李小食心虫GfunOBP2的原核表达及气味配体结合特性

doi:10.3864/j.issn.0578-1752.2023.12.006

摘要/Abstract

摘要：

【目的】通过测定李小食心虫（Grapholita funebrana）Plus-C气味结合蛋白2（odorant binding protein，GfunOBP2）结合性信息素和苹果树挥发化合物的能力，分析GfunOBP2的嗅觉功能，为阐释李小食心虫定位寄主植物的嗅觉分子机理打下基础。【方法】利用RT-PCR扩增GfunOBP2的ORF序列，通过同源注释和比对氨基酸序列中半胱氨酸（Cys）的分布模式确定GfunOBP2属于Plus-C OBP亚家族；利用RT-qPCR检测GfunOBP2在李小食心虫3日龄成虫触角、头、胸、足、翅、腹部和性腺中的相对表达量；构建pET30a(+)/GfunOBP2原核表达载体，在大肠杆菌（Escherichia coli）BL21(DE3)细胞中表达GfunOBP2重组蛋白。利用荧光竞争结合试验测定重组GfunOBP2蛋白对5种性信息素和35种苹果树挥发化合物的结合能力；通过分子对接预测GfunOBP2与具有强结合能力的气味配体的相互作用力及关键氨基酸残基。【结果】克隆获得GfunOBP2（GenBank登录号：OQ054799.1）的全长序列，共编码183个氨基酸，氨基酸序列中有12个保守的Cys，其分布模式表明GfunOBP2属于Plus-C OBP。GfunOBP2主要在成虫触角中表达，在雄虫触角中的相对表达量显著高于雌虫触角（P<0.05）。重组GfunOBP2蛋白对反-2-己烯-1-醇、苯甲醇、1-庚醇、1-癸醇、己醛、庚醛、乙酸-顺-3-己烯酯、2-甲基丁酸叶醇酯、α-罗勒烯、β-石竹烯、α-蒎烯和柠檬烯具有强结合能力，抑制常数K_i均小于5.0 μmol·L^-1。分子对接结果显示氢键、Donor-Donor相互作用和烷基相互作用是GfunOBP2结合反-2-己烯-1-醇、1-庚醇和1-癸醇的主要弱相互作用力，氢键和碳氢键是GfunOBP2结合乙酸-顺-3-己烯酯和2-甲基丁酸叶醇酯的主要弱相互作用力，烷基相互作用是GfunOBP2结合α-罗勒烯和β-石竹烯的唯一弱作用力。疏水性氨基酸Ile、Pro、Phe、Ala、Leu和Val在GfunOBP2结合气味配体中起着重要作用。【结论】GfunOBP2主要在李小食心虫成虫触角中表达，重组GfunOBP2蛋白对被测的35种苹果树挥发化合物中的12种具有强结合能力、对10种化合物具有中等结合能力，表明其在识别寄主植物挥发化合物的过程中起着重要作用。研究结果为证实Plus-C OBP参与李小食心虫外周嗅觉通讯提供了理论依据。

关键词: 李小食心虫, 化学感受, 气味结合蛋白, 寄主植物挥发物, 分子对接

Abstract:

