摘要
【目的】为了更好地了解昆虫气味结合蛋白(odorant binding proteins,OBPs)在梨小食心虫Grapholita molesta(Busck)嗅觉识别中的作用,并明确其与寄主挥发物的结合特性。【方法】利用RT-PCR和RACE技术克隆梨小食心虫OBP基因;采用RT-PCR和实时定量PCR对该基因在成虫不同组织和羽化后不同日龄成虫中的表达情况进行了测定;以N-phenyl-1-naphthylamine(1-NPN)为荧光探针,采用荧光竞争结合试验对GmolOBP3蛋白的结合特性进行了分析。【结果】得到梨小食心虫一个新的气味结合蛋白基因,命名为GmolOBP3(GenBank登录号:KF395363)。GmolOBP3开放阅读框全长492 bp,编码163个氨基酸残基,预测分子量和等电点分别为18.72 kDa和4.93,呈酸性,具有典型的6个半胱氨酸位点。GmolOBP3在雌、雄成虫触角和腹部均有表达,成虫在羽化后5 d内,雌蛾触角中GmolOBP3表达量随羽化后日龄而增加,但雄蛾在羽化后第5天触角中GmolOBP3表达量显著降低。通过构建GmolOBP3原核表达载体,在大肠杆菌Escherichia coli中诱导表达并获得了GmolOBP3重组蛋白。荧光竞争结合实验对GmolOBP3蛋白与16种寄主挥发物及4种性信息素类似物的结合力发现,在供试的4种梨小食心虫性信息素类似物中,GmolOBP3蛋白与反-8-十二碳烯醋酸酯和十二烷-1-醇不结合,而与顺-8-十二碳烯醋酸酯和顺-8-十二碳烯醇结合,但结合力较弱,结合常数分别为83.00和103.70μmol/L;与16种寄主挥发物结合能力也不强,其中结合最强的是β-紫罗酮,结合常数为49.36μmol/L。【结论】由此推断,GmolOBP3具有选择性识别和结合各种配基的特性。
【Aim】 To study the function and binding characteristics with plant volatiles of the odorantbinding proteins in the oriental fruit moth, Grapholita molesta (Busck).【Methods】 The OBP cDNA from G. molesta was cloned by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), its tissue and developmental expression profiles were detected by RT-PCR and Real-time PCR, and the protein binding property was analyzed using fluorescence competitive binding assay with N-phenyl-1-naphthylamine (1-NPN) as the fluorescent probe. 【Results】A novel OBP cDNA from G. molesta was obtained, which was named as GmolOBP3 (GenBank accession no.: KF395363). GmolOBP3 contains a 492 bp open reading frame encoding a 163-amino-acid residue peptide. The predicted molecular weight and isoelectric point are 18.72 kDa and 4.93, respectively. The mature protein GmolOBP3 includes six typical conservative cysteine residues, which are the hallmark of insect OBPs. GmolOBP3 was expressed in the antennae and abdomen of female and male adults. In five days after eclosion, the expression level of GmolOBP3 in antennae of female moths increased with day-old after eclosion, but on the 5th day after eclosion, the expression quantity in antennae of male moths significantly reduced. In order to obtain the recombinant protein GmolOBP3, we constructed the prokaryotic expression vector of GmolOBP3, which was successfully expressed in the optimized condition. The recombinant protein was purified by anion exchange and Superose-12. The binding affinity of GmolOBP3 with 16 plant volatiles and 4 sex pheromone analogs indicated that GmolOBP3 could not bind with (E)-8-dodecenyl acetate and 1-dodecanol, while had weaker affinity with (Z)-8-dodecenyl acetate and (Z)-8-dodecenol with the association constants of 83.00 and 103.70 μmol/L, respectively. GmolOBP3 could also weakly bind with 16 plant volatiles with the strongest affinity to β-ionone and with a association constant of 49.36 μmol/L. 【Conclusion】 We so inferred that GmolOBP3 protein have characteristics of selective binding with various ligands.
出处
《昆虫学报》
CAS
CSCD
北大核心
2014年第3期274-285,共12页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31272043)
国家公益性行业(农业)科研专项(201103024)
关键词
梨小食心虫
气味结合蛋白
CDNA克隆
组织表达谱
原核表达
荧光竞争结合
Grapholita molesta
odorant binding proteins
cDNA cloning
tissue expression pattern
prokaryotic expression
fluorescence competitive binding assay