PARTIAL CLONING AND CHARACTERIZATION OF THE cDNA OF GENERAL ODORANT BINDING PROTEIN 1 GENE IN THE ANTENNA OF HELICOVERPA ARMIGERA (HÜBNER)*

Abstract Using RT‐PCR and RACE techniques, part of the cDNA encoding the general odorant binding protein 1 gene (named as GOBP1‐Harra) from the antenna of Helwoverpa armigera (Hubner) has been cloned. The cDNA length of GOBP1‐Harm is 876 bp. The results of sequencing and structural analysis showed that the mature protein reading frame of GOBPl‐Harm is 435 base pairs in length and 145 amino acids encoded. The predicted MW and pl are 17.0 kD and 4.89, respectively. The deduced amino acid sequence showed a highly similarity to the sequence of GOBP1 from different moth species and shared several common structural features with odorant binding proteins from other insects.     

Cloning,expression and immunocytochemical localization of a general odorant-binding protein gene from Helicoverpa armigera (Hübner)

A cDNA clone coding for general odorant-binding protein2 was isolated from the antenna of Helicoverpa armigera by RT-PCR and (5'/3')-RACE technique. Results of sequencing and structural analyses showed that the full-length of GOBP2Harm was 636 bp, possessing 162 amino acid residues and a signal peptide of 21 amino acids. Its predicted molecular weight and isoelectric point were 18.2 kDa and 5.21, respectively. This deduced amino acid sequence shared some common structural features with odorant-binding proteins from several moth species, including the six conserved cysteine motif, typical of insect's OBPs. Northern blot showed that GOBP2Harm is specifically expressed in the antenna of Helicoverpa armigera at similar levels in both sexes. In order to obtain sufficient GOBP2 for further determining its biochemical and physiological properties, a bacterical expression vector of GOBP2 was constructed and successfully expressed. The protein was obtained mainly as insoluble inclusion bodies, that, however, could be solubilized and refolded. The rGOBP2 was purified by affinity chromatography and gel filtration. The rGOBP2 was shown to cross-react with an anti-GOBP antiserum from Antheraea polyphemus. Finally, polyclonal antibodies against GOBP2Harm were used to mark the distribution of the protein in olfactory sensilla and were tested by immuno-electron microscopy. In the male, GOBP2Harm is mainly expressed in sensilla basiconica, while in the female, it is equally expressed in sensilla basiconica and in sensilla trichodea.     

斜纹夜蛾普通气味结合蛋白GOBP1基因的表达定位分析

气味调控斜纹夜蛾Spodoptera litura（鳞翅目,夜蛾科）的交配和产卵行为,而嗅觉气味结合蛋白（OBP）是昆虫与外界环境进行化学信息交流中的一种重要蛋白。本研究基于已报道的斜纹夜蛾普通气味结合蛋白GOBP1基因cDNA序列（GenBank登录号为EF159978）设计引物,通过RT-PCR法分析了GOBP1 mRNA的组织特异性表达情况,并对GOBP1基因条带进行了克隆、测序和Blast比对。此外,再通过RNA原位杂交的方法进一步分析了GOBP1 mRNA在触角中的表达定位。结果表明:GOBP1只在斜纹夜蛾的触角组织中表达,而且GOBP1转录本较多分布于靠近嗅觉感受器即触角边缘部位。这些结果进一步说明GOBP1是斜纹夜蛾嗅觉过程中的重要蛋白,同时为深入研究GOBP1与其他蛋白的相互空间定位关系、GOBP1的功能等奠定了基础。     

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.     

cDNA cloning and recombinant expression of the general odorant binding protein II from Spodoptera litura

A cDNA encoding the general odorant binding protein II (GOBP II) was isolated from the antennae of Spodoptera litura (SlGOBP II, GenBank Accession No. EU086371) by homologous cloning and rapid amplification of cDNA ends (RACE). Sequencing and structural analyses revealed that the open reading frame (ORF) of SlGOBP II was 489 bp, encoding 162 amino acids with a predicted MW of 18.2 kD and pI of 5.72. SlGOPB II shared typical structural features of odorant binding proteins with other insects, including the six conservative cysteine residues. The deduced amino acid sequence of SlGOPB II shared significant identity with the GOBP II from S. frugiperda and S. exigua. RT-PCR and Northern blot analyses showed that SlGOBP II was specifically expressed in the antennae. cDNA encoding SlGOBP II was constructed into the pET-32a vector and the recombinant protein was highly expressed in Es-cherichia coli BL21 (DE3) after induction with IPTG. SDS electrophoresis and Western blot analysis confirmed the molecular weight of the recombinant SIGOBPII i.e, 32 kD, which has a 6×His tag at the N-terminus. The recombinant SlGOBP II was purified by single-step Ni-NTA affinity chromatography and used to raise antiserum in rabbits. ELISA showed that the titer of antiserum was 1︰12800, while Western blot analysis showed that the recombinant SlGOBP II was recognized as anti-SlGOBP II an-tiserum.     