【Objective】The objective of this study is to determine the binding affinities of the Plus-C odorant binding protein 2 of Grapholita funebrana (GfunOBP2) to sex pheromones and volatile compounds from apple trees, and to provide a basis for explaining the olfactory molecular mechanism of locating the host plants of G. funebrana.【Method】The ORF of GfunOBP2 was cloned by RT-PCR, and GfunOBP2 was identified as a Plus-C OBP subfamily protein through homology annotation and alignment of cysteine distribution patterns in amino acid sequences. The relative expression level of GfunOBP2 in the antenna, head, thorax, leg, wing, abdomen, and sex gland of the 3-day-old adults of G. funebrana was detected by RT-qPCR. The prokaryotic expression vector pET30a(+)/GfunOBP2 was constructed, and the recombinant GfunOBP2 protein was expressed in Escherichia coli BL21 (DE3) cells. The binding affinity of recombinant GfunOBP2 protein to five sex pheromones and 35 plant volatiles of apple trees was determined by using a fluorescence competitive binding assay. The interaction force and key amino acid residues of GfunOBP2 interacting with odorant ligands with strong binding affinities were predicted by molecular docking.【Result】The full-length ORF sequence of GfunOBP2 (GenBank number: OQ054799.1) was cloned, encoding 183 amino acids. It was found that GfunOBP2 has 12 conserved cysteines, and the distribution motif of cysteine residues indicated that GfunOBP2 belongs to the Plus-C OBP subfamily. GfunOBP2 was mainly expressed in the antennae of adults, and the relative expression level in male antennae was significantly higher than that in female antennae (P<0.05). Recombinant GfunOBP2 protein exhibited strong binding affinities to (E)-2-hexen-1-ol, benzyl alcohol, 1-heptanol, 1-decanol, hexanal, heptanal, cis-3-hexenyl acetate, cis-3-hexenyl 2-methylbutanoate, α-ocimene, β-caryophyllene, α-pinene and limonene, and the inhibition constant (K_i) for each ligand above was less than 5.0 μmol·L^-1. The molecular docking results showed that hydrogen bonds, donor-donor interactions, and alkyl interactions are the main weak interactions between GfunOBP2 and (E)-2-hexen-1-ol, 1-heptanol, and 1-decanol. The conventional hydrogen bonds and carbon hydrogen bonds are the main weak interactions between GfunOBP2 and cis-3-hexenyl acetate and cis-3-hexenyl 2-methylbutanoate. The alkyl interaction is the only weak force of GfunOBP2 interacting with α-ocimene and β-caryophyllene. Several hydrophobic amino acid residues, including Ile, Pro, Phe, Ala, Leu, and Val, play an important role in GfunOBP2’s binding to odorant ligands.【Conclusion】GfunOBP2 is mainly expressed in the antennae of adults of G. funebrana and the corresponding recombinant protein has strong binding affinities to 12 of the 35 volatile compounds of apple trees, and has moderate binding affinities to 10 compounds, indicating that GfunOBP2 plays an important role in the process of perceiving and recognizing the volatile compounds of host plants. This study provides a theoretical basis for confirming that Plus-C OBP was involved in the peripheral olfactory communication of G. funebrana.

Key words: Grapholita funebrana, chemoreception, odorant binding protein (OBP), host-plant volatile, molecular docking

年和粉, 张钰析, 李伯辽, 陈秀琳, 罗坤, 李广伟. 李小食心虫GfunOBP2的原核表达及气味配体结合特性[J]. 中国农业科学, 2023, 56(12): 2302-2316.

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.

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.0578-1752.2023.12.006

https://www.chinaagrisci.com/CN/Y2023/V56/I12/2302

图/表 9

表1

图1

图2

图3

表2

重组GfunOBP2蛋白对性信息素和苹果树挥发化合物的结合能力"

