沙葱萤叶甲气味结合蛋白GdauOBP20的基因克隆、 原核表达及其结合特性

doi:10.3864/j.issn.0578-1752.2019.20.020

摘要/Abstract

摘要：

【目的】 沙葱萤叶甲（Galeruca daurica）是近年来在内蒙古草原上暴发成灾的新害虫,本文旨在克隆沙葱萤叶甲气味结合蛋白基因GdauOBP20的cDNA全长序列,明确其重组蛋白与寄主植物挥发物的结合特性,为揭示沙葱萤叶甲嗅觉的分子机理打下基础。【方法】 基于沙葱萤叶甲转录组数据,利用RACE技术对沙葱萤叶甲气味结合蛋白基因GdauOBP20进行cDNA全长克隆;利用生物信息学软件预测分析其编码蛋白的理化特性和结构特征;通过原核表达系统表达目的蛋白,并使用Ni-NTA Agarose亲和层析柱进行重组蛋白纯化。最后采用荧光竞争结合的方法,以N-苯基-1-萘胺（N-phenyl-1-naphthylamine,1-NPN）为荧光探针,检测GdauOBP20重组蛋白与13种主要寄主挥发物的结合情况。【结果】 GdauOBP20的cDNA全长序列为567 bp（GenBank登录号MK250532）,其中5′末端非编码区长24 bp,3′末端非编码区长123 bp,具有ployA尾结构;开放阅读框（ORF）全长为420 bp,编码139个氨基酸。氨基酸序列中含有4个保守的半胱氨酸位点,属于Minus-C OBP亚家族。预测蛋白三级结构中含有6个α螺旋和两对由半胱氨酸形成的二硫键。成功构建了重组表达载体并获得了高纯度的重组蛋白。重组蛋白GdauOBP20与荧光探针1-NPN的结合常数为12.8 μmol·L ^-1,结合能力较好,可作为本试验的荧光报告子。在所测的13种主要寄主植物挥发物中,除与二烯丙基三硫醚无结合能力外,GdauOBP20重组蛋白与其他12种寄主植物挥发物均有不同程度的结合能力,其中与对二甲苯和环庚三烯的结合能力最强,解离常数分别为22.91和26.55 μmol·L ^-1,而与月桂烯结合能力最弱,解离常数为116.29 μmol·L ^-1。【结论】 沙葱萤叶甲GdauOBP20能与寄主植物的多种主要挥发性物质结合,推测其可能在沙葱萤叶甲对寄主植物的定位过程中发挥重要的作用。

关键词: 沙葱萤叶甲, 气味结合蛋白, RACE克隆, 原核表达, 荧光竞争结合试验

Abstract:

【Objective】 Galeruca daurica is a new pest with outbreak status in the Inner Mongolia grasslands in recent years. The objective of this study is to clone the full-length cDNA sequence of GdauOBP20, and clarify the binding property of the recombinant protein to main host plant volatiles, which will lay a necessary foundation for revealing the molecular mechanism of olfaction in G. daurica.【Method】 RACE technique was used to clone the full-length cDNA of GdauOBP20 based on the transcriptome database of G. daurica. The physicochemical properties and structural characteristics of the encoded protein were predicted and analyzed by bioinformatics software. The recombinant protein GdauOBP20 was induced to express by constructing prokaryotic expression system, and purified by using the Ni-NTA Agarose affinity column. Finally, the fluorescence competitive assay was applied, and N-phenyl-1-naphthylamine (1-NPN) was selected as the fluorescence probe to measure the binding profiles of GdauOBP20 recombinant protein with 13 main host plant volatiles.【Result】 The full-length cDNA of GdauOBP20 is 567 bp (GenBank accession number: MK250532), with the non-coding regions of 5′ and 3′ ends of 24 bp and 123 bp, respectively, and a ployA tail structure. The open reading frame (ORF) is 420 bp, encoding 139 amino acids. The amino acid sequence of GdauOBP20 contains 4 conserved cysteine residues, indicating that it belongs to Minus-C OBP subfamily. The three-dimensional structure prediction of GdauOBP20 contains six alpha helix and two pairs of disulfide bonds formed by cysteine. The recombinant expression vector was successfully constructed, and the recombinant protein with high purity was obtained. The binding capacity of the recombinant protein GdauOBP20 to the fluorescence probe 1-NPN was strong with a binding constant of 12.8 μmol·L ^-1, indicating that it could be used as the fluorescence reporter in this experiment. Affinities of recombinant protein GdauOBP20 with 13 main host plant volatiles were tested. Among them, except diallyl trisulfide, other 12 volatiles showed certain binding capacities with the recombinant protein, and p-xylene and 1,3,5-cycloheptatriene displayed the strongest affinity with the dissociation constants of 22.91 and 26.55 μmol·L ^-1, respectively, whereas myrcene exhibited the weakest binding affinity with the dissociation constant of 116.29 μmol·L ^-1.【Conclusion】 GdauOBP20 has a certain binding capacity with main host plant volatiles, suggesting that it may play an important role in the localization of host plants.

