[1]何兴菲 张嘉俊 乔梁 陈斌 何正波**.中华按蚊气味结合蛋白AsinOBP62基因的克隆、表达及与人体挥发物的结合特性[J].应用与环境生物学报,2021,27(04):1-14.[doi:10.19675/j.cnki.1006-687x.2020.08003]
HE Xingfei,ZHANG Jiajun,QIAO Liang,et al.Cloning and expression of odorant binding protein AsinOBP62 in Anopheles sinensis and the binding characteristics with human volatiles[J].Chinese Journal of Applied & Environmental Biology,2021,27(04):1-14.[doi:10.19675/j.cnki.1006-687x.2020.08003]
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中华按蚊气味结合蛋白AsinOBP62基因的克隆、表达及与人体挥发物的结合特性()
HE Xingfei,ZHANG Jiajun,QIAO Liang,et al.Cloning and expression of odorant binding protein AsinOBP62 in Anopheles sinensis and the binding characteristics with human volatiles[J].Chinese Journal of Applied & Environmental Biology,2021,27(04):1-14.[doi:10.19675/j.cnki.1006-687x.2020.08003]
《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]
- 卷:
- 27卷
- 期数:
- 2021年04期
- 页码:
- 1-14
- 栏目:
- 研究论文
- 出版日期:
- 2021-08-25
文章信息/Info
- Title:
- Cloning and expression of odorant binding protein AsinOBP62 in Anopheles sinensis and the binding characteristics with human volatiles
- Keywords:
- Anopheles sinensis ; odorant-binding protein 62; gene cloning; prokaryotic expression; human volatile; binding affinities
- 摘要:
- 嗅觉系统在蚊虫追寻宿主、寻找配偶、选择产卵场所等行为中起着重要的作用，气味结合蛋白与气味分子结合是昆虫嗅觉识别的第一步生化反应。为研究中华按蚊气味结合蛋白在追寻宿主中的作用，通过反转录PCR（RT-PCR）克隆气味结合蛋白62（Odorant binding protein 62, AsinOBP62）基因，利用定量PCR分析AsinOBP62在不同组织（触角、喙、下颚须及头胸腹部）、不同发育时期（蛹期、羽化1d、3d）和吸血前后的表达水平，通过荧光竞争结合实验测定AsinOBP62蛋白与16种人体气味物质和2种趋避剂的结合能力。结果显示，AsinOBP62基因开放阅读框长507 bp，编码168个氨基酸，具有气味结合蛋白典型的6个保守半胱氨酸残基，属于Classic亚家族成员。定量PCR结果显示，AsinOBP62主要在成蚊触角中表达，羽化后AsinOBP62的表达水平逐渐增强。吸食血液后，AsinOBP62的表达水平显著下降。荧光竞争结合实验显示，在测定的18种气味物质中，AsinOBP62与12种化合物具有结合活性，表明AsinOBP62对人体挥发物具有明显的选择性。其中，与2-甲基吡啶结合能力最强，解离常数Ki = 34.34 μmol/L。根据AsinOBP62基因的表达特点和重组蛋白的结合特性，推断AsinOBP62基因可能在雌蚊追寻宿主的过程中起着重要的作用。（图6 表2 参50）
- Abstract:
- The olfactory system of mosquitoes plays a crucial role in many types of behavior, including host seeking, mate selection and oviposition. The binding of odorant binding protein with odor molecules is the first biochemical reaction of insect olfactory recognition. To reveal the function of odorant binding protein of Anopheles sinensis in the host-seeking behavior, the odorant binding protein 62 (AsinOBP62) gene was cloned by reverse transcription PCR, its expression levels in different adult tissues (antennae, proboscis, maxillary palps, head without chemosensory tissues, thorax and abdomen), different developmental stages (pupal, 1 d and 3 d after eclosion), before and post blood meal of An. sinensis were measured by real time quantitative PCR (qPCR). The recombinant protein of AsinOBP62 was expressed and purified by prokaryotic expression and affinity chromatography, and the binding affinities of the AsinOBP62 recombinant protein with 16 human odors and 2 repellents were analyzed by fluorescence competitive binding assay. The results showed that the open reading frame of AsinOBP62 gene is 507 bp in length, encoding 168 amino acids. The matured protein possessed six conserved cysteines and could be classified into the Classic OBP subfamily. qPCR results showed that AsinOBP62 was mainly expressed in the antennae of adult mosquitoes, and the expression level of AsinOBP62 gradually increased after eclosion. After blood meal, the expression level of AsinOBP62 decreased significantly. The fluorescence competitive binding experiment showed that AsinOBP62 had binding activity with 12 compounds among 16 human odors, indicating that AsinOBP62 had obvious selectivity to human volatiles. Among them, 2-methylpyridine had the strongest binding ability, and the dissociation constant Ki = 34.34 μmol/L. Based on the expression characteristics of AsinOBP62 gene and the binding characteristics of the recombinant protein, it was concluded that AsinOBP62 gene may play an important role in the host-seeking behavior of female A. sinensis mosquitoes.
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[1]黄婷,冉永红,罗世惠,等.红千层精油的抗氧化活性及其对中华按蚊幼虫的杀虫活性测定[J].应用与环境生物学报,2015,21(06):1086.[doi:10.3724/SP.J.1145.2015.04058]
HUANG Ting,RAN Yonghong,LUO Shihui,et al.Antioxidant capacity and larvicidal activity of Callistemon rigidus essential oil[J].Chinese Journal of Applied & Environmental Biology,2015,21(04):1086.[doi:10.3724/SP.J.1145.2015.04058]
备注/Memo
- 备注/Memo:
-
收稿日期 Received: 2020-08-02 接受日期 Accepted: 2020-10-16
国家自然科学基金项目（31572332）、重庆市留创项目和重庆市自然科学基金项目（cstc2019jcyj-msxmX0242）资助
通讯作者 Corresponding author (E-mail: zhengbohe@cqnu.edu.cn)
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更新日期/Last Update:
2020-11-06