中国农业科学 ›› 2021, Vol. 54 ›› Issue (5): 945-958.doi: 10.3864/j.issn.0578-1752.2021.05.007
刘孝贺1(),仇贵生1,佟兆国2,张怀江1,闫文涛1,岳强1,孙丽娜1(
)
收稿日期:
2020-05-11
接受日期:
2020-06-22
出版日期:
2021-03-01
发布日期:
2021-03-09
通讯作者:
孙丽娜
作者简介:
刘孝贺,E-mail:基金资助:
XiaoHe LIU1(),GuiSheng QIU1,ZhaoGuo TONG2,HuaiJiang ZHANG1,WenTao YAN1,Qiang YUE1,LiNa SUN1(
)
Received:
2020-05-11
Accepted:
2020-06-22
Online:
2021-03-01
Published:
2021-03-09
Contact:
LiNa SUN
摘要:
【目的】桃小食心虫(Carposina sasakii)是我国北方果树生产中的重要害虫之一,本文通过体外表达桃小食心虫化学感受蛋白CsasCSP16,明确其与寄主挥发物分子和性信息素分子的结合特性,并通过生物信息学预测CsasCSP16与挥发物分子结合的关键氨基酸位点,为揭示桃小食心虫嗅觉的分子机理提供理论依据。【方法】通过原核表达系统获得CsasCSP16蛋白,并使用Ni-NTA柱纯化重组蛋白。采用荧光竞争结合的方法,以N-苯基-1-萘胺(N-pheny-1-naphthylamine,1-NPN)为荧光探针,从33种寄主挥发物和2种性信息素分子中筛选出与CsasCSP16结合亲和性高的配体分子。通过对CsasCSP16进行同源建模获得其三维结构模型,以CSPMbraA6(PDB ID:1N8U)为模板成功构建CsasCSP16的三维结构模型。使用Autodock Vina软件将CsasCSP16与亲和性高的配体分子进行对接,构建蛋白-配体复合物。然后使用GROMACS(2019.3)软件对复合物进行动力学模拟,从动力学模拟的平衡状态中选取100个构象,使用g_mmpbsa软件计算CsasCSP16与气味分子的结合能,并通过能量分解的方法预测关键氨基酸残基。【结果】成功构建了CsasCSP16的克隆表达载体,通过大肠杆菌原核表达系统获得高纯度的重组蛋白。与35种配体分子的荧光竞争结合表明,CsasCSP16与水杨酸甲酯、6-甲基-5-庚烯-2-酮、十五烷、庚酸丁酯和α-蒎烯有较强的结合活性,其Ki值分别为6.59、6.25、3.50、6.73和4.47 μmol·L-1。分子对接的结果表明,CsasCSP16与水杨酸甲酯、6-甲基-5-庚烯-2-酮、十五烷、庚酸丁酯和α-蒎烯的Vina Score值分别为-6.1、-5.3、-5.8、-5.2和-6.6。动力学模拟结果表明,CsasCSP16与气味分子复合物在50 ns内达到平衡状态,通过g_mmpbsa计算复合物的结合能,CsasCSP16-水杨酸甲酯、CsasCSP16-6-甲基-5-庚烯-2-酮、CsasCSP16-十五烷和CsasCSP16-庚酸丁酯的结合自由能分别为-50.264、-65.551、-136.035和-93.805 kJ·mol-1;最后,通过分解每个氨基酸贡献的结合自由能表明异亮氨酸49(Ile49)、缬氨酸71(Val71)、异亮氨酸72(Ile72)和酪氨酸90(Tyr90)贡献的结合能>3 kJ·mol-1,因此推测这4种氨基酸在CsasCSP16结合气味分子的过程中发挥关键作用。【结论】桃小食心虫CsasCSP16能与寄主植物的多种气味分子结合,推测其可能在桃小食心虫对寄主植物的定位过程中发挥重要作用。异亮氨酸49(Ile49)、缬氨酸71(Val71)、异亮氨酸72(Ile72)和酪氨酸90(Tyr90)可能是CsasCSP16与配体分子结合的关键氨基酸位点。
刘孝贺,仇贵生,佟兆国,张怀江,闫文涛,岳强,孙丽娜. 桃小食心虫化学感受蛋白CSP16配体结合特性[J]. 中国农业科学, 2021, 54(5): 945-958.
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.
