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【目的】ABC(ATP-binding cassette)转运蛋白是一类重要的跨膜蛋白超家族,某些ABC转运蛋白基因在一些害虫的抗药性品系中表达显著提高。本研究旨在克隆禾谷缢管蚜Rhopalosiphum padi ABC转运蛋白基因Rhpa ABCG9,Rhpa ABCG20和Rhpa ABCG23 c DNA全序列,分析这3个基因在该虫不同发育阶段和不同抗药性品系中的表达模式,为阐明ABC转运蛋白在禾谷缢管蚜抗药性中的作用和其他生理功能,以及深入分析该虫抗药性机理奠定基础。【方法】采用RT-PCR与RACE技术,克隆了基因c DNA全序列;利用实时荧光定量PCR技术,研究这3个基因在禾谷缢管蚜不同生长发育阶段和不同抗药性品系中的表达变化。【结果】获得了Rhpa ABCG9,Rhpa ABCG20和Rhpa ABCG23 3个基因c DNA全序列,其开放阅读框长度分别为2 103,2 436和2 082 bp,分别编码700,811和693个氨基酸。结构分析表明,3个蛋白均具有ABC转运蛋白家族典型的结构特征;系统进化分析结果显示,3个蛋白氨基酸序列分别与豌豆蚜Acyrthosiphon pisum各对应氨基酸的序列一致性最高。实时荧光定量PCR分析结果表明,这3个基因在禾谷缢管蚜不同发育时期均不同程度表达。Rhpa ABCG20在各个发育时期的表达变化差异不显著;Rhpa ABCG9表达量在4龄若蚜最高,在1龄若蚜最低;Rhpa ABCG23表达量在3龄若蚜最高,1龄若蚜最低,其他阶段差异不显著。禾谷缢管蚜异丙威抗性品系中,Rhpa ABCG20表达量显著高于敏感品系,而Rhpa ABCG9和Rhpa ABCG23表达量均低于敏感品系,但差异不显著;禾谷缢管蚜吡虫啉抗性品系中,Rhpa ABCG20和Rhpa ABCG23表达量显著高于敏感品系,而Rhpa ABCG9表达上调不显著。【结论】Rhpa ABCG9,Rhpa ABCG20和Rhpa ABCG23基因可能参与禾谷缢管蚜体内农药的运输,并与禾谷缢管蚜的抗药性具有一定的关系。本研究结果为进一步深入分析禾谷缢管蚜的抗药性机制,以及该虫的抗药性治理与综合防治奠定了基础。
【Objective】 ABC (ATP-binding cassette) transporter is an important transmembrane protein superfamily. Some ABC transporter genes were significantly increased in some pest resistant strains. In this study, we cloned the full-length cDNA sequence of Rhopalosiphum padi ABC transporter Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23c, and analyzed the expression pattern of these three genes in different developmental stages and different resistant strains. In order to elucidate the role of ABC transporter in the resistance of H. armigera and other physiological functions, and to lay a foundation for further analysis of the mechanism of drug resistance of this insect. 【Method】 The full-length cDNA sequence of the gene cDNA was cloned by RT-PCR and RACE. The expression of these three genes in different growth stages and resistant strains of Aphis gossypii were studied by real-time fluorescence quantitative PCR . 【Result】 The complete cDNA sequence of 3 genes of Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23 was obtained. The open reading frame of them was 2 103, 2 436 and 2 082 bp, encoding 700, 811 and 693 amino acids respectively. Structural analysis showed that all three proteins had the typical structural characteristics of ABC transporter family. Phylogenetic analysis showed that the amino acid sequences of the three proteins had the highest sequence identities with the corresponding amino acids of Acyrthosiphon pisum. The results of real-time quantitative PCR showed that the three genes were expressed to different extents in different developmental stages of Aphis gossypii. Rhpa ABCG9 expression in the various developmental stages was not significantly different; Rhpa ABCG9 expression in the fourth instar aphid the highest, the lowest in the first instar aphid; Rhpa ABCG23 expression in the third instar aphid highest, the first instar aphid lowest, other stages The difference was not significant. In Rhopalosiphum padii, the expression level of Rhpa ABCG20 in Rhizoctonia solani was significantly higher than that in the susceptible strain, while the expression levels of Rhpa ABCG9 and Rhpa ABCG23 were lower than those in the susceptible strain, but the difference was not significant. The expression of Rhpa ABCG20 and Rhpa ABCG23 was significantly higher than that of the susceptible strain, while the up-regulation of Rhpa ABCG9 was not significant. 【Conclusion】 Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23 genes may be involved in the transport of pesticides in Aphis gossypii, and have a certain relationship with the resistance of Aphis gossypii. The results of this study will provide a basis for further analysis of the resistance mechanism of H. avenae and its resistance management and integrated control.