulation of the flavonoid pathway in plants can effectively be used to produce altered phenolic profiles required for elucidating the roles of low-molecular weight phenolics and condensed tannins in plant-herbivore relationships, and suggest that phenolic secondary metabolites participate in regulation of plant growth.


Assuntos
Betula/química , Flavonoides/metabolismo , Mariposas/fisiologia , Plantas Geneticamente Modificadas/química , Taninos/metabolismo , Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Betula/enzimologia , Betula/parasitologia , Flavonoides/farmacologia , Herbivoria/efeitos dos fármacos , Interações Hospedeiro-Parasita , Larva/crescimento & desenvolvimento , Larva/fisiologia , Mariposas/crescimento & desenvolvimento , Oxigenases/antagonistas & inibidores , Oxigenases/genética , Oxigenases/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , Interferência de RNA , Taninos/farmacologia
6.
BMC Plant Biol ; 19(1): 552, 2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31830911

RESUMO

BACKGROUND: Understanding lignin biosynthesis and composition is of central importance for sustainable bioenergy and biomaterials production. Species of the genus Miscanthus have emerged as promising bioenergy crop due to their rapid growth and modest nutrient requirements. However, lignin polymerization in Miscanthus is poorly understood. It was previously shown that plant laccases are phenol oxidases that have multiple functions in plant, one of which is the polymerization of monolignols. Herein, we link a newly discovered Miscanthus laccase, MsLAC1, to cell wall lignification. Characterization of recombinant MsLAC1 and Arabidopsis transgenic plants expressing MsLAC1 were carried out to understand the function of MsLAC1 both in vitro and in vivo. RESULTS: Using a comprehensive suite of molecular, biochemical and histochemical analyses, we show that MsLAC1 localizes to cell walls and identify Miscanthus transcription factors capable of regulating MsLAC1 expression. In addition, MsLAC1 complements the Arabidopsis lac4-2 lac17 mutant and recombinant MsLAC1 is able to oxidize monolignol in vitro. Transgenic Arabidopsis plants over-expressing MsLAC1 show higher G-lignin content, although recombinant MsLAC1 seemed to prefer sinapyl alcohol as substrate. CONCLUSIONS: In summary, our results suggest that MsLAC1 is regulated by secondary cell wall MYB transcription factors and is involved in lignification of xylem fibers. This report identifies MsLAC1 as a promising breeding target in Miscanthus for biofuel and biomaterial applications.


Assuntos
Lacase/genética , Lignina/química , Proteínas de Plantas/genética , Poaceae/fisiologia , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/fisiologia , Lacase/metabolismo , Lignina/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Poaceae/química , Poaceae/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
7.
Molecules ; 24(24)2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31847134

RESUMO

BACKGROUND: In recent years, genetically modified technology has developed rapidly, and the potential impact of genetically modified foods on human health and the ecological environment has received increasing attention. The currently used methods for testing genetically modified foods are cumbersome, time-consuming, and expensive. This paper proposed a more efficient and convenient detection method. METHODS: Near-infrared diffuse reflectance spectroscopy (NIRDRS) combined with multivariate calibration methods, including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and support vector machines (SVM), were used for identification of different rice varieties and transgenic (Bt63)/non-transgenic rice. Spectral pretreatment methods, including Norris-Williams smooth (NWS), standard normal variate (SNV), multiplicative scatter correction (MSC), and Savitzky-Golay 1st derivative (SG 1st-Der), were used for spectral noise reduction and effective information enhancement. Accuracy was used to evaluate the qualitative discriminant models. RESULTS: The results showed that the SG 1st-Der pretreatment method, combined with the SVM, provided the optimal model to distinguish different rice varieties. The accuracy of the optimal model was 98.33%. For the discrimination model of transgenic/non-transgenic rice, the SNV-SVM model, MSC-SVM model, and SG 1st-Der-PLS-DA model all achieved good analysis results with the accuracy of 100%. CONCLUSION: The results showed that portable NIR spectroscopy combined with chemometrics methods could be used to identify rice varieties and transgenic characteristics (Bt63) due to its fast, non-destructive, and accurate advantages.


