二化螟Minus-C气味结合蛋白的分子克隆及功能鉴定

气味结合蛋白（odorant binding proteins, OBPs）在昆虫对寄主气味的感受中起重要作用, 但有关Minus-C OBP及其功能的报道很少。本研究通过基因组数据分析并利用RACE技术, 克隆和鉴定了二化螟Chilo suppressalis (Walker)的一个Minus-C OBP基因, 命名为CsupOBP1（GenBank登录号： KC492498）。CsupOBP1基因的开放阅读框长423 bp, 编码141个氨基酸, 其中N端的18个氨基酸为预测的信号肽序列, 成熟蛋白序列中具有4个保守的半胱氨酸位点。实时定量PCR分析显示, 该基因在幼虫头部及成虫雌雄足、 翅和雄性触角等化感组织中高表达, 其中在雄虫触角内的表达量显著高于雌虫触角。利用荧光竞争结合实验对CsupOBP1重组蛋白与38种化合物的结合特性的测定表明, 重组CsupOBP1与β 紫罗兰酮的结合能力最强（Ki=9.53 μmol/L）。触角电位测定表明, 二化螟成虫可对β-紫罗兰酮产生显著的触角电生理反应, 但雄虫反应明显强于雌虫, 与结合试验及雄虫触角中CsupOBP1的表达量显著高于雌虫触角的测定结果相一致。鉴于β-紫罗兰酮是水稻等植物中普遍存在的一种芳香气味组分, 推测CsupOBP1可能通过对该气味的结合和运输, 从而在二化螟对寄主植物的嗅觉定向中起作用。     

Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.     

花椒窄吉丁触角转录组及嗅觉相关基因分析

【目的】建立花椒窄吉丁Agrilus zanthoxylumi成虫触角转录组数据库,挖掘嗅觉相关基因,为今后研究其触角的化学感受机制及生物防控提供理论支撑。【方法】采用高通量测序平台IlluminaNovaSeq 6000对花椒窄吉丁雌雄成虫触角进行转录组测序,用Trinity软件对获得的高质量reads进行序列拼接与组装;使用BLAST软件将触角转录组数据比对NR, NT, Swiss-Prot, GO, KEGG, BLASTX,eggNOG, Pfam, TmHMM, SignalP, KO, Map, BLASTP和RNAMMER公共数据库;基于初步筛选到的花椒窄吉丁候选气味结合蛋白(odorant binding protein, OBP)和化学感受蛋白(chemosensory protein, CSP)以及其他鞘翅目昆虫的同源蛋白的核苷酸序列,利用MEGA软件进行系统进化分析。运用RPKM (reads perkilobase per million mapped reads)值对嗅觉相关基因表达量进行分析。【结果】花椒窄吉丁雌雄成虫触角转录组测序共获得36 209条基因和90 982条转录本,其N₅₀分别为2 103和2 523 bp,组装完整性较高。注释到NR数据库的基因最多(41.62%),其中与赤拟谷盗Tribolium castaneum相似基因所占比例最高(19%)。在GO数据库中比对到11 614个基因,按功能分为细胞组分、分子功能与生物学进程三大类57个分支,其中分子功能大类中的结合(70.57%)与催化活性(45.51%)相关基因占比最多;KEGG代谢途径分析表明,7 427条基因参与了5类代谢通路,其中涉及信号转导的基因最多,为815条;筛选到7个候选OBP基因和5个具有全长开放阅读框的CSP基因,其编码蛋白均具有化学感受蛋白家族的典型特征。系统进化分析表明,花椒窄吉丁OBPs和CSPs分别与苹果小吉丁A. mali的OBPs和CSPs氨基酸序列一致性最高。RPKM值表明,嗅觉相关基因AzanOBP1和AzanOBP2在雌成虫触角中不表达,在雄成虫触角中微量表达;AzanOBP3在雄成虫触角中高丰度表达。【结论】首次获得了花椒窄吉丁成虫触角转录组数据,筛选到了花椒窄吉丁OBP, CSP, Or, IR和SNMP等的嗅觉相关基因。推测触角中高丰度表达的OBPs对雄成虫识别同类异性释放的信息素或寄主植物释放的挥发物起关键作用。研究结果可为花椒窄吉丁化学感受基因功能分析及嗅觉感受机制研究奠定分子基础。     