气味及来源 Odorant and source	CAS登录号 CAS number	纯度 Purity	IC₅₀ (μmol·L^-1)	K_i (μmol·L^-1)
性信息素Sex pheromone
顺-8-十二碳烯醇(Z)-8-Dodecenyl alcohol^a	112-53-8	>98.0% (AR)	6.02±0.06	5.01±0.05
乙酸-顺-8-十二碳烯酯(Z)-8-Dodecenyl acetate^a	28079-04-1	>98.0% (AR)	9.29±0.36	7.73±0.30
乙酸-反-8-十二碳烯酯(E)-8-Dodecenyl acetate^a	38363-29-0	>98.0% (AR)	8.58±0.12	7.14±0.10
乙酸-顺-8-十四碳烯酯(Z)-8-Tetradecenyl acetate^a	35835-80-4	>90.0% (AR)	6.21±0.03	5.17±0.03
乙酸-顺-10-十四碳烯酯(Z)-10-Tetradecenyl acetate^a	35153-16-3	>90.0% (AR)	12.73±0.13	10.59±0.11
植物挥发化合物Plant volatile
反-2-己烯-1-醇(E)-2-Hexen-1-ol^b,c,d	928-95-0	97.0% (AR)	4.67±0.04	3.89±0.04
顺-3-己烯-1-醇(Z)-3-Hexen-1-ol^b,c,d	928-96-1	98.0% (AR)	6.26±0.10	5.21±0.09
苯甲醇Benzyl alcohol^b	100-51-6	≥99.0% (AR)	5.15±0.08	4.28±0.07
1-庚醇1-Heptanol^d	111-70-6	≥99.9% (GC)	4.42±0.06	3.68±0.05
2-丁氧基乙醇2-Butoxyethanol^d	111-76-2	97.0% (GC)	-	-
1-辛烯-3-醇1-Octen-3-ol^c,d	3391-86-4	>98.0% (AR)	15.25±0.28	12.69±0.24
异辛醇2-Ethyl-1-hexanol^c,d	104-76-7	>99.0% (GC)	15.12±0.43	12.58±0.36
1-癸醇1-Decanol^b,c,d	112-30-1	98.0% (GC)	4.37±0.35	3.64±0.29
芳樟醇Linalool^b,c,d	78-70-6	>98.0% (GC)	-	-
反-2-己烯醛(E)-2-Hexenal^b,c,d	85761-70-2	98.0% (AR)	7.32±0.03	6.09±0.03
己醛Hexanal^c,d	66-25-1	>95.0% (AR)	5.26±0.16	4.38±0.14
庚醛Heptanal^c,d	111-71-7	≥98.0% (GC)	5.89±0.28	4.91±0.23
辛醛Octanal^d	124-13-0	99.0% (AR)	6.88±0.30	5.73±0.25
壬醛Nonanal^b,d	124-19-6	95.0% (AR)	7.31±0.06	6.08±0.05
癸醛Decanal^b,d	112-31-2	97.0% (AR)	9.12±0.14	7.59±0.12
十一醛Undecanal^b,c,d	112-44-7	97.0% (AR)	-	-
苯甲醛Benzaldehyde^b,c,d	100-52-7	≥99.5% (GC)	-	-
乙酸丁酯Butyl acetate^b,d	123-86-4	99.0% (AR)	8.17±0.37	6.80±0.32
2-甲基丁酸乙酯Ethyl 2-methylbutyrate^d	7452-79-1	98.0% (AR)	11.35±0.07	9.45±0.06
戊酸乙酯Ethyl valerate^d	539-82-2	≥99.8% (GC)	-	-
乙酸-顺-3-己烯酯Cis-3-Hexenyl acetate^b,c,d	3681-71-8	>97.0% (GC)	4.71±0.01	3.92±0.01
丁酸丁酯Butyl butyrate^d	109-21-7	>99.0% (GC)	14.03±0.18	11.68±0.15
2-甲基丁酸叶醇酯Cis-3-Hexenyl 2-methylbutanoate^b,d	53398-85-9	97.0% (GC)	4.81±0.03	4.00±0.03
己酸戊酯Pentyl hexanoate^d	540-07-8	>98.0% (GC)	14.41±0.71	11.99±0.59
月桂酸丁酯Butyl laurate^d	106-18-3	>99.9% (GC)	7.34±0.05	6.11±0.03
肉豆蔻酸丁酯Butyl myristate^b,c,d	110-36-1	>97.0% (GC)	-	-
棕榈酸异丙酯Isopropyl palmitate^b,c,d	142-91-6	97.0% (AR)	-	-
β-莰烯β-Camphene^b,c,d	79-92-5	95.0% (AR)	-	-
α-蒎烯α-Pinene^b,c,d	80-56-8	98.0% (AR)	5.15±0.06	4.29±0.05
α-罗勒烯α-Ocimene^b,c,d	502-99-8	≥90.0% (AR)	3.52±0.05	2.93±0.04
柠檬烯Limonene^b,c,d	138-86-3	95.0% (AR)	5.64±0.05	4.69±0.04
3-篦烯3-Carene^b,c,d	13466-78-9	90.0% (AR)	11.05±0.01	9.20±0.01
β-石竹烯β-Caryophyllene^b,c,d	87-44-5	>90.0% (GC)	3.43±0.02	2.86±0.01
6-甲基-5-庚烯-2-酮6-Methyl-5-hepten-2-one^b,c,d	110-93-0	98.0% (AR)	11.30±0.15	9.41±0.12
2-甲基-6-亚甲基-1,7-辛二烯-3-酮 2-Methyl-6-methylene-1,7-octadien-3-one^d	41702-60-7	>95.0% (AR)	-	-