Assuntos
Oryza/classificação , Plantas Geneticamente Modificadas/classificação , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Análise Discriminante , Análise dos Mínimos Quadrados , Oryza/química , Oryza/genética , Plantas Geneticamente Modificadas/química , Análise de Componente Principal , Máquina de Vetores de Suporte
8.
J Agric Food Chem ; 67(49): 13506-13508, 2019 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-31725270

RESUMO

Since 2011, the European Food Safety Authority (EFSA) has implemented combined difference and equivalence testing of agronomic, phenotypic, and composition data in the risk assessment of genetically modified crops. A short perspective is provided on misunderstandings that have shown up in published criticisms of the approach to equivalence testing, different viewpoints regarding the questions to be answered, and new developments in statistical modeling.


Assuntos
Produtos Agrícolas/química , Análise de Alimentos/métodos , Plantas Geneticamente Modificadas/química , Qualidade de Produtos para o Consumidor , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Inocuidade dos Alimentos , Alimentos Geneticamente Modificados , Humanos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Medição de Risco
9.
J Agric Food Chem ; 67(45): 12590-12598, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31639305

RESUMO

Carotenoids play key roles in photosynthesis and photoprotection. Few multicellular plants produce the ketocarotenoid astaxanthin, a strong antioxidant; however, Arabidopsis thaliana lines overexpressing the Chlamydomonas reinhardtii ß-carotene ketolase (CrBKT) accumulated high amounts of astaxanthin in the leaves. In this study, we investigated the changed regulation of key metabolic pathways and the tolerance of the engineered plants to biotic and abiotic stresses resulting from the heterologous expression of CrBKT. Transcriptome analysis identified 1633 and 1722 genes that were differentially expressed in the leaves and siliques, respectively, of CrBKT-overexpressing plants (line CR5) as compared to wild-type Arabidopsis. These genes were enriched in the carotenoid biosynthetic pathways, and plant hormone biosynthesis and signaling pathways. In particular, metabolic profiling showed that, as compared to the wild-type leaves and siliques, overexpression of CrBKT increased the levels of most amino acids, but decreased the contents of sugars and carbohydrates. Furthermore, CR5 plants had lower sensitivity to abscisic acid (ABA) and increased tolerance to oxidative stress. CR5 plants also exhibited enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our study provides insight into the regulation of carotenoids and the related pathways, which may be involved in plant response to oxidative stress and pathogen infection.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/química , Arabidopsis/microbiologia , Regulação da Expressão Gênica de Plantas , Estresse Oxidativo , Doenças das Plantas/genética , Reguladores de Crescimento de Planta/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Pseudomonas syringae/fisiologia , Xantofilas/biossíntese
10.
Mikrochim Acta ; 186(11): 731, 2019 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-31659455

RESUMO

Fluorescent polymer dots (PDs) with maximum excitation/emission wavelengths of 410/515 nm were prepared in water solution from 1,4-benzoquinone and ethylenediamine. The green fluorescence of these PDs is screened off by the red-colored oxidation product (PPDox, maximum absorption at 510 nm) formed by horseradish peroxidase (HRP)-catalyzed oxidation of p-phenylenediamine (PPD). It causes the reduction of the fluorescence intensity of the PDs due to spectral overlap and an inner filter effect (IFE). If glucose is enzymatically oxidized under the formation of H2O2, the formed H2O2 can be quantified by the above IFE. The assay for HRP activity and glucose have detection limits of 0.2 U·L-1 and 0.1 µM, respectively. The nanoprobe was further extended to an immunosorbent assay (ELISA) for the determination of insecticidal Cry1Ab/Ac protein with a detection limit of 0.25 ng·mL-1. The ELISA was applied to rice leaf analysis. Graphic abstract Schematic representation of fluorometrict enzyme-linked immunosorbent assay for Cry1Ab/Ac protein detection based on horseradish peroxidase (HRP)-triggered fluorescence quenching of polymer dots (PDs). Quenching is caused by an inner filter effect (IFE) caused by PPDox, the oxidation product of p-phenylenediamine (PPD).


