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目的克隆淡色库蚊(Culex pipiens pallens)ATP合酶g亚基基因编码区序列并进行生物信息学分析,比较其在溴氰菊酯抗性品系和敏感品系中的表达方法利用PCR方法扩增ATP合酶g亚基基因编码区序列。利用生物信息学分析工具,预测ATP合酶g亚基基因编码蛋白的理化性质、蛋白结构域、跨膜区、信号肽和亚细胞定位;对淡色库蚊和其他物种的ATP合酶g亚基进行氨基酸序列多重比对,并构建系统发育树。通过实时定量PCR检测比较ATP合酶g亚基基因在淡色库蚊溴氰菊酯敏感品系和抗性品系中的表达。结果克隆出长306bp的淡色库蚊ATP合酶g亚基基因编码区序列(GenBank登录号:KY783435),该基因编码101个氨基酸。生物信息学分析ATP合酶g亚基基因编码蛋白理论分子质量单位(Mr)为11×10~3,等电点为9.56,在第6-98位氨基酸具有ATP合酶g亚基结构域,未发现信号肽和跨膜区,亚细胞定位预测以线粒体表达量最多(占39.1%)。实时定量PCR检测ATP合酶g亚基基因在淡色库蚊溴氰菊酯抗性品系中的表达量是敏感品系中表达量的3.14倍。结论获得淡色库蚊ATP合酶g亚基基因编码区序列,并进行了生物信息学分析,证实该基因在溴氰菊酯抗性品系中高表达。
Objective To clone and sequence the coding region of ATP synthase subunit g gene of Culex pipiens pallens and analyze its bioinformatics. The expression of ATPase gene in deltamethrin resistant strain and susceptible strain was amplified by PCR Synthase g subunit gene coding region sequence. Bioinformatics analysis tools were used to predict the physical and chemical properties, protein domain, transmembrane domain, signal peptide and subcellular localization of the ATP synthase g subunit gene. The ATP synthase g subunit of Culex pipiens pallens and other species Amino acid sequence multiple alignment, and the construction of phylogenetic tree. The expression of ATP synthase subunit gene in deltamethrin sensitive and resistant strains of Culex pipiens pallens was detected by real-time quantitative PCR. Results The coding sequence of ATP synthase g subunit gene of Culex pipiens pallens (GenBank accession number: KY783435) was cloned. The gene encoded 101 amino acids. Bioinformatics analysis of ATP synthase g subunit gene encoded protein theoretical molecular mass unit (Mr) of 11 × 10 ~ 3, the isoelectric point of 9.56, in the 6-98 amino acids with ATP synthase g subunit domain, No signal peptide and transmembrane region were found, and subcellular localization predicted the highest expression of mitochondria (39.1%). Quantitative real-time PCR assay showed that the expression level of ATP synthase subunit gene in deltamethrin-resistant strain of Culex pipiens pallens was 3.14 times that of the sensitive strain. Conclusion The coding sequence of ATP synthase g subunit gene of Culex pipiens pallens was obtained and analyzed by bioinformatics. The results showed that the gene was highly expressed in deltamethrin resistant strains.