表2

图4

表3

图5

图6

参考文献 65

[1]	刘海荣, 赵百丽, 赵文琦, 张武杰, 杨晓华, 齐玉新, 顾广军, 刘畅. 黑龙江省果树食心虫区域分布及防治. 黑龙江农业科学, 2010(5): 65-68.
	LIU H R, ZHAO B L, ZHAO W Q, ZHANG W J, YANG X H, QI Y X, GU G J, LIU C. Distribution and prevention of fruit trees borer in Heilongjiang Province. Heilongjiang Agricultural Sciences, 2010(5): 65-68. (in Chinese)
[2]	STEFANOVA D, VASILEV P, KUTINKOVA H, ANDREEV R, PALAGACHEVA N, TITYANOV M. Possibility for control of plum fruit moth Grapholita funebrana Tr. by pheromone dispensers. Journal of Biopesticides, 2019, 12(2): 153-156.
[3]	吴维钧. 两种果树害虫——旋杖潜叶蛾及李小食心虫. 昆虫学报, 1961, 10(4/6): 395-400.
	WU W J. A new record of two species of fruit tree pests from North China. Acta Entomologica Sinica, 1961, 10(4/6): 395-400. (in Chinese)
[4]	RIZZO R, FARINA V, SAIANO F, LOMBARDO A, RAGUSA E, VERDE G L. Do Grapholita funebrana infestation rely on specific plum fruit features?. Insects, 2019, 10(12): 444. doi: 10.3390/insects10120444
[5]	蒋玉宝, 张素梅, 祁光增, 周科清, 程文波. 陇东苹果主要食心虫和卷叶蛾成虫发生动态监测. 甘肃农业科技, 2014(2): 30-32.
	JIANG Y B, ZHANG S M, QI G Z, ZHOU K Q, CHENG W B. Dynamics monitoring of occurs of borers and leafrollers adult on apple in Longdong Areas. Gansu Agricultural Science and Technology, 2014(2): 30-32. (in Chinese)
[6]	ZHENG Y, WU R X, DORN S, CHEN M H. Diversity of tortricid moths in apple orchards: Evidence for a cryptic species of Grapholita (Lepidoptera: Tortricidae) from China. Bulletin of Entomological Research, 2017, 107(2): 268-280. doi: 10.1017/S0007485316000973
[7]	杨磊, 孙惠敏, 林河州, 陈刘生, 王少山. 石河子垦区果树食心虫种类及其种群动态. 北方园艺, 2017(12): 132-135.
	YANG L, SUN H M, LIN H Z, CHEN L S, WANG S S. Species and population dynamics of fruit borer in Shihezi Reclamation Zone. Northern Horticulture, 2017(12): 132-135. (in Chinese)
[8]	陈秀琳, 陈玉鑫, 包琳杰, 康乐, 孙勇, 李广伟. 延安地区苹果园食心虫种类及其种群消长动态调查. 植物保护, 2021, 47(2): 219-225.
	CHEN X L, CHEN Y X, BAO L J, KANG L, SUN Y, LI G W. Species and population dynamics of fruit-borer in apple orchards of Yan’an area. Plant Protection, 2021, 47(2): 219-225. (in Chinese)
[9]	李兴龙, 张馨月, 雷岳杰, 肖小容, 杨永棒, 张振兴, 孙惠敏, 王少山. 石河子垦区果树食心虫迷向技术. 新疆农业科学, 2020, 57(1): 173-180. doi: 10.6048/j.issn.1001-4330.2020.01.020
	LI X L, ZHANG X Y, LEI Y J, XIAO X R, YANG Y B, ZHANG Z X, SUN H M, WANG S S. Study on the mating disruption control technology of fruit borer in Shihezi Reclamation Area. Xinjiang Agricultural Sciences, 2020, 57(1): 173-180. (in Chinese) doi: 10.6048/j.issn.1001-4330.2020.01.020
[10]	金岩. 吉林地区“123”苹果蛀果害虫发生规律及药剂防治. 北方园艺, 2013(14): 123-124.
	JIN Y. Occurrence and chemical control of borer fruit pests of “123” apple. Northern Horticulture, 2013(14): 123-124. (in Chinese)
[11]	LI L L, XU B Q, LI C Q, LI B L, CHEN X L, LI G W. Different binding affinities of three general odorant-binding proteins in Grapholita funebrana (Treitscheke) (Lepidoptera: Tortricidae) to sex pheromones, host plant volatiles, and insecticides. Journal of Economic Entomology, 2022, 115(4): 1129-1145. doi: 10.1093/jee/toac063
[12]	LI L L, XU B Q, LI C Q, LI B L, LUO K, LI G W, CHEN X L. Functional disparity of four pheromone-binding proteins from the plum fruit moth Grapholita funebrana Treitscheke in detection of sex pheromone components. International Journal of Biological Macromolecules, 2023, 225: 1267-1279. doi: 10.1016/j.ijbiomac.2022.11.186
[13]	NASTAS T, RAILEANU N, CHEPTINARI V, ROSCA G. Methodological and technological methods for application of sexual pheromones against Grapholitha funebrana Tr.. Scientific Studies & Research, 2014, 23(2): 12-19.
[14]	VERDE G L, GUARINO S, BARONE S, RIZZO R. Can mating disruption be a possible route to control plum fruit moth in Mediterranean environments?. Insects, 2020, 11(9): 589. doi: 10.3390/insects11090589
[15]	DU J, LI G W, XU X L, WU J X. Development and fecundity performance of oriental fruit moth (Lepidoptera: Tortricidae) reared on shoots and fruits of peach and pear in different seasons. Environmental Entomology, 2015, 44(6): 1522-1530. doi: 10.1093/ee/nvv124 pmid: 26314026
[16]	KARLSSON M F, PROFFIT M, BIRGERSSON G. Host-plant location by the Guatemalan potato moth Tecia solanivora is assisted by floral volatiles. Chemoecology, 2017, 27(5): 187-198. doi: 10.1007/s00049-017-0244-2
