昆虫信息素结合蛋白及其分子运输机制和生理功能研究进展

昆虫信息素结合蛋白是气味结合蛋白多基因家族的一个分支,在昆虫识别性信息素过程中起重要作用。该文从信息素结合蛋白的分子特征、与信息素分子的结合及释放机制、生理功能和进化基因组学等方面进行了综述,针对鳞翅目昆虫进行了重点阐述。     

家蚕信息素结合蛋白BmPBP2和BmPBP3基因的初步鉴定及表达分析

嗅觉对昆虫的生存、繁殖等起着重要的作用。依据家蚕Bombyx mori全基因组序列设计特异引物,扩增得到了两个信息素结合蛋白BmPBP2和BmPBP3基因的cDNA片段。结合已报道的家蚕信息素结合蛋白BmPBP1和两个普通气味结合蛋白BmGOBP的信息,对其基因结构分析表明,这5个基因均由3个外显子组成,具有保守的外显子/内含子边界和典型的6个Cys残基,且3个PBP基因在基因组上串联分布。序列同源性分析表明,BmPBP2和BmPBP3与烟草天蛾的PBP2和PBP3的同源性高达69%和63%。半定量RT-PCR分析结果显示,BmPBP2和BmPBP3基因在成虫触角中特异表达,且雌雄表达水平相当。这些结果表明BmPBP2和BmPBP3可能起着性信息素识别的作用。     

绿盲蝽气味结合蛋白AlucOBP7的表达及气味结合特性

气味结合蛋白(odorant binding proteins,OBPs)在昆虫嗅觉识别中起着重要的作用,尤其是在运输外界脂溶性气味分子通过嗅觉感器淋巴液到达嗅觉受体(olfactory receptors,ORs)的过程中发挥关键作用。明确OBPs在昆虫同外界进行信息交流过程中的作用有利于阐明昆虫嗅觉识别的机制,同时可为利用干扰昆虫嗅觉识别来进行害虫防治奠定理论基础。本研究克隆了一个绿盲蝽Apolygus lucorum(Meyer-Dür)气味结合蛋白AlucOBP7基因(GenBank登录号:JQ675724),并进行了原核表达,以1-NPN为荧光探针采用荧光竞争结合实验研究了AlucOBP7蛋白和10种棉花挥发物及6种性信息素类似物的结合能力。结果表明:在10种棉花挥发物中,AlucOBP7能够和2-己酮及水杨酸甲酯有效结合,结合常数分别为55.13μmol/L和28.26μmol/L。在6种盲蝽性信息素类似物中,4-氧代-反-2-己烯醛和AlucOBP7具有较强的结合能力,结合常数为23.14μmol/L。丁酸乙酯、丁酸丁酯及己酸己酯也能够和AlucOBP7有效结合,但结合能力中等,结合常数分别为30.58,39.26和35.81μmol/L。初步推测,AlucOBP7可能是绿盲蝽性信息素结合蛋白(pheromone binding proteins,PBPs),并在感受性信息素和植物挥发物的过程中发挥双重功能。     

昆虫普通气味结合蛋白研究进展

昆虫气味结合蛋白(OBPs)参与昆虫识别环境中气味信息的第一步反应,在调控昆虫生命活动中起着重要的作用。普通气味结合蛋白(GOBPs)主要参与昆虫对寄主植物挥发物或信息素的识别。本文总结了GOBPs基因的分子特征和生理功能,以及GOBPs基因在不同组织、性别和发育阶段的表达特性;针对GOBPs与性信息素结合蛋白(PBPs)在染色体上串联形成的GOBPs/PBPs亚家族基因簇,阐述了GOBPs基因的进化起源、扩增与丢失现象;梳理了昆虫GOBPs基因在虫情监测和害虫防治等方面的应用前景,提出GOBPs基因是未来害虫生物防治的重要靶点。     

