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《农业科学学报》2016,(3)
Strategies for insect population control are currently targeting chemical communication at the molecular level. The diamondback moth Plutella xylostella represents one of the most serious pests in agriculture, however detailed information on the proteins mediating olfaction in this species is still poor. This species is endowed with a repertoire of a large number of olfactory receptors and odorant binding proteins(OBPs). As a contribution to map the specificities of these chemical sensors in the moth and eventually unrave l the complexity of chemodetection, we have measured the affinities of three selected OBPs to a series of potential odorants. Three proteins are highly divergent in their amino acid sequences and show markedly different expression profiles. In fact, PxylOBP3 is exclusively expressed in the antennae of both sexes, PxylOBP9 is male specific and present only in antennae and reproductive organs, while PxylOBP19, an unusual OBP with nine cysteines, is ubiquitously present in all the organs examined. Such expression pattern suggests that the last two proteins may be involved in non-chemosensory functions. Despite such differences, the three OBPs exhibit similar binding spectra, together with high selectivity. Among the 26 natural compounds tested, only two proved to be good ligands, retinol and coniferyl aldehyde. This second compound is particularly interesting being part of the chemical pathway leading to regeneration of lignin, one of the defense strategies of the plant against insect attack, and might find applications as a repellent for P. xylostella and other pests. 相似文献
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美洲斑潜蝇气味结合蛋白OBP13的鉴定与功能 总被引：2，自引：2，他引：0
【目的】克隆与鉴定美洲斑潜蝇（Liriomyza sativae）气味结合蛋白（odorant binding protein,OBP）基因,并对其序列特征、表达情况、系统发育及蛋白功能进行研究,为深入研究美洲斑潜蝇嗅觉机制提供依据。【方法】通过PCR技术克隆美洲斑潜蝇OBP13编码区全长,用DNAMAN对其进行序列分析,用BLAST进行同源性比较,并使用MEGA6.0构建进化树,进行系统发育分析。通过实时定量PCR对OBP13在美洲斑潜蝇不同组织中的表达情况进行分析。构建原核表达载体,进行原核表达及蛋白纯化,获取重组蛋白。设定荧光分光光度计的激发波长为337 nm,以1-NPN为荧光探针,研究OBP13与25种不同气味配基的结合特性。使用间接免疫荧光染色技术及制备的OBP13的特异性多克隆抗体,对其在组织中的分布情况进行定位。将美洲斑潜蝇触角进行包埋、切片、免疫染色,并在激光共聚焦显微镜下进行观察,了解OBP13在触角感器中的亚细胞分布。【结果】获得了一个美洲斑潜蝇气味结合蛋白基因,命名为LsatOBP13（GenBank登录号：KT250751）。LsatOBP13开放阅读框全长462 bp,编码153个氨基酸,预测成熟蛋白分子量为17.80 kD,等电点为5.75,N-末端的前17个氨基酸为信号肽序列。具有4个保守的半胱氨酸位点,为Minus-C OBP。系统发育分析显示,其与地中海实蝇CcapOBP99a-like位于同一小分支上,亲缘关系较近。检测LsatOBP13在不同组织的表达水平,发现LsatOBP13在触角中的表达量远远高于其他组织。成功构建了重组表达载体并获得了高纯度的重组蛋白。对25种气味配基的结合能力检测,发现LsatOBP13与反式-2-己烯醛、芳樟醇、1-辛烯-3-醇、α-紫罗兰酮、苯并噻唑、β-紫罗兰酮具有较强的结合能力,解离常数分别为12.592、10.995、11.165、11.224、10.336、9.218 μmol·L-1,其中与β-紫罗兰酮亲和能力最强。通过免疫荧光定位,发现其在触角中主要定位在毛形感器和锥形感器上,在触角嗅觉窝及触角芒连接处也有分布。【结论】美洲斑潜蝇OBP13为非典型的气味结合蛋白Minus-C OBP。表达情况、气味结合特性、免疫定位结果表明,OBP13主要存在于美洲斑潜蝇触角中,参与触角对绿叶植物中大量存在的气味物质的识别过程,推测其在美洲斑潜蝇嗅觉识别、寄主植物定位中发挥功能。 相似文献