Assuntos
Proteínas de Bactérias/análise , Endotoxinas/análise , Glucose/análise , Proteínas Hemolisinas/análise , Peroxidase do Rábano Silvestre/análise , Polímeros/química , Pontos Quânticos/química , Anticorpos Imobilizados/imunologia , Armoracia/enzimologia , Bacillus thuringiensis/química , Proteínas de Bactérias/imunologia , Endotoxinas/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Glucose/química , Glucose Oxidase/química , Proteínas Hemolisinas/imunologia , Peróxido de Hidrogênio/química , Limite de Detecção , Oryza/química , Fenilenodiaminas/química , Plantas Geneticamente Modificadas/química , Espectrometria de Fluorescência/métodos
11.
Proc Natl Acad Sci U S A ; 116(42): 20947-20952, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570578

RESUMO

Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterized by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or ß) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study, we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (vs. outer) positions by more than 20-fold (i.e., from ∼3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.


Assuntos
Arabidopsis/genética , Substitutos da Gordura/química , Gorduras/química , Leite Humano/química , Óleos Vegetais/química , Sementes/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Substitutos da Gordura/metabolismo , Humanos , Fórmulas Infantis/química , Óleos Vegetais/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/química , Sementes/genética , Estereoisomerismo
12.
Electrophoresis ; 40(22): 2921-2928, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31475363

RESUMO

Application of a microfluidic CE* device for CZE-MS allows for fast, rapid, and in-depth analysis of large sample sets. This microfluidic CZE-MS device, the 908 Devices ZipChip, involves minimal sample preparation and is ideal for small cation analytes, such as alkaloids. Here, we evaluated the microfluidic device for the analysis of alkaloids from Lobelia cardinalis hairy root cultures. Extracts from wild-type, transgenic, and selected mutant plant cultures were analyzed and data batch processed using the mass spectral processing software MZmine2 and the statistical software Prism 8. In total 139 features were detected as baseline resolved peaks via the MZmine2 software optimized for the electrophoretic separations. Statistically significant differences in the relative abundance of the primary alkaloid lobinaline (C27 H34 N2 ), along with several putative "lobinaline-like" molecules were observed utilizing this approach. Additionally, a method for performing both targeted and untargeted MS/MS experiments using the microfluidic device was developed and evaluated. Coupling data-processing software with CZE-MS data acquisition has enabled comprehensive metabolomic profiles from plant cell cultures to be constructed within a single working day.


Assuntos
Alcaloides/análise , Eletroforese Capilar/métodos , Lobelia , Plantas Geneticamente Modificadas , Espectrometria de Massas em Tandem/métodos , Biologia Computacional , Limite de Detecção , Modelos Lineares , Lobelia/química , Lobelia/citologia , Técnicas Analíticas Microfluídicas/métodos , Células Vegetais/química , Extratos Vegetais/química , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/citologia , Reprodutibilidade dos Testes
13.
J Sci Food Agric ; 99(15): 6903-6910, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31415094