中华蜜蜂信息素结合蛋白OBP10的基因克隆、原核表达和配基结合特性分析

【目的】气味结合蛋白在中华蜜蜂Apis cerana cerana嗅觉系统中起到重要作用,本实验拟研究中华蜜蜂一个新的信息素结合蛋白OBP10及其与蜜蜂信息素以及蜜源开花植物挥发物的结合特性。【方法】本实验通过RT-PCR扩增获得OBP10基因全长(Gen Bank登录号:KP717060.1),以p ET-30a构建原核表达载体,并以Ni2+琼脂糖柱进行重组蛋白表达和分离纯化,在N-苯基-1-萘胺(N-phenyl-1-naphthylamine,1-NPN)作为荧光报告子下利用荧光光谱法体外研究重组Acer OBP10与多种候选化学配基的结合特征。【结果】经多序列联配分析,发现Acer OBP10的多个同源基因均为信息素结合蛋白(pheromone binding proteins,PBPs)。配基结合特性分析显示,Acer OBP10对14种候选配基中的蜂王信息素成分对羟基苯甲酸甲酯(HOB)竞争力最大,相对荧光可降至6.06%,解离常数11.04μmol/L;进一步表明Acer OBP10属于一个新的中蜂PBPs。此外,Acer OBP10也能和包括工蜂信息素(香叶醇和橙花醇)、报警信息素(2-庚酮和乙酸异戊酯)等蜜蜂信息素以及蜜源开花植物挥发物之一的β-紫罗兰酮结合,表明Acer OBP10可能是一种以信息素结合为主的多功能结合蛋白。【结论】Acer OBP10是中蜂一个新的信息素结合蛋白,与此前我们鉴定的蜂王信息素结合蛋白Acer ASP1相比,Acer OBP10对蜜蜂信息素的结合谱更为广泛,这些结果将对进一步研究中华蜜蜂信息素识别和传递提供理论基础,对于深入了解中华蜜蜂嗅觉影响生理功能的行为机制具有重要意义。     

螟蛾总科昆虫性信息素的种类、分泌及感受机制研究

本文从螟蛾总科昆虫性信息素分泌腺体及分泌节律、性信息素受体及结合蛋白、性信息素的结构及组成特点、近缘种相似性及种内变异等方面综述了螟蛾总科昆虫性信息素的研究进展。最后对需要进一步研究的问题提出了展望。     

蛾类昆虫性信息素受体及其作用机理

蛾类昆虫性信息素受体首先从烟芽夜蛾Heliothis virescens和家蚕Bombyx mori中鉴定出来, 到目前为止已经克隆得到了19种蛾类昆虫的几十种性信息素受体基因, 并且这些基因在系统发育树中聚成一个亚群。性信息素受体从蛾类蛹期开始表达, 主要表达在雄性触角的毛形感器中, 少部分受体在雌性触角、 雄性触角其他感器以及身体其他部位中也有表达。大部分蛾类性信息素受体的配体并不是单一的, 而是能够对多种性信息素组分有反应, 部分性信息素受体还能够识别性信息素以外的其他物质, 还有一部分性信息素受体的识别配体目前尚不清楚。另外发现在雌性蛾类触角中也存在一些嗅觉受体能够识别雄性分泌的性信息素。在蛾类性信息素受体与性信息素识别的过程中, 性信息素结合蛋白不仅能够特异性地运送配体到嗅觉神经元树状突上, 还能够提高性信息素与性信息素受体之间的结合效率。另外, OrCo类受体与性信息素受体共表达在嗅觉神经元中, 在蛾类性信息素受体与配体的识别过程中扮演了重要角色。但是蛾类信息素对神经元刺激的终止并非由性信息素受体控制, 而是由细胞中的气味降解酶等其他因子调控。蛾类性信息素受体研究中还有很多疑问需要解答, 其过程可能比我们想象的更为复杂。     

