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萝卜蚜LeATPf基因的克隆及其RNA干扰制剂对萝卜蚜的防治效果研究
Cloning the LeATPf gene of Lipaphis erysimi and evaluating the effectiveness of RNA interference knockdown of this gene as a means of controlling L. erysimi
张 浩;王金彦;赵 杰;陈义娟;蒋杰贤;季香云
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DOI:10.7679/j.issn.2095-1353.2022.088
作者单位:上海市农业科学院生态环境保护研究所,上海 201403;上海市浦东新区农业技术推广中心,上海 201201
中文关键词:RNA干扰;萝卜蚜;三磷酸腺苷合酶f亚基;害虫防治;新型农药
英文关键词:RNA interference; Lipaphis erysimi; ATP synthases subunit f; pest control; new pesticide
中文摘要:
【目的】 克隆萝卜蚜Lipaphis erysimi (Kaltenbach) 的三磷酸腺苷合酶f亚基(ATP synthases subunit f,LeATPf)基因,并利用RNA干扰技术抑制该基因表达,分析复配RNA干扰剂对萝卜蚜的防治效果,推动以RNA干扰为核心的害虫绿色防控新技术。【方法】 利用同源克隆技术获得萝卜蚜LeATPf基因的全长cDNA,通过RNA干扰技术沉默其基因表达;采用浸叶法测定不同浓度的复配RNA干扰剂对萝卜蚜的杀虫效果,并通过田间小区试验测定RNA干扰剂对萝卜蚜的虫口减退率和田间防治效果。【结果】 萝卜蚜LeATPf基因编码长度为324 bp的cDNA序列,与其它5种蚜虫的ATPf基因同源性在88%以上,且LeATPf基因主要在2龄期萝卜蚜中表达;喷施RNA干扰剂可有效抑制LeATPf基因的表达,且高浓度的RNA干扰剂(200.00 mg/L)对萝卜蚜的室内杀虫效果与吡虫啉处理组无显著差异(P>0.05);在田间试验中,中高浓度的RNA干扰剂(50.00、100.00和200.00 mg/L)在处理后第3和第7天对萝卜蚜的虫口减退率和防治效果与吡虫啉处理组无显著差异(P>0.05),但在处理后14 d对萝卜蚜的虫口减退率和防治效果显著高于吡虫啉处理组(P<0.05)。【结论】 针对LeATPf基因设计并配置的中高浓度的RNA干扰剂对萝卜蚜的防治效果与吡虫啉相当,但是其药效持续性优于吡虫啉;LeATPf基因是开发萝卜蚜RNA干扰制剂的良好靶标基因。
英文摘要:
[Objectives]  To clone the ATP synthases subunit f gene of Lipaphis erysimi (Kaltenbach) (LeATPf) and assess the effectiveness of RNAi (RNA interference) as a more environmentally-friendly way of controlling L. erysimi. [Methods]  The full-length cDNA sequence of the LeATPf gene was obtained using the reverse transcription technique and expression of the gene silenced using RNA interference. The effect of exposing L. erysimi to a residual film of the RNAi preparation was measured under laboratory conditions, and the effect of spraying the RNAi preparation on L. erysimi in the field was measured in a field experiment. [Results]  The cDNA sequence encoding the LeATPf gene is 324 bp in length, and is more than 90% homologous with the corresponding gene in another five aphid species. The LeATPf gene was mainly expressed in 2nd instar L. erysimi. Spraying aphids with the RNAi preparation effectively inhibited expression of the LeATPf gene. The corrected mortality of L. erysimi following treatment with the RNAi preparation (200.00 mg/L) was not significantly different to that following treatment with 70% imidacloprid after 1, 3 or 5 days (P<0.05). A field experiment indicated that the rate and magnitude of a population’s decline following treatment with the RNAi preparation (50.00, 100.00 or 200.00 mg/L) were not significantly different from the result of spraying 70% imidacloprid after 1, 3 or 7 days (P<0.05). However, the rate and magnitude of population decline using the RNAi preparation were significantly higher than that achieved with 70% imidacloprid on the 14th day after treatment (P<0.05). [Conclusion]  The effectiveness of medium to high concentrations (50.00, 100.00 and 200.00 mg/L) of an RNAi preparation (dsLeATPf) to control L. erysimi was equivalent to imidacloprid, except that the effects of the RNAi preparation lasted longer than that of imidacloprid. The LeATPf gene is a good target for developing an RNA interference preparation to control L. erysimi in the field.
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