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西花蓟马气味结合蛋白的cDNA克隆、序列分析及时空表达 总被引：2，自引：1，他引：1
【目的】鉴定西花蓟马(Frankliniella occidentalis)气味结合蛋白（odorant binding protein,OBP）并对其序列特征及分布情况进行研究,为阐明该虫嗅觉识别的机制、利用干扰昆虫嗅觉识别来进行害虫防治提供依据。【方法】利用RT-PCR和RACE技术克隆西花蓟马气味结合蛋白基因,用DNAMAN软件进行序列分析,用BLAST进行同源性比较,并用MEGA6.0的邻接法（neighbor-joining）构建系统进化树,应用服务器Chou & Fasman预测蛋白的二级结构,通过实时荧光定量PCR检测该基因在西花蓟马不同发育期以及成虫不同组织中的分布情况。【结果】克隆了一个西花蓟马气味结合蛋白基因,命名为FoccOBP1（GenBank登录号：KM527948）;该基因cDNA序列全长660 bp,其中开放阅读框450 bp,编码149个氨基酸,3′端非编码区长172 bp,具有真核生物polyA加尾结构,5′末端非编码区长38 bp,预测成熟蛋白分子量为16.39 kD,等电点为7.46,N端有22个氨基酸组成的信号肽序列,具有气味结合蛋白典型的6个保守半胱氨酸位点特征,其排列方式为C1-X26-C2-X3-C3-X40-C4-X9-C5-X8-C6,符合典型OBP 6个保守的半胱氨酸位点结构模型。氨基酸序列中有3个亲脂性区域,第74-83位的氨基酸残基形成的亲脂性口袋尤为明显,可能是脂溶性气味分子的结合位点;FoccOBP1氨基酸序列与3个半翅目和10个同翅目昆虫OBP聚在同一分支,其中与绿盲蝽（Apolygus lucorum）的AlucOBP8（GenBank登录号为AFJ54049.1）、苜蓿盲蝽（Adelphocoris lineolatus）的AlinOBP5（GenBank登录号为ACZ58031.1）、牧草盲蝽（Lygus lineolaris）的LlinOBP2（GenBank登录号为AHF71029.1）同源相似性最高,其次是棉蚜（Aphis gossypii）的AgosOBP7（GenBank登录号为AGE97637.1）、大豆蚜（Aphis glycines）的AglyOBP7（GenBank登录号为AHJ80893.1）、禾谷缢管蚜（Rhopalosiphum padi）的RpadOBP7（GenBank登录号为AHL30243.1）等昆虫的OBP,表明FoccOBP1与这些基因的亲缘关系也较近;经FoccOBP1蛋白的二级结构预测,FoccOBP1以α-螺旋为主,其次是β-折叠,转角含量较少;经不同发育阶段检测发现,FoccOBP1在西花蓟马若虫期和初羽化成虫期的表达量较高,且随成虫羽化后天数的延长表达量逐渐降低,在雌雄成虫间的表达量也有差异;在初羽化成虫的不同组织检测发现,FoccOBP1几乎在初羽化成虫触角中特异性表达;构建了重组表达质粒pET-30a/FoccOBP1。【结论】明确了FoccOBP1的核苷酸、氨基酸序列特征,分析了该蛋白的二级结构特征,根据FoccOBP1在西花蓟马中的时空表达情况,推测该基因可能在西花蓟马嗅觉识别、定位寄主植物、信息素合成或性行为方面扮演重要角色;成功构建了重组表达质粒pET-30a/FoccOBP1,为下一步该蛋白表达纯化及其功能的深入研究打下基础。 相似文献
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Volatiles from Sophora japonica flowers attract Harmonia axyridis adults (Coleoptera: Coccinellidae)
The multicolored Asian lady beetle, Harmonia axyridis(Coleoptera: Coccinellidae), is a common generalist predator in China and is occasionally found gathering on the Chinese pagoda tree, Sophora japonica, in summer. In a field investigation, we found that H. axyridis adults preferred S. japonica during its flowering period even though their optimal prey(aphid) is absent at this time. In addition, male and female adults were attracted to S. japonica flowers to a similar extent in a Y-tube olfactometer assay. Using coupled gas chromatography-electroantennogram detection(GC-EAD), we identified a flower odor component(nonanal) that elicited a significant electrophysiological response in H. axyridis. Electroantennogram(EAG) dose-dependent responses revealed that the amplitude of the adult beetle's EAG response increased with increasing concentration of nonanal, peaking at 10 mg mL~(–1). In Y-tube olfactometer behavioral tests, H. axyridis adults preferred a 10 mg mL~(–1) nonanal source over a 100 mg mL~(–1) diluent. Under field conditions, the adults were significantly attracted to both concentrations(10 and 100 mg