烟夜蛾性信息素结合蛋白2(PBP2)基因的克隆、序列分析与时空表达

性信息素结合蛋白（pheromone binding proteins, PBPs）在昆虫雌雄间信息交流中起着重要作用。 本研究利用RT-PCR和RACE方法, 从烟夜蛾Helicoverpa assulta (Guenée)雄虫触角中克隆了性信息素结合蛋白2基因的开放阅读框及3′末端序列, 该基因被命名为HassPBP2(GenBank登录号为EU316186)。克隆和测序结果表明, HassPBP2开放阅读框全长450 bp, 编码149个氨基酸残基, 推测编码蛋白的分子量为16.9 kD, 等电点为5.56。HassPBP2基因结构分析表明, 该基因由3个外显子和2个内含子组成, 内含子的长度分别为90和261 bp。氨基酸序列联配分析表明, 此序列具有气味结合蛋白的典型特征, 与其他鳞翅目昆虫PBPs的一致性在34%~91%之间, 其中与棉铃虫Helicoverpa armigera PBP2和烟芽夜蛾Heliothis virescens PBP2的序列一致性高达91%。时间表达和组织表达分析显示, HassPBP2在卵期、幼虫期和蛹早期不表达, 在蛹中期开始表达, 并一直持续到成虫中期, 且只在雌、雄成虫触角中表达。     

草地贪夜蛾4个性信息素结合蛋白基因的克隆及表达模式分析

草地贪夜蛾是世界性的重大害虫,2019年1月入侵我国并迅速扩散到20多个省市。性诱剂是对草地贪夜蛾进行监测和诱杀的有效手段,但是其作用识别机制仍不清楚,限制了高效性诱剂的研发和应用。性信息素结合蛋白(PBPs)在鳞翅目昆虫包括草地贪夜蛾性信息素识别过程中起重要作用。本研究从草地贪夜蛾中克隆了4个编码PBPs的cDNA序列,命名为SfruPBP1-SfruPBP4。4个SfruPBPs均含有完整的开放阅读框,所编码的蛋白具有性信息素结合蛋白的典型特征:N-端有信号肽、具有保守的6个半胱氨酸残基。系统进化分析显示SfruPBPs与斜纹夜蛾和海灰翅夜蛾PBPs的亲缘关系最近,且4个SfruPBPs被聚在不同的进化分支。4个SfruPBPs基因均由3个外显子和2个内含子组成,内含子插入位点高度保守。此外,4个SfruPBPs在草地贪夜蛾基因组上呈串联排列。SfruPBP1、SfruPBP2和SfruPBP3在成虫触角中特异性表达,其中SfruPBP1和SfruPBP2在雄成虫触角中的表达水平显著高于雌虫,而SfruPBP3在雌雄触角中的表达水平无显著差异。SfruPBP4特异性表达于雄成虫腹部。本研究结果为揭示草地贪夜蛾性信息素识别机制奠定了基础。     

昆虫触角气味结合蛋白的研究进展

昆虫触角气味结合蛋白是一类亲水性的酸性蛋白,在触角感器淋巴液中浓度很高,主要分为4种,即性外激素结合蛋白、普通气味结合蛋白1、普通气味结合蛋白2和气味结合蛋白类似蛋白。由于它们在昆虫识别外界气味物质中起着重要的作用,近10年来,国外对其进行了广泛、深入的研究。该文从气味结合蛋白的研究方法、生化特性、分子结构和生理功能等方面进行综述。     

CRISPR/Cas9‐mediated PBP1 and PBP3 mutagenesis induced significant reduction in electrophysiological response to sex pheromones in male Chilo suppressalis

Pheromone-binding proteins (PBPs) are thought to bind and transport sex pheromones onto the olfactory receptors on the dendrite membrane of olfactory neurons, and thus play a vital role in sex pheromone perception. However, the function of PBPs has rarely been demonstrated in vivo.In this study, two PBPs (PBP1 and PBP3) of Chilo suppressalis, one of the most notorious pyralid pests, were in vivo functionally characterized using insects with the PBP gene knocked out by the CRISPR/Cas9 system. First, through direct injection of PBP-single guide RNA (sgRNA)/Cas9 messenger RNA into newly laid eggs, a high rate of target-gene editing (checked with polled eggs) was induced at 24 h after injection, 21.3% for PBPl-sgRNA injected eggs and 19.5% for PBP3-sgRNA injected eggs. Second, by an in-crossing strategy, insects with mutant PBP1 or PBP3 (both with a premature stop codon) were screened and homozygous mutants were obtained in the G3 generation. Third, the mutant insects were measured for electroantennogram (EAG) response to female sex pheromones. As a result, both PBP mutant males displayed significant reduction in EAG response, and this reduction in PBP1 mutants was higher than that in PBP3 mutants, indicating a more important role of PBP1. Finally, the relative importance of two PBPs and the possible off target effect induced by sgRNA-injection are discussed. Taken together, our study provides a deeper insight into the function of and interaction between different PBP genes in sex pheromone perception of C. suppressalis, as well as a valuable reference in methodology for gene functional study in other genes and other moth species.     