[17]	WOLFIN M S, CHILSON III R R, THRALL J, LIU Y X, VOLO S, CHA D H, LOEB G M, LINN JR C E. Proximate mechanisms of host plant location by a specialist phytophagous insect, the grape berry moth, Paralobesia viteana. Journal of Chemical Ecology, 2019, 45(11/12): 946-958. doi: 10.1007/s10886-019-01112-1
[18]	JIANG N J, TANG R, GUO H, NING C, LI J C, WU H, HUANG L Q, WANG C Z. Olfactory coding of intra- and interspecific pheromonal messages by the male Mythimna separata in North China. Insect Biochemistry and Molecular Biology, 2020, 125: 103439. doi: 10.1016/j.ibmb.2020.103439
[19]	VENTHUR H, ZHOU J J. Odorant receptors and odorant-binding proteins as insect pest control targets: A comparative analysis. Frontiers in Physiology, 2018, 9: 1163. doi: 10.3389/fphys.2018.01163 pmid: 30197600
[20]	LI T T, LIU W C, ZHU J, YANG Y H, MA C, LU C, ZHANG K X. Crystal structure and ligand identification of odorant binding protein 4 in the natural predator Chrysopa pallens. International Journal of Biological Macromolecules, 2019, 141: 1004-1012. doi: 10.1016/j.ijbiomac.2019.09.043
[21]	LAUE M, STEINBRECHT R A. Topochemistry of moth olfactory sensilla. International Journal of Insect Morphology and Embryology, 1997, 26(3/4): 217-228. doi: 10.1016/S0020-7322(97)00023-8
[22]	LEE D, DAMBERGER F F, PENG G, HORST R, GÜNTERT P, NIKONOVA L, LEAL W S, WÜTHRICH K. NMR structure of the unliganded Bombyx mori pheromone-binding protein at physiological pH. FEBS Letters, 2002, 531(2): 314-318. doi: 10.1016/S0014-5793(02)03548-2
[23]	STENGL M, FUNK N W. The role of the coreceptor Orco in insect olfactory transduction. Journal of Comparative Physiology A, 2013, 199(11): 897-909. doi: 10.1007/s00359-013-0837-3 pmid: 23824225
[24]	ZENG F F, XU P X, LEAL W S. Odorant receptors from Culex quinquefasciatus and Aedes aegypti sensitive to floral compounds. Insect Biochemistry and Molecular Biology, 2019, 113: 103213. doi: 10.1016/j.ibmb.2019.103213
[25]	VOGT R G, RIDDIFORD L M. Pheromone binding and inactivation by moth antennae. Nature, 1981, 293(5828): 161-163. doi: 10.1038/293161a0
[26]	CHEN X L, LI G W, XU X L, WU J X. Molecular and functional characterization of odorant binding protein 7 from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae). Frontiers in Physiology, 2018, 9: 1762. doi: 10.3389/fphys.2018.01762
[27]	LARTIGUE A, GRUEZ A, SPINELLI S, RIVIÈRE S, BROSSUT R, TEGONI M, CAMBILLAU C. The crystal structure of a cockroach pheromone-binding protein suggests a new ligand binding and release mechanism. The Journal of Biological Chemistry, 2003, 278(32): 30213-30218. doi: 10.1074/jbc.M304688200
[28]	CHRISTAIN C, SILVIA S, AMANDINE L, MARIELLA T. Structural diversity of insects odorant binding proteins around a conserved core. Chemical Senses, 2011, 31: A11-A12.
[29]	ZHOU J J, HUANG W S, ZHANG G A, PICKETT J A, FIELD L M. “Plus-C” odorant-binding protein genes in two Drosophila species and the malaria mosquito Anopheles gambiae. Gene, 2004, 327(1): 117-129. doi: 10.1016/j.gene.2003.11.007
[30]	ZHENG Z C, LI D Z, ZHOU A M, YI S C, LIU H, WANG M Q. Predicted structure of a minus-C OBP from Batocera horsfieldi (Hope) suggests an intermediate structure in evolution of OBPs. Scientific Reports, 2016, 6: 33981. doi: 10.1038/srep33981
[31]	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. doi: 10.1111/ins.2017.24.issue-5
[32]	CHEN X F, LEI Y B, LI H F, XU L, YANG H, WANG J J, JIANG H B. CRISPR/Cas9 mutagenesis abolishes odorant-binding protein BdorOBP56f-2 and impairs the perception of methyl eugenol in Bactrocera dorsalis (Hendel). Insect Biochemistry and Molecular Biology, 2021, 139: 103656. doi: 10.1016/j.ibmb.2021.103656
[33]	ZHAN H X, DEWER Y, ZHANG J P, TIAN J H, LI D, QU C, YANG Z, LI F Q, LUO C. Odorant-binding protein 1 plays a crucial role in the olfactory response of Bemisia tabaci to R-curcumene. Journal of Agricultural and Food Chemistry, 2021, 69(43): 12785-12793. doi: 10.1021/acs.jafc.1c03825
[34]	HAN W K, YANG Y L, SI Y X, WEI Z Q, LIU S R, LIU X L, YAN Q, DONG S L. Involvement of GOBP2 in the perception of a sex pheromone component in both larval and adult Spodoptera litura revealed using CRISPR/Cas9 mutagenesis. Insect Biochemistry and Molecular Biology, 2022, 141: 103719. doi: 10.1016/j.ibmb.2022.103719
[35]	VANDESOMPELE J, DE PRETER K, PATTYN F, POPPE B, VAN ROY N, DE PAEPE A, SPELEMAN F. Accurate normalization of real-time quantitative RT-PCR data by genometric averaging of multiple internal control genes. Genome Biology, 2002, 3(7): research0034.1-0034.11.
