关键词: 橘小实蝇, 纳米载体, 星形阳离子聚合物, hsp68, RNAi

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.

Key words: Bactrocera dorsalis, nanocarrier, star polycation, hsp68, RNAi