Table2_Five Visual and Olfactory Target Genes for RNAi in Agrilus Planipennis.DOC

RNA interference (RNAi) is a widely used technique for gene function researches and recently pest controls. It had been applied in emerald ash borer (EAB Agrilus planipennis) larvae and adults, and achieved significant interference effects, whether by ingesting or microinjecting. Feeding in the phloem and cambial regions, the larvae of A. planipennis are difficult to be controlled by conventional insecticides, so adult stage is the critical stage for EAB control. However, the target genes of adult stage of A. planipennis need to be further screened. Here, we preliminarily screened five potential target genes of vision and olfaction for RNAi in A. planipennis. Three odorant binding proteins (OBPs) and three opsins, which expressed significantly different between newly emerged and sexually mature EABs (OBP5, OBP7, OBP10, LW opsin 1 and UV opsin 2) or highly in sexually mature male EAB (UV opsin 3), were selected as targets to design primers for gene silencing. After dsRNA injection, the gene expression levels were determined by real-time quantitative PCR. We found that the expression levels of five genes were significantly down-regulated, during the 4 days after dsRNA injection. Among these genes, the expression of LW opsin 1 was down-regulated the most, causing a reduction of 99.1% compared with the control treated with EGFP dsRNA, followed by UV opsin 3 (97.4%), UV opsin 2 (97.0%), OBP7 (96.2%), and OBP10 (88.7%). This study provides a basis for further RNAi-based new controlling method development of A. planipennis at adult stage.

RNA干扰（RNA interference, RNAi）是一项广泛应用于基因功能研究，且近年来被用于害虫防治的常用生物技术。该技术已应用于白蜡窄吉丁（Agrilus planipennis，简称EAB）的幼虫与成虫，无论是取食法还是显微注射法均取得了显著的干扰效果。由于白蜡窄吉丁幼虫取食韧皮部与形成层区域，传统杀虫剂难以对其起到防控作用，因此成虫期是该害虫防治的关键时期。但目前针对白蜡窄吉丁成虫期的RNAi靶基因仍有待进一步筛选。本研究初步筛选出5个可用于白蜡窄吉丁RNAi防控的视觉与嗅觉相关潜在靶基因：选取3个气味结合蛋白（odorant binding proteins, OBPs）与3个视蛋白作为靶标设计引物以进行基因沉默，其中OBP5、OBP7、OBP10、长波视蛋白1（LW opsin 1）与紫外线视蛋白2（UV opsin 2）在初羽化与性成熟的白蜡窄吉丁成虫间存在显著表达差异，而紫外线视蛋白3（UV opsin 3）则在性成熟雄虫中高表达。通过双链RNA（dsRNA）注射处理后，采用实时定量PCR检测各靶基因的表达水平。结果显示，在dsRNA注射后的4天内，5个靶基因的表达水平均显著下调。其中长波视蛋白1的下调效果最为显著，相较于增强绿色荧光蛋白双链RNA（EGFP dsRNA）处理的对照组，其表达量降低了99.1%；其次依次为紫外线视蛋白3（97.4%）、紫外线视蛋白2（97.0%）、OBP7（96.2%）与OBP10（88.7%）。本研究为后续开发基于RNAi技术的白蜡窄吉丁成虫期新型防控方法提供了理论依据。