表1
重组蛋白CsasCSP16 与不同寄主挥发物和性信息素分子结合的数据"
寄主挥发物和性信息素分子 Host volatile and sex pheromone molecule | CSP16 | 结合能 Binding energy (kJ·mol-1) | ||
---|---|---|---|---|
IC50 (μmol·L-1) | Ki (μmol·L-1) | |||
醇类 Alcohols | 2-己烯醇 2-hexenol | >50 | >50 | - |
苯乙醇 Phenethyl alcohol | >50 | >50 | - | |
醛类 Aldehydes | 反-2-己烯醛 (E)-2-hexenal | >50 | >50 | - |
己醛 Hexanal | >50 | >50 | - | |
苯甲醛 Benzaldehyde | >50 | >50 | - | |
庚醛 Heptanal | >50 | >50 | - | |
辛醛 Octanal | >50 | >50 | - | |
壬醛 Nonanal | >50 | >50 | - | |
癸醛 Decanal | >50 | >50 | - | |
酯类 Esters | 丁酸乙酯 Ethyl butyrate | >50 | >50 | - |
乙酸丁酯 Butyl acetate | >50 | >50 | - | |
乙酸异戊酯 Isoamyl acetate | >50 | >50 | - | |
乙酸-顺-3-己烯酯 (Z)-3-hexenyl acetate | >50 | >50 | - | |
丁酸丁酯 Butyl butyrate | >50 | >50 | - | |
己酸乙酯 Ethyl hexanoate | >50 | >50 | - | |
乙酸己酯 Hexyl acetate | >50 | >50 | - | |
水杨酸甲酯 Methyl salicylate | 18.69 | 6.59 | -25.52 | |
庚酸乙酯 Ethyl heptanoate | >50 | >50 | - | |
己酸丁酯 Butyl hexanoate | >50 | >50 | - | |
茉莉酸甲酯 Methyl jasmonate | >50 | >50 | - | |
乙酸二甲基丁酯 Dimethyl butyl acetate | >50 | >50 | - | |
辛酸正丁酯 Butyl octanoate | >50 | >50 | - | |
庚酸丁酯 Butyl heptanoate | 19.74 | 6.73 | -21.76 | |
辛酸丙酯 Propyl octanoate | >50 | >50 | - | |
萜类 Terpenoids | α-蒎烯 α-pinene | 11.50 | 4.47 | -27.61 |
α-罗勒烯 α-ocimene | >50 | >50 | - | |
腈类 Benzonitriles | 苯甲腈 Benzonitrile | >50 | >50 | - |
烷烃类 Alkanes | 癸烷 Decane | >50 | >50 | - |
十四烷 Tetradecane | >50 | >50 | - | |
十五烷 Pentadecane | 7.90 | 3.50 | -24.27 | |
十六烷 Hexadecane | >50 | >50 | - | |
十八烷 Octadecane | >50 | >50 | - | |
酮类物质 Ketone | 6-甲基-5-庚烯-2-酮 6-methyl-5-hepten-2-one | 17.83 | 6.25 | -22.16 |
Z-7-二十烯-11-酮 Z-7-eicosen-11-one | >50 | >50 | - | |
Z-7-十九烯-11-酮 Z-7-nonadecen-11-one | >50 | >50 | - |
表2
CsasCSP16与配体分子的结合自由能和组分"
配体 Ligand name | 范德华力 Val der Waals (kJ·mol-1) | 静电作用力 Electrostatic force (kJ·mol-1) | 极性溶剂化能 Polar solvation energy (kJ·mol-1) | 非极性溶剂化能 Nonpolar solvation energy (kJ·mol-1) | 结合能 Binding energy (kJ·mol-1) |
---|---|---|---|---|---|
水杨酸甲酯 Methyl salicylate | -82.466 (0.682) | -18.084 (0.850) | 61.336 (0.750) | -11.103 (0.055) | -50.264 (0.702) |
6-甲基-5-庚烯-2酮 6-methyl-5-hepten-2-one | -93.570 (0.927) | -16.158 (1.325) | 55.604 (0.973) | -11.428 (0.061) | -65.551 (1.080 ) |
十五烷 Pentadecane | -164.748 (0.845) | -0.969 (0.069) | 49.259 (0.848) | -19.537 (0.089) | -136.035 (1.047) |
庚酸丁酯 Butyl heptanoate | -109.800 (0.787) | -1.567 (0.211) | 33.365 (0.782) | -15.787 ( 0.079) | -93.805 (1.191) |
表3
主要残基对结合自由能的贡献"
气味分子 Volatile ligand | 相互作用能 Interaction energy (kJ·mol-1) | |||
---|---|---|---|---|
范围 Range (>2.0 ) | 范围 Range (-1.0—-3.0 ) | 范围 Range (-3.0—-5.0) | 范围 Range (<-5.0 ) | |
水杨酸甲酯 Methyl salicylate | Tyr28, Gln64, Thr68 | Leu45, Ala52, Cys57, Cys60, Val71 | - | Ile49 |
6-甲基-5-庚烯-2酮 6-methyl-5-hepten-2-one | Arg50 | Met32, Ile49, Leu53, Ile72, Leu75, Trp83, Leu86, Thr87, Tyr96, Val 97, Tyr100 | Thr90 | - |
十五烷 Pentadecane | Asp91, His94 | Leu15, Ile18, Leu19, Met32, Leu45, Leu53, Leu75, Trp83, Leu86, Thr87, Tyr96, Val97 | Ile72, Tyr90 | Ile49, Val71 |
庚酸丁酯 Butyl heptanoate | - | Leu15, Leu25, Ile29, Met32, Leu45, Leu53, Cys57, Thr68, Val71, Trp83, Leu86, Val97, Tyr100 | Ile72, Tyr90 | Ile49 |
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