绿盲蝽毒死蜱抗性和敏感品系乙酰胆碱酯酶-2基因的克隆、序列分析及表达水平

Cloning,sequence analysis and expression levels of the acetylcholinesterase-2 gene in the chlorpyrifos-resistant and susceptible strains of Apolygus lucorum( Hemiptera: Miridae)

【目的】为了明确绿盲蝽Apolygus lucorum乙酰胆碱酯酶-2(acetylcholinesterase-2,ACh E2)与毒死蜱抗性的关系。【方法】本研究利用RACE技术获得了绿盲蝽ACh E2基因(Al-ace2)的全长序列,并检测了Al-ace2在绿盲蝽毒死蜱抗性品系(BZ-R)及敏感品系(SLF和BZ)中的氨基酸序列多态性及mRNA相对表达量。【结果】Al-ace2开放阅读框长1 935 bp,编码644个氨基酸残基;推导的氨基酸序列含有ACh E的典型结构,如催化三联体、氧阴离子洞、胆碱结合位点及围绕活性位点丝氨酸的保守结构域FGESAG。与两个敏感品系(SLF和BZ)相比,绿盲蝽毒死蜱抗性品系BZ-R Al-ace2基因中存在T552M氨基酸替换,发生频率为5%,但此位点氨基酸并不保守,也不靠近活性位点,推测其与抗性无关。在绿盲蝽SLF,BZ和BZ-R品系中,Al-ace1表达量均约为Al-ace2的3倍,且Al-ace2转录水平在3个品系间无显著差异。【结论】结果说明,在绿盲蝽体内ACh E2是次要表达的乙酰胆碱酯酶,该ACh E2可能与绿盲蝽BZ-R品系对毒死蜱的抗性无关。

【Aim 】 To investigate the relationship between acetylcholinesterase-2（ ACh E2） and chlorpyrifos-resistance in Apolygus lucorum. 【Methods】The ACh E2 gene（ Al-ace2） was cloned using RACE method. The amino acid polymorphism and the transcription level of Al-ace2 in the chlorpyrifosresistant（ BZ-R） and susceptible（ SLF and BZ） strains of A. lucorum were detected. 【Results】The Alace2 gene contains a 1 935 bp open reading frame,which encodes 644 amino acid residues. The deduced amino acid sequence shows the common properties of ACh E,such as the catalytic triad amino acids,the characteristic motif ‘FGESAG＇surrounding the active Ser,oxyanion hole and the choline binding sites.T552 M amino acid substitution was only found in Al-ace2 in the resistant strain（ BZ-R） with the mutation frequency of 5%,but not in the two susceptible strains（ SLF and BZ）. The amino acid at position 552 is neither conserved nor near to active sites,suggesting that the T552 M substitution has much higher possibility to cause polymorphism among individuals than to contribute to chlorpyrifos resistance in A.lucorum. The transcription level of Al-ace1 was approximately three-fold as high as that of Al-ace2 in SLF,BZ and BZ-R strains. The transcription level of Al-ace2 showed no significant difference among the three strains. 【Conclusion】The results suggest that ACh E2 is likely the minor ACh E and might not be involved in chlorpyrifos resistance in the BZ-R strain of A. lucorum.