绿豆象气味结合蛋白基因的克隆及表达分析

[目的]对绿豆象Callosobruchus chinensis气味结合蛋白(odorant binding proteins,OBPs)基因进行克隆、鉴定和组织表达分析,为研究OBPs在绿豆象嗅觉感受过程中的功能奠定基础.[方法]基于绿豆象触角转录组数据,通过RT-PCR克隆绿豆象6个OBP基因并进行生物信息学分析;...     

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.     

Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus (GOEZE)

In the insect phylum, the relationships between individuals and their environment are often modulated by chemical communication. Odorant binding proteins (OBPs) are widely and robustly expressed in insect olfactory organs and play a key role in chemosensing and transporting hydrophobic odorants across the sensillum lymph to the olfactory receptor neuron. In this study, a novel OBP gene (AlinOBP1) in the lucerne plant bug, Adelphocoris lineolatus was identified, cloned and expressed. Real-time PCR results indicated that the expression level of AlinOBP1 gene differed in each developmental stage (from first instar to adult) and was predominantly expressed in the antennae of adults. The expression level of AlinOBP1 was 1.91 times higher in male antennae than in female antennae. The binding properties of AlinOBP1 with 114 odorants were measured using a fluorescence probe, N-phenyl-1-naphthylamine (1-NPN), with fluorescence competitive binding. The results revealed that AlinOBP1 exhibits high binding abilities with two major putative pheromone components, ethyl butyrate and trans-2-hexenyl butyrate. In addition, it was observed that six volatiles released from cotton, octanal, nonanal, decanal, 2-ethyl-1-hexanol, β-caryophyllene and β-ionone also bind to AlinOBP1. Immunocytochemistry analysis showed that AlinOBP1 was expressed in the sensillum lymph of sensilla trichodica and sensilla basiconca. Our results demonstrate that AlinOBP1 may function as a carrier in the chemoperception of the lucerne plant bug.     

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.     

Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

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。     

Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A.arabiensis

~~Identification and expression profiling of putative odorant-binding proteins in the malaria mosquitoes, Anopheles gambiae and A. arabiensis1. Curtis, C. F., Introduction 1: An overview of mosquito biology, behaviour and importance, in Olfaction in Mosquito-Host Interactions (eds. Bock, G. R.. Cardew, G.), New York: Wiley, 1996, 3-7. 2. Nighom, A., Hildebrand. J. G.. Dissecting the molecular mechanisms of olfaction in a malaria-vector mosquito, PNAS, 2002, 99(3): 1113-…