Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 16.857
Filtrar
1.
BMC Plant Biol ; 19(1): 371, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31438856

RESUMO

BACKGROUND: Propamocarb (PM) is one of the main pesticides used for controlling cucumber downy mildew. However, due to its volatility and internal absorption, PM can easily form pesticide residues on cucumber fruits that seriously endanger human health and pollute the environment. The breeding of new cucumber varieties with a low abundance of PM residues via genetic methods constitutes an effective strategy for reducing pesticide residues and improving cucumber safety and quality. To help elucidate the molecular mechanism resulting in a low PM residue abundance in cucumber, we used the cucumber cultivar 'D0351' (which has the lowest PM residue content) as the test material and identified genes related to low PM residue abundance through high-throughput tag-sequencing (Tag-Seq). RESULTS: CsMAPEG was constitutively expressed and showed both varietal and organizational differences. This gene was strongly expressed in 'D0351'. The expression levels of CsMAPEG in different cucumber tissues under PM stress were as follows: fruit>leaf>stem>root. CsMAPEG can respond to salicylic acid (SA), gibberellin (GA) and Corynespora cassiicola Wei (Cor) stress and thus plays an important regulatory role in plant responses to abiotic and biological stresses. The PM residue abundance in the fruits of CsMAPEG-overexpressing plants was lower than those found in antisense CsMAPEG plants and wild-type plants at all tested time points. The results revealed that CsMAPEG played a positive role in reducing the PM residue abundance. A CsMAPEG sense construct increased the contents of SOD, POD and GST in cucumber fruits, enhanced the degradation and metabolism of PM in cucumber, and thus effectively reduced the pesticide residue abundance in cucumber fruits. CONCLUSIONS: The expression patterns of CsMAPEG in cucumber cultivars with high and low pesticide residue abundances and a transgenic verification analysis showed that CsMAPEG can actively respond to PM stress and effectively reduce the PM residue abundance in cucumber fruits. The results of this study will help researchers further elucidate the mechanism responsible for a low PM residue abundance in cucumber and lay a foundation for the breeding of new agricultural cucumber varieties with low pesticide residue abundances.


Assuntos
Carbamatos/farmacologia , Cucumis sativus/genética , Fungicidas Industriais/farmacologia , Genes de Plantas , Resíduos de Praguicidas , Clonagem Molecular , Cucumis sativus/efeitos dos fármacos , Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Perfilação da Expressão Gênica , Vetores Genéticos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transformação Genética
2.
World J Microbiol Biotechnol ; 35(7): 109, 2019 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-31280382

RESUMO

Echinocandin B (ECB) is an important lipohexapeptide used for chemical manufacture of the antifungal agent anidulafungin. Sterigmatocystin (ST) is a polyketide mycotoxin produced by certain species of Aspergillus such as Aspergillus delacroxii SIPIW15, which could produce both ECB and ST. However, the presence of the potent carcinogen ST will greatly affect the quality and safety of ECB production. Therefore, it is essential to eliminate the ST biosynthesis and increase ECB titers in Asp. delacroxii SIPIW15. In this study, the polyketide synthase gene (stcA) required for biosynthesis of ST and its flanking region in Asp. delacroxii SIPIW15 were cloned, sequenced and analyzed firstly. Based on Agrobacterium-mediated transformation, the ΔstcA mutant AMT-1 was obtained and its yield of ECB was increased by 40% without ST detected at the same time as compared to the original strain. The results of the fed-batch experiments showed that the ECB yield of the ΔstcA strain AMT-1 was increased to 2163 ± 31 mg/l and no ST was detected in the 50 l bioreactor. This work suggested that the ΔstcA strain AMT-1 has the potential for application in ECB production improvement, and more importantly, to eliminate ST-related environmental pollution in ECB fermentation industry.


Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Equinocandinas/biossíntese , Equinocandinas/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genes Fúngicos/genética , Policetídeo Sintases/genética , Esterigmatocistina/biossíntese , Agrobacterium/genética , Anidulafungina , Antifúngicos , Sequência de Bases , Técnicas de Cultura Celular por Lotes , Reatores Biológicos , DNA Fúngico/isolamento & purificação , Fermentação , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Metabolismo Secundário/genética , Transformação Genética
3.
Microbiol Res ; 226: 55-64, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31284945

RESUMO

Functional association between genomic loci and specific biological traits remains lacking in many fungi, including the African tree pathogen Ceratocystis albifundus. This is mainly because of the absence of suitable transformation systems for allowing genetic manipulation of this and other fungi. Here, we present an optimized protocol for Agrobacterium tumefaciens-mediated transformation of C. albifundus. Strain AGL-1 of A. tumefaciens and four binary T-DNA vectors (conferring hygromycin B or geneticin resistance and/or expressing the green fluorescent protein [GFP]) were used for transforming germinated conidia of three isolates of C. albifundus. Stable expression of these T-DNA-encoded traits was confirmed through sequential sub-culturing of fungal transformants on selective and non-selective media and by using PCR and sequence analysis. Single-copy integration of the respective T-DNAs into the genomes of these fungi was confirmed using Southern hybridization analysis. The range of experimental parameters determined and optimised included: (i) concentrations of hygromycin B and geneticin required for inhibiting growth of the wild type fungus and (ii) the dependence of transformation on acetosyringone for inducing the bacterium's virulence genes, as well as (iii) the duration of fungus-bacterium co-cultivation periods and (iv) the concentrations of fungal conidia and bacterial cells used for the latter. The system developed in this study is stable with a high-efficiency, yielding up to 400 transformants per 106 conidia. This is the first report of a transformation protocol for C. albifundus and its availability will be invaluable for functional studies in this important fungus.


Assuntos
Agrobacterium tumefaciens/genética , Ascomicetos/genética , Transformação Genética , Ascomicetos/citologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Southern Blotting , Carbenicilina/farmacologia , Técnicas de Cocultura , DNA Bacteriano , Regulação Fúngica da Expressão Gênica , Gentamicinas/farmacologia , Proteínas de Fluorescência Verde/genética , Higromicina B/farmacologia , Canamicina/farmacologia , Reação em Cadeia da Polimerase , Análise de Sequência , Virulência/genética
4.
World J Microbiol Biotechnol ; 35(8): 119, 2019 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-31332541

RESUMO

The microalgal genus of Nannochloropsis is considered one of the most promising organisms for the production of biofuels due to their high lipid content. Transformation systems for marine Nannochloropsis species have been established in the recent decade, however, genetic manipulation of Nannochloropsis limnetica, the only known freshwater species in this genus, is not yet available. Based on established marine Nannochloropsis species electrotransformation protocol, nuclear genetic transformation was established in N. limnetica, meanwhile the appropriate antibiotic selection concentration and electric field strength of electroporation were determined. For the selection of transformants in N. limnetica on plates, 0.07 µg mL-1 of zeocin or 5 µg mL-1 of hygromycin B was proved sufficient, and the transformation efficiency was < 2 × 10-8 with a single pulse ranging from 2200 to 2600 V using 2-mm electroporation cuvettes. Pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times, and the highest transformation efficiency of 10-11 × 10-6 was obtained with an electric field strength of 12,000 V/cm. Our results help to expand the biotechnological applications of this freshwater species and provide means for successful electrotransformation of other microalgae as well. High-efficiency transformation of freshwater Nannochloropsis pretreatment of N. limnetica with 10 mM lithium acetate and 3 mM dithiothreitol before electroporation increased transformation efficiency hundreds of times.


Assuntos
Eletroporação , Água Doce/microbiologia , Microalgas/metabolismo , Estramenópilas/metabolismo , Acetatos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microalgas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Estramenópilas/genética , Transformação Genética
5.
BMC Plant Biol ; 19(1): 246, 2019 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-31182023