[36]	KELMANSKY D M, MARTÍNEZ E J, LEIVA V. A new variance stabilizing transformation for gene expression data analysis. Statistical Applications in Genetics and Molecular Biology, 2013, 12(6): 653-666. doi: 10.1515/sagmb-2012-0030 pmid: 24133097
[37]	CALVELLO M, GUERRA N, BRANDAZZA A, D’AMBROSIO C, SCALONI A, DANI F R, TURILLAZZI S, PELOSI P. Soluble proteins of chemical communication in the social wasp Polistes dominulus. Cellular and Molecular Life Sciences, 2003, 60(9): 1933-1943. doi: 10.1007/s00018-003-3186-5
[38]	ZHANG T T, MEI X D, FENG J N, BERG B G, ZHANG Y J, GUO Y Y. Characterization of three pheromone-binding proteins (PBPs) of Helicoverpa armigera (Hübner) and their binding properties. Journal of Insect Physiology, 2012, 58(7): 941-948. doi: 10.1016/j.jinsphys.2012.04.010
[39]	GUERIN P M, ARN H, BUSER H R, CHARMILLOT P, TÓTH M, SZIRÁKI G. Sex pheromone of Grapholita funebrana occurrence of Z-8- and Z-10-tetradecenyl acetate as secondary components. Journal of Chemical Ecology, 1986, 12(6): 1361-1368. doi: 10.1007/BF01012355
[40]	VALLAT A, DORN S. Changes in volatile emissions from apple trees and associated response of adult female codling moths over the fruit-growing season. Journal of Agricultural and Food Chemistry, 2005, 53(10): 4083-4090. pmid: 15884843
[41]	CASADO D, GEMENO C, AVILLA J, RIBA M. Day-night and phenological variation of apple tree volatiles and electroantennogram responses in Cydia pomonella (Lepidoptera: Tortricidae). Environmental Entomology, 2006, 35(2): 258-267. doi: 10.1603/0046-225X-35.2.258
[42]	CHEN X L, SU L, LI B L, LI G W, WU J X. Molecular and functional characterization of three odorant binding proteins from the oriental fruit moth Grapholita molesta (Busck) (Lepidoptera: Tortricidae). Archives of Insect Biochemistry and Physiology, 2018, 98(2): e21456. doi: 10.1002/arch.v98.2
[43]	LI G W, CHEN X L, LI B L, ZHANG 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
[44]	HUANG G Z, LIU J T, ZHOU J J, WANG Q, DONG J Z, ZHANG Y J, LI X C, LI J, GU S H. Expressional and functional comparisons of two general odorant binding proteins in Agrotis ipsilon. Insect Biochemistry and Molecular Biology, 2018, 98: 34-47. doi: 10.1016/j.ibmb.2018.05.003
[45]	YAN Q Y, YANG J W, YAO Y W, JIA Z, WANG Y Q, CHENG M, YAN X B, XU Y F. Research of the active components and potential mechanisms of Qingfei Gujin Decoction in the treatment of osteosarcoma based on network pharmacology and molecular docking technology. Computational and Mathematical Methods in Medicine, 2022, 2022: 7994425.
[46]	LAGARDE A, SPINELLI S, QIAO H L, TEGONI M, PELOSI P, CAMBILLAU C. Crystal structure of a novel type of odorant-binding protein from Anopheles gambiae, belonging to the C-plus class. The Biochemical Journal, 2011, 437(3): 423-430. doi: 10.1042/BJ20110522
[47]	SANDLER B H, NIKONOVA L, LEAL W S, CLARDY J. Sexual attraction in the silkworm moth: Structure of the pheromone- binding-protein-bombykol complex. Chemistry and Biology, 2000, 7(2): 143-151. doi: 10.1016/S1074-5521(00)00078-8
[48]	FALCHETTO M, CIOSSANI G, SCOLARI F, DI COSIMO A, NENCI S, FIELD L M, MATTEVI A, ZHUO J J, GASPERI G, FORNERIS F. Structural and biochemical evaluation of Ceratitis capitata odorant-binding protein 22 affinity for odorants involved in intersex communication. Insect Molecular Biology, 2019, 28(3): 431-443. doi: 10.1111/imb.v28.3
[49]	XU W, LEAL W S. Molecular switches for pheromone release from a moth pheromone-binding protein. Biochemical and Biophysical Research Communications, 2008, 372(4): 559-564. doi: 10.1016/j.bbrc.2008.05.087 pmid: 18503757
[50]	XU W, XU X, LEAL W S, AMES J B. Extrusion of the C-terminal helix in navel orangeworm moth pheromone-binding protein (AtraPBP1) controls pheromone binding. Biochemical and Biophysical Research Communications, 2011, 404(1): 335-338. doi: 10.1016/j.bbrc.2010.11.119 pmid: 21130734
[51]	DAMBERGER F F, ISHIDA Y, LEAL W S, WÜTHRICH K. Structural basis of ligand binding and release in insect pheromone- binding proteins: NMR structure of Antheraea polyphemus PBP1 at pH 4.5. Journal of Molecular Biology 2007, 373(4): 811-819. doi: 10.1016/j.jmb.2007.07.078
