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

Abstract

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

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.

Figures/Tables 6

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Table 2

References 30

[1]	PILPEL Y, LANCET D . Olfaction: Good reception in fruitfly antennae. Nature, 1999, 398(6725): 285, 287.
[2]	PELOSI P, IOVINELLA I, FELICIOLI A, DANI F R . Soluble proteins of chemical communication: an overview across arthropods. Frontier in Physiology, 2014,5:320.
[3]	VOGT R G . Biochemical diversity of odor detection: OBPs, ODEs and SNMPs//BLOMQUIST G J, VOGT R G. Insect Pheromone Biochemistry and Molecular Biology. London: Elsevier Academic Press, 2003: 391-445.
[4]	PELOSI P, ZHOU J J, BAN L P, CALVELLO M . Soluble proteins in insect chemical communication. Cellular and Molecular Life Sciences, 2006,63(14):1658-1676.
[5]	LEAL W S . Odorant reception in insects: roles of receptors, binding proteins, and degrading enzymes. Annual Review of Entomology, 2013,58:373-391.
[6]	昊翔, 周晓榕, 庞保平, 张卓然, 马崇勇 . 寄主植物对沙葱萤叶甲幼虫生长发育及取食的影响. 草地学报, 2014,22(4):854-858. doi: 10.11733/j.issn.1007-0435.2014.04.027
	HAO X, ZHOU X R, PANG B P, ZHANG Z R, MA C Y . Effects of host plants on feeding amount, growth and development of Galeruca daurica Joannis larvae (Coleoptera: Chrysomelidae). Acta Agrestia Sinica, 2014,22(4):854-858. (in Chinese) doi: 10.11733/j.issn.1007-0435.2014.04.027
[7]	昊翔, 周晓榕, 庞保平, 张卓然, 包祥 . 沙葱萤叶甲的形态特征和生物学特性研究. 草地学报, 2015,23(5):1106-1108.
	HAO X, ZHOU X R, PANG B P, ZHANG Z R, BAO X . Morphological and biological characteristics of Galeruca daurica Joannis. Acta Agrestia Sinica, 2015,23(5):1106-1108. (in Chinese)
[8]	LI L, ZHOU Y T, TAN Y, ZHOU X R, PANG B P . Identification of odorant-binding protein genes in Galeruca daurica (Coleoptera: Chrysomelidae) and analysis of their expression profiles. Bulletin of Entomological Research, 2017,107(4):550-561.
[9]	HE P, LI Z Q, ZHANG Y F, CHEN L, WANG J, XU L, ZHANG Y N, HE M . Identification of odorant-binding and chemosensory protein genes and the ligand affinity of two of the encoded proteins suggest a complex olfactory perception system in Periplaneta americana. Insect Molecular Biology, 2017,26(6):687-701.
[10]	李红亮, 张林雅, 庄树林, 倪翠侠, 韩宝瑜, 商晗武 . 中华蜜蜂普通气味结合蛋白ASP2的气味结合功能模式分析. 中国农业科学, 2013,46(1):154-161. doi: 10.3864/j.issn.0578-1752.2013.01.018
	LI H L, ZHANG L Y, ZHUANG S L, NI C X, HAN B Y, SHANG H W . Interpretation of odorant binding function and mode of general odorant binding protein ASP2 in Chinese honeybee (Apis cerana cerana). Scientia Agricultura Sinica, 2013,46(1):154-161. (in Chinese) doi: 10.3864/j.issn.0578-1752.2013.01.018
