西花蓟马化学感受蛋白的cDNA克隆、时空表达分析及组织定位

【目的】研究西花蓟马Frankliniella occidentalis化学感受蛋白(chemosensory proteins,CSPs)在其嗅觉及化学感受系统中的作用。【方法】利用RT-PCR和RACE技术克隆西花蓟马化学感受蛋白基因,用DNAMAN软件进行序列分析,使用BLAST进行同源性比较,采用MEGA6的Neighbor-joining法构建了进化树。通过实时定量PCR(real-time quantitative PCR,qRT-PCR)检测了西花蓟马不同发育期以及成虫不同组织(触角、头、足、胸、腹)中该基因的表达谱。免疫新西兰大白兔制备了Focc CSP1蛋白抗体,与样品切片中Focc CSP1蛋白以及10 nm胶体金颗粒偶联的羊抗兔二抗反应,经透射电镜观察,对该蛋白在西花蓟马成虫组织中进行免疫定位。【结果】克隆并鉴定了一个西花蓟马化学感受蛋白基因,命名为Focc CSP1(Gen Bank登录号:KM527949)。该基因c DNA序列全长597 bp,完整开放阅读框(ORF)288 bp,编码95个氨基酸,成熟蛋白分子量11.377 k D,等电点4.72,具有化学感受蛋白典型的4个保守半胱氨酸位点特征。Focc CSP1与东亚飞蝗Locusta migratoria Lmig CSP(Gen Bank登录号:CAJ01476.1)的氨基酸序列一致性最高,进化关系最近。Focc CSP1在西花蓟马不同发育阶段和成虫不同组织中均有表达,在羽化1 d的雌虫中相对表达量最高,其次是2龄若虫,蛹和成虫后期表达量最低;在触角和足中相对表达量较高。成功构建了重组表达质粒p ET-30a/Focc CSP1,并诱导表达,经Ni柱纯化,获得目的蛋白;免疫定位表明,该蛋白在西花蓟马触角、足、头等部位血淋巴中均大量存在。【结论】明确了西花蓟马化学感受蛋白基因Focc CSP1的核苷酸、氨基酸序列特征。Focc CSP1广泛分布在西花蓟马多个组织及各个发育期,据此推测该基因可能在西花蓟马嗅觉识别、感受机械刺激以及调节生长发育等方面扮演重要角色。     

Molecular analysis of NlugCSP3 with behaviorally active ligands of the brown planthopper,Nilaparvata lugens (Hemiptera: Delphacidae)

The insect olfactory system is sensitive to the complicated chemical environment. Insect’s chemosensory proteins (CSPs) supposedly act as transport of plant volatiles across the sensillar lymph and competitive fluorescent binding assay was commonly used to test the binding affinities with various plant volatiles. However, extensive research to determine the physiological role of CSPs is necessary and helpful through binding interaction of a protein with the plant volatiles. In this comparative study, we employed phylogenetic analysis, fluorescence spectra, quenching mode, and thermodynamic force to characterize Nilaparvata lugens CSP3 (NlugCSP3). The phylogenetic tree revealed that amino acid sequence of NlugCSP3 showed extremely close similarities with Laodelphax striatella (LstrCSP10 & LstrCSP11) and Sogatella furcifera (SurCSP3). The Stern-Volmer (SV) curve of NlugCSP3 fluorescence quenching indicated that nonadecane and 2-tridecanone clearly quenched NlugCSP3 fluorescence as a stable static quenching mode. Meanwhile, α-terpinene and farnesene collided with NlugCSP3, instead of forming stable complexes. The thermodynamic analysis of NlugCSP3 revealed that spontaneous binding interaction occurred in nonadecane and 2-tridecanone and is driven primarily by hydrophobic interactions. This study not only provides the information about the binding interaction of protein with the volatiles, but also improves the efficient recognition of behaviorally elicited volatiles that could be used in the management of brown planthopper.     

Characterization of two genes expressed in the salivary glands of the Hessian fly, Mayetiola destructor (Say)

Two genes, SSGP-11A1 and SSGP-12A1, have been isolated that encodes proteins with a secretion signal peptide at theN-terminals from the Hessian fly (Mayetiola destructor (Say)). The SSGP-11A1 gene contains one small intron (89 bp) and encodes a putative protein with 79 amino acids. The first 18 amino acids constitute a putative secretion signal peptide. The SSGP-12A1 gene contains three small introns and encodes a putative protein with 234 amino acids. The first 19 amino acids constitute a putative secretion signal peptide. Northern blot analysis revealed that both of the genes are primarily expressed in the salivary glands of Hessian fly larvae, the feeding stage of the insect. These observations are consistent with the possibility that the proteins encoded by them are secreted into host plants during feeding. Even though both genes are exclusively expressed in Hessian fly larvae, the expression profiles between them were quite different in insects at different instars. The SSGP-11A1 gene was expressed in all instars of larvae while the SSGP-12A1 gene was almost exclusively expressed in the first instar larvae. The differential expression suggests that the proteins encoded by these two genes may perform different functions. In situ hybridization revealed that the SSGP-11A1 gene is located on the short arm of chromosome A1 while SSGP-12A1 gene is on the long arm of chromosome A2.     

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.     