[52]	THODE A B, KRUSE S W, NIX J C, JONES D N M. The role of multiple hydrogen-bonding groups in specific alcohol binding sites in proteins: Insights from structural studies of LUSH. Journal of Molecular Biology, 2008, 376(5): 1360-1376. doi: 10.1016/j.jmb.2007.12.063 pmid: 18234222
[53]	ZHOU J J, ROBERTSON G, HE X, DUFOUR S, HOOPER A M, PICKETT J A, KEEP N H, FIELD L M. Characterization of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components. Journal of Molecular Biology, 2009, 389(3): 529-545. doi: 10.1016/j.jmb.2009.04.015
[54]	TSITSANOU K E, DRAKOU C E, THIREOU T, VITLIN GRUBER A, KYTHREOTI G, AZEM A, FESSAS D, ELIOPOULOS E, IATROU K, ZOGRAPHOS S E. Crystal and solution studies of the “Plus-C” odorant-binding protein 48 from Anopheles gambiae: Control of binding specificity through three-dimensional domain swapping. The Journal of Biological Chemistry, 2013, 288(46): 33427-33438. doi: 10.1074/jbc.M113.505289
[55]	孙亚兰, 吕琪卉, 杨海博, 胡镇杰, 李定旭, 董钧锋. 疆夜蛾Plus-C气味结合蛋白PsauOBP7的组织表达谱及配体结合特性分析. 昆虫学报, 2020, 63(7): 807-816.
	SUN Y L, LÜ Q H, YANG H B, HU Z J, LI D X, DONG J F. Tissue expression profiling and ligand binding characterization of the Plus-C odorant binding protein PsauOBP7 of Peridroma saucia (Lepidoptera: Noctuidae). Acta Entomologica Sinica, 2020, 63(7): 807-816. (in Chinese)
[56]	ZHANG H, CHEN J L, LIN J H, LIN J T, WU Z Z. Odorant-binding proteins and chemosensory proteins potentially involved in host plant recognition in the Asian citrus psyllid, Diaphorina citri. Pest Management Science, 2020, 76(8): 2609-2618. doi: 10.1002/ps.5799 pmid: 32083388
[57]	QU Y F, LIU X Y, ZHAO X, QIN J H, CAO Y Z, LI K B, ZHOU J J, WANG S S, YIN J. Evidence of the involvement of a Plus-C odorant-binding protein HparOBP14 in host plant selection and oviposition of the scarab beetle Holotrichia parallela. Insects, 2021, 12(5): 430. doi: 10.3390/insects12050430
[58]	JING D P, ZHANG T T, PRABU S, BAI S X, HE K L, WANG Z Y. Molecular characterization and volatile binding properties of pheromone binding proteins and general odorant binding proteins in Conogethes pinicolalis (Lepidoptera: Crambidae). International Journal of Biological Macromolecules, 2020, 146: 263-272. doi: 10.1016/j.ijbiomac.2019.12.248
[59]	ZHANG X Q, MANG D Z, LIAO H, YE J, QIAN J L, DONG S L, ZHANG Y N, HE P, ZHANG Q H, PURBA E R, ZHANG L W. Functional disparity of three pheromone binding proteins to different sex pheromone components in Hyphantria cunea (Drury). Journal of Agricultural and Food Chemistry, 2021, 69(1): 55-66. doi: 10.1021/acs.jafc.0c04476
[60]	QU C, YANG Z K, WANG S, ZHAO H P, LI F Q, YANG X L, LUO C. Binding affinity characterization of four antennae-enriched odorant- binding proteins from Harmonia axyridis (Coleoptera: Coccinellidae). Frontiers in Physiology, 2022, 13: 829766. doi: 10.3389/fphys.2022.829766
[61]	TANG H Y, XIE J X, LIU J T, KHASHAVEH A, LIU X X, YI C Q, ZHAO D Y, HE L, SUN Y, ZHANG Y J. Odorant-binding protein HvarOBP5 in ladybird Hippodamia variegata regulates the perception of semiochemicals from preys and habitat plants. Journal of Agricultural and Food Chemistry, 2023, 71(2): 1067-1076. doi: 10.1021/acs.jafc.2c07355
[62]	MEILLOUR P N L, LAGANT P, CORNARD J P, BRIMAU F, DANVIC C L, VERGOTEN G, MICHALSKI J C. Phenylalanine 35 and tyrosine 82 are involved in the uptake and release of ligand by porcine odorant-binding protein. Biochimica et Biophysica Acta, 2009, 1794(8): 1142-1150.
[63]	NORTHEY T, VENTHUR H, DE BIASIO F, CHAUVIAC F X, COLE A, JUNIOR K A, GROSSI G, FALABELLA P, FIELD L M, KEEP N H, ZHOU J J. Crystal structures and binding dynamics of odorant-binding protein 3 from two aphid species Megoura viciae and Nasonovia ribisnigri. Scientific Reports, 2016, 6: 24739. doi: 10.1038/srep24739
[64]	JIANG Q Y, WANG W X, ZHANG Z D, ZHANG L. Binding specificity of locust odorant binding protein and its key binding site for initial recognition of alcohols. Insect Biochemistry and Molecular Biology, 2010, 39(7): 440-447. doi: 10.1016/j.ibmb.2009.04.004
[65]	ZHANG Y N, ZHANG X Q, ZHANG X C, XU J W, LI L L, ZHU X Y, WANG J J, WEI J Y, MANG D Z, ZHANG F, YUAN X H, WU X M. Key amino acid residues influencing binding affinities of pheromone-binding protein from Athetis lepigone to two sex pheromones. Journal of Agricultural and Food Chemistry, 2020, 68(22): 6092-6103. doi: 10.1021/acs.jafc.0c01572

