禾谷缢管蚜ABC转运蛋白基因的克隆、分子特性及表达分析

Molecular cloning, characterization and expression analysis of ATPbinding cassette transporter genes in the bird cherry-oat aphid,Rhopalosiphum padi(Hemiptera: Aphididae)

【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。

【Aim】The ABC（ ATP-binding cassette） transporters are important transmembrane proteins encoded by a supergene family. The expression of some ABC transporter genes significantly increase in some insecticide resistant strains of insect species. This study aims to clone the full c DNA of ABC transporter genes Rhpa ABCG9, Rhpa ABCG20 and Rhpa ABCG23 of the bird cherry-oat aphid,Rhopalosiphum padi,and to analyze their expression patterns in different developmental stages and different insecticide resistant strains. The results will provide a theoretical knowledge to clarify the biological functions of ABC transporters of R. padi,especially their roles in the resistance to insecticides of this insect. 【Methods】RT-PCR and RACE were used to clone the full-length c DNAs of three ABC transporter genes from R. padi. The expression levels of the three genes in different developmental stages and different insecticide resistant strains of this aphid were detected by real-time quantitative PCR.【Results】The c DNAs of Rhpa ABCG9,Rhpa ABCG20 and Rhpa ABCG23 have an open reading frame（ ORF） of 2 103,2 436 and 2 082 bp with the deduced amino acid sequence of 700,811 and 693 residues,respectively. Structural analysis showed that all the three proteins have the typical structural features of ABC transporter family. Phylogenetic analysis indicated that the amino acid sequences of these three proteins show high identity to those of the corresponding proteins from Acyrthosiphon pisum. Realtime quantitative PCR analysis showed that the three genes were expressed differently in different developmental stages. The expression level of Rhpa ABCG20 was not significantly different in various developmental stages. The expression level of Rhpa ABCG9 was the highest in the 4th instar nymphs and the lowest in the 1st instar nymphs. The expression level of Rhpa ABCG23 was significantly higher in the3 rd instar nymphs and significantly lower in the 1st instar nymphs than those in other instar nymphs,while there was no significant difference among the other instar nymphs. Compared with that in the susceptible strain,the expression level of Rhpa ABCG20 in the isoprocarb resistant strain of R. padi was significantly up-regulated, whilst the expression levels of Rhpa ABCG9 and Rhpa ABCG23 were insignificantly down-regulated. The expression levels of Rhpa ABCG20 and Rhpa ABCG23 in the imidacloprid resistant strain were significantly higher than in the susceptible strain,whilst the expression level of Rhpa ABCG9 was not significantly up-regulated in the resistant strain. 【Conclusions】Rhpa ABCG9,Rhpa ABCG20 and Rhpa ABCG23 may play roles in the transport of insecticides in R. padi,and be related to the resistance of this pest to insecticides. The results provide a foundation for further study of the resistance mechanisms,and for the resistance management and integrated pest management of R. padi.