扶桑绵粉蚧组织蛋白酶B基因的克隆、原核表达和不同发育阶段表达分析

昆虫组织蛋白酶B在昆虫代谢过程中发挥重要作用。本研究利用RACE技术克隆了扶桑绵粉蚧Phenacoccus solenopsis Tinsley组织蛋白酶B基因的开放阅读框（ORF）序列, 命名为PsCb （GenBank登录号: JQ727999）。生物信息学分析表明, 该基因的开放阅读框包含927 bp的片段, 编码308个氨基酸。多序列比对表明, 该基因编码的蛋白在N端变异较大, 在C端保守性高。组织蛋白酶B基因的系统进化树结果表明扶桑绵粉蚧组织蛋白酶独自成为一支。原核表达电泳检测到一条大约35 kDa的目的条带, 与预测的蛋白分子量相符。组织蛋白酶B基因在扶桑绵粉蚧各个虫态均有表达, 卵期表达量相对较低, 2龄若虫期达到最高峰, 然后下降。本研究为进一步研究该基因的功能并开发出组织蛋白酶抑制剂, 从而研制出扶桑绵粉蚧杀卵剂和胚胎发育抑制剂等提供理论依据。     

小菜蛾化学感受蛋白基因PxylCSP1的克隆和表达

利用RT-PCR和RACE技术克隆到小菜蛾Plutella xylostella 化学感受蛋白（CSP）基因PxylCSP1（GenBank登录号: FJ361903）,其核苷酸序列全长405 bp,编码134个氨基酸残基,预测N-末端包含19个氨基酸组成的信号肽序列,估测其成熟蛋白分子量为13.56 kD,等电点为6.12。该基因编码氨基酸序列和其他鳞翅目昆虫CSP的氨基酸序列比对同源性较高（70%~80%）。RT-PCR结果表明PxylCSP1不仅存在于小菜蛾的触角中,还存在于头、足、腹和翅中。Real-time PCR结果表明PxylCSP1的表达水平因被测小菜蛾的性别、日龄、组织不同和交配与否而异。     

Solution structure of a chemosensory protein from the desert locust Schistocerca gregaria

Chemical stimuli, generally constituted by small volatile organic molecules, are extremely important for the survival of different insect species. In the course of evolution, insects have developed very sophisticated biochemical systems for the binding and the delivery of specific semiochemicals to their cognate membrane-bound receptors. Chemosensory proteins (CSPs) are a class of small soluble proteins present at high concentration in insect chemosensory organs; they are supposed to be involved in carrying the chemical messages from the environment to the chemosensory receptors. In this paper, we report on the solution structure of CSPsg4, a chemosensory protein from the desert locust Schistocerca gregaria, which is expressed in the antennae and other chemosensory organs. The 3D NMR structure revealed an overall fold consisting of six alpha-helices, spanning residues 13-18, 20-31, 40-54, 62-78, 80-90, and 97-103, connected by loops which in some cases show dihedral angles typical of beta-turns. As in the only other chemosensory protein whose structure has been solved so far, namely, CSP from the moth Mamestra brassicae, four helices are arranged to form a V-shaped motif; another helix runs across the two V's, and the last one is packed against the external face. Analysis of the tertiary structure evidenced multiple hydrophobic cavities which could be involved in ligand binding. In fact, incubation of the protein with a natural ligand, namely, oleamide, produced substantial changes to the NMR spectra, suggesting extensive conformational transitions upon ligand binding.     

Identification and genomic structure of chemosensory proteins (CSP) and odorant binding proteins (OBP) genes expressed in foreleg tarsi of the swallowtail butterfly Papilio xuthus

Chemoreception is a key feature for selection of host plants by phytophagous insects. Female swallowtail butterflies recognize their host plants using chemosensilla present on foreleg tarsi. We constructed a cDNA library of female tarsi and a genome library of Papilio xuthus. We identified 11 chemosensory protein (CSP) genes and three odorant binding proteins (OBP) genes from the cDNA library and eight additional CSP genes from the genome library using the ESTs as probes. A sequence similarity tree of insect CSPs showed that lepidopteran CSPs constructed big branches of the order. Small numbers of CSPs have been identified from the whole genomes of several insect orders which belong to branches separated from those of Lepidoptera. The CSP gene family of Lepidoptera may have diverged in at least two steps, the first on a small scale and the second on a large scale before and after the diversification of insect orders, respectively. Seventeen of 19 CSP genes of P. xuthus clustered in a specific region of the genome, suggesting that they were diversified by gene duplication from a common ancestral gene.     

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.     

Chemosensory proteins in the honey bee: Insights from the annotated genome, comparative analyses and expressional profiling

Small chemosensory proteins (CSPs) belong to a conserved, but poorly understood protein family that has been implicated in transporting chemical stimuli within insect sensilla. However, their expression patterns suggest that these molecules are also critical for other functions including early development. Here we used both bioinformatics and experimental approaches to characterize the CSP gene family in a social insect, the Western honey bee Apis mellifera, and then compared its members to CSPs in other arthropods. The number of CSPs in the honey bee genome (six) is similar to that found in the sequenced dipteran species (four-seven), but is much lower than the number of CSPs in the moth or in the beetle (around 20 each). These differences seem to be the result of lineage specific expansions. Our analysis of CSPs in a number of arthropods reveals a conserved gene family found in both Mandibulates and Chelicerates. Expressional profiling in diverse tissues and throughout development reveals broader than expected patterns of expression with none of the CSPs restricted to the antennae and one found only in the queen ovaries and in embryos. We conclude that CSPs are multifunctional context-dependent proteins involved in diverse cellular processes ranging from embryonic development to chemosensory signal transduction. Some CSPs may function in cuticle synthesis, consistent with their evolutionary origins in the arthropods.