mL~(–1)), and high concentrations generally had greater attraction. All these results suggest that nonanal, a volatile compound of S. japonica flowers, greatly attracts H. axyridis adults. This study provides a basis for the development of synthetic attractants of H. axyridis, with the potential to promote biocontrol services of this generalist predator in the native area(e.g., China) and to suppress its population by mass trapping in its invasive areas. 相似文献
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《农业科学学报》2012,11(3):430-438
The full-length sequence of the odorant binding protein 5 gene, HarmOBP5, was obtained from an antennae cDNA library of cotton bollworm, Helicoverpa armigera (Hübner). The cDNA contains a 444 bp open reading frame, encoding a protein with 147 amino acids, namely HarmOBP5. HarmOBP5 was expressed in Escherichia coli and the recombinant protein was purified by affinity chromatography. SDS-PAGE and Western blot analysis demonstrated that the purified protein can be used for further investigation of its binding characteristics. Competitive binding assays with 113 odorant chemicals indicated that HarmOBP5 has strong affinity to some special plant volatiles, including (E)-ß-farnesene, ethyl butyrate, ethyl heptanoate, and acetic acid 2-methylbutyl ester. Based on three-dimensional (3D) model of AaegOBP1 from Aedes aegypti, a 3D model of HarmOBP5 was predicted. The model revealed that some key binding residues in HarmOBP5 may play important roles in odorant perception of H. armigera. This study provides clues for better understanding physiological functions of OBPs in H. armigera and other insects. 相似文献
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A larval specific OBP able to bind the major female sex pheromone component in Spodoptera exigua(Hübner) 
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下载免费PDF全文 Odorant binding proteins(OBPs) in insects are postulated to solubilize and transport the hydrophobic odorants across the hydrophilic antennal lymph to the olfactory receptors(ORs) located on the dendrite membrane of the sensory neurons. OBPs in adult insects have been intensively reported, but those in larvae are rarely addressed. In our study, a full-length OBP c DNA, namely Sexi OBP13, was cloned by RT-PCR and RACE strategy from the heads of Spodoptera exigua larvae. The quantitative real-time PCR(q PCR) measurement indicated that Sexi OBP13 was highly expressed in larval head, but very low in other parts of larva and was not detected in any tissues of adult. The binding affinities of Sexi OBP13 to plant volatiles and female sex pheromone components were measured by competitive binding assays. Interestingly, Sexi OBP13 displayed a high binding affinity(Ki=3.82 μmol L–1) to Z9,E12–14:Ac, the major sex pheromone