AMPKα基因介导白纹伊蚊对四氟甲醚菊酯抗药性

Resistance of AMPKα gene mediated Aedes albopictus to dimefluthrin

  • 摘要: 【目的】探究白纹伊蚊(Aedes albopictus)腺苷酸活化蛋白激酶(AMPK)基因(AalAMPKα)与四氟甲醚菊酯的抗药性关系,揭示白纹伊蚊对四氟甲醚菊酯的抗性形成机制,为白纹伊蚊的综合治理提供理论基础。【方法】克隆AalAMPKα基因编码区(CDS)序列,通过Protparam、SMART、SWISS-MODEL和GSD_v2.0等在线软件进行生物信息学分析;使用实时荧光定量PCR技术检测AalAMPKα基因在白纹伊蚊不同组织和不同发育时期的表达情况;采用微量点滴法测定白纹伊蚊对四氟甲醚菊酯的抗药性;使用RNA干扰技术探究AalAMPKα基因在白纹伊蚊抗药性中的功能。【结果】AalAMPKα基因全长1653 bp,编码545个氨基酸,蛋白质的相对分子量为61.63 kD,理论等电点(pI)为8.03,为不稳定的亲水性蛋白;序列分析结果显示,AalAMPKα基因编码的氨基酸序列与其他物种相似,有着高度保守的结构域;AalAMPKα基因编码的氨基酸序列与埃及伊蚊(Aedes aegyptiAMPKα基因编码的氨基酸序列的相似性最高,与其他双翅目昆虫之间有着较近的亲缘关系;基因组结构分析显示,AMPKα基因在不同物种中的基因组结构差异较大,表现出物种特异性。AalAMPKα基因在白纹伊蚊的不同组织和不同发育时期均有不同程度的表达,其中在脂肪体和1日雄蚊中表达量最高。白纹伊蚊抗性品系AalAMPKα基因的表达量是敏感品系的3.98倍。四氟甲醚菊酯对白纹伊蚊敏感品系和抗性品系的半致死剂量(LD50)分别为0.354和8.010 ng/头,抗性品系的抗性倍数为22.63,为高水平抗性;对白纹伊蚊抗性品系雌蚊显微注射双链核糖核酸(dsRNA)沉默AalAMPKα基因24 h后,注射dsGFP的对照组LD50为7.876 ng/头,注射dsAalAMPKα 组的LD50为2.631 ng/头,沉默AalAMPKα基因后抗性倍数从22.25下降到7.43,抗药性极显著减弱(P<0.001)。【结论】AalAMPKα基因可介导白纹伊蚊对四氟甲醚菊酯的抗药性。

     

    Abstract: 【Objective】To explore the relationship between adenosine monophosphate activated protein kinase(AMPK) gene(AalAMPKα)and dimefluthrin resistance in Aedes albopictus,and to explore the mechanism of resistance formation to dimefluthrin in Aedes albopictus,which could provide scientific basis for comprehensive control of Aedes albopictus. 【Method】The sequence of coding region(CDS)of AalAMPKα was cloned,and bioinformatics analysis was carried out by online softwares such as Protparam,SMART,SWISS-MODEL and GSD_v2.0. Real-time fluorescence quantitative PCR was used to detect the expression of AalAMPKα in different tissues and developmental stages of Aedes albopictus. The resistance of Aedes albopictus to dimefluthrin was determined by the micro-drop method,the function of AalAMPKα gene in drug resistance of Aedes albopictus was investigated using RNA interference technology.【Result】The full length sequence of AalAMPKα was 1653 bp and encoded 545 amino acids,the molecular weight of the protein was 61.63 kD, and the theoretical isoelectric point(pI)was 8.03,which was a hydrophilic protein. Sequence analysis revealed that the amino acid sequence encoded by the AalAMPKα gene contained highly conserved domains shared with other species. The AalAMPKα gene encoded amino acid sequence showed the highest similarity with that of the AMPKα gene from Aedes aegypti and exhibited close phylogenetic relationships with other Dipteran insects. Genome structure analysis showed that the genome structure of AMPKα gene varied greatly among different species,showing species specificity. AalAMPKα gene was expressed to varying degrees in different tissues and at different developmental stages of Aedes albopictus,with the highest expression in fat body and one-day male mosquitoes. The expression of AalAMPKα gene in resistant Aedes albopictus was as 3.98 times as that of susceptible strain. The lethal medium dose(LD50)of dimefluthrin to Aedes albopictus susceptible strain and resistant strain were 0.354 and 8.010 ng/insect respectively,and the resistance times was 22.63, indicating a high level of resistance. After 24 h microinjection of double-strand ribonucleic acid (dsRNA) silent AalAMPKα gene on resistant strain of Aedes albopictus,the LD50 of dsGFP group was 7.876 ng/insect,and that of dsAalAMPKα group was 2.631 ng/insect. The resistance times decreased from 22.25 to 7.43 after silenced AalAMPKα gene, drug resistance was extremely significantly decreased(P<0.001).【Conclusion】AalAMPKα gene mediates the resistance of Aedes albopictus to dimefluthrin.

     

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