甜菜夜蛾细胞色素P450(CYP9A11)与3种杀虫剂的结合机理研究

细胞色素P450(以下简称CYP)与昆虫的抗药性密切相关。本研究运用AutoDock分子对接技术和分子力学泊松-波尔兹曼表面积法(molecular mechanics Poisson-Boltzmann surface area,MM-PBSA)结合自由能计算方法,分析了甜菜夜蛾CYP9A11与3种杀虫剂结合的作用位点、作用力类型和大小。结果表明:CYP9A11与毒死蜱结合形成两个氢键,有8个氨基酸残基参与形成疏水作用力,二者结合自由能为-3659.80 kJ/mol;CYP9A11与灭多威结合形成5个氢键,有3个氨基酸残基形成疏水作用力,结合自由能为-470.92 kJ/mol;CYP9A11中有7个氨基酸残基与氯氰菊酯结合形成疏水作用力,结合自由能为-473.44 kJ/mol。范德华力是CYP9A11与毒死蜱结合的主要驱动力,极性溶剂化能是CYP9A11与氯氰菊酯和灭多威结合的主要驱动力,这些结果为阐明甜菜夜蛾CYP9A11与3种杀虫剂的结合机理提供了参考。     

甜菜夜蛾化学感受蛋白SexiCSP2结合特性和机理研究

为阐明甜菜夜蛾Spodoptera exigua化学感受蛋白识别外源化合物的特性和作用机制,运用Western blotting法分析SexiCSP2蛋白在成虫各组织的表达谱,荧光竞争结合试验法测定SexiCSP2蛋白与25种外源配基的结合特性,运用分子对接、分子动力学、 结合自由能计算等方法研究SexiCSP2与配基的结合模式。结果显示SexiCSP2在成虫接触和非接触性器官中均有表达,与己醛结合能力最强,解离常数为1.3 μmol/L;分子模拟结果显示SexiCSP2与己醛结合形成1个氢键,两者结合自由能为-32.94 kJ/mol,主要驱动力为范德华力,主要抑制力为非极性溶剂化能。这些结果说明SexiCSP2在甜菜夜蛾识别外源化合物中起重要作用,可以作为防控甜菜夜蛾的有效靶标。     

入侵害虫松树蜂气味结合蛋白与其相关信息化学物质的分子对接

为探究入侵我国的重大林业检疫性害虫松树蜂Sirex noctilio的气味结合蛋白(odorant binding protein,OBP)与其相关信息化学物质的结合模式和结合能力,采用Swiss-model在线服务器对松树蜂触角中高表达的4种OBP序列进行同源模建,采用Procheck、Verify_3D和ERRAT程序对模建质量进行评价;并应用Autodock软件对OBP模型和相关信息化学物质进行分子对接分析。结果显示,在松树蜂触角中高表达的4种蛋白为SnocOBP4、SnocOBP6、SnocOBP9和SnocOBP12,其同源模建所得模型质量较好:均满足拉氏构象图中氨基酸位于最佳合理区的数量大于90%的条件;三维结构与一级结构的兼容性评分大于0.2;所得ERRAT值分别为85.71%、95.10%、98.15%和91.82%。分子对接显示,与松树挥发物α-蒎烯和β-蒎烯结合最好的蛋白是SnocOBP12,结合能分别为-5.36 kJ/mol和-5.48 kJ/mol;与雌性信息素成分顺-9-二十九烯结合最好的蛋白是SnocOBP4,结合能为-5.42 kJ/mol;与雄性信息素成分顺-3-癸烯醇和顺-4-癸烯醇结合最好的蛋白是SnocOBP9,结合能分别为-4.37 kJ/mol和-4.45 k J/mol;而3-蒈烯、顺-7-二十七烯和反-2,4-癸二烯醛等气味分子与SnocOBP6的结合能较低,结合能最低的是顺-7-二十七烯,为-6.08 k J/mol。雄蜂主要信息素成分顺-3-癸烯醇与其同分异构体反-3-癸烯醇相比,与SnocOBP9的结合能较低;但是反-3-癸烯醇能以较强的氢键竞争SnocOBP9结合位点。研究表明这4种松树蜂OBP与相应植物挥发物或雌雄信息素具有较好的亲和力,参与了松树蜂对不同信息化学物质的识别过程。     

氟虫腈与昆虫γ-氨基丁酸受体相互作用研究

利用荧光标记法及分子模拟法,研究了氟虫腈与昆虫γ-氨基丁酸(aminobutyric acid,GABA)受体的相互作用。荧光标记试验结果显示,氟虫腈与家蝇脑内GABA受体有较强的相互作用,其最大结合量[RT]值和亲和常数Kd值分别为(21.3±2.5) pmol/mg protein和(109±9) nmol/L。分子模拟结果显示:氟虫腈与果蝇RDL受体间形成3条氢键;两者之间的CDOCKER的相互作用能为-137.93 kJ/mol。试验和理论两方面均证实,氟虫腈对昆虫GABA受体的强亲和性是导致氟虫腈对昆虫产生高毒性的重要原因。     

