大劣按蚊Torso⁃like基因鉴定分子结构及表达特征

Identification,molecular structure and expression characteristics of Torso⁃like gene in Anopheles dirus

目的鉴定大劣按蚊Torso⁃like(tsl)基因及表达特征,为深入研究tsl基因功能提供理论基础。方法根据黑腹果蝇和冈比亚按蚊tsl基因,检索大劣按蚊全基因组并鉴别大劣按蚊tsl基因,设计特异性引物,并采用PCR和逆转录PCR技术扩增目的基因。利用生物信息学软件分析tsl基因编码TSL蛋白的理化性质、信号肽、跨膜结构、蛋白质二级结构和三级结构,并进行系统发育进化分析。利用实时荧光定量PCR技术检测该基因在大劣按蚊各组织中的表达水平。结果大劣按蚊tsl基因全长16751 bp,CDS区长1134 bp,编码377个氨基酸。TSL蛋白属于亲水性稳定蛋白。经生物信息学预测,TSL蛋白为位于膜外的分泌蛋白,且包含信号肽;其二级结构含α⁃螺旋(51.72%)、延伸链(12.20%)、β⁃折叠(4.78%)和无规则卷曲(31.30%),且能以5cj9.1.A为模板进行3D同源建模。系统发育进化分析发现大劣按蚊TSL蛋白与法老按蚊TSL蛋白亲缘关系较近。实时荧光定量PCR检测显示,tsl基因在大劣按蚊头、胸、腹、足均有表达,且在头部表达量最高、足部表达量较低。结论本研究从基因组水平鉴定了大劣按蚊tsl基因,分析了其蛋白结构及组织特异性表达特征,为进一步深入研究该基因功能奠定了基础。

Objective To characterize Torso⁃like(tsl)gene and investigate its expression characteristics in Anopheles dirus,so as to provide a theoretical basis for subsequent functional studies of the tsl gene.Methods According to the coding sequences of Drosophila melanogaster and An.gambiae tsl genes,the complete genome of An.dirus was retrieved and the An.dirus tsl gene was characterized.Specific primers were designed and the target gene was amplified using PCR and reverse⁃transcription PCR assays.The physicochemical properties,signal peptide,transmembrane structure,secondary structure and tertiary structure of the encoded protein TSL were analyzed using bioinformatics tools,and a phylogenetic analysis was performed.In addition,the specific expression of the tls gene was detected in various tissues of An.dirus using a quantitative real⁃time PCR assay.Results The An.dirus tsl gene was 16751 bp in length with a CDS region of 1134 bp,encoding 377 amino acids,and the encoded TSL protein was a stably hydrophilic protein.The TSL protein was predicted to be a secretory protein that was located in extra⁃mem⁃brane regions containing signal peptides.The secondary structure of the TSL protein containedα⁃helix(51.72%),extended strand(12.20%),β⁃bridge(4.78%)and random coil(31.30%)in the secondary structure,and a 3D homology model was generat⁃ed using 5cj9.1.A as a template.Phylogenetic analysis revealed a close genetic relationship in the TSL protein between An.dirus and An.farauti.In addition,quantitative real⁃time PCR assay detected the tsl gene expression in the head,chest,abdomen and foot of An.dirus,with the highest expression in the head and low expression in the foot.Conclusion The tsl gene is character⁃ized in An.dirus at a genomic level,and the prediction of the TSL protein structure and the elucidation of the tissue⁃specific tsl gene expression in An.dirus provide a basis for the further studies on the gene functions.