淡色库蚊XND-P450基因cDNA全长克隆及表达

Cloning and expression of XND-P450 gene from Culex pipiens pallens

【目的】克隆淡色库蚊氯菊酯抗性相关XND-P450基因,并对其进行生物信息学分析,研究其在敏感品系和抗性品系淡色库蚊中表达量的差异,为阐明XND-P450的功能和抗性机制奠定基础。【方法】依据淡色库蚊抗性品系与敏感品系差异表达的EST片段设计PCR引物,采用RACE技术克隆淡色库蚊氯菊酯抗性相关基因XND-P450全长cDNA序列,并对其进行生物信息学分析;采用半定量PCR技术,对XND-P450基因在敏感品系和抗性品系淡色库蚊中的表达量差异进行检测。【结果】经过克隆获得长度为1 679bp的淡色库蚊氯菊酯抗性相关基因XND-P450全长cDNA,编码523个氨基酸。生物信息学分析表明,该基因与致倦库蚊、埃及伊蚊CYP450基因的同源性在70%以上,其编码蛋白相对分子质量为33 244.9u,理论等电点为8.10,属于不稳定的亲水蛋白,氨基酸序列与致倦库蚊及冈比亚按蚊CYP450蛋白的同源性在40%以上。半定量PCR电泳分析显示,抗性品系淡色库蚊中的XND-P450基因表达量较敏感品系高。【结论】克隆了淡色库蚊氯菊酯抗性相关XND-P450基因,该基因在抗性品系淡色库蚊中的表达量高于敏感品系,推测其与氯菊酯抗性相关。

[Objective] The full length cDNA sequence of permethrin-resistance associated XND-P450 gene of Culex pipiens pallens was obtained and analyzed using bioinformatics methods. Its expression levels in susceptible and resistant strains were detected for clarification of the function and resistance mechanism of XND-P450 gene. [Method] PCR primers were designed according to expressed sequence tag from the different expressions of permethrin-susceptible and resistant strains of Culex quinquefasciatus. Full length cDNA of permethrin-resistance associated gene XND-P450 of Culex pipiens pallens was cloned using RACE method and bioinformatics analysis was conducted. The expression of XND-P450 gene in susceptible and resistant strains was detected using semi-quantitative RT-PCR. [Result] The obtained se- quence of cDNA of XND-P450 gene of Culex pipiens pallens was 1 679 bp coding 523 amino acids. Bioinformatics analysis showed that the full length cDNA of XND-P450 gene of Culex pipiens pallens shared more than 70~ identity in nucleotide sequence with CYP450 of Culex fatigans and Aedes aegypti. The XND-P450 gene was an unstable hydrophilic protein with a relative molecular mass of 33 244.9 u and an i- soelectric point of 8.10. Phylogenetic analysis showed that the full length cDNA of XNDP450 gene of Culez pipiens pallens shared more than 40% identity in amino acids sequence with CYP450 of Culecc fatigans and Anopheles gambiae. Semi-quantitative PCR electrophoresis showed that mRNA expression levels of XND-P450 gene in resistant strains were higher than in susceptible strains. [Conclusion] The permethrinresistance associated gene XND-P450 of Cule：c pipiens pallens was cloned. Its mRNA expression levels in resistant strains were higher than in susceptible strains,indicating that XND-P450 gene was related to permethrin resistance.