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目的 Orco是嗅觉功能的重要组成元件。本研究旨在克隆淡色库蚊的orco基因,分析其编码蛋白的氨基酸序列和orco基因组织表达谱。方法利用RT-PCR技术克隆Cpporco基因,对其编码蛋白的氨基酸序列进行生物信息学分析;利用软件MEGA 5.0对获得的Cpporco进行系统进化树分析;利用HMMTOP version 2.0和TMHMM server version 2.0软件预测CppOrco的跨膜结构域;利用RT-PCR检测雌、雄淡色库蚊的触角、下颚须、喙、头、体中的orco基因表达水平。结果从淡色库蚊中克隆出一个嗅觉受体共表达基因Cpporco,编码区全长1 443bp,编码481个氨基酸,Cpporco与其他蚊种的orco的氨基酸一致性为83.63%,与同属于库蚊属的致倦库蚊Cqorco基因编码的氨基酸序列一致性为98.96%。系统进化树分析CppOrco与CqOrco的亲缘关系较近。CppOrco属于TM7的GPCR,其N末端在胞内,C末端在胞外。Cpporco基因在雌、雄蚊的不同组织中表达量不同,其中在雌蚊触角和喙高表达。结论成功克隆了淡色库蚊orco基因序列全长,其编码蛋白主要在雌、雄蚊触角和喙高表达,为进一步研究蚊虫的嗅觉系统及其功能奠定了基础。
Orco is an important component of olfactory function. The aim of this study was to clone the orco gene of Culex pipiens pallens and to analyze the amino acid sequence of the encoded protein and orco gene expression profile. Methods The Cpporco gene was cloned by RT-PCR and the amino acid sequence of the protein was analyzed by bioinformatics. The phylogenetic tree of Cpporco was analyzed by software MEGA 5.0. The prediction of CppOrco by using HMMTOP version 2.0 and TMHMM server version 2.0 Transmembrane domain; using RT-PCR detection of female and male Culex pipiens antennae, mandibular beard, the beak, head, body orco gene expression levels. Results Cpporco was cloned from Culex pipiens pallens. The coding region of Cpporco was 1 443 bp in length and encoded 481 amino acids. The amino acid identity of Cpporco and other mosquito species was 83.63% Of the Culex pipiens pallens Cqorco gene encoded amino acid sequence identity was 98.96%. Phylogenetic tree analysis CppOrco is closely related to CqOrco. CppOrco belongs to the TM7 GPCR with its N-terminal intracellular and C-terminal extracellular. The Cpporco gene was expressed differently in different tissues of female and male mosquitoes, with high expression in the antennal and beaked females. Conclusion The full-length cDNA sequence of orco gene of Culex pipiens pallens was successfully cloned. Its coding protein is mainly expressed in the antennae and the beak of the female and male mosquitoes, which lays the foundation for further study on the olfactory system and its function in mosquitoes.