RESUMO
The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabrotica virgifera virgifera LeConte) via overexpression of an inverted repeat sequence bearing a portion of the putative corn rootworm orthologue of yeast Snf7 (DvSnf7), an essential component of insect cell receptor sorting. A quantitative assay was developed to detect DvSnf7 sense strand-containing dsRNA transcripts that is based on the QuantiGene Plex 2.0 RNA assay platform from Affymetrix. The QuantiGene assay utilizes cooperative binding of multiple oligonucleotide probes with specificity for the target sequence resulting in exceptionally high assay specificity. Successful implementation of this assay required heat denaturation in the presence of the oligonucleotide probes prior to hybridization, presumably to dissociate primary transcripts carrying the duplex dsRNA structure. The dsRNA assay was validated using a strategy analogous to the rigorous enzyme-linked immunosorbent assay evaluations that are typically performed for foreign proteins expressed in transgenic plants. Validation studies indicated that the assay is sensitive (to 10 pg of dsRNA/g of fresh tissue), highly reproducible, and linear over â¼2.5 logs. The assay was validated using purified RNA from multiple maize tissue types, and studies indicate that the assay is also quantitative in crude tissue lysates. To the best of our knowledge, this is the first report of a non-polymerase chain reaction-based quantitative assay for dsRNA-containing transcripts, based on the use of the QuantiGene technology platform, and will broadly facilitate characterization of dsRNA in biological and environmental samples.
Assuntos
Besouros/genética , Proteínas de Insetos/genética , Hibridização de Ácido Nucleico/métodos , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , RNA de Cadeia Dupla/genética , Zea mays/genética , Animais , Besouros/fisiologia , Proteínas de Insetos/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/prevenção & controle , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/parasitologia , RNA de Cadeia Dupla/metabolismo , Transgenes , Zea mays/metabolismo , Zea mays/parasitologiaRESUMO
Transgenic lines of soybean, Glycine max (L.) Merrill, expressing a synthetic cry1A gene (tic107) from Bacillus thuringiensis (Bt), were evaluated in screenhouse and conventional field trials for efficacy against lepidopteran pests. In screenhouse trials, Bt soybean and negative checks (isogenic segregants and parental lines) were evaluated against Anticarsia gemmatalis Hübner and Pseudoplusia includens (Walker) in the United States and against A. gemmatalis, Epinotia aporema (Walsingham), Rachiplusia nu (Guenée), and Spilosoma virginica (F.) in Argentina. Bt soybean exhibited virtually complete efficacy against each of these pests, whereas negative checks suffered significant damage. Bt soybean and negative checks also were evaluated in conventional trials against native populations of A. gemmatalis and P. includens in the southeastern United States. Each of these insects caused significant damage to negative checks in one or more locations, whereas Bt soybean exhibited virtually complete efficacy against these pests. In the laboratory, lyophilized leaf tissues from Bt soybean incorporated in artificial diet at a concentration representing a 25-fold dilution of fresh tissue caused complete mortality of A. gemmatalis and near complete mortality of P. includens neonates after 11 d, whereas mortality on negative checks did not exceed 10% for either insect. Average TIC107 expression approached or exceeded 50 microg/g fresh weight at V3 stage of growth and 200 microg/g by R6 stage of growth. These results demonstrate that expression of TIC107 in soybean can not only achieve highly efficacious control of several lepidopterans under field conditions but also provide a high dose for effective insect resistance management.
Assuntos
Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Endotoxinas/genética , Expressão Gênica , Glycine max/genética , Lepidópteros , Controle Biológico de Vetores , Plantas Geneticamente Modificadas/genética , Animais , Argentina , Toxinas de Bacillus thuringiensis , Proteínas Hemolisinas , Estados UnidosRESUMO
The present study reports the structural and functional changes of myocardial muscarinic receptor during early and late septic shock, Septic shock was induced by caecum ligation and puncture (CLP). The results showed that the number of M receptor on sarcolemma (SL) increased and that on the light vesicle (LV) decreased during early septic shock. The (3)H-QNB binding of M receptor on SL increased by 33.37%. By contrast during late septic shock, the number of M receptor on LV increased and that on SL decreased. The (3)H-QNB binding of LV M receptor was increased by 29.26%. At the same time phosphorylation of the M receptor was decreased during early septic shock and increased during late septic shock. These results suggest that the changes of M receptors may be related to the myocardial dysfunction during septic shock.