Comparative genomics and transcriptomics in ants provide new insights into the evolution and function of odorant binding and chemosensory proteins

Background

The complex societies of ants and other social insects rely on sophisticated chemical communication. Two families of small soluble proteins, the odorant binding and chemosensory proteins (OBPs and CSPs), are believed to be important in insect chemosensation. To better understand the role of these proteins in ant olfaction, we examined their evolution and expression across the ants using phylogenetics and sex- and tissue-specific RNA-seq.

Results

We find that subsets of both OBPs and CSPs are expressed in the antennae, contradicting the previous hypothesis that CSPs have replaced OBPs in ant olfaction. Both protein families have several highly conserved clades with a single ortholog in all eusocial hymenopterans, as well as clades with more dynamic evolution and many taxon-specific radiations. The dynamically evolving OBPs and CSPs have been hypothesized to function in chemical communication. Intriguingly, we find that seven members of the conserved clades are expressed specifically in the antennae of the clonal raider ant Cerapachys biroi, whereas only one dynamically evolving CSP is antenna specific. The orthologs of the conserved, antenna-specific C. biroi genes are also expressed in antennae of the ants Camponotus floridanus and Harpegnathos saltator, indicating that antenna-specific expression of these OBPs and CSPs is conserved across ants. Most members of the dynamically evolving clades in both protein families are expressed primarily in non-chemosensory tissues and thus likely do not fulfill chemosensory functions.

Conclusions

Our results identify candidate OBPs and CSPs that are likely involved in conserved aspects of ant olfaction, and suggest that OBPs and CSPs may not rapidly evolve to recognize species-specific signals.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-718) contains supplementary material, which is available to authorized users.     

Analysis of odorant-binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, which produces lepidopteran Type II sex pheromone components

Information on the olfactory system in antennae of Geometridae moths is very limited, and odorant-binding proteins (OBPs) working as transporters of lipophilic odors have not been identified. In the first investigation on this family of insects, we examined antennal OBPs of the Japanese giant looper, Ascotis selenaria cretacea. RT-PCR experiments using several pairs of degenerate primers designed from known cDNA sequences encoding lepidopteran OBPs successfully amplified partial sequences of two pheromone-binding proteins (PBPs), named AscrPBP1 and AscrPBP2 in reference to their corresponding nucleotide sequence homologies with other PBPs. Using 5′- and 3′-rapid amplification of cDNA end strategies, a cDNA clone for AscrPBP1 encoding a protein of 141 amino acids was isolated. Western blotting with the antiserum against recombinant AscrPBP1 overexpressed in Escherichia coli showed that the AscrPBP1 gene was more strongly expressed in male antennae than in female antennae. Furthermore, natural AscrPBP1was isolated by immunoprecipitation with the antiserum, and its binding ability was evaluated by using synthetic sex pheromonal compounds with a C₁₉ chain. The result indicated that AscrPBP1 bound not only the pheromone components, 3,6,9-nonadecatriene and its 3,4-epoxy derivative, but also unnatural 6,7- and 9,10-epoxy derivatives. While no general odorant-binding proteins (GOBPs) were amplified in the RT-PCR experiments, two antisera prepared from GOBP1 and GOBP2 of Bombyx mori suggested the occurrence of at least two GOBPs in the A. s. cretacea antennae.     

