铜绿丽金龟气味结合蛋白AcorOBP11的表达纯化及功能分析

doi:10.3864/j.issn.0578-1752.2021.14.008

摘要/Abstract

摘要：

【目的】通过研究铜绿丽金龟（Anomala corpulenta）气味结合蛋白11（odorant binding protein 11,OBP11）与寄主植物挥发物的结合特性,探讨AcorOBP11的功能,为阐明铜绿丽金龟识别寄主植物的嗅觉分子机制打下基础。【方法】设计特异性引物,通过RT-PCR克隆AcorOBP11,分析其序列特征并与相似序列进行对比;将目的基因OBP11连入原核表达载体pET28a后,重组质粒转入大肠杆菌感受态细胞BL21(DE3)。利用IPTG诱导AcorOBP11重组蛋白的表达,超声破碎菌体,取上清液过镍柱,利用重组肠激酶切除His标签后再次过镍柱纯化获得目的蛋白;纯化后的蛋白以1-NPN为荧光探针开展荧光竞争结合试验,测定AcorOBP11蛋白与37种寄主植物挥发物的结合特性;利用Modeller对AcorOBP11进行同源建模,以冈比亚按蚊气味结合蛋白AgamOBP48（PDB ID：4ij7）作为模板,获得其三维结构;利用Autodock半柔性对接将蛋白与化合物对接,模拟AcorOBP11与6种候选寄主植物挥发物的结合情况。【结果】克隆获得了AcorOBP11全长基因,开放阅读框共639 bp,N末端有17个氨基酸组成的信号肽,具有8个保守的半胱氨酸位点,属于Plus-C OBP亚家族,进化树结果表明其与HparOBP9进化关系最近。AcorOBP11成功连入pET28a表达载体,在0.25 mmol·L^-1 IPTG、37℃条件下诱导表达,并通过两次镍柱纯化得到目的蛋白。竞争结合试验结果表明,AcorOBP11结合谱较广,对α-紫罗兰酮、异戊醛、丙烯酸-2-乙基己酯、乙酸龙脑酯、柠檬烯、反-2-己烯醛、2-乙基己醛、异戊酸等13种寄主植物挥发物有较好的结合能力。其中,与寄主植物挥发物α-紫罗兰酮结合能力最好,竞争解离常数为22.78 μmol·L^-1。构建了AcorOBP11三维结构图并进行蛋白构象评估后,与6种化合物进行分子对接,结果表明AcorOBP11与α-紫罗兰酮结合自由能最低,为-24.74 kJ·mol^-1,表明其与α-紫罗兰酮的结合能力最强,与荧光竞争结合结果一致;从对接图还可以看出,α-紫罗兰酮不仅与AcorOBP11在Cys163处形成氢键,而且其疏水端进入了蛋白的疏水腔,两个甲基与蛋白表面高度契合,因此,α-紫罗兰酮与蛋白间结合能力最强。【结论】AcorOBP11能够识别多种寄主植物挥发物,推测其在铜绿丽金龟成虫定位寄主植物中发挥重要作用,此研究结果为生态防控铜绿丽金龟提供了新思路。

关键词: 铜绿丽金龟, 气味结合蛋白11, 原核表达, 荧光竞争结合, 同源建模, 分子对接

Abstract:

【Objective】The objective of this research is to explore the function of Anomala corpulenta odorant binding protein 11 (AcorOBP11) by studying the binding characteristics of AcorOBP11 with the host plant volatiles, and to lay the foundation for clarifying the olfactory molecular mechanism of AcorOBP11.【Method】Reverse transcription PCR (RT-PCR) was used to clone the full-length ORF ofAcorOBP11 by specific primers, and sequences with high similarity were downloaded for sequence comparison and analysis by BLAST. Prokaryotic expression of AcorOBP11 was conducted by Escherichia coliprotein expression system, the OBP11 was inserted into the expression vector pET28a, and the recombinant plasmid was transferred into E. coli competent cells BL21 (DE3). The target protein was purified by using recombinant enterokinase to remove the His-tag and purified again by using a nickel column. The purified protein was diluted with 50 mmol·L^-1 Tris-HCl (pH 7.4) to a dilution of 2 μmol·L^-1, and the odorant was diluted with chromatographic grade methanol to a final concentration of 1 mmol·L^-1, and 1-NPN was used as a fluorescent probe to determine the binding characteristics of AcorOBP11 to 37 host plant volatiles by fluorescence competitive binding assay. Modeller was used to obtain the three-dimensional structure of AcorOBP11 by using AgamOBP48 (PDB ID: 4ij7) as a template. Autodock semi-flexible docking was used to simulate the binding of AcorOBP11 to host plant volatiles.【Result】The full-length ORF ofAcorOBP11 was amplified, which is 639 bp in total, including 17 amino acids of signal peptide at the N-terminal. With eight conserved cysteine sites, AcorOBP11 belongs to the Plus-C OBP subfamily. The evolutionary tree results indicated that it had the closest evolutionarily relationship with HparOBP9. AcorOBP11 was successfully inserted into pET-28a and expressed at 0.25 mmol·L^-1 IPTG and induced expression at 37℃. The target protein was obtained by nickel column purification twice. The results of competitive binding experiments showed that AcorOBP11 has a good binding ability to 13 kinds of host plant volatiles, such as α-ionone, 3-methylbutanal, 2-ethylhexyl acrylate, bornyl acetate, cinene, trans-2-hexenal, 2-ethylhexanal, isovaleric acid. Among them, α-ionone has the best binding ability, and its competitive dissociation constant is 22.78 μmol·L^-1. The molecular docking results showed that AcorOBP11 has the lowest binding free energy with α-ionone, which is -24.74 kJ·mol^-1, and forms a hydrogen bond at Cys163, indicating that it has the strongest binding ability with α-ionone, which is consistent with the fluorescence competition binding result. 【Conclusion】AcorOBP11 can recognize a variety of host plant volatiles, and it is speculated that it plays an important role in locating host plants for A. corpulenta. The results will provide new insights forA. corpulenta ecological control.

Key words: Anomala corpulenta, odorant binding protein 11 (OBP11), prokaryotic expression, fluorescence competitive binding, homology modeling, molecular docking

秦健辉,李金桥,赵旭,李克斌,曹雅忠,尹姣. 铜绿丽金龟气味结合蛋白AcorOBP11的表达纯化及功能分析[J]. 中国农业科学, 2021, 54(14): 3017-3028.

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.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.14.008

https://www.chinaagrisci.com/CN/Y2021/V54/I14/3017

图/表 8

图1

图2

Table 1

Binding properties of AcorOBP11 with different ligands"

