RESUMO
Development of RNAi-based therapeutics is a fast growing field of pharmaceutical industry. Using plants for production of pharmaceutically valuable siRNAs may have significant advantages of cost-effectiveness, scalability and low risk of contamination with human pathogens. If edible plant species are genetically engineered to synthesize siRNAs, the costly stage of target product purification may be omitted. We describe the establishment of transgenic lettuce plants producing shRNA targeting delta isoform of protein kinase C (PKC-delta), an effective target for RNAi-based treatment of arterial hypertension. Transgenic lettuce plants were obtained by Agrobacterium-mediated transformation with genetic constructs harboring antiPKC and scrambled (control) shRNA genes. The presence of transgenes was proved by PCR analysis, and the accumulation of antiPKC shRNA was estimated using RT-qPCR technique. Six transgenic lettuce lines showed varying levels of antiPKC shRNA expression with the highest value reaching 14 ± 9 % of highly abundant endogenous lettuce micro RNA (miR156a), or 12.7 fmol/g dry weight. Plants carrying either antiPKC or scrambled shRNA genes flowered normally, but did not produce seeds. The described transgenic lettuce plants accumulating antiPKC siRNA are the subject for animal testing and can be considered as a raw material for the development of novel antihypertensive drugs.
Assuntos
Agrobacterium/genética , Anti-Hipertensivos/metabolismo , Engenharia Genética/métodos , Lactuca/genética , Proteína Quinase C-delta/genética , RNA Interferente Pequeno/genética , Agrobacterium/metabolismo , Animais , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Hipertensão/enzimologia , Hipertensão/genética , Hipertensão/patologia , Hipertensão/terapia , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Plantas Geneticamente Modificadas , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , RNA Interferente Pequeno/metabolismo , Transformação GenéticaRESUMO
In order to investigate the high temperature tolerance of spring canola plants (Brassica napus L.) constitutively expressing cyp11A1 gene which encodes bovine cytochrome P450(scc) the growth features were analyzed under short time heat stress (42 degrees C) in growth chamber. Earlier it was documented that results of the heat tolerance test positively correlated with improvement of high temperature resistance in field trial. Higher relative water content (by 13%) and superoxide dismutase (SOD) activity, lower electrolyte leakage (up 1.4-fold) and smaller increase in chlorophyll a and carotenoid contents in cyp11A1 canola leaves in comparison with wild-type plants under stress allowed to conclude cyp11A1 plants are more tolerant to high temperature than the control ones. We suppose that SOD activity increase which revealed in our transgenic canola in normal condition plays the defining role in the biochemical alterations in plant metabolism for the thermotolerance improvement. SOD activity increment could be caused by heterologous cytochrome P450(scc) activity which resulted in the superoxide radical formation. Cyp11A1 canola plants might be resistant to the other stress conditions of different origin.
Assuntos
Brassica napus/enzimologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Temperatura Alta , Plantas Geneticamente Modificadas/enzimologia , Estresse Fisiológico , Animais , Antioxidantes/metabolismo , Brassica napus/genética , Brassica napus/crescimento & desenvolvimento , Bovinos , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Fotossíntese/fisiologia , Pigmentos Biológicos/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Estresse Fisiológico/genéticaRESUMO
Superoxide dismutase (SOD) activity was investigated in leaves of transgenic canola plants which expressed heterologous genes of different origin, namely 1) herbicide resistance genes (bar and simultaneously bar and epsps); 2) DesC desaturase gene (desC) of cyanobacterium Synechococcus vulcanus; 3) human interferon alpha2b gene (huLFN-alpha2b); 4) esxA::fbpB(deltaTMD) fused gene, encoding ESAT-6 and Ag85b Mycobacterium tuberculosis proteins, inducing immune response against tuberculosis; 5) cyp11A1 gene of cytochrome P450(scc) from bovine adrenal cortex mitochondria. Introduction of herbicide resistance genes as well as desaturase gene of cyanobacterium and mycobacterium's genes did not change leaf SOD activity. At the same time it was shown that cyp11A1 and huIFN-alpha2b canola have increased leaf SOD activity up 58 and 33%, respectively, compared with control ones in non-stress conditions. It may be a prerequisite for improved resistance of these plants to the stressors of different origin.
Assuntos
Brassica napus/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Superóxido Dismutase/metabolismo , Transgenes , Brassica napus/genética , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Transformação GenéticaRESUMO
During the last decade interferons are regarded as potent candidates for generation of plant-based edible vaccines because of broad spectrum of antiviral activities and adjuvant properties. Establishment and certification of numerous interferon producing plant systems requests development of fast and efficient multiplex PCR protocol for the transgene detection in GM plants. Here we represent a protocol for simultaneous amplification in one assay of fragments of hIFN alpha 2b gene and two control genes, namely virD1 of Agrobacterium tumefaciens and conservative region of plant actin gene.
Assuntos
Brassica napus/genética , Genes Bacterianos , Genes de Plantas , Interferon-alfa/genética , Nicotiana/genética , Vacinas Virais/biossíntese , Actinas/análise , Actinas/genética , Agrobacterium tumefaciens/genética , Antivirais , Primers do DNA/genética , Eletroforese em Gel de Ágar , Humanos , Interferon alfa-2 , Interferon-alfa/biossíntese , Reação em Cadeia da Polimerase Multiplex/métodos , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Transformação Genética , Transgenes , Vacinas Virais/genéticaRESUMO
Spring rapeseed transgenic lines expressing human interferon alpha 2b were created by Agrobacterium-mediated transformation of aseptic plant leaf explants. The maximum antiviral activity of the leaf extracts reached 4500 IU/g fresh weight. It was determined that the antioxidant activity and the activity of an enzyme of plant antioxidant system--superoxide dismutase (SOD)--in the leaf tissues of transgenic plants increased compared to controls. There were no correlations between the interferon and antioxidant activities, as well as between SOD and interferon activities. Using the obtained transgenic rapeseed plants with high interferon and antioxidant activities as a feed additive for animals might have preventive effect on their body, increasing resistance to infections of various origins.
Assuntos
Brassica napus/genética , Interferon-alfa/genética , Plantas Geneticamente Modificadas/genética , Animais , Antioxidantes/metabolismo , Brassica napus/química , Brassica napus/enzimologia , Células Cultivadas , Efeito Citopatogênico Viral/efeitos dos fármacos , Humanos , Interferon alfa-2 , Extratos Vegetais/farmacologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/enzimologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Superóxido Dismutase/metabolismo , Suínos , Transformação Genética , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacosRESUMO
Phosphinothricin (PPT) resistant hybrid plants between Brassica napus L. cv. Kalinovsky and Orychophragmus violaceus (L.) O.E. Shulz. were obtained as a result of somatic hybridization experiments. The hybrids inherited PPT resistance from O. violaceus plants which were previously transformed by the vector containing Spm/dSpm Zea mays transposon system with bar gene located within the nonautonomous transposon. The obtained plants had intermediate morphology. Their hybrid nature has been confirmed by isozyme (esterase and amilase activity) and PCR (bar, gus, Spm/dSpm integration) analyses. The hybrids combined B. napus plastom and O. violaceus mithochondrion that was revealed by PCR-RFLP. The hybrid plants might be included to rapeseed breeding programme after examination of their oil quality as well as to chloroplast transformation experiments that is still urgent for B. napus.