Volatile pheromone signalling in Drosophila

Once captured by the antenna, the male‐specific pheromone 11‐cis vaccenyl acetate (cVA) binds to an extracellular binding protein called LUSH that undergoes a conformational shift upon cVA binding. The stable LUSH–cVA complex is the activating ligand for pheromone receptors present on the dendrites of the aT1 neurones, comprising the only class of neurones that detect volatile cVA pheromone. This mechanism can explain the single molecule sensitivity of pheromone detection. The receptor that recognizes activated LUSH consists of a complex of several proteins, including Or67d, a member of the tuning odourant receptor family, Orco, a co‐receptor ion channel, and SNMP, a CD36 homologue that may be an inhibitory subunit. In addition, genetic screens and reconstitution experiments reveal additional factors that are important for pheromone detection. Identification and functional dissection of these factors in Drosophila melanogaster Meigen should permit the identification of homologous factors in pathogenic insects and agricultural pests, which, in turn, may be viable candidates for novel classes of compounds to control populations of target insect species without impacting beneficial species.     

Beyond chemoreception: diverse tasks of soluble olfactory proteins in insects

Odorant‐binding proteins (OBPs) and chemosensory proteins (CSPs) are regarded as carriers of pheromones and odorants in insect chemoreception. These proteins are typically located in antennae, mouth organs and other chemosensory structures; however, members of both classes of proteins have been detected recently in other parts of the body and various functions have been proposed. The best studied of these non‐sensory tasks is performed in pheromone glands, where OBPs and CSPs solubilise hydrophobic semiochemicals and assist their controlled release into the environment. In some cases the same proteins are expressed in antennae and pheromone glands, thus performing a dual role in receiving and broadcasting the same chemical message. Several reports have described OBPs and CSPs in reproductive organs. Some of these proteins are male specific and are transferred to females during mating. They likely carry semiochemicals with different proposed roles, from inhibiting other males from approaching mated females, to marking fertilized eggs, but further experimental evidence is still needed. Before being discovered in insects, the presence of binding proteins in pheromone glands and reproductive organs was widely reported in mammals, where vertebrate OBPs, structurally different from OBPs of insects and belonging to the lipocalin superfamily, are abundant in rodent urine, pig saliva and vaginal discharge of the hamster, as well as in the seminal fluid of rabbits. In at least four cases CSPs have been reported to promote development and regeneration: in embryo maturation in the honeybee, limb regeneration in the cockroach, ecdysis in larvae of fire ants and in promoting phase shift in locusts. Both OBPs and CSPs are also important in nutrition as solubilisers of lipids and other essential components of the diet. Particularly interesting is the affinity for carotenoids of CSPs abundantly secreted in the proboscis of moths and butterflies and the occurrence of the same (or very similar CSPs) in the eyes of the same insects. A role as a carrier of visual pigments for these proteins in insects parallels that of retinol‐binding protein in vertebrates, a lipocalin structurally related to OBPs of vertebrates. Other functions of OBPs and CSPs include anti‐inflammatory action in haematophagous insects, resistance to insecticides and eggshell formation. Such multiplicity of roles and the high success of both classes of proteins in being adapted to different situations is likely related to their stable scaffolding determining excellent stability to temperature, proteolysis and denaturing agents. The wide versatility of both OBPs and CSPs in nature has suggested several different uses for these proteins in biotechnological applications, from biosensors for odours to scavengers for pollutants and controlled releasers of chemicals in the environment.     