气味配体 Ligand	分子式 Formula	IC₅₀(μmol·L^-1)	Ki (μmol·L^-1)
α-紫罗兰酮 α-Ionone	C₁₃H₂₀O	25.36	22.78
异戊醛 3-Methylbutanal	C₅H₁₀O	27.27	24.49
丙烯酸-2-乙基己酯 2-Ethylhexyl acrylate	C₁₁H₂₀O₂	27.96	25.11
乙酸龙脑酯 Bornyl acetate	C₁₂H₂₀O₂	29.35	26.36
柠檬烯 Cinene	C₁₀H₁₆	30.39	27.29
反-2-己烯醛 Trans-2-hexenal	C₆H₁₀O	32.54	29.23
2-乙基己醛 2-Ethylhexanal	C₈H₁₆O	33.33	29.94
异戊酸 Isovaleric acid	C₅H₁₀O₂	33.53	30.12
4-羟基-4-甲基-2-戊酮 4-Hydroxy-4-methyl-2-pentanone	C₆H₁₂O₂	33.55	30.13
庚醛 Heptanal	C₇H₁₄O	36.34	32.64
丁醛 Butyraldehyde	C₄H₈O	38.42	34.51
苯乙酸乙酯 Ethyl phenylacetate	C₁₀H₁₂O₂	38.54	34.61
辛醛 Octanal	C₈H₁₆O	38.87	34.91
α-松油醇 α-Terpineol	C₁₀H₁₈O	41.44	37.22
水杨酸甲酯 Methyl salicylate	C₈H₈O₃	41.83	37.57
顺-3-己烯基丁酯 Cis-3-hexenyl buyrate	C₁₀H₁₈O₂	42.84	38.48
1-辛烯-3-醇 1-Octen-3-ol	C₈H₁₆O	44.37	39.85
戊醛 Pentanal	C₅H₁₀O	44.40	39.88
己二酸二异丁酯 Diisobutyl adipate	C₁₄H₂₆O₄	45.49	40.86
反-2-壬烯醛 Trans-2-nonenal	C₉H₁₆O	45.55	40.91
苯甲酸甲酯 Methyl benzoate	C₈H₈O₂	46.01	41.32
反式-2-己烯-1-醇 Trans-2-hexen-1-ol	C₆H₁₂O	47.72	42.86
芳樟醇 Linalool	C₁₀H₁₈O	48.53	43.59
β-石竹烯 β-Caryophyllene	C₁₅H₂₄	48.71	43.75
β-罗勒烯 β-Ocimene	C₁₀H₁₆	48.76	43.79
甲基庚烯酮 6-Methyl-5-hepten-2-one	C₈H₁₄O	49.02	44.03
3,4-二甲基苯乙酮 3,4-Dimethyl phenylketone	C₁₀H₁₂O	49.58	44.53
乙酸叶醇酯 Cis-3-hexenyl acetate	C₈H₁₄O₂	50.95	45.76
苯甲醛 Benzaldehyde	C₇H₆O	51.45	46.21
2-壬酮 2-Nonanone	C₉H₁₈O	53.77	48.29
2-辛醇 2-Octanol	C₈H₁₈O	54.59	49.03
异丁醛 Isobutyraldehyde	C₄H₈O	59.34	53.30
壬醛 Nonanal	C₉H₁₈O	70.65	63.45
己酸 Caproic acid	C₆H₁₂O₂	152.96	137.37
α-丁香酚 α-Eugenol	C₁₀H₁₂O₂	N	N
法尼烯 Farnesene	C₁₅H₂₄	N	N
十二碳烯 Propylene tetramer	C₁₂H₂₄	N	N

