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      Transcriptome Analysis and Screening for Potential Target Genes for RNAi-Mediated Pest Control of the Beet Armyworm, Spodoptera exigua

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          Abstract

          The beet armyworm, Spodoptera exigua (Hübner), is a serious pest worldwide that causes significant losses in crops. Unfortunately, genetic resources for the beet armyworm is extremely scarce. To improve these resources we sequenced the transcriptome of S. exigua representing all stages including eggs, 1 st to 5 th instar larvae, pupae, male and female adults using the Illumina Solexa platform. We assembled the transcriptome with Trinity that yielded 31,414 contigs. Of these contigs, 18,592 were annotated as protein coding genes by Blast searches against the NCBI nr database. It has been shown that knockdown of important insect genes by dsRNAs or siRNAs is a feasible mechanism to control insect pests. The first key step towards developing an efficient RNAi-mediated pest control technique is to find suitable target genes. To screen for effective target genes in the beet armyworm, we selected nine candidate genes. The sequences of these genes were amplified using the RACE strategy. Then, siRNAs were designed and chemically synthesized. We injected 2 µl siRNA (2 µg/µl) into the 4 th instar larvae to knock down the respective target genes. The mRNA abundance of target genes decreased to different levels (∼20–94.3%) after injection of siRNAs. Knockdown of eight genes including chitinase7, PGCP, chitinase1, ATPase, tubulin1, arf2, tubulin2 and arf1 caused a significantly high level of mortality compared to the negative control (P<0.05). About 80% of the surviving insects in the siRNA-treated group of five genes (PGCP, chitinase1, tubulin1, tubulin2 and helicase) showed retarded development. In chitinase1-siRNA and chitinase7-siRNA administered groups, 12.5% survivors exhibited “half-ecdysis”. In arf1-siRNA and arf2-siRNA groups, the body color of 15% became black 48 h after injections. In summary, the transcriptome could be a valuable genetic resource for identification of genes in S. exigua and this study provided putative targets for RNAi pest control.

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          Most cited references 27

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          Mechanisms of gene silencing by double-stranded RNA.

          Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA silencing or RNA interference. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA silencing machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.
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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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              Mechanisms of dsRNA uptake in insects and potential of RNAi for pest control: a review.

              RNA interference already proved its usefulness in functional genomic research on insects, but it also has considerable potential for the control of pest insects. For this purpose, the insect should be able to autonomously take up the dsRNA, for example through feeding and digestion in its midgut. In this review we bring together current knowledge on the uptake mechanisms of dsRNA in insects and the potential of RNAi to affect pest insects. At least two pathways for dsRNA uptake in insects are described: the transmembrane channel-mediated uptake mechanism based on Caenorhabditis elegans' SID-1 protein and an 'alternative' endocytosis-mediated uptake mechanism. In the second part of the review dsRNA feeding experiments on insects are brought together for the first time, highlighting the achievement of implementing RNAi in insect control with the first successful experiments in transgenic plants and the diversity of successfully tested insect orders/species and target genes. We conclude with points of discussion and concerns regarding further research on dsRNA uptake mechanisms and the promising application possibilities for RNAi in insect control. Copyright (c) 2009 Elsevier Ltd. All rights reserved.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                18 June 2013
                : 8
                : 6
                Affiliations
                [1 ]Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China
                [2 ]Key Laboratory of Integrated Management of Crop Diseases and Pests, Ministry of Education, Nanjing Agricultural University, Nanjing, China
                Swedish University of Agricultural Sciences, Sweden
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: HL FL. Performed the experiments: HL YX. Analyzed the data: HL ZZ. Contributed reagents/materials/analysis tools: WJ FL. Wrote the paper: HL FL.

                Article
                PONE-D-12-37666
                10.1371/journal.pone.0065931
                3688801
                23823756

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 10
                Funding
                This work was in partial supported by the National High Technology Research and Development Program (“863”Program, http://www.863.gov.cn) of China (2012AA101505), the Basic Research Development Program of China (973 Program, 2012CB114100, http://www.973.gov.cn/), the National Science Foundation of China (31171843, http://www.nsfc.gov.cn/) and the Jiangsu Science Foundation for Distinguished Young Scholars (BK2012028, http://www.jstd.gov.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Agriculture
                Pest Control
                Integrated Control
                Biology
                Computational Biology
                Molecular Genetics
                Gene Identification and Analysis
                Sequence Analysis
                Plant Science
                Plant Pathology
                Plant Pests
                Zoology
                Entomology

                Uncategorized

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