component of S. exigua, while low affinities to the tested host plant volatiles(Ki27 μmol L–1). The behavioral tests further confirmed that Z9,E12–14:Ac was indeed active to elicit the behavioral activity of the third instar larvae of S. exigua. Taken together, our results suggest that Sexi OBP13 may play a role in reception of female sex pheromone in S. exigua larvae. The ecological significance of the larvae preference to the adult female sex pheromone was discussed. 相似文献
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金龟子嗅感器及嗅觉机理的研究进展 总被引：4，自引：0，他引：4
昆虫利用触角上的嗅觉感受器来识别化学信号。金龟子触角的嗅觉感受器为板形感器和锥形感器。与鳞翅目昆虫一样,金龟子嗅感器对化学气味具有选择性,并且雌雄两性间常常存在嗅觉差异。目前金龟子嗅觉信号转导的分子生物学研究也开展起来,已鉴定了多种金龟子的气味结合蛋白(OBP)和信息素结合蛋白(PBP),同一类群金龟子的PBPs相互之间同源性很高。 相似文献
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Su LIU Yuan-jie HUANG Fei QIAO Wen-wu ZHOU Zhong-jun GONG Jia-an CHENG Zeng-rong ZHU 《农业科学学报》2013,12(10):1816-1825
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Expression profiles and functional prediction of ionotropic receptors in Asian corn borer,Ostrinia furnacalis (Lepidoptera: Crambidae) 下载免费PDF全文
下载免费PDF全文 Genes involved in chemosensation are essential for odorant-mediated insect behaviors. Odorant receptors (ORs) bind and respond to pheromones and plant volatiles, regulating insect behaviors such as mating and host-plant selection, while ionotropic receptors (IRs), which are present at lower levels in insects than ORs, influence ion channels, especially in agricultural pests. Asian corn borer, Ostrinia furnacalis, is the main pest of maize that causes huge economic losses in Asia. Twenty-one OfurIRs have been identified, but none has been characterized. In this study, tissue-specific expression profiling, phylogenetic analysis, and electroantennography (EAG) analysis were applied to characterize the evolution, expression, and the potential function of OfurIRs. It was found that 20 OfurIRs were highly expressed in the antennae, except for OfurIR75p3, whereas 10 and nine OfurIRs were highly expressed in the proboscis and genitalia, respectively, indicating that these OfurIRs were functionally associated with feeding and oviposition. EAG results showed that seven acids elicited responses in the antennae of O. furnacalis and that 2-oxopentanoic acid displayed a significant female-biased response. Combined with the phylogenetic analysis, 10 OfurIRs in clade 4 were roughly predicted to be candidate receptors for 2-oxopentanoic acid and other tested acids. These results provide basic information about OfurIRs and may help advance the knolwedge on the olfactory system of O. furnacalis. 相似文献
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小菜蛾是为害十字花科蔬菜的重要害虫,干扰其嗅觉识别功能是实现小菜蛾有效治理的重要途径。本研究克隆并鉴定了1个小菜蛾气味结合蛋白基因,命名为PxylOBP13(GenBank登录号为KT156679);该基因开放阅读框全长387bp,编码128个氨基酸;预测蛋白分子量为14.33KDa,等电点为7.55,N端无信号肽序列,有3个疏水性区域,具有气味结合蛋白家族的保守结构域和6个保守的半胱氨酸残基,与6种鳞翅目昆虫的气味结合蛋白氨基酸序列一致性达50%以上。研究结果可为进一步研究小菜蛾气味结合蛋白功能,阐明小菜蛾嗅觉机制,制定科学有效的治理措施提供依据。 相似文献
