| 李广伟，陈秀琳，尚天翠，姚付龙,2017,光肩星天牛气味结合蛋白AglaOBP1的克隆、表达及结合特性[J].环境昆虫学报,(4):919-929 |
| 光肩星天牛气味结合蛋白AglaOBP1的克隆、表达及结合特性 |
| Coning, expression, and binding properties of odorant binding protein 1 from Anoplophora glabripennis Motschulsky (Coleoptera: Cerambycidae) |
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| DOI： |
| 中文关键词: 光肩星天牛 气味结合蛋白 嗅觉 荧光竞争结合实验 挥发物 |
| 英文关键词:Anoplophora glabripennis odorant binding protein olfactory fluorescence binding assay volatiles |
| 基金项目:新疆维吾尔自治区高校科研计划项目（XJEDU2014S062）；新疆维吾尔自治区自然科学基金（2015211C299） |
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| 中文摘要: |
| 利用RT-PCR技术克隆了光肩星天牛Anoplophora glabripennis Motschulsky气味结合蛋白（odorant binding proteins，OBPs）基因AglaOBP1（GenBank登录号：KX660670），AglaOBP1的开放阅读框长435 bp，编码144个氨基酸，其中N端有21个氨基酸组成的信号肽序列，成熟蛋白序列具有4个保守的半胱氨酸残基，AglaOPB1属于Minus-C OBP亚家族基因。AglaOBP1主要在成虫触角中表达，且雌虫触角中的表达量显著高于雄虫触角。重组蛋白AglaOBP1与34种气味配体的竞争结合实验表明，AglaOBP1具有广泛的结合谱，能与五角枫挥发物中的醇类、醛类、萜烯类和酮类物质结合，其中与顺-3-己烯-1-醇、顺-2-己烯-1-醇、1-己醇、反-2-己烯醛、反-2-癸烯醛、β-石竹烯、(+)-桧烯、α-蒎烯、1-(2,3-二甲基苯基)-乙酮的结合能力较强，结合常数分别为5.88、8.33、12.29、12.55、11.90、7.47、9.07、10.29和13.12 μM。此外，配体的官能团、碳链长度、空间构型也影响AglaOBP1对气味分子的结合。AglaOBP1基因在成虫触角中高丰度表达，其重组蛋白能够与五角枫挥发物的多种组分结合，表明AglaOBP1在成虫寄主定位选择中起重要作用。 |
| 英文摘要: |
| Odorant binding protein gene AglaOBP1 from Anoplophora glabripennis was cloned via reverse-transcription PCR, The sequence of AglaOBP1 has ORF of 435 bp that encodes 144 amino acids, including signal peptides of 21 amino acids at the N-terminal. The matured protein possessed four conserved cysteine and can classified Minus-C OBP subfamily genes. The results of RT-qPCR showed that AglaOBP1 was highly expressed in antennae, and the expression quantity in female antennae was significantly higher than that in male antennae. Recombinant AglaOBP1 (rAglaOBP1)was successfully expressed in a prokaryotic expression system and purified via Ni-NTA His·Bind Resin. The results of fluorescence competitive binding assays demonstrated that rAglaOBP1 exhibited broad binding properties and can bind aliphatic alcohols, aldehydes, terpenes and ketones. rAglaOBP1 showed higher binding abilities to Cis-3-Hexen-1-ol, Cis-2-Hexen-1-ol, 1-Hexanol, (E)-2-Hexenal, (E)-2-Decenal, β-Caryophyllene, (+)-Sabinene, α-Pinene, and 2,3-Dimethylacetophenone with the Ki value of 5.88, 8.33, 12.29, 12.55, 11.90, 10.29, 7.47, 9.07 and 13.12 μM, respectively. Besides, the functional group, the length of carbon chain and space isomerism of ligands were all influence the binding affinities of AglaOBP1. AglaOBP1 was highly expressed in antennae, and the recombinant protein showed high binding affinities with components emitted from Acer mono, illuminating AglaOBP1 play an important role in seeking host plants of the adults. |
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