Table 1

图3

图4

Table 2

图5

图6

参考文献 26

[1]	MUELLER D, PIERCE L, BENEZET H, KRISCHIK V. Practical application of pheromone traps in food and tobacco industry. Journal of the Kansas Entomological Society, 1990, 63(4):548-553.
[2]	KLEIN M G, TUMLINSON J H, LADD T L, DOOLITTLE R E. Japanese beetle (Coleoptera: Scarabaeidae): Response to synthetic sex attractant plus phenethyl propionate: Eugenol. Journal of Chemical Ecology, 1981, 7(1):1-7. doi: 10.1007/BF00988630
[3]	LEAL W S, ONO M, HASEGAWA M, SAWADA M. Kairomone from dandelion, Taraxacum officinale, attractant for scarab beetleAnomala octiescostata. Journal of Chemical Ecology, 1994, 20(7):1697-1704. doi: 10.1007/BF02059891
[4]	李晓峰, 曹雅忠, 尹姣, 张帅, 李金桥, 李克斌. 华北大黑鳃金龟植物源引诱剂配方筛选及引诱效果. 植物保护学报, 2020, 47(1):35-45.
	LI X F, CAO Y Z, YIN J, ZHANG S, LI J Q, LI K B. Prescription screening and trapping effect of plant volatile attractants to northern China scarab beetle Holotrichia oblita . Journal of Plant Protection, 2020, 47(1):35-45. (in Chinese)
[5]	谢明惠, 陈浩梁, 衣建坤, 李克斌, 张杰, 苏卫华. 铜绿丽金龟对植物源挥发物的触角电位和行为反应. 植物保护, 2015, 41(1):33-38.
	XIE M H, CHEN H L, YI J K, LI K B, ZHANG J, SU W H. Electroantennographic and behavioral responses of Anomala corpulenta to plant volatiles . Plant Protection, 2015, 41(1):33-38. (in Chinese)
[6]	FRANCIS F, LOGNAY G, HAUBRUGE E. Olfactory responses to aphid and host plant volatile releases: (E)-beta-farnesene an effective kairomone for the predator Adalia bipunctata. Journal of Chemical Ecology, 2004, 30(4):741-755. doi: 10.1023/B:JOEC.0000028429.13413.a2
[7]	LEAL W S. Odorant reception in insects: Roles of receptors, binding proteins, and degrading enzymes. Annual Review of Entomology, 2013, 58:373-391. doi: 10.1146/annurev-ento-120811-153635
[8]	VOGT R G, RIDDIFORD L M. Pheromone binding and inactivation by moth antennae. Nature, 1981, 293(5828):161-163. doi: 10.1038/293161a0
[9]	JU Q, QU M J, WANG Y, JIANG X J, LI X, DONG S L, HAN Z J. Molecular and biochemical characterization of two odorant-binding proteins from dark black chafer, Holotrichia parallela. Genome, 2012, 55(7):537-546. doi: 10.1139/g2012-042
[10]	房迟琴, 张鑫鑫, 刘丹丹, 李克斌, 张帅, 曹雅忠, 樊东, 尹姣. 暗黑鳃金龟气味结合蛋白HparOBP15a基因的克隆及功能分析. 昆虫学报, 2016, 59(3):260-268.
	FANG C Q, ZHANG X X, LIU D D, LI K B, ZHANG S, CAO Y Z, FAN D, YIN J. Cloning and functional analysis of an odorant-binding protein HparOBP15a gene from Holotrichia parallela (Coleoptera: Melolonthidae) . Acta Entomologica Sinica, 2016, 59(3):260-268. (in Chinese)
[11]	DENG S S, YIN J, ZHONG T, CAO Y Z, LI K B. Function and immunocytochemical localization of two novel odorant-binding proteins in olfactory sensilla of the scarab beetleHolotrichia oblita Faldermann (Coleoptera: Scarabaeidae). Chemical Senses, 2012, 37(2):141-150. doi: 10.1093/chemse/bjr084
[12]	WANG B, GUAN L, ZHONG T, LI K B, YIN J, CAO Y Z. Potential cooperations between odorant-binding proteins of the scarab beetle Holotrichia oblita Faldermann (Coleoptera: Scarabaeidae). PLoS ONE, 2013, 8(12):e84795. doi: 10.1371/journal.pone.0084795
[13]	YIN J, WANG C Q, FANG C Q, ZHANG S, CAO Y Z, LI K B, LEAL W S. Functional characterization of odorant-binding proteins from the scarab beetleHolotrichia oblita based on semiochemical- induced expression alteration and gene silencing. Insect Biochemistry and Molecular Biology, 2019, 104:11-19. doi: 10.1016/j.ibmb.2018.11.002
