小菜蛾气味结合蛋白OBP2基因的克隆、表达谱及其结合特性分析

Cloning,expression profiling and binding characterization of the OBP2 gene in the diamondback moth,Plutella xylostella( Lepidoptera: Plutellidae)

【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。

【Aim 】 Odorant binding proteins（ OBPs） play crucial roles,functioning as transporting vectors of odors,in host location and oviposition selection for many insects. Cloning and identification of OBP genes and clarifying their ligand-binding characteristics of the compounds may help address the molecular mechanisms in P. xylostella. 【Methods】OBP2 was cloned from P. xylostella based on PCR approach. Signal peptide and transmembrane region were predicted and the nucleotide sequence of P.xylostella OBP2（ Pxyl OBP2） was aligned with that of the homologs from other insects using DNAMAN.A neighbor-joining tree was constructed using MEGA5. 0. Stage- and tissue-specific expressions of Pxyl OBP2 were profiled by using real-time quantitative PCR（ qRT-PCR）. Prokaryotic expression vector was constructed to express the recombinant protein,and the purified protein was detected by SDS-PAGE.The protein binding affinity of Pxyl OBP2 with 39 compounds was analyzed using fluorescence competitive binding assay. 【Results】Pxyl OBP2 was successfully cloned and sequenced. Its ORF is 546 bp in length（ Gen Bank accession no. KT070562）,encoding 182 amino acids,and the encoded protein has six cysteine conserved domains,with the predicted molecular mass of 22. 24 k Da and isoelectric point of5. 69. SDS-PAGE analysis showed that the fusion protein was successfully expressed. The expression profiling of Pxyl OBP2 exhibited a higher expression level in virgin male adults than in female adults and mated male adults,and the expression levels of Pxyl OBP2 in legs of male and female adults were higher than those in other tissues. The binding affinity test of Pxyl OBP2 to three sex pheromones and 36 different plant volatiles showed that Pxyl OBP2 could bind with Z-11-16： Ald,with a dissociation constant of48. 951 μmol / L,and with 11 plant volatiles,exhibiting a stronger capability of binding linalool and 1-nonanol with the dissociation constants of 4. 733 and 6. 861 μmol / L,respectively. 【Conclusion】The nucleotide and amino acid sequences of Pxyl OBP2 were characterized. Based on qRT-PCR and competitive binding test results,we infer that Pxyl OBP2 may play important roles in adult courtship of the moth,and host plant volatiles play a synergism role.