2.5%高效氟氯氰菊酯水乳剂在苹果叶片表面的润湿性能

采用光学视频接触角测量仪测定了苹果叶片的表面自由能,以及2.5%高效氟氯氰菊酯水乳剂(beta-cyfluthrin EW)稀释液的表面张力及静态接触角、0~60 s动态接触角。结果表明:苹果叶片近轴面的表面自由能为52.76 m J/m2,以极性分量为主导;而远轴面的表面自由能为21.79 m J/m2,且以色散力分量(非极性分量)为主导。高效氟氯氰菊酯水乳剂2 000倍稀释液的表面张力为52.45 m N/m,与25 g/L高效氟氯氰菊酯乳油3 000倍稀释液的相近,远低于去离子水的表面张力74.65 m N/m;其静态接触角与25 g/L高效氟氯氰菊酯乳油3 000倍稀释液的无显著差异,且在0~60 s内接触角下降趋势最快。研究表明:苹果叶片的近轴面比远轴面更易被药液润湿;在苹果园喷施2.5%高效氟氯氰菊酯水乳剂时,药液的稀释倍数不宜太高,以2 000倍为最佳。     

基于柑橘绿霉菌CYP51同源模建的定点突变与抗性机制分析

为阐明杀菌剂与PdCYP51的相互作用及其抗性机制,基于最新解析的真核生物人类的CYP51晶体结构,同源模建了PdCYP51的三维结构,并选取商品化的杀菌剂烯唑醇进行分子对接,预测影响PdCYP51与烯唑醇相互作用的关键氨基酸。采用定点突变技术获得了PdCYP51-Y112H、F120L、F120D和S309A 4种突变体（PdCYP51m）。结果表明,突变体蛋白PdCYP51-Y112H、F120L、F120D和S309A表达量均有不同程度的变化,PdCYP51-Y112H和F120L与未突变蛋白表达量相当,而PdCYP51-F120D和S309A表达量增加。4种突变体与杀菌剂的结合能力降低,其结合常数分别为1.28、0.18、1.03和1.31μmol/L,均大于未突变PdCYP51的0.12μmol/L。表明这些氨基酸是杀真菌剂烯唑醇与PdCYP51结合的关键氨基酸,PdCYP51与烯唑醇形成的疏水性空腔和稳定配体杀菌剂分子的作用力是酶蛋白与杀菌剂结合的重要因素。     

新型除草剂1-(4-甲基嘧啶-2-基)-3-(2-硝基苯磺酰)脲的溶液构象研究

应用系统构象搜索及分子动力学模拟方法,研究了新型超高效、"对环境友好"的除草剂1-(4-甲基嘧啶-2-基)-3-(2-硝基苯磺酰)脲分子在常态以及溶液中的构象特征。在极性溶剂水形成的溶液中,分子呈现伸展形和卷曲形,而在非极性溶剂CCl4形成的溶液中则只呈现伸展形。通过计算协同旋转的各势能面,得到不同构象相互转换所需克服的能垒为21 kJ/mol。分子在溶液中很容易转换到活性构象与乙酰乳酸合成酶作用。     

除草剂与乙酰乳酸合成酶相互作用的分子机理研究进展

综述了酵母乙酰乳酸合成酶(ScALS)与磺酰脲类除草剂氯嘧磺隆(chlorimuron-ethyl,CE)形成的复合物在0.28 nm分辨率下的晶体结构及拟南芥乙酰乳酸合成酶(AtALS)与磺酰脲类和咪唑啉酮类除草剂复合物的三维结构。除草剂的分子结构与酶、底物并不相似,但它们与酶形成的复合物可阻断底物进入酶活性位点通路而起抑制作用。连接磺酰脲的10个氨基酸残基同样连接咪唑喹啉酸,另有6个残基只与磺酰脲而不与咪唑喹啉酸相连,有2个残基只与咪唑喹啉酸而不与磺酰脲相连,即两种抑制剂占据了特别的重叠位点,但以不同方式连接。抗性杂草的产生是因为突变株ALS的残基位点变异,从而引起除草剂与ALS结合方式的变化。这些研究对进一步理解除草剂与靶分子的作用方式及除草剂的分子药物设计具有重要的指导作用。     

