苹果黄蚜细胞色素P450基因AcCYP6CY14的表达及其在抵抗吡虫啉中的作用
Expression of the cytochrome P450 gene AcCYP6CY14 and its role in imidacloprid resistance in Aphis citricola
范继巧1, 2** 韩鹏飞1, 3 高 越1, 2 刘中芳1, 2 张鹏九1, 2 杨
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DOI：10.7679/j.issn.2095-1353.2019.037
作者单位：1. 农业有害生物综合治理山西省重点实验室，太原 030031；2. 山西省农业科学院植物保护研究所，太原 030031；3. 山西大学应用生物学研究所，太原 030006
中文关键词：苹果黄蚜；吡虫啉；细胞色素P450；RNA干扰；解毒代谢
英文关键词：Aphis citricola; imidacloprid; cytochrome P450; RNA interference; metabolic detoxification
中文摘要：【目的】 研究苹果黄蚜Aphis citricola细胞色素P450基因AcCYP6CY14的表达及其在抵抗吡虫啉中的作用。【方法】 搜索苹果黄蚜转录组数据库，获得细胞色素P450基因AcCYP6CY14的cDNA部分序列，采用RT-PCR技术克隆目的基因cDNA序列全长。利用实时定量PCR（Real-time quantitative PCR, RT-qPCR）技术测定目的基因在苹果黄蚜不同发育阶段的表达水平。采用浸虫法，用浓度为LC30的吡虫啉对苹果黄蚜3龄若蚜进行诱导，RT-qPCR检测吡虫啉诱导不同时间后P450基因的表达情况。利用RNA干扰（RNAi）技术沉默苹果黄蚜3龄若蚜的P450基因，并研究该基因沉默后，苹果黄蚜对吡虫啉的敏感性。【结果】 获得苹果黄蚜细胞色素P450基因的cDNA全长序列，为1 542 bp，编码513个氨基酸，命名为AcCYP6CY14（GenBank登录号：MH717248）。研究发现该基因在苹果黄蚜各个发育阶段均有表达，在3龄若蚜中表达量最高。经吡虫啉诱导12 h后，该基因的相对表达量显著高于对照。dsRNA饲喂结果显示，苹果黄蚜取食dsAcCYP6CY14 24 h后，将其转接到新鲜的苹果嫩稍上，24、48、72 h后，该基因的表达量显著下调。沉默该P450基因后，苹果黄蚜对吡虫啉的敏感性极显著升高（P<0.01），死亡率提高了65.2%。【结论】 克隆得到了苹果黄蚜P450基因AcCYP6CY14的cDNA全长序列，该基因在苹果黄蚜整个生育期均有表达，且在3龄若蚜中表达量最高，推测该基因可能参与了苹果黄蚜对吡虫啉的解毒代谢。
英文摘要：
[Objectives] To study the developmental expression of the cytochrome P450 gene AcCYP6CY14 and its role in imidacloprid resistance in Aphis citricola. [Methods] Partial cDNA sequences of AcCYP6CY14 were obtained by searching the transcriptome database of A. citricola. Full-length cDNA of the target gene was cloned using RT-PCR, and its relative expression in different developmental stages measured using real-time quantitative PCR (RT-qPCR). The 3rd instar of A. citricola was chosen for an induction experiment using the insect-soaking method and the relative expression of AcCYP6CY14 was detected by RT-qPCR after exposing insects to imidacloprid for different periods of time. The target gene was silenced using RNA interference (RNAi) technology by feeding nymphs dsRNA, and the susceptibility of the nymphs to imidacloprid was assessed. [Results] The length of the full-length cDNA sequence of AcCYP6CY14 was 1 542 bp, encoding 513 amino acids (GenBank accession No.: MH717248). AcCYP6CY14 was expressed in all developmental stages of A. citricola but was highest in 3rd instar nymphs. After exposure to an LC30 dose of imidacloprid for 12 h, the relative expression of the AcCYP6CY14 was significantly higher than that of the control group. Feeding nymphs dsAcCYP6CY14 for 24 h significantly decreased transcripts of AcCYP6CY14 when nymphs were transferred to tender stems for 24, 48 and 72 h. A. citricola became more sensitive to imidacloprid (P < 0.01) when AcCYP6CY14 was silenced after which mortality increased by 65.2%. [Conclusion] The full-length cDNA sequence of AcCYP6CY14 was cloned and was found to be expressed in all developmental stages of A. citricola. Expression was highest in 3rd instar nymphs. This gene may play a role in imidacloprid resistance in A. citricola.