论文部分内容阅读
【目的】气味结合蛋白(odorant binding proteins,OBPs)在昆虫寄主定位、产卵地选择等行为中发挥重要作用,克隆与鉴定小菜蛾Plutella xylostella OBP基因、明确其与配体化合物的结合特性有助于阐明小菜蛾嗅觉识别的分子机制。【方法】利用PCR技术克隆小菜蛾OBP2,对获得的编码序列全长进行信号肽及跨膜区域预测,用DNAMAN与其他昆虫的OBP2进行多序列比对,采用MEGA5.0邻接法(neighbor-joining method,NJ)构建进化树。通过实时定量PCR(qRT-PCR)分析Pxyl OBP2在小菜蛾不同发育阶段和不同组织中的表达模式。构建原核表达载体p ET28aPxyl OBP2,进行原核表达及蛋白纯化。利用荧光竞争结合实验对Pxyl OBP2蛋白与39种配基化合物的结合特性进行分析。【结果】成功获得小菜蛾OBP2基因Pxyl OBP2(Gen Bank登录号:KT070562)的编码序列全长,其完整开放阅读框大小为546 bp,编码182个氨基酸,具有气味结合蛋白典型的6个保守半胱氨酸结合位点。荧光定量PCR结果表明,发育表达模式显示,Pxyl OBP2在未交配雄性成虫中的表达量均明显高于雌性成虫和已交配雄虫;组织表达模式显示,Pxyl OBP2在足中的表达量高于其他组织。经预测成熟蛋白大小为22.24 k Da,等电点5.69。SDS-PAGE结果显示融合蛋白成功表达。荧光竞争结合实验对3种性信息素和36种植物挥发物结合发现,Pxyl OBP2与性信息素Z-11-16:Ald可以结合,解离常数48.951μmol/L;可以和11种寄主植物挥发物有效结合,其中,与芳樟醇、正壬醇结合能力最强,解离常数分别为4.733和6.861μmol/L。【结论】本研究明确了Pxyl OBP2的核苷酸、氨基酸序列,并根据qRT-PCR和荧光竞争结合实验结果,推断Pxyl OBP2与小菜蛾雄虫寻求配偶有关,且寄主挥发物芳樟醇、正壬醇起协同促进作用。
【OBJECTIVE】 Odorant binding proteins (OBPs) play an important role in insect host location and spawning site selection. Cloning and identification of Plutella xylostella OBP gene and its binding properties with ligand compounds are helpful To elucidate the molecular mechanism of olfactory traits in Plutella xylostella. 【Method】 OBP2 was cloned by PCR and the signal peptide and transmembrane region were predicted. The DNAMAN and OBP2 of other insects were compared with each other by using MEGA5.0 neighbor-joining method, NJ) to build an evolutionary tree. The expression pattern of Pxyl OBP2 in different developmental stages and different tissues of Plutella xylostella was analyzed by real-time quantitative PCR (qRT-PCR). The prokaryotic expression vector p ET28aPxyl OBP2 was constructed for prokaryotic expression and protein purification. The binding properties of Pxyl OBP2 protein to 39 ligands were analyzed using fluorescence-competitive binding assays. 【Result】 The full length of the OBP2 gene Pxyl OBP2 (Gen Bank accession number: KT070562) was successfully obtained. The complete open reading frame (ORF) of the OBP2 gene was 546 bp, encoding a polypeptide of 182 amino acids with six conservative half Cystine binding site. The results of real-time PCR showed that the expression pattern of Pxyl OBP2 was significantly higher than that of the female and the mated male in the unpaired male adults. The expression pattern of Pxyl OBP2 in the foot was higher than that in the other organization. The predicted size of mature protein was 22.24 kDa, isoelectric point 5.69. SDS-PAGE results showed that the fusion protein was successfully expressed. Fluorescence competition binding experiment showed that Pxyl OBP2 and pheromone Z-11-16: Ald could bind and dissociation constant 48.951μmol / L with three sex pheromones and 36 kinds of plant volatiles. It could volatilize with 11 kinds of host plants Among them, Linalool and n-nonyl alcohol had the strongest binding ability and the dissociation constants were 4.733 and 6.861μmol / L, respectively. 【Conclusion】 In this study, the nucleotide and amino acid sequences of Pxyl OBP2 were determined. According to the results of qRT-PCR and fluorescence competition, it was concluded that Pxyl OBP2 was associated with male diamondback moth seeking spouses, and that the host volatile linalool, Nonanol play a synergistic role in promoting.