关键词: 朱砂叶螨, V-ATP酶, 原核表达, RNA干扰, 纳米载体

Abstract: 【Aim】 To explore the toxic effect of star polycation (SPc)-mediated RNAi targeting the V-ATPase H subunit gene TcV-ATPase-H on female adults of Tetranychus cinnabarinus. 【Methods】TcV-ATPase-H of T. cinnabarinus was cloned by PCR. The expression levels of TcV-ATPase-H in T. cinnabarinus at different developmental stages (egg, larva, nymph and female adult) were detected using RT-qPCR. RNAi of TcV-ATPase-H in female adult of T. cinnabarinus was carried out via the leaf-disc method and nanocarrier-mediated method, respectively, and the corrected mortality rate was measured. Through strawberry pot experiments, the toxic effect of the combined application of dsTcV-ATPase-H/SPc with matrine on female adult of T. cinnabarinus on strawberry leaves was detected, the chlorophyll content in the apical new leaves of strawberries and the above-ground dry weight of strawberry plants were determined. At the same time, the toxicity of the combined application of dsTcV-ATPase-H/SPc with matrine to the non-target organism Neoseiulus californicus was measured by the slide-dipping method.【Results】 The cDNA sequence of TcV-ATPase-H of T. cinnabarinus was cloned. TcV-ATPase-H was expressed in the egg, larva, nymph and female adult of T. cinnabarinus, with the highest expression level in female adult and the lowest expression level in larva. Feeding dsTcV-ATPase-H to T. cinnabarinus by the leaf-disc method could effectively reduce the expression level of TcV-ATPase-H by 59% and caused the corrected mortality rate of female adult T. cinnabarinus to reach 37.99% at 72 h after feeding, which was significantly improved compared with the dsEGFP control group. Nanocarrier-mediated RNAi of TcV-ATPase-H could significantly reduce the expression level of TcV-ATPase-H by 20% and significantly increased the corrected mortality rate by 28.25% compared with non-nanocarrier-mediated RNAi. At the same time,the combined application of nanocarrier-mediated dsTcV-ATPase-H with matrine can significantly reduce the survival number of female adults of T. cinnabarinus and had a remarkable inhibitory effect on the population growth of T. cinnabarinus, but had no significant effect on the chlorophyll content in strawberry leaves, the above-ground dry weight of strawberry plants, or the mortality rate of N. californicus.【Conclusion】Nanocarrier-mediated dsTcV-ATPase-H enhances the inhibition of the expression of TcV-ATPase-H, improves the lethal effect on T. cinnabarinus, which provide a foundation for the development of RNA-based pesticides of dsV-ATPase-H encapsulated in nanomaterials.

Key words: Tetranychus cinnabarinus, V-ATPase, prokaryotic expression, RNA interference, nanocarrier