RESUMO

BACKGROUND: Rapid-cycling Brassica rapa (RCBr), also known as Wisconsin Fast Plants, are small robust plants with a short lifecycle that are widely used in biology teaching. RCBr have been used for decades but there are no published reports of RCBr genetic transformation. Agrobacterium-mediated vacuum infiltration has been used to transform pakchoi (Brassica rapa ssp. chinensis) and may be suitable for RCBr transformation. The floral dip transformation method, an improved version of vacuum infiltration, could make the procedure easier. RESULTS: Based on previous findings from Arabidopsis and pakchoi, plants of three different ages were inoculated with Agrobacterium. Kanamycin selection was suboptimal with RCBr; a GFP screen was used to identify candidate transformants. RCBr floral bud dissection showed that only buds with a diameter less than 1 mm carried unsealed carpels, a key point of successful floral dip transformation. Plants across a wide range of inflorescence maturities but containing these immature buds were successfully transformed, at an overall rate of 0.1% (one per 1000 T1 seeds). Transformation was successful using either vacuum infiltration or the floral dip method, as confirmed by PCR and Southern blot. CONCLUSION: A genetic transformation system for RCBr was established in this study. This will promote development of new biology teaching tools as well as basic biology research on Brassica rapa.


Assuntos
Agrobacterium/fisiologia , Brassica rapa/genética , Brassica rapa/microbiologia , Engenharia Genética/métodos , Transformação Genética , Southern Blotting , Flores/genética , Reação em Cadeia da Polimerase
6.
Prep Biochem Biotechnol ; 49(8): 800-806, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31156029

RESUMO

In this study, azurin, a bacteriocin with anticancer property, was produced by food-grade Lactococcus lactis using the Nisin Controlled Gene Expression (NICE) System. In addition, the antibacterial and cytotoxic properties of recombinant azurin in the culture supernatant were also investigated. Azurin gene from Pseudomonas aeruginosa was cloned into the pNZ8149 vector and the resulting recombinant DNA was transformed into food grade L. lactis NZ3900. The expression of azurin protein was induced by the optimum concentration of nisin for 3 h. Inhibition zones for Escherichia coli and Bacillus cereus were observed at 5.0 and 10 mg/mL concentrations of lyophilized supernatants containing azurin, but no inhibition zone at azurin-free lyophilized supernatants. When HUVEC, HT29, HCT116, and MCF7 cell lines were treated with lyophilized culture supernatants with azurin or without azurin, cell viability decreased with increasing concentrations of the supernatant. Furthermore, the supernatants containing azurin showed more anti-proliferative effect than the azurin-free supernatants. This work provides a practicable method to produce recombinant azurin in the food grade L. lactis strain. As a result, the recombinant L. lactis strain, producing azurin, can be used in the investigation of food biopreservatives and in the development of a therapeutic probiotic.


Assuntos
Azurina/genética , Clonagem Molecular/métodos , Lactococcus lactis/genética , Pseudomonas aeruginosa/genética , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Azurina/farmacologia , Bacillus cereus/efeitos dos fármacos , Linhagem Celular , Escherichia coli/efeitos dos fármacos , Amplificação de Genes , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transformação Genética
7.
Plant Mol Biol ; 100(4-5): 481-494, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31073810

RESUMO

KEY MESSAGE: Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and reliable resistance against a major poplar pathogen, Septoria musiva. Host defense peptides (HDPs), also known as cationic antimicrobial peptides, represent a diverse group of small membrane-active molecules that are part of the innate defense system of their hosts against pathogen invasion. Here we describe a strategy for development of poplar plants with enhanced HDP production and resistance to the commercially significant fungal pathogen Septoria musiva. The naturally occurring linear amphipathic α-helical HDP dermaseptin B1, which has 31 residues and originated from the skin secretion of arboreal frogs, was N-terminally modified (MsrA2) and evaluated in vitro for antifungal activity and phytotoxicity. The MsrA2 peptide inhibited germination of S. musiva conidia at physiologically relevant low micromolar concentrations that were non-toxic to poplar protoplasts. The nucleotide sequence of MsrA2, optimized for expression in plants, was introduced into the commercial hybrid poplar Populus nigra L. × P. maximowiczii A. Henry (NM6) via Agrobacterium-mediated transformation. Transgene expression was regulated by the pathogen-inducible poplar promoter win3.12T, a part of the poplar innate defense system. Most importantly, the induced accumulation of MsrA2 peptide in poplar leaves was sufficient to confer resistance against S. musiva. The antifungal resistance of plants with high MsrA2 expression and MsrA2 accumulation was strong and reproducible, and without deleterious effects on plant growth and development. These results provide an insight into development of new technologies for engineering durable disease resistance against major pathogens of poplar and other plants.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Ascomicetos/imunologia , Resistência à Doença/genética , Populus/imunologia , Genoma de Planta , Plantas Geneticamente Modificadas/imunologia , Populus/genética , Populus/microbiologia , Regiões Promotoras Genéticas , Transformação Genética , Transgenes
8.
BMC Plant Biol ; 19(1): 181, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060493

