淡色库蚊抗性相关基因--NYD-GBE基因的克隆与分析

目的:获取淡色库蚊抗药性相关NYD-GBE基因cDNA全长序列并验证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性相关NYD-GBE基因片段设计引物,采用快速扩增cDNA末端法(rapidamplificationofcDNAends,RACE)扩增糖原分支酶基因5′、3′端,经对位拼接获得全长序列,并用相应的软件进行生物信息学分析。结果:获得淡色库蚊NYD-GBEcDNA全长序列,开放阅读框为2070bp(GeneBank/NCBIDQ102393),编码689个氨基酸。蛋白质序列分析显示,NYD-GBE与冈比亚按蚊(Anophelesgambiae)和拟暗果蝇(Drosophilapseudoobscura)一个未知功能的蛋白同源性最高,为82%和72%,与人糖原分支酶的基因同源性次之为60%。荧光定量PCR结果显示,NYD-GBE在淡色库蚊溴氰菊酯抗性品系中的表达量是敏感品系的19.7倍。结论:获得淡色库蚊抗药性相关NYD-GBE基因全长cDNA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-GBE与淡色库蚊溴氰菊酯抗性相关,值得进一步研究。

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

Objective: To acquire the full length of NYD-GBE cDNA of Cx pipiens pallens from the deltamethrin-resistant strain and identify the expression levels in resistant and susceptible strains.Methods:Through RACE(rapid amplifyication of cDNA ends) method,the full length NYD-GBE cDNA of Cx.pipiens pallens was acquired.The expression levels of NYD-GBE between the resistant and the susceptible strains were identified by real time quantitative PCR.Results: A sequence of 2 337 base pairs including an open reading frame of 2 070 base pairs was gotten named NYD-GBE(GenBank/NCBI DQ102 393).NYD-GBE was acquired and expressed 19.7 fold in the resistant strain more than that in the susceptible strain by real time quantitative PCR.The encoded protein of Cx.Pipiens pallens had 689 amino acid with 82%?72% and 60% homology of An.Gambiae?D.pseudoobscura and Glycogen branching enzyme of human,respectively.Conclusion: NYD-GBE expressed higher in the resistant strain than that in the susceptible strain of the Cx.pipiens pallens,indicating that NYD-GBE may be associated with the deltamethrin resistance of Cx.Pipiens pallens.The results are important for further study the relationship between NYD-GBE and insect pesticide resistance.