反枝苋乙酰乳酸合成酶与烟嘧磺隆分子结合模式分析及抗性位点预测

为研究反枝苋对乙酰乳酸合成酶 (ALS) 抑制剂的抗性机制,本研究根据反枝苋的ALS氨基酸序列,利用同源模建的方法构建了其三维结构,并采用分子对接和分子动力学模拟的方法预测了反枝苋ALS与烟嘧磺隆分子的结合模式。根据结合模式对已报道的Pro 197和Trp 574等位点突变产生抗性的原因进行了分析。结果发现:Pro 197和Trp 574等位点的残基与烟嘧磺隆分子之间存在重要的疏水作用和π-π作用等其他相互作用,或该位点的残基具有特殊结构影响着通道形状。分析表明,ALS与烟嘧磺隆之间的氢键、疏水作用等非共价相互作用以及通道形状的改变都有可能影响二者结合稳定性,从而使杂草产生抗性。基于此结论,本研究预测Val 196、Met 200、Phe 206和Lys 256突变同样可能使杂草对ALS抑制剂敏感度发生变化。本研究利用计算机模拟技术分析了ALS抗性机制,为反抗性除草剂的分子设计提供了指导。     

花椒窄吉丁气味结合蛋白AzanOBP3与其寄主挥发物的分子对接

为明确花椒窄吉丁Agrilus zanthoxylumi气味结合蛋白AzanOBP3与其寄主花椒Zanthoxylum bungeanum主要气味挥发物的结合模式及结合能力,利用Signal P 5.0和ProParam Tool-ExPASy软件对AzanOBP3的基本理化性质进行生物信息学分析,采用Swiss-Model对AzanOBP3序列进行同源建模,通过SAVES 5.0软件中Verify-3D、ERRAT以及ProCheck模块评价模型,应用AutoDock Tool 1.5.6软件对AzanOBP3模型和46种主要寄主挥发物进行分子对接分析。结果显示,花椒窄吉丁气味结合蛋白AzanOBP3同源建模所得模型质量较好,其Verify-3D、ERRAT以及ProCheck得分均符合同源建模要求。AzanOBP3与46种花椒气味挥发物主要以氢键、疏水作用和范德华力相结合,其中, 13种链状配体与AzanOBP3的结合能力总体上弱于33种环状配体,链状配体中的芳樟醇和环状配体中的荜澄茄油烯与AzanOBP3表现出较好的结合能力,自由结合能分别为-5.2 kJ/mol和-6.6 kJ/mol。最终筛选出4种链状配体（乙酸芳樟酯、芳樟醇、乙酸香叶酯和别罗勒烯）和10种环状配体（乙酸松油酯、β-石竹烯、葎草烯、3-蒈烯、荜澄茄油烯、右旋大根香叶烯、β-榄香烯、乙酸叶醇酯、可巴烯和倍半水芹烯）可与AzanOBP3稳定结合。表明花椒窄吉丁气味结合蛋白AzanOBP3能与寄主植物花椒的多种挥发性物质结合,推测AzanOBP3可能在其寻找寄主与取食行为中发挥着重要作用。     

双条杉天牛OBP与其嗅觉相关化学物质的分子对接及分子动力学分析

为探究双条杉天牛Semanotus bifasciatus气味结合蛋白（odorant binding protein,OBP）与信息化合物的识别结合机理，筛选出与OBP结合效果最佳的化学配体，选择在双条杉天牛触角中高表达的OBP2、OBP6、OBP7序列，通过Swiss-model在线软件对其进行同源模建，使用ERRAT程序、Procheck和Verify＿3D进行建模质量评估；采用Autodock软件对OBP模型和相关化学物质进行分子对接分析，通过GROMACS 2019.6软件包进行目的OBP与结合效果最佳配体复合体的分子动力学模拟。结果显示，OBP2、OBP6和OBP7模型所得到的ERRAT值分别为97.03%、94.06%和97.39%，三维结构与一级结构的兼容性所得评分大于0.2的氨基酸百分比均大于80.00%，且拉氏构象图中氨基酸位于最佳合理区的数量均大于90.00%，说明所建模型合理；在11个化合物中，柏木醇与OBP2、OBP6和OBP7的结合性能最好，结合能分别为-7.04、-6.86和-6.62 kJ/mol，在结合过程中，疏水作用力和氢键作用力共同作用使蛋白与配体...     

The investigation of the interaction between 5-Iodouracil and human serum albumin by spectroscopic and modeling methods and determination of protein by synchronous fluorescence technique

The interaction of 5-Iodouracil with human serum albumin (HSA) was investigated in aqueous solution by fluorescence quenching spectrum in combination with UV absorption spectrum and modeling method. The quenching mechanism and binding characteristics of 5-Iodouracil with HSA were obtained from the fluorescence measurement. The binding constants were calculated according to the Lineweaver-Burk equation at different temperatures. And the thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS), were calculated by thermodynamic equations. The results showed that the hydrophobic interaction played a major role in the binding of 5-Iodouracil with HSA. The binding distance was obtained according to Föster’s non-radiative energy transfer theory. The effect of some normal ions on the binding constants was discussed. Modeling method was applied to elucidate the interaction mode between 5-Iodouracil and HSA, which was agreed with the calculated result by thermodynamic method. Moreover, the synchronous fluorescence technique was successfully applied to quantify total protein in human body fluids including serum, urine, and saliva samples. The results showed that this method was stable, reliable, selective, sensitive, and practical for the determination of HSA.     

