Vyas Meenal, Raza Amir, Ali Muhammad Yousaf, Ashraf Muhammad Aleem, Mansoor Shahid, Shahid Ahmad Ali, Brown Judith K
School of Plant Sciences, University of Arizona, Tucson, Arizona, United States of America.
National Institute for Biotechnology & Genetic Engineering (NIBGE), Faisalabad, Pakistan.
PLoS One. 2017 Jan 3;12(1):e0168921. doi: 10.1371/journal.pone.0168921. eCollection 2017.
Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly 'gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit α, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalase1, and Trehalose transporter1. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport-associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit α and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.
对农业害虫和植物病毒载体烟粉虱（Bemisia tabaci (Genn.)）的防治依赖于化学杀虫剂的使用。RNA干扰（RNAi）是真核生物（包括昆虫）中一种依赖同源性的先天免疫反应，在双链RNA（dsRNA）识别具有100%序列同源性的相应转录本后，导致其降解。在本研究中，从烟粉虱B型烟粉虱消化道或“肠道”构建的电子注释转录组文库中选择了六个粉虱“肠道”基因，并测试了与每个相应基因靶点具有100%序列同源性的dsRNA摄入后敲低效率。候选基因有：乙酰胆碱受体亚基α、α-葡萄糖苷酶1、水通道蛋白1、热休克蛋白70、海藻糖酶1和海藻糖转运蛋白1。在基因特异性功能生物测定中进一步测试了RNAi敲低的效果，并在处理后六天内每隔24小时记录死亡率。基于定量PCR分析，所有六个测试基因的表达均显著降低。中到高的粉虱死亡率与渗透调节、糖代谢和糖运输相关基因的下调有关，表明粉虱的生存能力与RNAi结果相关。沉默的乙酰胆碱受体亚基α和热休克蛋白70基因最初显示粉虱死亡率较低，然而，分别在杀虫剂处理或高温处理后，敲低效率显著提高并观察到死亡，表明敲低后敏感性增强可能与系统性沉默有关。肠道特异性dsRNA的口服给药，结合基因表达的定量PCR分析和将敲低与死亡率相关联的相应基因特异性生物测定，为功能基因组学分析和发现潜在的dsRNA生物农药靶点提供了一种可行的方法。该方法可应用于功能基因组学分析，以促进物种特异性dsRNA介导的对其他非模式半翅目的控制。