RESUMO

BACKGROUND: Castor (Ricinus communis L.) is an important seed oil crop. Castor oil is a highly demanded oil for several industrial uses. Current castor bean varieties suffer from low productivity and high risk of insect pests and diseases. High productive and pest/disease resistance varieties are needed. Lignin has been associated to the resistance for pest, disease and lodging. Lignin is produced from several metabolites of the phenylpropanoid pathway. PAL is the key enzyme of the phenylpropanoid pathway. The gene PAL may assist in the improvement of resistance of castor bean. RESULTS: The RcPAL CDs was amplified and its function was examined by transgenic overexpression and antisense expression, lignin histochemical staining, real-time PCR, lignin content measurement and morphological investigation. Its full length was 2145 bp, encoding 714 amino acids. The overexpression of RcPAL (7.2 times) increased significantly the PAL activity, dyeing depth of xylem cells and lignin content (14.44%), resulting in a significantly lower plant height, deeper and thicker blade, more green leaves, shorter internode, thicker stem diameter, and opposite in antisense expression plants (lignin content lowered by 27.1%), demonstrated that the gene RcPAL was a key gene in castor lignin biosynthesis. CONCLUSIONS: The gene RcPAL is a key gene in castor lignin biosynthesis and can be induced to express under mechanical damage stress. When up-regulated, it increased the lignin content significantly and dwarfed the plant height, and opposite when down-regulated. The gene RcPAL may assist in the improvement of resistance and plant type of castor bean.


Assuntos
Vias Biossintéticas/genética , Genes de Plantas , Lignina/biossíntese , Fenilalanina Amônia-Liase/genética , Ricinus/genética , Ricinus/metabolismo , Cinamatos/farmacologia , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas , Higromicina B/análogos & derivados , Higromicina B/farmacologia , Fenilalanina Amônia-Liase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Estresse Mecânico , Transformação Genética
9.
World J Microbiol Biotechnol ; 35(5): 77, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31069553

RESUMO

Ethylene is a volatile alkene which is used in large commercial scale as a precursor in plastic industry, and is currently derived from petroleum refinement. As an alternative production strategy, photoautotrophic cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 have been previously evaluated as potential biotechnological hosts for producing ethylene directly from CO2, by the over-expression of ethylene forming enzyme (efe) from Pseudomonas syringae. This work addresses various open questions related to the use of Synechococcus as the engineering target, and demonstrates long-term ethylene production at rates reaching 140 µL L-1 h-1 OD750-1 without loss of host vitality or capacity to produce ethylene. The results imply that the genetic instability observed earlier may be associated with the expression strategies, rather than efe over-expression, ethylene toxicity or the depletion of 2-oxoglutarate-derived cellular precursors in Synechococcus. In context with literature, this study underlines the critical differences in expression system design in the alternative hosts, and confirms Synechococcus as a suitable parallel host for further engineering.


Assuntos
Etilenos/biossíntese , Engenharia Metabólica/métodos , Fotossíntese/fisiologia , Synechococcus/genética , Synechococcus/metabolismo , Biotecnologia , Dióxido de Carbono/metabolismo , Clonagem Molecular , Tolerância a Medicamentos , Escherichia coli/genética , Etilenos/toxicidade , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Instabilidade Genômica , Ácidos Cetoglutáricos/metabolismo , Liases/genética , Liases/metabolismo , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Synechococcus/efeitos dos fármacos , Synechococcus/crescimento & desenvolvimento , Transformação Genética
10.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
11.
Plant Cell Rep ; 38(7): 825-833, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31139894

RESUMO

Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts. This has proven to be a powerful approach for the production of proteins at high levels. The second approach is based on nuclear transformation, followed by post-translational import of proteins from the cytosol into chloroplasts. In the nuclear transformation approach, foreign genes are stably integrated into the nuclear genome or transiently expressed in the nucleus by non-integrating T-DNA. Although this approach also has great potential for protein production at high levels, it has not been thoroughly investigated. In this review, we focus on nuclear transformation-based protein expression and its subsequent sequestration in chloroplasts, and summarize the different strategies used for high-level production of recombinant proteins. We also discuss future directions for further improvements in protein production in chloroplasts through nuclear transformation-based gene expression.