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异色瓢虫对草地贪夜蛾的捕食行为观察与评价 总被引：1，自引：1，他引：0
【目的】探究天敌昆虫异色瓢虫Harmonia axyridis对草地贪夜蛾Spodoptera frugiperda的捕食潜力。【方法】在室内条件下评价异色瓢虫成虫和4龄幼虫对草地贪夜蛾1～2龄幼虫的捕食能力,观察并总结异色瓢虫的捕食行为特点。【结果】通过观察,发现异色瓢虫捕食草地贪夜蛾幼虫主要经过静止、搜寻、试探、取食和爬行5种行为,其中异色瓢虫成虫捕食3龄草地贪夜蛾幼虫时出现3对足抱握猎物的现象。异色瓢虫成虫和4龄幼虫对草地贪夜蛾1～2龄幼虫的捕食功能反应符合HollingⅡ模型。异色瓢虫成虫和4龄幼虫对草地贪夜蛾1龄幼虫的日最大捕食量分别为196.08和161.29头,瞬时攻击率分别为1.390 6和1.451 4,处理时间分别为0.005 1和0.006 2 d;异色瓢虫成虫和4龄幼虫对草地贪夜蛾2龄幼虫的日最大捕食量分别为65.36和41.84头,瞬时攻击率分别为1.145 7和1.271 0,处理时间分别为0.015 3和0.023 9 d,即异色瓢虫成虫对草地贪夜蛾幼虫的捕食能力强于4龄幼虫。【结论】异色瓢虫对草地贪夜蛾有一定的潜在控害能力,可用于防控草地贪夜蛾。 相似文献
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为合理保护和利用天敌及科学地选取抽样方法,开展合肥地区"518"油桃园山楂叶螨和李肖叶甲与其天敌之间空间关系研究,运用地学统计学方法求得天敌和害虫各自半变异函数的变程,用灰色关联度分析方法分析害虫与天敌变程的关联度,关联度值越大的天敌在空间上对害虫的跟随关系越密切。分析了山楂叶螨和李肖叶甲种群数量在全年最多时段5月23日—7月17日的油桃园中8种主要天敌对两种害虫空间跟随关系的密切程度。结果表明,与山楂叶螨空间上跟随关系密切的前3位天敌依次是异色瓢虫、草间小黑蛛和八点球腹蛛;与李肖叶甲空间上跟随关系密切的前3位天敌依次是草间小黑蛛、龟纹瓢虫和八点球腹蛛。用生态位分析法分析油桃树冠不同方位上与山楂叶螨空间上跟随关系密切的前3位天敌依次是草间小黑蛛、异色瓢虫和八点球腹蛛;与李肖叶甲空间上跟随关系密切的前3位天敌依次是草间小黑蛛、异色瓢虫和八点球腹蛛。 相似文献
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昆虫化学感受蛋白研究进展 总被引：1，自引：0，他引：1
为了研究昆虫嗅觉系统中化学感受蛋白(CSPs)在昆虫体内所发挥的作用,综述了国内外30年来对CSPs蛋白的研究。发现CSPs蛋白不仅存在于昆虫的嗅觉器官触角中,而且在非嗅觉器官中也被鉴定出来,如在小地老虎的性腺中。CSPs蛋白除了行使嗅觉功能外,还具有抗药性、视觉色素载体、营养吸收、再生和发育等功能。在一些昆虫中,同一种CSPs蛋白在性腺和触角中同时表达,因此具有接受信号和释放信号的双重功能。然而,研究还存在不足,今后还需要研究CSPs参与嗅觉识别的具体过程和发挥的作用,以及CSPs参与提高昆虫抗药性的功能。 相似文献
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Genome-wide characterization and expression analysis of WRKY family genes during development and resistance to Colletotrichum fructicola in cultivated strawberry (Fragaria×ananassa Duch.) 下载免费PDF全文
下载免费PDF全文 Based on the recently published whole-genome sequence of cultivated strawberry ‘Camarosa’, in this study, 222 FaWRKY genes were identified in the ‘Camarosa’ genome. Phylogenetic analysis showed that the 222 FaWRKY candidate genes were classified into three groups, of which 41 were in group I, 142 were in group II, and 39 were in group III. The 222 FaWRKY genes were evenly distributed among the seven chromosomes. The exon–intron structures and motifs of the WRKY genes had evolutionary diversity in different cultivated strawberry genomes. Regarding differential expression, the expression of FaWRKY133 was relatively high in leaves, while FaWRKY63 was specifically expressed in roots. FaWRKY207, 59, 46, 182, 156, 58, 39, 62 and 115 were up-regulated during achene development from the green to red fruit transition. FaWRK181, 166 and 211 were highly expressed in receptacles at the ripe fruit stage. One interesting finding was that FaWRKY179 and 205 were significantly repressed after Colletotrichum fructicola inoculation in both ‘Benihoppe’ and ‘Sweet Charlie’ compared with Mock. The data reported here provide a foundation for further comparative genomics and analyses of the distinct expression patterns of FaWRKY genes in various tissues and in response to C. fructicola inoculation. 相似文献
