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      ARN interferente: Potenciales usos en genómica funcional y control genético de Hypothenemus hampei (Coleoptera: Scolytinae) Translated title: RNA interference: potential uses on functional genomics and genetic control of Hypothenemus hampei (Coleoptera: Scolytinae)

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          Abstract

          Los organismos eucarióticos, incluidos los insectos, poseen un mecanismo para el silenciamiento de genes específicos mediante pequeñas secuencias de ARN de doble cadena (ARNdc), proceso conocido como ARN de interferencia (ARNi). El objetivo de esta investigación fue implementar esta herramienta para evaluar genes candidatos que puedan usarse como estrategia genética de control de la broca del café. Se utilizó una metodología de ingesta de gotas con diferentes ARNdc y diferentes concentraciones depositadas en la cavidad preoral de larvas de primer instar de la broca. Luego, las larvas se criaron en una dieta artificial suplementada con los respectivos ARNdc. Previamente, se comprobó la efectividad de la ingesta por vía oral mediante dos estrategias de control: a) ingesta de un compuesto fluorescente (Calcofluor®) en el tracto digestivo del insecto y b) ingesta de una mezcla comercial de inhibidores de proteasas. Los genes seleccionados para el silenciamiento fueron los que codifican en la broca las proteínas mananasa, xilanasa, citocromo P450 mono-oxigenasa (2 genes), ATPasa D, α-tubulina y actina. Se encontró que los ARNdc de los citocromos P450 produjeron el mayor efecto causando una mortalidad de las larvas de un 64% y 52% respectivamente, seguido de los ARNdc de los genes mananasa y xilanasa, con un porcentaje de mortalidad de 37% y 33%, respectivamente, para la máxima concentración evaluada.

          Translated abstract

          Eukaryotic organisms, including insects, have a mechanism for specific gene silencing by short sequences of double-stranded RNA (dsRNA), such a process is known as RNA interference (RNAi). The objective of this research was to follow up this technique to screen candidate genes for the genetic control of the coffee berry borer Hypothenemus hampei. A methodology of feeding by oral drops containing different dsRNAs and dsRNAs´ dosages that were placed in the preoral cavity of first instar larvae was used. Then, the larvae were reared on an artificial diet supplemented with the respective dsRNA. Previously, we verified the effectiveness of the dsRNA feeding using two control strategies: a) intake of a fluorescent compound (CalcofluorTM) and b) intake of a commercial mixture of protease inhibitors. The genes coding for mannanase, xylanase, cytochrome P450 mono-oxygenase (two genes), ATPase D, α-tubulin and actin, expressed in the coffee berry borer, were selected for silencing. We found that dsRNA of cytochromes P450 produced the greatest effect, causing larval mortality of 64% and 52% respectively, followed by dsRNA of mannanase and xylanase, with a mortality rate of 37% and 33%, respectively, for the highest concentration tested.

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          Most cited references25

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          Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.

          Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
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            Control of coleopteran insect pests through RNA interference.

            Commercial biotechnology solutions for controlling lepidopteran and coleopteran insect pests on crops depend on the expression of Bacillus thuringiensis insecticidal proteins, most of which permeabilize the membranes of gut epithelial cells of susceptible insects. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. Toward this end, we demonstrate that ingestion of double-stranded (ds)RNAs supplied in an artificial diet triggers RNA interference in several coleopteran species, most notably the western corn rootworm (WCR) Diabrotica virgifera virgifera LeConte. This may result in larval stunting and mortality. Transgenic corn plants engineered to express WCR dsRNAs show a significant reduction in WCR feeding damage in a growth chamber assay, suggesting that the RNAi pathway can be exploited to control insect pests via in planta expression of a dsRNA.
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              Silencing a cotton bollworm P450 monooxygenase gene by plant-mediated RNAi impairs larval tolerance of gossypol.

              We identify a cytochrome P450 gene (CYP6AE14) from cotton bollworm (Helicoverpa armigera), which permits this herbivore to tolerate otherwise inhibitory concentrations of the cotton metabolite, gossypol. CYP6AE14 is highly expressed in the midgut and its expression correlates with larval growth when gossypol is included in the diet. When larvae are fed plant material expressing double-stranded RNA (dsRNA) specific to CYP6AE14, levels of this transcript in the midgut decrease and larval growth is retarded. Both effects are more dramatic in the presence of gossypol. As a glutathione-S-transferase gene (GST1) is silenced in GST1 dsRNA-expressing plants, feeding insects plant material expressing dsRNA may be a general strategy to trigger RNA interference and could find applications in entomological research and field control of insect pests.
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                Author and article information

                Journal
                rcen
                Revista Colombiana de Entomología
                Rev. Colomb. Entomol.
                Sociedad Colombiana de Entomología (Bogotá, Distrito Capital, Colombia )
                0120-0488
                2665-4385
                December 2011
                : 37
                : 2
                : 167-172
                Affiliations
                [04] Caldas orgnameCENICAFE Colombia josed.rubio@ 123456cafedecolombia.com
                [05] Caldas orgnameCENICAFE Colombia Ricardo.Acuna@ 123456cafedecolombia.com
                [03] Caldas orgnameCENICAFE Colombia claudia.florez@ 123456cafedecolombia.com
                [02] Caldas orgnameCENICAFE Colombia beatrizelena.padilla@ 123456cafedecolombia.com
                [01] Caldas orgnameUniversidad Nacional de Colombia Colombia carolina.aguilera@ 123456cafedecolombia.com
                Article
                S0120-04882011000200001 S0120-0488(11)03700201
                31894db5-f286-4c68-bf2f-e57e51890d56

                This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

                History
                : 11 November 2010
                : 22 September 2011
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 26, Pages: 6
                Product

                SciELO Colombia

                Self URI: Texto completo solamente en formato PDF (ES)
                Categories
                Sección agrícola

                RNAi,Mannanase,Xylanase,Cytochromes P450,ARNi,Mananasa,Xilanasa,Citrocromos P450

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