淡色库蚊抗药性相关基因——NYD-MLC2基因的克隆与分析

Cloning and sequence analysis of a novel deltamethrin-resistance associated gene NYD-MLC2 from Culex pipiens pallens

目的:获取淡色库蚊抗药性相关基因NYD-MLC2的cDNA全长序列并鉴定其在抗性品系和敏感品系中的表达差异。方法:根据抑制性差减杂交(SSH)结合cDNA芯片分离获得的淡色库蚊抗药性相关NYD-MLC2基因片段序列设计引物,采用快速扩增cDNA末端法(rapidamplificationofcDNAends,RACE)扩增NYD-MLC2基因3′、5′端,经对位拼接获得全长序列,并进行生物信息学分析;荧光定量PCR验证此基因在抗性和敏感品系的表达差异。结果:获得淡色库蚊NYD-MLC2cDNA全长序列,开放阅读框为630bp(GenBank/NCBIDQ140391),编码210个氨基酸。蛋白质序列分析显示,NYD-MLC2与冈比亚按蚊(Anophelesgambiae)一个未知功能的蛋白同源性最高,为91%;实时荧光定量PCR结果显示,NYD-MLC2在淡色库蚊溴氰菊酯抗性品系中的表达量是敏感品系的4.08倍。结论:获得淡色库蚊抗药性相关NYD-MLC2基因cDNA全长序列,并证实其在淡色库蚊溴氰菊酯抗性品系中高表达,提示NYD-MLC2与淡色库蚊溴氰菊酯抗药性相关,值得进一步研究。

Objective： To obtain a full length cDNA of novel deltarnethrin-resistance related gene from Culex pipiens pallens and identify differential expression levels between resistant and susceptible strains. Methods： By RACE （rapid amplifyication of eDNA ends）, the full length of NYD-MLC2 eDNA of Cx. pipiens pallens was acquired,then sequenced and analyzed by softwares of bioin- formaties.The expression levels of NYD-MLC2 in resistant and susceptible strains were detected by real time quantitative PCR. Re- suits： A sequence of 1 002 base pairs including an open reading frame of 630 base pairs was obtained and named as NYD-MLC2 （GenBank/NCBI accession number DQ140391 ）. NYD-MLC2 was found to be expressed 4.08 fold higher in the resistant strain than in the susceptible strain. The encoded protein of Cx. pipiens pallens had 210 amino acids with 91% homology of Anopheles gambiae. Conclusion： NYD-MLC2 expressed higher in resistant strain than in susceptible strain of the Cx. pipiens pallens ,indicating that NYD-MLC2 may be associated with the deltamethfin resistance of Cx. pipiens pallens. The results are valuable for further study of the relationship between NYD-MLC2 gene and insect resistance.