【摘要】 【目的】提供一种用于提高橘小实蝇Bactrocera dorsalis中的RNA干扰(RNA interference, RNAi)效率和双链RNA(double-stranded RNA, dsRNA)稳定性的纳米复合物及其制备方法与应用手段。【方法】采用琼脂糖凝胶阻滞实验(agarose gel shifting test)检测橘小实蝇成虫血淋巴对dseGFP的降解能力，星形阳离子聚合物(star polycation, SPc)与dseGFP结合的最佳质量比，以及SPc对dseGFP的保护作用。通过向橘小实蝇成虫腹部注射常用于dsRNA递送体系的表面活性剂烷基糖苷(alkyl polyglucoside, APG),计算死亡率，评估其毒性。在体外合成的dshsp68与纳米载体SPc混合后，用显微注射的方法将纳米复合物注射入橘小实蝇成虫的腹部，运用RT-qPCR检测RNAi效率。【结果】琼脂糖凝胶电泳结果显示橘小实蝇血淋巴能迅速降解dseGFP,当SPc与dseGFP的质量比大于或等于1∶1时能自发形成SPc/dseGFP纳米复合物，减缓橘小实蝇血淋巴对dseGFP的降解。表面活性剂APG对橘小实蝇成虫毒性较强，注射APG后48 h时橘小实蝇的平均死亡率为78.33%,不适宜作为注射法的助剂。单独注射dshsp68后48 h时的RNAi效率为49.53%,在72 h时失去RNAi效果，而注射SPc/dshsp68纳米复合物的RNAi效率更高，48 h时达到了85.99%,且在72 h时的RNAi效率为40.87%,具有更长的RNAi持效时间。【结论】SPc可以结合dsRNA形成复合物，减缓dsRNA被橘小实蝇血淋巴RNase的降解，从而实现持续高效的RNAi敲降效果。本研究建立了一种通过纳米载体SPc实现高效RNAi的方法，可以用于橘小实蝇基因功能研究。更多还原

【Abstract】 【Aim】 To develop a nanocomplex-mediated delivery system for enhancing RNA interference(RNAi) efficiency and dsRNA stability in Bactrocera dorsalis, along with its preparation method and application strategies. 【Methods】Agarose gel shifting test was used to detect the ability of the adult B. dorsalis hemolymph to degrade dseGFP, the optimal mass ratio for the combination of star polycation(SPc) and dseGFP, and the protective effect of SPc on dseGFP. The mortality rate of adult B. dorsalis was calculated to assess the toxicity of the surfactant alkyl polyglucoside(APG), commonly used in dsRNA delivery systems, by injecting APG into the abdomen of adult B. dorsalis. After mixing in vitro synthesized dshsp68 with the nanocarrier SPc, the nanocomplex was injected into the abdomen of adult B. dorsalis using microinjection method, and the RNAi efficiency was detected using RT-qPCR. 【Results】Agarose gel electrophoresis result showed that B. dorsalis hemolymph was able to degrade dseGFP rapidly. When the mass ratio of SPc to dseGFP was greater than or equal to 1∶1, SPc/dseGFP nanocomplex was spontaneously formed, thus protecting dseGFP from being degraded by B. dorsalis hemolymph. The surfactant APG showed strong toxicity towards adult B. dorsalis. At 48 h after injection of APG, the average mortality rate of B. dorsalis was 78.33%, making it unsuitable as an adjuvant for injection methods. The RNAi efficiency of dshsp68 injected alone at 48 h was 49.53% and faded away at 72 h. In contrast, the RNAi efficiency of SPc/dshsp68 nanocomplex was higher, reaching 85.99% at 48 h and remaining at 40.87% at 72 h, offering prolonged duration of RNAi effect. 【Conclusion】SPc can bind dsRNA to form complex and protect dsRNA from being degraded by RNase in hemolymph, thereby achieving sustained and efficient RNAi effects. This study established a method to achieve high-efficiency RNAi with the nanocarrier SPc, which can be used to examine gene function of B. dorsalis.更多还原