[11]	LI N, SUN X, WANG M Q . Expression pattern and ligand-binding properties of odorant-binding protein 13 from Monochamus alternatus Hope. Journal of Applied Entomology, 2017,141(9):751-757.
[12]	LI Y, ZHOU P, ZHANG J, YANG D, LI Z, ZHANG X, ZHU S, YU Y, CHEN N . Identification of odorant binding proteins in Carpomya vesuviana and their binding affinity to the male-borne semiochemicals and host plant volatiles. Journal of Insect Physiology, 2017,100:100-107.
[13]	李兆群, 张帅, 周淑芬, 雒珺瑜, 崔金杰 . 棉铃虫气味结合蛋白HarmOBP16的组织表达谱及配基结合特性分析. 昆虫学报, 2017,60(8):891-899.
	LI Z Q, ZHANG S, ZHOU S F, LUO J Y, CUI J J . Tissue expression profiling and ligand-binding properties of HarmOBP16 of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Acta Entomologia Sinica, 2017,60(8):891-899. (in Chinese)
[14]	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. 中国农业科学, 2018,51(5):893-904. doi: 10.3864/j.issn.0578-1752.2018.05.008
	CHEN D K, ZHANG L Y, XING Z L, LEI Z R . Identification and function of the OBP13 protein from the leafminer (Liriomyza sativae). Scientia Agricultura Sinica, 2018,51(5):893-904. (in Chinese) doi: 10.3864/j.issn.0578-1752.2018.05.008
[15]	陈秀琳, 苏丽, 陈丽慧, 李怡萍, 仵均祥, 李广伟 . 梨小食心虫Minus-C气味结合蛋白的分子克隆、表达谱及结合特性分析. 昆虫学报, 2018,61(7):771-783.
	CHEN X L, SU L, CHEN L X, LI Y P, WU J X, LI G W . Molecular cloning, expression profiling and binding characterization of a Minus-C odorant binding protein from the oriental fruit moth, Grapholita molesta (Lepidoptera: Tortricidae). Acta Entomologia Sinica, 2018,61(7):771-783. (in Chinese)
[16]	孙佳斌, 刘乃勇, 李双美, 闫祺, 董双林 . 斜纹夜蛾信息素结合蛋白SlitPBP4的分子克隆、组织表达谱及结合特性分析. 昆虫学报, 2018,61(6):657-667.
	SUN J B, LIU N Y, LI S M, YAN Q, DONG S L . Molecular cloning, tissue expression profiling and binding characterization of the pheromone binding protein SlitPBP4 from Spodoptera litture (Lepidoptera: Noctuidae). Acta Entomologia Sinica, 2018,61(6):657-667. (in Chinese)
[17]	PELLETIER J, GUIDOLIN A, SYED Z, CORNEL A J, LEAL W S . Knockdown of a mosquito odorant-binding protein involved in the sensitive detection of oviposition attractants. Journal of Chemical Ecology, 2010,36(3):245-248.
[18]	DONG K, SUN L, LIU J T, GU S H, ZHOU J J, YANG R N, DHILOO K H, GAO X W, GUO Y Y, ZHANG Y J . RNAi-induced electrophysiological and behavioral changes reveal two pheromone binding proteins of Helicoverpa armigera involved in the perception of the main sex pheromone component Z11-16:Ald. Journal of Chemical Ecology, 2017,43(2):207-214.
[19]	ZHANG X Y, ZHU X Q, GU S H, ZHOU Y L, WANG S Y, ZHANG Y J, GUO Y Y . Silencing of odorant binding protein gene AlinOBP4 by RNAi induces declining electrophysiological responses of Adelphocoris lineolatus to six semiochemicals. Insect Science, 2017,24(5):789-797.