引物名称 Primer name	序列 Primer sequence (5′-3′)	产物大小 Product size (bp)	引物用途 Use of primers
GfunOBP2-F	ATGTTCAGAGGCAGTGTATTGTTC	552	基因克隆 Gene cloning
GfunOBP2-R	TTAGTTAGCGAAAGGAGGTCTGCAC	552	基因克隆 Gene cloning
qGfunOBP2-F	ACGAAGAGTTCCAAGCAGCA	114	表达量检测 Expression level detection
qGfunOBP2-R	TAGAGCTGACGCAGAACACG	114
qGfunEF-1α-F	AGGAGATCGAGCAACAGGAA	244
qGfunEF-1α-R	CACGACTCTCGGGACTTCTC	244
qGfunβ-actin-F	CTTTCACCACCACCGCTG	156
qGfunβ-actin-R	CGCAAGATTCCATACCCA	156
GfunOBP2-eF	CGGGATCCCAATTTCCACCAAGTTTGCCT (BamH I)	507	原核表达 Prokaryotic expression
GfunOBP2-eR	CCAAGCTTGGATAAAAAAATTAGTTAGCGAA (Hind III)	507	原核表达 Prokaryotic expression

配体 Ligand	结构式 Structural formula	结合能Mean binding energy (kJ·mol^-1)
反-2-己烯-1-醇 (E)-2-Hexen-1-ol		-5.20
1-庚醇 1-Heptanol		-5.16
1-癸醇 1-Decanol		-5.66
乙酸-顺-3-己烯酯 Cis-3-Hexenyl acetate		-6.17
2-甲基丁酸叶醇酯 Cis-3-Hexenyl 2-methylbutanoate		-6.26
α-罗勒烯 α-Ocimene		-6.65
β-石竹烯 β-Caryophyllene		-6.78