›› 2017, Vol. 60 ›› Issue (10): 1141-1154.doi: 10.16380/j.kcxb.2017.10.005

• 研究论文 • 上一篇    下一篇


李广伟*, 陈秀琳, 尚天翠   

  1. (伊犁师范学院生物与地理科学学院, 新疆伊宁 835000)
  • 出版日期:2017-10-20 发布日期:2017-10-20

cDNA cloning, expression and ligand binding properties of the odorant binding protein AglaOBP12 in the Asian longhorned beetle, Anoplophora glabripennis (Coleoptera: Cerambycidae)

LI Guang-Wei*, CHEN Xiu-Lin, SHANG Tian-Cui   

  1. (College of Biology and Geography, Yili Normal University, Yining, Xinjiang 835000, China)
  • Online:2017-10-20 Published:2017-10-20

摘要: 【目的】克隆和鉴定光肩星天牛Anoplophora glabripennis气味结合蛋白(odorant binding proteins, OBPs)基因,明确其表达特点及与寄主植物挥发物的结合特性,有助于阐明光肩星天牛嗅觉识别的分子机制。【方法】根据光肩星天牛雌成虫触角转录组数据,利用RT-PCR克隆OBP12基因,并进行生物信息学分析。通过实时定量PCR(qRT-PCR)测定OBP12在光肩星天牛成虫触角、头(移除触角)、胸、腹、足、翅中的转录水平。利用原核表达系统和Ni离子亲和层析技术表达和纯化OBP12重组蛋白,荧光竞争结合实验测定重组蛋白与39种气味配体的结合能力。【结果】获得光肩星天牛气味结合蛋白基因AglaOBP12(GenBank登录号: KX890109)的完整编码序列,其开放阅读框长414 bp,编码137个氨基酸,N末端具有18个氨基酸组成的信号肽序列,蛋白序列具有6个保守的半胱氨酸残基,AglaOPB12属于Classical OBPs亚家族基因。qRT-PCR测定结果表明,AglaOBP12主要在成虫触角中表达,在其他组织微量表达。在待测的39种寄主植物挥发物中,重组蛋白AglaOBP12仅与19种化合物具有结合活性,表明AglaOBP12对寄主植物挥发物具有明显的选择结合特性。重组蛋白AglaOBP12与十二烷醇、十四烷醇、法尼醇、十二醛、乙酸-顺-3-己烯酯和β石竹烯的结合能力较强,结合常数分别为1.96, 0.96, 1.03, 0.82, 0.77和0.74 μmol/L。【结论】明确了AglaOBP12的核苷酸和氨基酸序列组成,重组AglaOBP12蛋白与主链有12个碳原子的醇类、醛类和萜烯类挥发物有特异性的结合活性。根据AglaOBP12基因的表达特点和重组蛋白的结合特性,推测AglaOBP12在光肩星天牛成虫定位补充营养寄主植物中发挥重要作用。

关键词: 光肩星天牛, 气味结合蛋白, 嗅觉, 基因表达, 荧光竞争结合实验

Abstract: 【Aim】 Cloning and identification of the odorant binding protein (OBP) genes and clarifying their expression features and ligand-binding characteristics with host-plant volatiles are helpful to address the molecular mechanisms of olfaction in the Asian longhorned beetle, Anoplophora glabripennis. 【Methods】 Based on the antenna transcriptome data of A. glabripennis female adult, the complete coding sequence of OBP12 was cloned using RT-PCR and bioinformatically analyzed. The expression levels of OBP12 in the antenna, head (without antennae), thorax, abdomen, leg and wing were assayed by realtime quantitative PCR (qRT-PCR). The recombinant OBP12 protein was prokaryotically expressed and then purified by Ni ion affinity chromatography. The binding affinities of the recombinant OBP12 with 39 ligands were assessed by using fluorescent competitive binding assay. 【Results】 AglaOBP12 of A. glabripennis was successfully cloned and sequenced (GenBank accession no.: KX890109). Its ORF is 414 bp in length, encoding 137 amino acids with the signal peptide of 18 amino acids at the N-terminal. The matured protein possessed six conserved cysteines and could be classified into the Classical OBP subfamily. qRT-PCR results showed that AglaOBP12 primarily expressed in the antenna and slightly expressed in other tissues. Among the 39 chemicals tested, the recombinant AglaOBP12 had only binding activities to 19 compounds, suggesting that AglaOBP12 has obvious selective binding characteristics to host plant volatiles. The recombinant AglaOBP12 showed higher binding affinities to dodecanol, tetradecanol, farnesol, dodecanal, cis-3-hexenyl acetate and β-caryophyllene, with the Ki values of 196, 0.96, 1.03, 0.82, 0.77 and 0.74 μmol/L, respectively. 【Conclusion】 The nucleotide and deduced amino acid sequences of AglaOBP12 were characterized in this study. AglaOBP12 shows particularly strong binding affinities to alcohols, aldehydes and terpenes with 12 carbon atoms in the main-chain. Based on the results of qRT-PCR and fluorescent competitive binding assay, we speculated that AglaOBP12 in the Asian longhorned beetle plays an important role in locating trophic host plants.

Key words: Anoplophora glabripennis, odorant binding protein, olfactory, gene expression, fluorescence competitive binding assay