[20]	REBIJITH K B, ASOKAN R, HANDE H R, KUMAR N K K, KRISHNA V, VINUTHA J, BAKTHAVATSALAM N . RNA interference of odorant-binding protein 2 (OBP2) of the cotton aphid, Aphis gossypii (Glover), resulted in altered electrophysiological responses. Applied Biochemistry and Biotechnology, 2016,178(2):251-266.
[21]	SUN X, ZENG F F, YAN M J, ZHANG A, LU Z X, WANG M Q . Interactions of two odorant-binding proteins influence insect chemoreception. Insect Molecular Biology, 2016,25(6):712-723.
[22]	ZHU G H, XU J, CUI Z, DONG X T, YE Z F, NIU D J, HUANG Y P, DONG S L . Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing. Insect Biochemistry and Molecular Biology, 2016,75:1-9.
[23]	BAN L, SCALONI A, BRANDAZZA A, ANGELI S, ZHANG L, YAN Y, PELOSI P . Chemosensory proteins of Locusta migratoria. Insect Molecular Biology, 2003,12(2):125-134.
[24]	BENKERT P, BIASINI M, SCHWEDE T . Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics, 2011,27(3):343-350.
[25]	何洪巨, 王希丽, 张建丽 . GC-MS法测定大葱、细香葱、小葱中的挥发性物质. 分析测试学报, 2004,23(增刊):98-100, 103.
	HE H J, WANG X L, ZHANG J L . Analysis of volatile components of shallots by GC-MS. Journal of Instrumental Analysis, 2004,23(Suppl.):98-100, 103. (in Chinese)
[26]	CHENG L, LUO J, LI P, YU H, HUANG J F, LUO L X . Microbial diversity and flavor formation in onion fermentation. Food and Function, 2014,5(9):2338-2347.
[27]	LI M F, LI T, LI W, YANG L D . Changes in antioxidant capacity, levels of soluble sugar, total polyphenol, organosulfur compound and constituents in garlic clove during storage. Industrial Crops and Products, 2015,69:137-142.
[28]	魏丹, 叶占峰, 高建清, 董双林 . 二化螟Minus-C气味结合蛋白的分子克隆及功能鉴定. 昆虫学报, 2013,56(7):754-764.
	WEI D, YE Z F, GAO J Q, DONG S L . Molecular cloning and functional identification of a Minus-C odorant binding protein from the rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae). Acta Entomologica Sinica, 2013,56(7):754-764. (in Chinese)
[29]	王然, 张晓曼, 李峰齐, 吴帆, 李红亮, 罗晨 . 烟粉虱MED隐种气味结合蛋白OBP的克隆、原核表达及植物挥发物的结合特性. 植物保护学报, 2016,43(1):32-39.
	WANG R, ZHANG X M, LI F Q, WU F, LI H L, LUO C . Cloning and prokaryotic expression of odorant binding protein OBP8 in MED cryptic species Bemisia tabaci and the binding characteristics with plant volatiles. Journal of Plant Protection, 2016,43(1):32-39. (in Chinese)
[30]	曹凤勤, 刘万学, 范中南, 万方浩, 程立生 . B型烟粉虱对三种寄主植物及其挥发物的行为反应. 昆虫学报, 2008,51(8):830-838.
	CAO F Q, LIU W X, FAN Z N, WAN F H, CHENG L S . Behavioural responses of Bemisia tabaci B-biotype to three host plants and their volatiles. Acta Entomologica Sinica, 2008,51(8):830-838. (in Chinese)