RESUMO

BACKGROUND: Bacillus thuringiensis (Bt) synthesizes Cry1Ac protein, which is toxic to many lepidopteran pests, and the cry1ac gene has been expressed in several transgenic crop plants. The Cry1Ac protein has been isolated from Bt kurstaki HD73 and purified to homogeneity. Polyclonal antibodies were raised against purified Cry1Ac in rabbits and goat. Sandwich ELISA was developed for Cry1Ac using goat IgG as a coating antibody, and affinity-purified rabbit IgG as the primary antibody. RESULTS: The sensitivity of the assay was in the range of 0.47-1000 ng. It was subsequently employed in validating biological samples. Fifteen different cotton-seed samples were screened: 12 were found to be Bt positive and 3 Bt negative. The CS7 seeds showed the highest Bt content of 8.51 ± 0.45 µg g-1 , followed by CS8 (6.0 ± 0.02 µg g-1 ), CS15 (5.9 ± 0.03 µg g-1 ), CS9 (5.5 ± 0.05 µg g-1 ), and CS10 (4.83 ± 0.013 µg g-1 ). The CS5 seeds showed Bt content of 3.6 ± 0.21 µg g-1 . The F2 generation, CS6 (Kaveri seeds) showed lower Bt content (2.9 ± 0.06 µg g-1 ). The CL5 samples showed Cry1Ac content of 0.99 ± 0.009 µg g-1 . The amount of Cry1Ac protein in leaves, stem, and roots of germinated Bt cotton plants (CS10 and CS4) were 1.76 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , 2.0 ± 0.1 µg g-1 , and 1.6 ± 0.15 µg g-1 , 1.9 ± 0.01 µg g-1 , and 2.0 ± 0.01 µg g-1 dry tissue, respectively. CONCLUSION: The method developed can be used for screening the expression levels of Cry1Ac in different transgenic Bt cultivars and also spurious Bt cotton seeds procured by farmers. © 2019 Society of Chemical Industry.


Assuntos
Bacillus/química , Endotoxinas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Gossypium/química , Plantas Geneticamente Modificadas/química , Animais , Anticorpos/análise , Anticorpos/imunologia , Bacillus/metabolismo , Endotoxinas/imunologia , Endotoxinas/metabolismo , Gossypium/genética , Gossypium/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Coelhos , Sementes/química , Sementes/genética , Sementes/metabolismo
14.
Nat Commun ; 10(1): 3548, 2019 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-31391460

RESUMO

Microbial fermentation of lignocellulosic biomass to produce industrial chemicals is exacerbated by the recalcitrant network of lignin, cellulose and hemicelluloses comprising the plant secondary cell wall. In this study, we show that transgenic poplar (Populus trichocarpa) lines can be solubilized without any pretreatment by the extreme thermophile Caldicellulosiruptor bescii that has been metabolically engineered to shift its fermentation products away from inhibitory organic acids to ethanol. Carbohydrate solubilization and conversion of unpretreated milled biomass is nearly 90% for two transgenic lines, compared to only 25% for wild-type poplar. Unexpectedly, unpretreated intact poplar stems achieved nearly 70% of the fermentation production observed with milled poplar as the substrate. The nearly quantitative microbial conversion of the carbohydrate content of unpretreated transgenic lignocellulosic biomass bodes well for full utilization of renewable biomass feedstocks.


Assuntos
Clostridiales/metabolismo , Fermentação , Microbiologia Industrial , Engenharia Metabólica , Populus/metabolismo , Biomassa , Celulose/metabolismo , Clostridiales/genética , Etanol/metabolismo , Lignina/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Polissacarídeos/metabolismo , Populus/química , Populus/genética
15.
J Agric Food Chem ; 67(35): 9877-9884, 2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31398030

RESUMO

Heavy metal contaminants and nutrient deficiencies in soil negatively affect crop growth and human health. The plant cadmium resistance (PCR) protein transports heavy metals. The abundance of PCR is correlated with that of cell number regulator (CNR) protein, and the two proteins have similar conserved domains. Hence, CNR might also participate in heavy metal transport. We isolated and analyzed TaCNR5 from wheat (Triticum aestivum). The expression level of TaCNR5 in the shoots of wheat increased under cadmium (Cd), zinc (Zn), or manganese (Mn) treatments. Transgenic plants expressing TaCNR5 showed enhanced tolerance to Zn and Mn. Overexpression of TaCNR5 in Arabidopsis increased Cd, Zn, and Mn translocation from roots to shoots. The concentrations of Zn and Mn in rice grains were increased in transgenic plants expressing TaCNR5. These roles of TaCNR5 in the translocation and distribution of heavy metals mean that it has potential as a genetic biofortification tool to fortify cereal grains with micronutrients.