Human serum albumin interaction with paraquat studied using spectroscopic methods

The interaction between herbicide paraquat and human serum albumin (HSA) was investigated by fluorescence and UV/Vis absorption spectroscopy. Paraquat can strongly quench the intrinsic fluorescence of HSA by static quenching and nonradiative energy transferring; The hydrophobic and electrostatic interactions play a major role in stabilizing the complex. The binding site number n and apparent binding constant K_A, corresponding thermodynamic parameters ΔG, ΔH, ΔS at different temperatures, were calculated. The distance r between donor (HSA) and acceptor (paraquat) was obtained according to fluorescence resonance energy transfer. The effect of paraquat on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy.     

游离氨基酸含量与茶树抗螨性的关系

分析了茶树新梢中游离氨基酸的含量与茶树品种对茶橙瘿螨（Ａｃａｐｈｙｌｌａ ｔｈｅａｅ）的抗性关系。抗性品种游离氨基酸总量以及茶氨酸（Ｔｈｅａ）、谷氨酸（Ｇｌｕ）、丙氨酸（Ａｌａ）、甘氨酸（Ｇｌｙ）、天冬氨酸（Ａｓｐ）、丝氨酸（Ｓｅｒ）、缬氨酸（Ｖａｌ）等的含量显著蒿于感性品种。抗性品种新梢中与氨基酸合成有关的谷氨酸脱氢酶（ＧＤＨ）和苯丙氨酸解氨酸（ＰＡＬ）的活性分别是感性品种的２．３倍和３．２倍,     

氰氟草酯水解动力学研究

研究了氰氟草酯在不同温度、不同pH条件下于水溶液中的降解动态,并通过液相色谱法对水解产物进行了鉴定。结果表明,氰氟草酯在酸性条件下比较稳定,在微酸性及中性条件下水解速度加快,在碱性条件下水解迅速;温度升高水解反应加速,水解反应温度效应系数为2.1~3.0,活化能为74.90 kJ/mol。水解产物主要是(R)-2- -丙酸、(R)-2- -丙酸和(R)-2- -丙酸。     

In vivo biochemical changes in liver and gill of Clarias batrachus during cypermethrin exposure and following cessation of exposure

The sublethal effect of a synthetic pyrethroid, cypermethrin on total protein, amino acids, ammonia, glycogen, and enzymes like aminotransaminases (AIAT, AAT), glutamate dehydrogenase, and glycogen phosphorylases (a and ab) was studied in physiological important tissues viz; liver and gill tissues of freshwater teleost air breathing fish, Clarias batrachus. The study was conducted during exposure of 1/3 (33%) of LC₅₀ concentration and followed by cessation of exposure. Thirty-six fish were exposed to 0.07 mg/L cypermethrin for 10 days. After 10 days, 18 fish were released to freshwater and kept in the same for 10 days (recovery group). Thirty-six fish were kept in freshwater as control batch. Protein content in liver tissues decreased at the end of 1st and 5th day followed by slight increase at the end of 10th day. Gill tissue showed statistical significant decrease (P < 0.001) in protein content during exposure period of 10 days. Recovery in protein content was observed to a large extent in both the tissues. Total free amino acids were increased in liver and gill tissues throughout the treatment period, recovery response was seen after cessation of exposure. Ammonia level was decrease in both the tissues throughout the exposure period except in liver tissue at the end of 1st day of exposure. Recovery response was exhibited by both the tissues. A decreased in glycogen content of liver tissue was observed during exposure period, gill tissue also showed decrease in glycogen at the end of 1st and 5th day followed by increase at the end of 10th day of exposure period. When the fish were transferred to freshwater, recovery in glycogen content was noted. The activity level of alanine, aspartate aminotransaminase, glutamate dehydrogenase, and phosphorylases (a, ab) was increased in both the tissues, followed by recovery response after released of fish into freshwater. The present study showed that cypermethrin caused alterations in certain biochemical mechanisms of C. batrachus. This fish indicated recovery response when transferred to cypermethrin free water.     

小地老虎化学感受蛋白AipsCSP2配体结合特性分析

为了明确小地老虎化学感受蛋白基因AipsCSP2在雌、雄成虫各组织中的表达情况,解析AipsCSP2蛋白的配体结合特性并探讨其功能.本文基于小地老虎性腺转录组数据,利用PCR技术克隆AipsCSP2基因,并进行生物信息学和系统进化分析;采用qPCR技术测定该基因在小地老虎雌、雄成虫不同组织中的表达水平;利用原核表达技术...