Key words: Galeruca daurica, odorant binding protein, RACE cloning, prokaryotic expression, fluorescence competitive binding assay

李玲,谭瑶,周晓榕,庞保平. 沙葱萤叶甲气味结合蛋白GdauOBP20的基因克隆、原核表达及其结合特性[J]. 中国农业科学, 2019, 52(20): 3705-3712.

Ling LI,Yao TAN,XiaoRong ZHOU,BaoPing PANG. Molecular Cloning, Prokaryotic Expression and Binding Characterization of Odorant Binding Protein GdauOBP20 in Galeruca daurica[J]. Scientia Agricultura Sinica, 2019, 52(20): 3705-3712.

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名称 Name	引物序列Primer sequence (5′-3′)	引物用途 Use of primers
GdauOBP20-5′ GSP	CACGGTCGAAAATTCCTTTTTTGCC	RACE末端扩增 RACE end amplification
GdauOBP20-3′ GSP	TTGGCCCCATCTAAAGTTGCCGACG	RACE末端扩增 RACE end amplification
GdauOBP20-5′ NGSP	TGGATCCGTAATCTCCATTAAGGGA	巢氏PCR Nested PCR
GdauOBP20-3′ NGSP	TGGAGCTTGTCACAACAAAGTCGCA	巢氏PCR Nested PCR
GdauOBP20-F1	ATGTTTCGGGAGCTTCTA	中间片段扩增 Middle fragment amplification
GdauOBP20-R1	TTAGAATATTATAAAGAGTTC	中间片段扩增 Middle fragment amplification
GdauOBP20-F2	AGTCACACACCATGTTTCG	cDNA全长验证 Verification of full-length cDNA
GdauOBP20-R2	GATTTTTATTTATTTCTACAAAGAG	cDNA全长验证 Verification of full-length cDNA
GdauOBP20-F3	GAATTCAATCCCTTAATGGAGAT	原核表达 Prokaryotic expression
GdauOBP20-R3	CTCGAGTTAGAATATTATAAAGAGTT	原核表达 Prokaryotic expression

化学配基 Chemical ligand	IC₅₀ (μmol·L^-1)	解离常数Ki Dissociation constant (μmol·L^-1)
二烯丙基硫醚 Diallyl sulfide	42.30	42.10
二烯丙基二硫 Diallyl disulphide	50.97	50.73
二烯丙基三硫醚 Diallyl trisulfide	—	—
二甲基二硫醚 Dimethyl disulfide	31.79	31.64
二甲基三硫醚 Dimethyl trisulfide	31.16	31.01
1,3-二噻烷 1,3-Dithiane	49.86	49.63
己醛Hexanal	49.05	48.83
2-己烯醛2-Hexenal	37.97	37.80
苯甲酸甲酯 Methyl benzoate	37.37	37.19
对二甲苯p-Xylene	23.07	22.91
顺-2-己烯-1-醇 (Z)-2-Hexen-1-ol	47.01	46.80
月桂烯Myrcene	116.83	116.29
环庚三烯 1,3,5-Cycloheptatriene	26.68	26.55