Assuntos
Manganês/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Triticum/genética , Zinco/metabolismo , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Biofortificação , Transporte Biológico , Cádmio/análise , Cádmio/metabolismo , Manganês/análise , Oryza/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Triticum/química , Triticum/metabolismo , Zinco/análise
16.
J Agric Food Chem ; 67(28): 7898-7907, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31282664

RESUMO

This study aimed to explore the effects of silencing HB12 and TT8 genes on protein utilization characteristics of alfalfa. Ground samples of 11 HB12-silenced (HB12i), 5 TT8-silenced (TT8i) and 4 wild type (WT) were incubated in a Daisy II incubator with N15 labeled ammonium sulfate for 0, 4, 8, 12, and 24 h. CP degradation and degradational kinetics, microbial nitrogen fractions, and protein metabolic profiles were determined. Moreover, relationships between protein profiles and FTIR spectral parameters were estimated. Results showed that transgenic alfalfa had lower CP degradation, microbial protein, and total available protein compared with WT, especially for HB12i. In addition, CP degradation and protein metabolic profiles were closely correlated with FTIR spectral parameters and thereby could be predicted from spectral parameters. In conclusion, silencing of HB12 and TT8 genes in alfalfa decreased protein degradational and metabolic profiles, which were predictable with FTIR spectral parameters.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Inativação Gênica , Proteínas de Homeodomínio/genética , Medicago sativa/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rúmen/metabolismo , Ração Animal/análise , Animais , Bactérias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bovinos , Digestão , Proteínas de Homeodomínio/metabolismo , Cinética , Medicago sativa/química , Medicago sativa/metabolismo , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteólise , Rúmen/química , Rúmen/microbiologia
17.
J Agric Food Chem ; 67(31): 8559-8572, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31298518

RESUMO

Avenin-like b protein is rich in cysteine residues, providing the possibility to form intermolecular disulfide bonds and then participate in glutenin polymerization. Site-directed mutagenesis was adopted to produce mutant avenin-like b gene encoding mutant avenin-like b protein, in which one tyrosine codon at the C-terminal is substituted by a cysteine codon. Compared with the control lines, both transgenic lines with wild-type and mutant avenin-like b genes demonstrated superior dough properties. While compared within the transgenic lines, the mutant lines showed relative weaker dough strength and decreased sodium-dodecyl-sulfate sedimentation volumes (from 69.7 mL in line WT alb-1 to 41.0 mL in line Mut alb-4). These inferior dough properties were accompanied by the lower contents of large-sized glutenin polymers, the decreased particle diameters of glutenin macropolymer (GMP), due to the lower content of intermolecular ß-sheets (from 39.48% for line WT alb-2 to 30.21% for line Mut alb-3) and the varied contents of disulfide bonds (from 137.37 µmol/g for line WT alb-1 to 105.49 µmol/g for line Mut alb-4) in wheat dough. The extra cysteine might alter the original disulfide bond structure, allowing cysteine residue usually involved in an intermolecular disulfide bond to become available for an intrachain disulfide bond. Avenin-like b proteins were detected in glutenin macropolymers, providing further evidence for this protein to participate in the polymerization of glutenin. This is the first time to investigate the effect of a specific cysteine residue in the avenin-like b protein on flour quality.


Assuntos
Cisteína/genética , Farinha/análise , Plantas Geneticamente Modificadas/genética , Prolaminas/genética , Triticum/genética , Pão/análise , Cisteína/metabolismo , Dissulfetos/química , Manipulação de Alimentos , Mutagênese Sítio-Dirigida , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/metabolismo , Prolaminas/metabolismo , Triticum/química , Triticum/metabolismo
18.
Nat Plants ; 5(7): 715-721, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31285558

