Dias Naymã, Cagliari Deise, Kremer Frederico Schmitt, Rickes Leticia Neutzling, Nava Dori Edson, Smagghe Guy, Zotti Moisés
Molecular Entomology and Applied Bioinformatics Laboratory, Faculty of Agronomy, Department of Crop Protection, Federal University of Pelotas, Pelotas, Brazil.
Bioinformatics and Proteomics Laboratory, Technological Development Center, Federal University of Pelotas, Pelotas, Brazil.
Front Physiol. 2019 Jun 27;10:794. doi: 10.3389/fphys.2019.00794. eCollection 2019.
RNA interference (RNAi) technology has been used in the development of approaches for pest control. The presence of some essential genes, the so-called "core genes," in the RNAi machinery is crucial for its efficiency and robust response in gene silencing. Thus, our study was designed to examine whether the RNAi machinery is functional in the South American (SA) fruit fly (Diptera: Tephritidae) and whether the sensitivity to the uptake of double-stranded RNA (dsRNA) could generate an RNAi response in this fruit fly species. To prepare a transcriptome database of the SA fruit fly, total RNA was extracted from all the life stages for later cDNA synthesis and Illumina sequencing. After the transcriptome assembly and gene annotation, the transcriptome was screened for RNAi pathway genes, as well as the duplication or loss of genes and novel target genes to dsRNA delivery bioassays. The dsRNA delivery assay by soaking was performed in larvae to evaluate the gene-silencing of , and the upregulation of and after dsRNA delivery was analyzed to verify the activation of siRNAi machinery. We tested the stability of dsRNA using dsGFP with an incubation of larvae body fluid (hemolymph). We identified 55 genes related to the RNAi machinery with duplication and loss for some genes and selected 143 different target genes related to biological processes involved in post-embryonic growth/development and reproduction of . Larvae soaked in dsRNA (dsV-ATPase) solution showed a strong knockdown of V-ATPase after 48 h, and the expression of and responded with an increase upon the exposure to dsRNA. Our data demonstrated the existence of a functional RNAi machinery in the SA fruit fly, and we present an easy and robust physiological bioassay with the larval stages that can further be used for screening of target genes at organisms' level for RNAi-based control of fruit fly pests. This is the first study that provides evidence of a functional siRNA machinery in the SA fruit fly.
RNA干扰（RNAi）技术已被用于害虫防治方法的开发。RNAi机制中某些必需基因（即所谓的“核心基因”）的存在对于其在基因沉默中的效率和强大反应至关重要。因此，我们的研究旨在检验RNAi机制在南美果蝇（双翅目：实蝇科）中是否具有功能，以及对双链RNA（dsRNA）摄取的敏感性是否能在这种果蝇物种中产生RNAi反应。为了制备南美果蝇的转录组数据库，从其所有生命阶段提取总RNA，用于后续的cDNA合成和Illumina测序。在进行转录组组装和基因注释后，筛选转录组中的RNAi途径基因，以及基因的复制或缺失和dsRNA递送生物测定的新靶基因。通过浸泡进行dsRNA递送测定，以评估幼虫中的基因沉默，并分析dsRNA递送后某些基因的上调情况，以验证siRNAi机制的激活。我们使用dsGFP在幼虫体液（血淋巴）中孵育来测试dsRNA的稳定性。我们鉴定了55个与RNAi机制相关的基因，其中一些基因存在复制和缺失，并选择了143个与南美果蝇胚胎后生长/发育和繁殖所涉及的生物学过程相关的不同靶基因。浸泡在dsRNA（dsV-ATPase）溶液中的幼虫在48小时后显示出V-ATPase的强烈敲低，并且某些基因的表达在暴露于dsRNA后随着增加而响应。我们的数据证明了南美果蝇中存在功能性RNAi机制，并且我们提出了一种简单而强大的幼虫阶段生理生物测定方法，该方法可进一步用于在生物体水平筛选基于RNAi的果蝇害虫防治的靶基因。这是第一项提供南美果蝇中功能性siRNA机制证据的研究。
