中文摘要
埃及伊蚊浓核病毒AeDNV属短浓核病毒属，可特异性感染蚊子类，具有蚊虫生物防治的潜在价值。为增强其杀虫效果发展其为生物杀虫剂，我们将东亚钳蝎昆虫毒素BmK IT1插入包含野生AeDNV全基因组的质粒载体pUCA中，C'末端融合表达绿色荧光蛋白GFP为示踪分子，构建成可表达蝎毒蛋白的重组病毒表达载体。重组病毒载体与野生病毒表达载体共转染包装细胞-白纹伊蚊C6/36细胞获得重组病毒。qPCR法定量病毒，以重组病毒引入白纹伊蚊幼虫孳生水体感染幼虫，以野生病毒为对照，评估各组半数死亡时间LT(50) 和半数致死量 LD(50)，结果显示重组病毒对白纹伊蚊具有更有效的杀伤效果。我们进一步测试AeDNV作为RNAi载体的能力与其应用于蚊媒控制的潜在效果，白纹伊蚊V-ATPase基因作为靶基因，将特异性siRNA表达框插入重组病毒，构建成表达siRNA的重组病毒载体，使用重组病毒载体转染C6/36细胞，qPCR测定V-ATP基因抑制率 于96 h达到90% 以上。重组病毒感染白纹伊蚊幼虫，幼虫体内V-ATPase 抑制率达到70% 以上，幼虫死亡率相对于野生病毒对照组显著性升高。
英文摘要
The Aedes aegypti densovirus (AeDNV) has previously shown potential in mosquito control. To improve its efficacy as a biopesticide, the gene for an excitatory insect-specific toxin from Buthus martensii Karsch (BmK IT1) was inserted into the AeDNV genome and cloned into pUCA plasmid. The coding sequence for green fluorescent protein was ligated to the C-terminus of the BmK IT1 gene as a screening marker. Recombinant and helper plasmids were cotransfected into C6/36 cells; wild-type viruses were the controls. The recombinant viruses were identified and quantified by real-time polymerase chain reaction and exposed to Ae. albopictus larvae for the evaluation of its bioinsecticidal activity. LT(50) and LD(50) bioassays showed that the recombinant AeDNV had stronger and faster pathogenic effects on Ae. albopictus than the wild-type virus. This is the first report on the recombinant AeDNA containing the insect-specific toxin, BmK IT1, which may be used to develop a novel type of insecticide. Furthermore, we investigated the ability of plasmids containing AeDNV recombinant viral transducing genome to induce RNA interference (RNAi) effects in C6/C36 cells. We then evaluated the efficiency of a recombinant AeDNV vector to induce RNAi in Aedes albopictus larvae. We found that the expression of V-ATPase was inhibited by up to 90% at 96 h post-transfection in transfected C6/C36 cells. In addition, the bioinsecticidal activities of various RNAi-expressing AeDNV vectors used to infect Ae. albopictus larvae were also tested. We found that when Ae. albopictus larvae were infected with recombinant AeDNV, expression of V-ATPase was downregulated by nearly 70% compared to controls. Furthermore, the median survival time bioassays demonstrated that recombinant AeDNV caused more serious pathogenic effects than the wild type virus. This is the first report showing that recombinant virus plasmid and corresponding recombinant AeDNV can be used as an effective in vitro and in vivo RNAi delivery system, respectively.
结题摘要
埃及伊蚊浓核病毒AeDNV属短浓核病毒属，可特异性感染蚊子类，具有蚊虫生物防治的潜在价值。为增强其杀虫效果发展其为生物杀虫剂，我们将东亚钳蝎昆虫毒素BmK IT1插入包含野生AeDNV全基因组的质粒载体pUCA中，C'末端融合表达绿色荧光蛋白GFP为示踪分子，构建成可表达蝎毒蛋白的重组病毒表达载体。重组病毒载体与野生病毒表达载体共转染包装细胞-白纹伊蚊C6/36细胞获得重组病毒。qPCR法定量病毒，以重组病毒引入白纹伊蚊幼虫孳生水体感染幼虫，以野生病毒为对照，评估各组半数死亡时间LT(50) 和半数致死量 LD(50)，结果显示重组病毒对白纹伊蚊具有更有效的杀伤效果。我们进一步测试AeDNV作为RNAi载体的能力与其应用于蚊媒控制的潜在效果，白纹伊蚊V-ATPase基因作为靶基因，将特异性siRNA表达框插入重组病毒，构建成表达siRNA的重组病毒载体，使用重组病毒载体转染C6/36细胞，qPCR测定V-ATP基因抑制率 于96 h达到90% 以上。重组病毒感染白纹伊蚊幼虫，幼虫体内V-ATPase 抑制率达到70% 以上，幼虫死亡率相对于野生病毒对照组显著性升高。