烟实夜蛾触角普通气味结合蛋白Ⅱ cDNA的克隆、序列分析及在大肠杆菌中的表达

利用RT PCR技术扩增了编码烟实夜蛾Helicoverpa assulta雌、雄虫触角普通气味 结合蛋白Ⅱ的Cdna片段,将其克隆至Pgem-T Easy载体,获得了普通气味结合蛋白Ⅱ基因成熟蛋白阅读框序列。将该基因重组到表达型质粒Pet-30a(+)中,并转化入原核细胞中表达。序列 测定结果表明,烟实夜蛾触角普通气味结合蛋白基因的成熟蛋白阅读框全长489 bp,编码162个 氨基酸残基,预测分子量和等电点分别为18.2 kD和5.35。推导的氨基酸序列与已报道的10种昆虫普通气味结合蛋白Ⅱ高度同源（73%～98%）,并具有气味结合蛋白的典型特征。SDS-PAGE和Western印迹分析表明,经IPTG诱导,普通气味结合蛋白Ⅱ基因能在大肠杆菌BL21(DE3)中表达,电泳检测到一条约23 kD大小的外源蛋白,与预测的融合蛋白分子量大小相应。     

Odorant-binding-protein subfamilies associate with distinct classes of olfactory receptor neurons in insects

The olfactory receptors of terrestrial animals exist in an aqueous environment, yet detect odorants that are primarily hydrophobic. The aqueous solubility of hydrophobic odorants is thought to be greatly enhanced via odorant binding proteins (OBP) which exist in the extracellular fluid surrounding the odorant receptors. We have isolated and partially sequenced 14 candidate OBPs from six insect (moth) species. All 14 represent a single homologous family based on conserved sequence domains. The 14 proteins can be divided into three subfamilies based on differences in tissue specific expression and similarities in amino acid sequences. All 14 proteins are specifically expressed in antennal olfactory tissue. Subfamily I represents previously described pheromone binding proteins (PBP), which are male-specific, associate with pheromone-sensitive neurons, and are highly variable in their sequences when compared among species. Subfamilies II and III are expressed in both male and female antennae, appear to associate with general-odorant-sensitive neurons, and are highly conserved when compared among species. The properties of the subfamily II and III proteins suggest these are general-odorant binding proteins (GOBP). The properties of the respective insect OBP subfamilies suggest that they have different odorant binding specificities. The association of different insect OBP subfamilies with distinct classes of olfactory neurons having different odorant specificities suggests that OBPs can act as selective signal filters, peripheral to the actual receptor proteins.     

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。     

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.     

Construction and analysis of cDNA libraries from the antennae of male and female cotton bollworms Helicoverpa armigera (Hübner) and expression analysis of putative odorant-binding protein genes

Two high-quality cDNA libraries were constructed from female and male antennae of the cotton bollworm Helicoverpa armigera (Hübner). The titers were approximately 2.0 × 10⁶ pfu/ml for females and 2.3 × 10⁶ pfu/ml for males, and this complies with the test requirement. From the libraries, 1750 male ESTs and 1640 female ESTs were sequenced and further analyzed. We identified 15 olfactory genes (12 are new), and 14 of them have the characteristic six conserved cysteine residues. With the exception of OBP9, all the genes were classified as classical OBP genes. By alignment and cluster analysis, the 14 classical OBPs were divided into pheromone binding protein (PBP) genes, odorant binding protein (OBP) genes, general odorant binding protein 1 (GOBP1) genes, general odorant binding protein 2 (GOBP2) genes and antennae binding protein (ABP) genes. Among these genes, we obtained three PBP genes (PBP1–PBP3) including two new PBP genes, one new ABP gene, nine new OBP genes (OBP1–OBP9), one known GOBP1 gene and one known GOBP2 gene. Furthermore, the expression patterns of these 14 classical OBP genes were investigated in various tissues by real-time quantitative polymerase chain reaction (qPCR). The results indicated that some OBP genes are expressed differently in different sexes and tissues, but most of them are highly expressed in antennae.     

Construction and analysis of cDNA libraries from the antennae of male and female cotton bollworms Helicoverpa armigera (Hübner) and expression analysis of putative odorant-binding protein genes

Two high-quality cDNA libraries were constructed from female and male antennae of the cotton bollworm Helicoverpa armigera (Hübner). The titers were approximately 2.0 × 10? pfu/ml for females and 2.3 × 10? pfu/ml for males, and this complies with the test requirement. From the libraries, 1750 male ESTs and 1640 female ESTs were sequenced and further analyzed. We identified 15 olfactory genes (12 are new), and 14 of them have the characteristic six conserved cysteine residues. With the exception of OBP9, all the genes were classified as classical OBP genes. By alignment and cluster analysis, the 14 classical OBPs were divided into pheromone binding protein (PBP) genes, odorant binding protein (OBP) genes, general odorant binding protein 1 (GOBP1) genes, general odorant binding protein 2 (GOBP2) genes and antennae binding protein (ABP) genes. Among these genes, we obtained three PBP genes (PBP1-PBP3) including two new PBP genes, one new ABP gene, nine new OBP genes (OBP1-OBP9), one known GOBP1 gene and one known GOBP2 gene. Furthermore, the expression patterns of these 14 classical OBP genes were investigated in various tissues by real-time quantitative polymerase chain reaction (qPCR). The results indicated that some OBP genes are expressed differently in different sexes and tissues, but most of them are highly expressed in antennae.     

Pheromone-binding protein and general odorant-binding protein of Sesamia nonagrioides: sex- and diel-dependent expression

The expression of a pheromone‐binding protein (PBP) and a general odorant‐binding protein (GOBP) in Sesamia nonagrioides (Lef.) (Lepidoptera: Noctuidae) was studied. Lymantria dispar's PBP1 antibody yielded an immunoreactive band with an apparent MW of approximately 14.8 kDa, present specifically in the antennae of both sexes, with males having approximately three‐fold the quantity found in females. Furthermore, Manduca sexta's odorant‐binding protein‐2 (GOBP2) antibody recognized a band at approximately 14.5 kDa in the antennae of both sexes. Levels of both proteins were compared between scotophase and photophase periods in insects that were raised under L16:D8 or under constant light. The level of GOPB2 was significantly lower in both sexes during photophase and continuous light; whereas the level of the PBP was significantly lower in females’ antennae, in males’ antennae it remained at the same level as that found during the scotophase.