Molecular characterization,expression pattern and ligand‐binding properties of the pheromone‐binding protein gene from Cyrtotrachelus buqueti

Pheromone‐binding proteins (PBPs) play important roles in the information exchange between insect sexes, specifically in the process of transporting fat‐soluble odour molecules from the external environment to olfactory receptors through the olfactory sensillum lymph. The PBP functions in this process may explain the sex pheromone identification mechanism used by insects, laying a theoretical foundation for the prevention and control of pests by interfering with olfactory recognition. In the present study, a PBP gene of Cyrtotrachelus buqueti (GenBank accession number: KU845733) is cloned for prokaryotic expression. Using N‐phenyl‐1‐naphthylamine as the fluorescent probe in a competitive binding assay, the ability of CbuqPBP1 to bind 12 sex pheromone analogues and three volatiles of Neosinocalamus affinis shoots is examined. Of the 12 C. buqueti sex pheromone analogues, dibutyl phthalate gives the greatest displacement (inhibitory constant value of 11.1 μm ), whereas the other sex pheromone components show much smaller displacements. Consistent with other PBPs, the three plant volatiles (linalool, benzaldehyde and indole) show only a limited displacement of CbuqPBP1. However, the binding abilities of 1 : 1 ratios of each of the three plant volatiles with dibutyl phthalate show increases of 62.3%, 65.1% and 51.7% over the binding abilities of the three plant volatiles alone. CbuqPBP1 has dual roles in the processes of sensing sex pheromones and plant volatiles.     

中华蜜蜂信息素结合蛋白ASP1 cDNA的克隆及时空表达

信息素结合蛋白（pheromone binding proteins, PBPs）在昆虫信息素的识别、传递和处理过程中具有重要作用。本研究首次克隆了中华蜜蜂Apis cerana cerana的一个PBP基因Ac-ASP1（GenBank序列号为DQ449670）,其预测蛋白具有典型的气味结合蛋白（OBPs）标志（即成熟肽含有6个保守的半胱氨酸）。利用real-time PCR技术对Ac-ASP1在中蜂不同组织和发育历期的时空表达谱进行了鉴定。绝对定量结果显示Ac-ASP1高丰度地表达于工蜂触角（2.07×10⁶ 拷贝数/μg）,而在其他组织（如头、胸、腹、翅及足）中呈低丰度表达（10²拷贝数/μg）; 相对定量结果显示Ac-ASP1在各发育历期如幼虫、蛹以及成虫发育早期（1～6日龄）均有大量表达,而在21日龄前后具有另外一个高丰度表达时期。这些结果可为明确Ac-ASP1在中蜂蜂王信息素信号识别传递过程中的作用提供参考。     

A receptor and binding protein interplay in the detection of a distinct pheromone component in the silkmoth Antheraea polyphemus

Male moths respond to conspecific female-released pheromones with remarkable sensitivity and specificity, due to highly specialized chemosensory neurons in their antennae. In Antheraea silkmoths, three types of sensory neurons have been described, each responsive to one of three pheromone components. Since also three different pheromone binding proteins (PBPs) have been identified, the antenna of Antheraea seems to provide a unique model system for detailed analyzes of the interplay between the various elements underlying pheromone reception. Efforts to identify pheromone receptors of Antheraea polyphemus have led to the identification of a candidate pheromone receptor (ApolOR1). This receptor was found predominantly expressed in male antennae, specifically in neurons located beneath pheromone-sensitive sensilla trichodea. The ApolOR1-expressing cells were found to be surrounded by supporting cells co-expressing all three ApolPBPs. The response spectrum of ApolOR1 was assessed by means of calcium imaging using HEK293-cells stably expressing the receptor. It was found that at nanomolar concentrations ApolOR1-cells responded to all three pheromones when the compounds were solubilized by DMSO and also when DMSO was substituted by one of the three PBPs. However, at picomolar concentrations, cells responded only in the presence of the subtype ApolPBP2 and the pheromone (E,Z)-6,11-hexadecadienal. These results are indicative of a specific interplay of a distinct pheromone component with an appropriate binding protein and its related receptor subtype, which may be considered as basis for the remarkable sensitivity and specificity of the pheromone detection system.