[14]	WANG C Q, LI J Q, LI E T, NYAMWASA I, LI K B, ZHANG S, PENG Y, WEI Z J, YIN J. Molecular and functional characterization of odorant-binding protein genes in Holotrichia oblita Faldermann. International Journal of Biological Macromolecules, 2019, 136:359-367. doi: 10.1016/j.ijbiomac.2019.06.013
[15]	WEI H S, DUAN H X, LI K B, ZAHNG S, WEI Z J, YIN J. The mechanism underlying OBP heterodimer formation and the recognition of odors in Holotrichia oblita Faldermann. International Journal of Biological Macromolecules, 2020, 152:957-968. doi: 10.1016/j.ijbiomac.2019.10.182
[16]	王超群, 赵莹, 曹雅忠, 魏红爽, 李克斌, 张帅, 彭宇, 尹姣. 铜绿丽金龟气味结合蛋白AcorOBP1的表达和结合特性分析. 昆虫学报, 2017, 60(4):363-371.
	WANG C Q, ZHAO Y, CAO Y Z, WEI H S, LI K B, ZHANG S, PENG Y, YIN J. Expression and binding characterization of odorant binding protein 1 (AcorOBP1) in Anomala corpulenta (Coleoptera: Scarabaeoidea) . Acta Entomologica Sinica, 2017, 60(4):363-371. (in Chinese)
[17]	LASKOWSKI R A, MACARTHUR M W, MOSS D S, THORNTON J M. PROCHECK: A program to check the stereochemical quality of protein structures. Journal of Applied Crystallography, 1993, 26(2):283-291. doi: 10.1107/S0021889892009944
[18]	LASKOWSKI R A, RULLMANNN J A, MACARTHUR M W, KAPTEIN R, THORNTON J M. AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. Journal of Biomolecular NMR, 1996, 8(4):477-486.
[19]	王桂荣, 郭予元, 吴孔明. 昆虫触角气味结合蛋白的研究进展. 昆虫学报, 2002, 45(1):131-137.
	WANG G R, GUO Y Y, WU K M. Progress in the studies of antenna odorant binding proteins of insects. Acta Entomologica Sinica, 2002, 45(1):131-137. (in Chinese)
[20]	FIELD L M, PICKETT J A, WADHAMS L J. Molecular studies in insect olfaction. Insect Molecular Biology, 2010, 9(6):545-551. doi: 10.1046/j.1365-2583.2000.00221.x
[21]	鞠倩, 郭晓强, 李晓, 蒋相国, 倪婉莉, 曲明静. 铜绿丽金龟对寄主植物挥发物的触角电生理及行为反应. 植物保护学报, 2016, 43(2):281-287.
	JU Q, GUO X Q, LI X, JIANG X G, NI W L, QU M J. EAG and behavioral responses of copper-green chafer Anomala corpulentaMotschulsky (Coleoptera: Rutelidae) . Journal of Plant Protection, 2016, 43(2):281-287. (in Chinese)
[22]	YIN J, FENG H L, SUN H Y, XI J H, CAO Y Z, LI K B. Functional analysis of general odorant binding protein 2 from the meadow moth, Loxostege sticticalis L. (Lepidoptera: Pyralidae). PLoS ONE, 2012, 7(3):e33589. doi: 10.1371/journal.pone.0033589
[23]	LI S, PICIMBON J F, JI S D, KAN Y C, QIAO C L, ZHOU J J, PELOSI P. Multiple functions of an odorant-binding protein in the mosquito Aedes aegypti. Biochemical and Biophysical Research Communications, 2008, 372(3):464-468. doi: 10.1016/j.bbrc.2008.05.064
[24]	LI G W, CHEN X L, LI B L, ZAHNG G H, LI Y P, WU J X. Binding properties of general odorant binding proteins from the oriental fruit moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae). PLoS ONE, 2016, 11(5):e0155096. doi: 10.1371/journal.pone.0155096
[25]	李广伟, 陈秀琳, 尚天翠. 光肩星天牛气味结合蛋白AglaOBP12的基因克隆、表达及配体结合特征. 昆虫学报, 2017, 60(10):1141-1154.
	LI G W, CHEN X L, SHANG T C. cDNA cloning, expression and ligand binding properties of the odorant binding protein AglaOBP12 in the Asian longhorned beetle,Anoplophora glabripennis (Coleoptera: Cerambycidae) . Acta Entomologica Sinica, 2017, 60(10):1141-1154. (in Chinese)
[26]	KOSHLAND D E. Application of a theory of enzyme specificity to protein synthesis. Proceeding of the National Academy of Sciences of the United States of America, 1958, 44(2):98-104.