Expression of odorant-binding proteins and chemosensory proteins in some Hymenoptera

The expression of chemosensory proteins (CSPs) and odorant-binding proteins (OBPs) in individuals of different castes and ages have been monitored in three species of social hymenopterans, Polistes dominulus (Hymenoptera, Vespidae), Vespa crabro (Hymenoptera, Vespidae) and Apis mellifera (Hymenoptera, Apidae), using PCR with specific primers and polyclonal antibodies. In the paper wasp P. dominulus, OBP is equally expressed in antennae, wings and legs of all castes and ages, while CSP is often specifically present in antennae and in some cases also in legs. In the vespine species V. crabro CSP is antennal specific, while OBP is also expressed in legs and wings. The three CSPs and the five OBPs of A. mellifera show a complex pattern of expression, where both classes of proteins include members specifically expressed in antennae and others present in other parts of the body. These data indicate that at least in some hymenopteran species CSPs are specifically expressed in antennae and could perform roles in chemosensory perception so far assigned only to OBPs.     

Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori

Insect chemosensory proteins (CSPs) as well as odorant-binding proteins (OBPs) have been supposed to transport hydrophobic chemicals to receptors on sensory neurons. Compared with OBPs, CSPs are expressed more broadly in various insect tissues. We performed a genome-wide analysis of the candidate CSP gene family in the silkworm. A total of 20 candidate CSPs, including 3 gene fragments and 2 pseudogenes, were characterized based on their conserved cysteine residues and their similarity to CSPs in other insects. Some of these genes were clustered in the silkworm genome. The gene expression pattern of these candidates was investigated using RT-PCR and microarray, and the results showed that these genes were expressed primarily in mature larvae and the adult moth, suggesting silkworm CSPs may be involved in development. The majority of silkworm CSP genes are expressed broadly in tissues including the antennae, head, thorax, legs, wings, epithelium, testes, ovaries, pheromone glands, wing disks, and compound eyes.     

Ultrastructural characterization of olfactory sensilla and immunolocalization of odorant binding and chemosensory proteins from an ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae)

The three-dimensional structures of two odorant binding proteins (OBPs) and one chemosensory protein (CSP) from a polyphagous ectoparasitoid Scleroderma guani (Hymenoptera: Bethylidae) were resolved bioinformatically. The results show that both SguaOBP1 and OBP2 are classic OBPs, whereas SguaCSP1 belongs to non-classic CSPs which are considered as the "Plus-C" CSP in this report. The structural differences between the two OBPs and between OBP and CSP are thoroughly described, and the structural and functional significance of the divergent C-terminal regions (e.g., the prolonged C-terminal region in SguaOBP2 and the additional pair of cysteines in SguaCSP1) are discussed. The immunoblot analyses with antisera raised against recombinant SguaOBP1, OBP2, and CSP1, respectively, indicate that two SguaOBPs are specific to antennae, whereas SguaCSP1, which are more abundant than OBPs and detected in both male and female wasps, expresses ubiquitously across different tissues.We also describe the ultrastructure of the antennal sensilla types in S. guani and compare them to 19 species of parasitic Hymenoptera. There are 11 types of sensilla in the flagellum and pedicel segments of antennae in both male and female wasps. Seven of them, including sensilla placodea (SP), long sensilla basiconica (LSB), sensilla coeloconica (SC), two types of double-walled wall pore sensilla (DWPS-I and DWPS-II), and two types of sensilla trichodea (ST-I and ST-II), are multiporous chemosensilla. The ultralsturctures of these sensilla are morphologically characterized. In comparison to monophagous specialists, the highly polyphagous generalist ectoparasitoids such as S. guani possess more diverse sensilla types which are likely related to their broad host ranges and complex life styles. Our immunocytochemistry study demonstrated that each of the seven sensilla immunoreacts with at least one antiserum against SguaOBP1, OBP2, and CSP1, respectively. Anti-OBP2 is specifically labeled in DWPS-II, whereas the anti-OBP1 shows a broad spectrum of immunoactivity toward four different sensilla (LSB, SP, ST-I and ST-II). On the other hand, anti-CSP1 is immunoactive toward SP, DWPS-I and SC. Interestingly, a cross co-localization pattern between SguaOBP1 and CSP1 is documented for the first time. Given that the numbers of OBPs and CSPs in many insect species greatly outnumber their antennal sensilla types, it is germane to suggest such phenomenon could be the rule rather than the exception.