Related Articles 15

[1]	LIU ChuanXia, CHEN Xin, WANG Xiao, LI XueWen, LI TingTing, WENG ChangJiang, ZHENG Jun. Preparation and Application of Polyclonal Antibodies Against Pig CD1d Protein [J]. Scientia Agricultura Sinica, 2024, 57(8): 1620-1628.
[2]	WANG ZhiXiong, XU Dong, TIAN XiaoLi, WAN Peng, XIA Gen, SONG XuRong, WANG FuLian, GUI LianYou, ZHANG GuoHui. Cloning, Prokaryotic Expression and Ligand Binding Property of BminMinusOBP1 and BminPlusOBP1 from Bactrocera minax [J]. Scientia Agricultura Sinica, 2024, 57(24): 4894-4906.
[3]	QIAN YanHong, SONG Shuai, WEN XiaoHui, NIU RuiHui, YANG YanQiu, ZHENG BoBin, YUAN ZiGuo, LUO ShengJun. Establishment and Application of a Tube-Based Chemiluminescence Immunoassay Method for Detecting Antibodies Against Trichinella spiralis in Pigs [J]. Scientia Agricultura Sinica, 2024, 57(22): 4578-4588.
[4]	BIAN XianYu, LI SuFen, WANG JianXin, HAN Nan, LU HongTing, CHENG Xi, ZHOU JinZhu, TAO Ran, ZHU XueJiao, DONG HaiLong, ZHANG XueHan, LI Bin. Prokaryotic Expression, Antibody Preparation and Application of Major Non-Structural Proteins of Porcine Rotavirus [J]. Scientia Agricultura Sinica, 2024, 57(17): 3494-3506.
[5]	YANG Ling, TIAN XiaoLi, GUI LianYou, WANG FuLian, ZHANG GuoHui. Interaction Mechanisms Between Bactrocera minax Odorant-Binding Protein BminOBP6 and Its Ligands [J]. Scientia Agricultura Sinica, 2023, 56(7): 1311-1321.
[6]	YANG HuiZhen, YANG Huan, WU ZiXuan, FAN KuoHai, YIN Wei, SUN PanPan, ZHONG Jia, SUN Na, LI HongQuan. Prokaryotic Expression and Metal Binding Characterization of Metallothionein 1A and 2A of Sus scrofa [J]. Scientia Agricultura Sinica, 2023, 56(17): 3461-3478.
[7]	NIAN HeFen, ZHANG YuXi, LI BoLiao, CHEN XiuLin, LUO Kun, LI GuangWei. Expression and Ligand Binding Characteristics of GfunOBP2 from Grapholita funebrana [J]. Scientia Agricultura Sinica, 2023, 56(12): 2302-2316.
[8]	ZHAO HuiTing,PENG Zhu,JIANG YuSuo,ZHAO ShuGuo,HUANG Li,DU YaLi,GUO LiNa. Expression and Binding Properties of Odorant Binding Protein AcerOBP7 in Apis cerana cerana [J]. Scientia Agricultura Sinica, 2022, 55(3): 613-624.
[9]	Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG. Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids [J]. Scientia Agricultura Sinica, 2021, 54(6): 1143-1153.
[10]	XiaoHe LIU,GuiSheng QIU,ZhaoGuo TONG,HuaiJiang ZHANG,WenTao YAN,Qiang YUE,LiNa SUN. Ligands Binding Characteristics of Chemosensory Protein CsasCSP16 of Carposina sasakii [J]. Scientia Agricultura Sinica, 2021, 54(5): 945-958.
[11]	QIN JianHui,LI JinQiao,ZHAO Xu,LI KeBin,CAO YaZhong,YIN Jiao. Expression, Purification and Functional Analysis of Odorant Binding Protein 11 (OBP11) in Anomala corpulenta [J]. Scientia Agricultura Sinica, 2021, 54(14): 3017-3028.
[12]	XIE KunLun,LIU LiMing,LIU Mei,PENG Bin,WU HuiJie,GU QinSheng. Prokaryotic Expression of dsRNA of Zucchini yellow mosaic virus and Its Control Efficacy on ZYMV [J]. Scientia Agricultura Sinica, 2020, 53(8): 1583-1593.
[13]	BI KeRan,LI Yin,HAN KaiKai,ZHAO DongMin,LIU QingTao,LIU YuZhuo,HUANG XinMei,YANG Jing. Prokaryotic Expression and Polyclonal Antibody Preparation of Duck Oligoadenylate Synthase-Like Protein [J]. Scientia Agricultura Sinica, 2019, 52(23): 4429-4436.
[14]	LI Du, NIU ChangYing, LI FengQi, LUO Chen. Binding Characterization of Odorant Binding Protein OBP56h in Drosophila suzukii with Small Molecular Compounds [J]. Scientia Agricultura Sinica, 2019, 52(15): 2616-2623.
[15]	WANG Hui,CHAI ZhiXin,ZHU JiangJiang,ZHONG JinCheng,ZHANG ChengFu,Xin JinWei. Cloning and Identification of Long-Chain Non-Coding RNA Linc24063 and Its Correlation with the Expression Level of miRNAs in Yak [J]. Scientia Agricultura Sinica, 2019, 52(14): 2538-2547.

Metrics

Viewed

Full text

Abstract

Cited

Shared

Discussed

Comments

Recommended 0

No Suggested Reading articles found!

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

Molecular Cloning, Prokaryotic Expression and Binding Characterization of Odorant Binding Protein GdauOBP20 in Galeruca daurica

RichHTML

PDF