瓜实蝇普通气味结合蛋白基因的克隆及原核表达  

Cloning and prokaryotic expression of cDNA encoding general odorant binding protein from Bactrocera cucurbitae(Coquillett)

作　　者：申建梅[1] 胡黎明[1] 宾淑英[1] 廖泓之[1] 林进添[1] 

机构地区：[1]仲恺农业工程学院植物保护系,广州510225

出　　处：《华中农业大学学报》2011年第4期455-460,共6页Journal of Huazhong Agricultural University

基　　金：国家自然科学基金项目(30971923)

摘　　要：利用RT-PCR和RACE方法克隆获得瓜实蝇Bactrocera cucurbitae(Coquillett)普通气味结合蛋白(general odorant binding protein,GOBP)基因的cDNA全长序列,命名为BcucOBP。测序结果表明,BcucOBP开放阅读框全长447 bp,编码149个氨基酸。氨基酸序列比对及三维结构同源建模表明,此序列具有OBPs的典型特征,序列中具有6个保守的半胱氨酸和6个α螺旋区域。构建了重组表达载体pET28a(+)-BcucOBP,并转化大肠杆菌BL21(DE3)中。SDS-PAGE及Western blotting分析表明,在IPTG进行诱导下目标蛋白以His-标签融合蛋白的形式在宿主菌中得到稳定表达。荧光定量PCR分析表明,BcucOBPmRNA在除雌虫腹节外的各个组织中都有表达,但在触角中的表达量最高。另外,BcucOBPmRNA在昆虫前足和生殖节中具有明显的性别差异表达特征,推测其不仅具有普通气味蛋白的功能,还可能参与了信息素的运输过程,在瓜实蝇交配行为中发挥重要作用。In this study,OBP from Bactrocera cucurbitae was cloned and named as BcucOBP.The full-length of open reading frame（ORF）is 447 bp,encoding 149 amino acid residues.Amino acid sequence alignment and 3D-Homology Modeling analysis showed that BcucOBP shared the typical structural feature of OBP with other insects,including 6 conservative Cys residues and 6α-helixes in the sequence.A recombinant plasmid pET28a（＋）-BcucOBP was constructed and transformed into Escherichia coli BL21（DE3）.Western blotting indicated that the His-tagged BcucOBP stably expressed in DE3 induced by IPTG.Real-time PCR analysis shows that BcucOBP mRNA is expressed nearly in all tissues except for female visceral segment.The relative concentration of BcucOBP mRNA is the highest in antenna of B.cucurbitae compared with other tissues.BcucOBP mRNA shows clear gender-differential expression pattern in general section and the foreleg.The special expression pattern suggested that BcucOBP not only acted as a general odor protein but also was involved in the transport of pheromone in the mating behavior of B.cucurbitae.

关 键 词：瓜实蝇 气味结合蛋白 基因克隆 组织表达特征 原核表达 

分 类 号：Q966[生物学—昆虫学]