美洲斑潜蝇气味结合蛋白OBP13的鉴定与功能

doi:10.3864/j.issn.0578-1752.2018.05.008

摘要/Abstract

摘要： 【目的】克隆与鉴定美洲斑潜蝇（Liriomyza sativae）气味结合蛋白（odorant binding protein，OBP）基因，并对其序列特征、表达情况、系统发育及蛋白功能进行研究，为深入研究美洲斑潜蝇嗅觉机制提供依据。【方法】通过PCR技术克隆美洲斑潜蝇OBP13编码区全长，用DNAMAN对其进行序列分析，用BLAST进行同源性比较，并使用MEGA6.0构建进化树，进行系统发育分析。通过实时定量PCR对OBP13在美洲斑潜蝇不同组织中的表达情况进行分析。构建原核表达载体，进行原核表达及蛋白纯化，获取重组蛋白。设定荧光分光光度计的激发波长为337 nm，以1-NPN为荧光探针，研究OBP13与25种不同气味配基的结合特性。使用间接免疫荧光染色技术及制备的OBP13的特异性多克隆抗体，对其在组织中的分布情况进行定位。将美洲斑潜蝇触角进行包埋、切片、免疫染色，并在激光共聚焦显微镜下进行观察,了解OBP13在触角感器中的亚细胞分布。【结果】获得了一个美洲斑潜蝇气味结合蛋白基因，命名为LsatOBP13（GenBank登录号：KT250751）。LsatOBP13开放阅读框全长462 bp，编码153个氨基酸，预测成熟蛋白分子量为17.80 kD，等电点为5.75，N-末端的前17个氨基酸为信号肽序列。具有4个保守的半胱氨酸位点，为Minus-C OBP。系统发育分析显示，其与地中海实蝇CcapOBP99a-like位于同一小分支上，亲缘关系较近。检测LsatOBP13在不同组织的表达水平，发现LsatOBP13在触角中的表达量远远高于其他组织。成功构建了重组表达载体并获得了高纯度的重组蛋白。对25种气味配基的结合能力检测，发现LsatOBP13与反式-2-己烯醛、芳樟醇、1-辛烯-3-醇、α-紫罗兰酮、苯并噻唑、β-紫罗兰酮具有较强的结合能力，解离常数分别为12.592、10.995、11.165、11.224、10.336、9.218 μmol·L^-1，其中与β-紫罗兰酮亲和能力最强。通过免疫荧光定位，发现其在触角中主要定位在毛形感器和锥形感器上，在触角嗅觉窝及触角芒连接处也有分布。【结论】美洲斑潜蝇OBP13为非典型的气味结合蛋白Minus-C OBP。表达情况、气味结合特性、免疫定位结果表明，OBP13主要存在于美洲斑潜蝇触角中，参与触角对绿叶植物中大量存在的气味物质的识别过程，推测其在美洲斑潜蝇嗅觉识别、寄主植物定位中发挥功能。

关键词: 美洲斑潜蝇, 气味结合蛋白, 序列分析, 表达情况, 气味结合特性, 免疫荧光定位

Abstract: 【Objective】The objective of this study is to clone and clarity odorant binding protein gene in the leafminer (Liriomyza sativae), analyze the sequence properties, gene expression, phylogeny and protein function, which would provide a basis for further study of the olfactory mechanism.【Method】The whole coding region of OBP gene from L. sativae was cloned by PCR strategy. Nucleotide sequence was analyzed using DNAMAN and homology comparison was analyzed using BLAST. An evolutionary tree was constructed by MEGA 6.0 to analyze phylogeny. Expression situation of LsatOBP13 in different tissues was assayed by the real-time quantitative PCR. Prokaryotic expression vector was constructed and then the recombinant protein was expressed and purified. The excitation wavelength of the fluorescence spectrophotometer was 337 nm and the 1-NPN was used as fluorescent probe to study the binding properties of OBP13 with 25 different odor ligands. The distribution of the LsatOBP13 was located by the indirect immuno-fluorescent staining and specific polyclonal antibody which was already prepared. The antenna of L.sativae was embedded, sliced up and observed under laser scanning confocal microscope to study the subcellular distribution of OBP13.【Result】An OBP gene in L.sativae was obtained and named as LsatOBP13. The accession number in GenBank ID is KT250751. The whole length of LsatOBP13 coding region is 462 bp that encodes a putative protein of 153 amino acids with a molecular mass of 17.80 kD and an isoelectric point of 5.75, and deduced amino acid sequence possesses a putative signal peptide of 17 amino acid residues at the N terminus. There are four conservative cysteine sites in the protein sequence, so that the LsatOBP13 is a Minus-C OBP. Phylogenetic analysis showed that the OBP13 and CcapOBP99a-like were clustered into one branch and had a high homology. According to the expression level in different tissues, the expression level in the antenna was much higher than that in other tissues. The recombinant expression vector was successfully constructed and the recombinant protein of high purity was obtained. After testing combining capacity of 25 odor ligands, it was found that LsatOBP13 had a good combining capacity with trans-2-hexenal, linalool, 1-octen-3-ol, α-ionone, benzothiazole and β-ionone, and the dissociation constants were 12.592, 10.995, 11.165, 11.224, 10.336, 9.218 μmol·L^-1, respectively. In these 6 kinds of odors, the affinity of β-ionone was the strongest. By immunofluorescence localization, it was found that it was mainly located in trichoid sensilla and basiconic sensilla on the crosscut flagellum section, and also in the olfactory pit and the joint of arista and funicular section.【Conclusion】The LsatOBP13 is a Minus-C OBP which belongs to the atypical OBPs. Expression situation, odorant binding characteristic and immunofluorescence localization showed that OBP13 existed mainly in the antenna of L. sativae and involved in the recognition process of odors in green leafed plants. It is presumed that it plays a role in olfactory recognition and host plant location of L.sativae.

Key words: Liriomyza sativae, odorant binding protein, sequence analysis, expression situation, odorant binding characteristic, immunofluorescence localization

陈东凯，张林雅，邢振龙，雷仲仁. 美洲斑潜蝇气味结合蛋白OBP13的鉴定与功能[J]. 中国农业科学, 2018, 51(5): 893-904.

CHEN DongKai, ZHANG LinYa, XING ZhenLong, LEI ZhongRen. Identification and Function of the OBP13 Protein from the Leafminer (Liriomyza sativae)[J]. Scientia Agricultura Sinica, 2018, 51(5): 893-904.

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