摘要
【目的】探索纳米载体星形阳离子聚合物(star polycation,SPc)介导V-ATP酶H亚基基因TcV-ATPase-H的RNAi对朱砂叶螨Tetranychus cinnabarinus雌成螨的毒效。【方法】通过PCR克隆朱砂叶螨TcV-ATPase-H;利用RT-qPCR检测TcV-ATPase-H在朱砂叶螨不同发育阶段(卵、幼螨、若螨和雌成螨)的表达量;分别通过叶碟法和纳米载体介导对朱砂叶螨雌成螨的TcV-ATPase-H进行RNAi,检测校正死亡率;通过草莓盆栽实验,检测ds TcV-ATPase-H/SPc和苦参碱联合施用对草莓叶片上朱砂叶螨雌成螨的毒效,测定草莓顶端新叶叶绿素含量和草莓植株地上部位干重,同时以玻片浸渍法测定ds TcV-ATPase-H/SPc和苦参碱联合施用对非靶标生物加州新小绥螨Neoseiulus californicus的毒力。【结果】克隆获得朱砂叶螨TcV-ATPase-H cDNA序列;TcV-ATPase-H在朱砂叶螨卵、幼螨、若螨和雌成螨中均表达,其中以在雌成螨中表达量最高,在幼螨中表达量最低。叶碟法对朱砂叶螨饲喂ds TcV-ATPase-H能够有效降低TcV-ATPase-H的表达量59%,72 h时引起的朱砂叶螨雌成螨的校正死亡率达到37.99%,与对照组ds EGFP相比有显著提高;纳米载体介导比非纳米载体介导TcV-ATPase-H的RNAi能够显著降低TcV-ATPase-H的表达量20%,同时使校正死亡率显著提高了28.25%。纳米载体介导ds TcV-ATPase-H和苦参碱的联合施用能显著降低朱砂叶螨雌成螨的存活数,对朱砂叶螨种群生长有明显抑制效果,但对草莓植株叶绿素含量及草莓植株地上部位干重和加州新小绥螨的死亡率均不产生显著影响。【结论】纳米载体介导ds TcV-ATPase-H能够提高对TcV-ATPase-H表达的抑制,提高对朱砂叶螨的致死效果,该研究为后期研发纳米材料包裹ds V-ATPase-H的RNA农药提供了实验基础。
【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 ds TcV-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 ds TcV-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 ds TcV-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 ds EGFP 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 ds TcV-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 ds TcV-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 ds V-ATPase-H encapsulated in nanomaterials.
作者
王谦稳
杨任铎
李安琪
卜春亚
WANG Qian-Wen;YANG Ren-Duo;LI An-Qi;BU Chun-Ya(Key Laboratory for Northern Urban Agriculture,Ministry of Agriculture and Rural Affairs,College of Biological and Resource Environment,Beijing University of Agriculture,Beijing 102206,China)
出处
《昆虫学报》
北大核心
2025年第11期1570-1579,共10页
Acta Entomologica Sinica
基金
北京市自然科学基金项目(6212004)
国家自然科学基金项目(31670648)。
关键词
朱砂叶螨
V-ATP酶
原核表达
RNA干扰
纳米载体
Tetranychus cinnabarinus
V-ATPase
prokaryotic expression
RNA interference
nanocarrier