Assuntos
Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Cloroplastos/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Genética/genética
12.
Gene ; 709: 8-16, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31132514

RESUMO

Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboring cas9 gene and a guide RNA targeting umps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool for A. pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.


Assuntos
Ascomicetos/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genoma Fúngico , Protoplastos/metabolismo , Transformação Genética , Organismos Geneticamente Modificados , Saccharomyces cerevisiae/genética
13.
Mol Biotechnol ; 61(6): 461-468, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997667

RESUMO

Synthetic biology and genetic engineering in algae offer an unprecedented opportunity to develop species with traits that can help solve the problems associated with food and energy supply in the 21st century. In the green alga Chlamydomonas reinhardtii, foreign genes can be expressed from the chloroplast genome for molecular farming and metabolic engineering to obtain commodities and high-value molecules. To introduce these genes, selectable markers, which rely mostly on the use of antibiotics, are needed. This has risen social concern associated with the potential risk of horizontal gene transfer across life kingdoms, which has led to a quest for antibiotic-free selectable markers. Phosphorus (P) is a scarce nutrient element that most organisms can only assimilate in its most oxidized form as phosphate (Pi); however, some organisms are able to oxidize phosphite (Phi) to Pi prior to incorporation into the central metabolism of P. As an alternative to the use of the two positive selectable makers already available for chloroplast transformation in C. reinhardtii, the aadA and the aphA-6 genes, that require the use of antibiotics, we investigated if a phosphite-based selection method could be used for the direct recovery of chloroplast transformed lines in this alga. Here we show that following bombardment with a vector carrying the ptxD gene from Pseudomonas stutzeri WM88, only cells that integrate and express the gene proliferate and form colonies using Phi as the sole P source. Our results demonstrate that a selectable marker based on the assimilation of Phi can be used for chloroplasts transformation in a biotechnologically relevant organism. The portable selectable marker we have developed is, in more than 18 years, the latest addition to the markers available for selection of chloroplast transformed cells in C. reinhardtii. The ptxD gene will contribute to the repertoire of tools available for synthetic biology and genetic engineering in the chloroplast of C. reinhardtii.


Assuntos
Proteínas de Bactérias/genética , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , NADH NADPH Oxirredutases/genética , Fosfitos/metabolismo , Fósforo/metabolismo , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Proteínas de Bactérias/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Engenharia Genética/métodos , Marcadores Genéticos , Vetores Genéticos/química , Vetores Genéticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Fosfitos/farmacologia , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genética , Seleção Genética , Transformação Genética
14.
Bioengineered ; 10(1): 87-97, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-30957636

RESUMO

Expression of recombinant proteins fused to a novel glycomodule tag, termed hydroxyproline (Hyp)-O-glycosylated peptides (HypGP), was earlier found to boost secreted protein yields up to 500-fold in plant cell culture. Here, this technology was applied to the expression of human protease inhibitor α1-antitrypsin (AAT) in tobacco BY-2 cell culture. A designer HypGP tag composed of a 'Ala-Pro' motif of 20 units, or (AP)20, was engineered either at the N- or C-terminal end of AAT. The (AP)20 tag substantially increased the secreted yields of the recombinant AAT up to 34.7 mg/L. However, the (AP)20-tagged AAT products were frequently subjected to proteolytic processing. The intact AAT-(AP)20 along with some of the truncated AAT domains exhibited desired biological activity in inhibiting elastase. The results from this research demonstrated that the designer (AP)20 module engineered in BY-2 cells could function as a molecular carrier to substantially enhance the secreted yields of the recombinant AAT.