RESUMO

High accumulation of heterologous proteins expressed from the plastid genome has sometimes been reported to result in compromised plant phenotypes. Comparisons of transplastomic plants to wild-type (WT) are typically made in environmentally controlled chambers with relatively low light; little is known about the performance of such plants under field conditions. Here, we report on two plastid-engineered tobacco lines expressing the bacterial cellulase Cel6A. Field-grown plants producing Cel6A at ~20% of total soluble protein exhibit no loss in biomass or Rubisco content and only minor reductions in photosynthesis compared to WT. These experiments demonstrate that, when grown in the field, tobacco possesses sufficient metabolic flexibility to accommodate high levels of recombinant protein by increasing total protein synthesis and accumulation and/or by reallocating unneeded endogenous proteins. Based on current tobacco cultivation practices and readily achievable recombinant protein yields, we estimate that specific proteins could be obtained from field-grown transgenic tobacco plants at costs three orders of magnitude less than current cell culture methods.


Assuntos
Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Cloroplastos/metabolismo , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tabaco/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Celulase/análise , Celulase/genética , Cloroplastos/química , Cloroplastos/genética , Fotossíntese , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Tabaco/química , Tabaco/genética , Tabaco/metabolismo
19.
J Agric Food Chem ; 67(28): 7986-7994, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31282158

RESUMO

Compositional analyses were performed on samples of rice grain, straw, and derived bran obtained from golden rice event GR2E and near-isogenic control PSBRc82 rice grown at four locations in the Philippines during 2015 and 2016. Grain samples were analyzed for key nutritional components, including proximates, fiber, polysaccharides, fatty acids, amino acids, minerals, vitamins, and antinutrients. Samples of straw and bran were analyzed for proximates and minerals. The only biologically meaningful difference between GR2E and control rice was in levels of ß-carotene and other provitamin A carotenoids in the grain. Except for ß-carotene and related carotenoids, the compositional parameters of GR2E rice were within the range of natural variability of those components in conventional rice varieties with a history of safe consumption. Mean provitamin A concentrations in milled rice of GR2E can contribute up to 89-113% and 57-99% of the estimated average requirement for vitamin A for preschool children in Bangladesh and the Philippines, respectively.


Assuntos
Oryza/genética , Plantas Geneticamente Modificadas/química , Sementes/química , Aminoácidos/análise , Aminoácidos/metabolismo , Bangladesh , Ácidos Graxos/análise , Ácidos Graxos/metabolismo , Alimentos Geneticamente Modificados , Engenharia Genética , Valor Nutritivo , Oryza/química , Oryza/metabolismo , Filipinas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Provitaminas/análise , Provitaminas/metabolismo , Sementes/genética , Sementes/metabolismo , Vitamina A/análise , Vitamina A/metabolismo , beta Caroteno/análise , beta Caroteno/metabolismo
20.
J Biol Chem ; 294(32): 12091-12098, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31217277

RESUMO

Thiol-based redox regulation via ferredoxin-thioredoxin (Trx) reductase/Trx controls various functions in chloroplasts in response to light/dark changes. Trx is a key factor of this regulatory system, and five Trx subtypes, including 10 isoforms, have been identified as chloroplast-localized forms in Arabidopsis thaliana These subtypes display distinct target selectivity, and, consequently, they form a complicated redox regulation network in chloroplasts. In this study, we developed a FRET-based sensor protein by combining CFP, YFP, and the N-terminal region of CP12, a redox-sensitive regulatory and Trx-targeted protein in chloroplasts. This sensor protein enabled us to monitor the redox change of chloroplast thioredoxin in vivo, and we therefore designated this protein "change in redox state of Trx" (CROST). Using CP12 isoforms, we successfully prepared two types of CROST sensors that displayed different affinities for two major chloroplast Trx isoforms (f-type and m-type). These sensor proteins helped unravel the real-time redox dynamics of Trx molecules in chloroplasts during the light/dark transition.


Assuntos
Proteínas de Arabidopsis/química , Cloroplastos/metabolismo , Proteínas Luminescentes/genética , Tiorredoxinas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transferência Ressonante de Energia de Fluorescência , Luz , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Oxirredução , Folhas de Planta/química , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Tiorredoxinas/química
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