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【目的】克隆斑翅果蝇（Drosophila suzukii）气味结合蛋白56h（odorant binding protein 56h,OBP56h）基因,诱导表达斑翅果蝇OBP56h重组蛋白(DsuzOBP56h),研究其与小分子化合物的结合特性。【方法】通过RT-PCR并设计特异性引物克隆斑翅果蝇OBP56h ORF全长,从NCBI数据库中下载相似度高的昆虫气味结合蛋白序列,进行序列比对和分析。以NdeⅠ和XhoⅠ为酶切位点,将OBP56h连入pET-30a(+)原核表达载体,将重组质粒转入BL21(DE3)大肠杆菌感受态细胞。扩大培养阳性菌株,并用IPTG诱导表达DsuzOBP56h重组蛋白。收集菌液,通过超声波破碎细胞得到蛋白,利用Ni-NTA柱纯化蛋白,进行Tris-HCl透析,用BCA法测定蛋白浓度。蛋白用50 mmol·L -1 Tris-HCl（pH 7.4）稀释至终浓度2 μmol·L -1,配基用色谱级甲醇稀释至终浓度1 mmol·L -1,以4,4′-二苯胺基-1,1′-联萘-5,5′-二磺酸二钾盐（4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt,bis-ANS）荧光探针为报告子,利用荧光竞争结合试验检测DsuzOBP56h蛋白与18种候选小分子化合物配基的结合特性。 【结果】克隆获得了斑翅果蝇OBP56h的ORF全长,共405 bp,N-端含有19个氨基酸组成的信号肽,具有6个保守半胱氨酸位点,符合OBP的典型特征,与其同属的黑腹果蝇OBP56h进化关系最近。成功连入pET-30a(+)表达载体,在1 mmol·L -1IPTG、28℃条件下诱导DsuzOBP56h蛋白表达,并过柱纯化得到目的蛋白。荧光光谱试验显示,荧光探针bis-ANS与DsuzOBP56h的解离常数为0.9568 μmol·L -1,适合作为本试验中竞争性荧光结合试验的报告子;进一步的荧光竞争结合试验表明,在18种候选配基中,苦味物质盐酸小蘖碱和香豆素与DsuzOBP56h的结合亲和性较强,解离常数分别为12.16和17.93 μmol·L -1,柚皮素与DsuzOBP56h的解离常数为25.32 μmol·L -1,草莓叶片产生的一种对斑翅果蝇具有吸引作用的挥发性气味物质β-环柠檬醛也能与DsuzOBP56h结合,其解离常数为31.37 μmol·L -1。【结论】斑翅果蝇气味结合蛋白OBP56h能与测试的多种植物苦味物质和挥发性气味物质结合,表明DsuzOBP56h很有可能参与斑翅果蝇对食物味觉和嗅觉的识别行为,研究结果可为理解斑翅果蝇的取食行为提供理论依据,并为开展斑翅果蝇的生态防控提供新思路。 相似文献
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A gene encoding a novel G protein β subunit of β1 subclass, GβMmed was isolated from Microplitis mediator （Hymenoptera： Braconidae）. The full-length sequence of GβMmed is 1 119 bp, the cDNA contains a 1 023 bp open reading frame that encodes a protein with 340 amino acids, and the predicted molecular weight of GβMmed is 37.23 kDa and isoelectric point is 5.86. By the quantitative real-time RT-PCR method, the tissue-specific expression and quantitative changes in the developmental expression profile of GβMmed were detected. It was found that GβMmed was abundantly expressed in M. mediator antennae, head （without antennae）, thorax, abdomen, legs and the wings, and especially at high levels in abdomen. In antennae, expression varied through 1st day before emergence to 5-d-old adults, and had equal expression levels detected in females and males in total. In head, GβMmed expresses while initially high in females, and have another peaked in stage 4 and 1st day, in males showed a peak of GβMmed expression prior to emergence and relatively low levels after emergence. In female abdomen GβMmed expression levels have two peaks in stage 1 and the 5th d, but just have one peak in male abdomen in stage 1. In all other tissues expression was low and stable. 相似文献