Assuntos
Elastase Pancreática/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Secretadas Inibidoras de Proteinases/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , alfa 1-Antitripsina/biossíntese , Sequência de Bases , Técnicas de Cultura de Células , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Glicosilação , Humanos , Elastase Pancreática/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Células Vegetais/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/isolamento & purificação , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/farmacologia , Tabaco/citologia , Tabaco/metabolismo , Transformação Genética , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/isolamento & purificação , alfa 1-Antitripsina/farmacologia
15.
Phytochemistry ; 162: 99-108, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30877900

RESUMO

A single-chain variable antibody fragment (scFv) library tested against the non-structural NSP5 protein of human rotavirus A was screened by a yeast two-hybrid system against three proteins derived from the RNA-dependent RNA polymerase (RdRp) of cucumber mosaic virus (CMV), with the aim of blocking their function and preventing viral infection once expressed in planta. The constructs tested were (i) '2a' consisting of the full-length 2a gene (839 amino acids, aa), (ii) 'Motifs' covering the conserved RdRp motifs (IV-VII) (132 aa) and (iii) 'GDD' located within the conserved RdRp motif VI (GDD, 22 aa). In yeast two-hybrid (Y2H) selection assays the '2a' and 'Motifs' constructs interacted with 96 and 25 library constructs, respectively, while the 'GDD' construct caused transactivation. Y2H-interacting scFvs were analyzed in vivo for their interaction with the 2a and Motifs proteins in a mammalian transient expression system. Eighteen tobacco lines stably transformed with four selected scFvs were produced and screened for resistance against two different CMV isolates. Different levels of resistance and rate of recovery were observed with CMV of both groups I and II, particularly in lines expressing intrabodies against the full-length 2a protein. This work describes for the first time the use of intrabodies against the RdRp of CMV to obtain plants that reduce infection of a pandemic virus, showing that the selected scFvs can modulate virus infection and induce premature recovery in tobacco plants.


Assuntos
Especificidade de Anticorpos , Cucumovirus/fisiologia , Engenharia Genética/métodos , RNA Replicase/imunologia , Anticorpos de Cadeia Única/imunologia , Tabaco/genética , Tabaco/fisiologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Cucumovirus/enzimologia , Plantas Geneticamente Modificadas , Anticorpos de Cadeia Única/química , Transformação Genética
16.
Gene ; 700: 176-178, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-30898720

RESUMO

The recent disclosure of a human embryo subjected to a genetic transformation using the CRISPR/cas9 system give rise to several concerns on ethical questions about its uncontrolled use in humans. Although CRISPR/cas9 has demonstrated its efficiency, this system still lacks the capability to avoid the introduction of undesirable mutations through the target genome. In this Letter, we present several undesirable impacts that CRISPR/cas9 system have in the genetic transformation of the human genome. We briefly discuss, using the very recent literature from distinct high impact journals, the main concerns related to CRISPR/cas9 to deal with off-target effects and how the research community has treated it.


Assuntos
Edição de Genes/métodos , Transformação Genética , Sistemas CRISPR-Cas , Edição de Genes/ética , Genoma Humano , Humanos , RNA Guia
17.
Vet Microbiol ; 230: 14-22, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827380

RESUMO

An increasing number of studies suggest that cutaneous papillomaviruses (PVs) might be involved in skin carcinogenesis. However, only a few animal PVs have been investigated regard to their transformation properties. Here, we investigate and compare the oncogenic potential of 2 ovine Delta and Dyokappa PVs, isolated from ovine skin lesions, in vitro and ex vivo. We demonstrate that both OaPV4 (Delta) and OaPV3 (Dyokappa) E6 and E7 immortalize primary sheep keratinocytes and efficiently deregulate pRb pathway, although they seem unable to alter p53 activity. Moreover, OaPV3 and OaPV4-E6E7 expressing cells show different shape, doubling time, and clonogenic activities, providing evidence for a stronger transforming potential of OaPV3 respect to OaPV4. Also, similarly to high-risk mucosal and cutaneous PVs, the OaPV3-E7 protein, constantly expressed in sheep squamous cell carcinomas, binds pRb with higher affinity compared to the E7 encoded by OaPV4, a virus associated to fibropapilloma. Finally, we found that OaPV3 and OaPV4-E6E7 determine upregulation of the pro-proliferative proteins cyclin A and cdk1 in both human and ovine primary keratinocytes. Collectively, results provide evidence for implication of ovine PVs in cutaneous proliferative lesions and skin cancer progression, and indicate sheep as a possible animal model for the study of cutaneous lesions and malignancies.


