淡色库蚊嗅觉受体基因orco的克隆及组织表达谱分析

Cloning and tissue expression profiling of the olfactory receptor gene orco from Culex pipiens pallens

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。

Objective orco is an important component of olfactory functions.The aims of the present study were to clone and identity the orco gene in Culex pipiens pallens and to analyze its tissue expression profiles. Methods Cpporco was cloned using RT-PCT and analyzed bioinformatically.Amino acid sequences of orco were aligned using the program ClustalW and a neighbor-joining tree was constructed using the software MEGA 5.0.The transmembrane region of Cpporco was predicted using HMMTOP version 2.0and TMHMM server version 2.0.Expression profiles in different tissues（antennae,the proboscis,the maxillary palp,and the head and body）were determined using RT-PCR. Results In this study,the full-length cDNA sequence of orco was obtained fromCulex pipiens pallens.Cpporco is 1,443 bp in length and encodes 481 amino acids.The amino acid sequence of Cpporco was 83.63% similar to that of orco from other mosquito species,and Cpporco was most similar（98.96%）to Cqorco.Phylogenetic tree analysis indicated that Cpporco is closely related to Cqorco.Cpporco has seven transmembrane domains with an intracellular N-terminus and an extracellular C-terminus.Levels of Cpporco expression differed in different tissues of male and female mosquitoes,and the highest level of Cpporco expression was in the antennae and proboscis of females. Conclusion Cloning of the full length of the Cpporco gene and its expression profiles have provided a basis for future study of the olfactory system of mosquitoes and its functions.