Assuntos
Queratinócitos/virologia , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas E7 de Papillomavirus/genética , Pele/virologia , Transformação Genética , Animais , Proteína Quinase CDC2/genética , Células Cultivadas , Ciclina A/genética , Deltapapillomavirus/genética , Deltapapillomavirus/isolamento & purificação , Humanos , Camundongos , Células NIH 3T3 , Ovinos , Pele/patologia , Regulação para Cima
18.
BMC Res Notes ; 12(1): 191, 2019 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-30925937

RESUMO

OBJECTIVES: The fission yeast Schizosaccharomyces pombe is predicted to encode ~ 200 proteins of < 100 amino acids, including a number of previously uncharacterised proteins that are found conserved in related Schizosaccharomyces species only. To begin an investigation of the function of four of these so-called microproteins (designated Smp1-Smp4), CRISPR-Cas9 genome editing technology was used to delete the corresponding genes in haploid fission yeast cells. RESULTS: None of the four microprotein-encoding genes was essential for viability, meiosis or sporulation, and the deletion cells were no more sensitive to a range of cell stressors than wild-type, leaving the function of the proteins unresolved. During CRISPR-Cas9 editing however, a number of strains were isolated in which additional sequences were inserted into the target loci at the Cas9 cut site. Sequencing of the inserts revealed these to be derived from the chum salmon Oncorhynchus keta, the source of the carrier DNA used in the S. pombe transformation.


Assuntos
Sistemas CRISPR-Cas , DNA/genética , Deleção de Genes , Edição de Genes/métodos , Genoma Fúngico/genética , Schizosaccharomyces/genética , Animais , Sequência de Bases , Genes Fúngicos/genética , Oncorhynchus keta/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Análise de Sequência de DNA/métodos , Homologia de Sequência do Ácido Nucleico , Transformação Genética
19.
Mol Biotechnol ; 61(5): 332-344, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30830513

RESUMO

The successful introduction of isopentenyl transferase (IPT) gene into perennial ryegrass, cultivars Numan and Grassland using Agrobacterium tumefaciens via three explants (callus, seed and meristem tip) under three individual experiment was evaluated. In the first experiment, the calli were inoculated with LBA4404 Agrobacterium strain under vacuum, heat and in combination of both at 42 °C for 5 min followed by vacuum treatment (390 mm Hg pressure) for 15 min. Sonication-assisted Agrobacterium-mediated transformation (SAAT) was applied for seed and meristem tip transformation of perennial ryegrass for the first time. Results showed positive effects of heat treatment on transformation efficiency during Agro-infection in both cultivars. However, heat shock treatment was more effective in 'Grassland' than 'Numan' (14.2% vs 9.2%). In addition, high transformation efficiency of about 46.65% and 29.15% was observed using meristem tip explants of 'Grassland' and 'Numan' based on IPT and RD29A positive PCR results, respectively. Seed transformation efficiency in 'Grassland' and 'Numan' under SAAT method reached to 37.5% and 16.65%, respectively. Results of these experiments revealed that LBA4404 strain was more efficient than GV3101 in transformation of both perennial ryegrass cultivars. The DNA-blot analysis confirmed that a single T-DNA copy of the IPT gene was integrated into the genomic DNA of the positive transgenic T0 plants which obtained from callus and meristem tip explants of 'Grassland' after heat and SAAT treatment, respectively. Because monocots are not the host of Agrobacterium tumefaciens, this novel protocol can be used in further experiments on genetic transformation of perennial ryegrass cultivars.


Assuntos
Agrobacterium tumefaciens/genética , Alquil e Aril Transferases/genética , Lolium/genética , Transfecção/métodos , Alquil e Aril Transferases/metabolismo , Temperatura Alta , Lolium/crescimento & desenvolvimento , Lolium/microbiologia , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sonicação , Transformação Genética , Vácuo
20.
Appl Microbiol Biotechnol ; 103(8): 3239-3248, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30877356

RESUMO

Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes , Microalgas/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica , Marcação de Genes , Engenharia Genética , RNA Guia/genética , Transformação Genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA