Expressed Sequence Tag Clones Research Articles

Allergen Homologues, Pathogenesis-Related 1, Polygalacturonase, and Pectin Methyl Esterase from a Japanese Hop.

Japanese hop is an important cause of weed pollinosis in East Asia. Its pollen is abundant in autumn. This pollen is known to be the cause of many allergic diseases. However, molecular characteristics of its allergens have not been elucidated. In this study, we produced recombinant proteins of allergen homologues from Japanese hop by the analysis of expressed sequence tags (EST), and evaluated its allergenicity. cDNA library was constructed using as little as 50 ng of total RNA from Japanese hop pollen. Allergen homologues were identified by the initial screening of 963 EST clones. Recombinant proteins were overexpressed in the E. coli expression system and purified using Ni-nitrilotriacetic acid-agarose. Purified proteins were analyzed by ELISA. Japanese hop pathogenesis-related 1 protein (PR-1) shares 37.0 to 44.4% of amino acid sequence identity with Art v 2, Cuc m 3, and Cyn d 24. Pectin methyl esterase (PME) shows 23.2 to 50.2% of identities to Act d 7, Ole e 11, and Sal k 1. Polygalacturonase (PGs) shows 16.7 to 19.3% of identities to Phl p 13, Cry j 2, Cha o 2, Jun a 2, Pla a 2, and Pla or 2. IgE antibodies from Japanese hop allergy patients' sera recognized PR-1 (3.4%), PME (13.8%), PGs (3.7%), and profilin (13.8%), respectively. Novel allergenic components were identified, even though low IgE reactivity was displayed reflecting the low degree of cross-reactivity with other pollen allergens. We believe that these molecules have worth further studies.

Overexpression of ginseng patatin-related phospholipase pPLAIIIβ alters the polarity of cell growth and decreases lignin content in Arabidopsis

BackgroundThe patatin-related phospholipase AIII family (pPLAIIIs) genes alter cell elongation and cell wall composition in Arabidopsis and rice plant, suggesting diverse commercial purposes of the economically important medicinal ginseng plant. Herein, we show the functional characterization of a ginseng pPLAIII gene for the first time and discuss its potential applications. MethodspPLAIIIs were identified from ginseng expressed sequence tag clones and further confirmed by search against ginseng database and polymerase chain reaction. A clone showing the highest homology with pPLAIIIβ was shown to be overexpressed in Arabidopsis using Agrobacterium. Quantitative polymerase chain reaction was performed to analyze ginseng pPLAIIIβ expression. Phenotypes were observed using a low-vacuum scanning electron microscope. Lignin was stained using phloroglucinol and quantified using acetyl bromide. ResultsThe PgpPLAIIIβ transcripts were observed in all organs of 2-year-old ginseng. Overexpression of ginseng pPLAIIIβ (PgpPLAIIIβ-OE) in Arabidopsis resulted in small and stunted plants. It shortened the trichomes and decreased trichome number, indicating defects in cell polarity. Furthermore, OE lines exhibited enlarged seeds with less number per silique. The YUCCA9 gene was downregulated in the OE lines, which is reported to be associated with lignification. Accordingly, lignin was stained less in the OE lines, and the expression of two transcription factors related to lignin biosynthesis was also decreased significantly. ConclusionOverexpression of pPLAIIIβ retarded cell elongation in all the tested organs except seeds, which were longer and thicker than those of the controls. Shorter root length is related to auxin-responsive genes, and its stunted phenotype showed decreased lignin content.

Characterization of Der f 22 - a paralogue of the major allergen Der f 2

We previously identified an expressed sequence tag clone, Der f 22, showing 41% amino acid identity to published Der f 2, and show that both genes are possible paralogues. The objective of this study was to characterize the genomic, proteomic and immunological functions Der f 22 and Der f 2. The full-length sequence of Der f 2 and Der f 22 coded for mature proteins of 129 and 135 amino acids respectively, both containing 6 cysteine residues. Phylogenetic analysis of known group 2 allergens and their homologues from our expressed sequence tag library showed that Der f 22 is a paralogue of Der f 2. Both Der f 2 and Der f 22 were single gene products with one intron. Both allergens showed specific IgE-binding to over 40% of the atopic patients, with limited of cross-reactivity. Both allergens were detected at the gut region of D. farinae by immunostaining. Der f 22 is an important allergen with significant IgE reactivity among the atopic population, and should be considered in the diagnostic panel and evaluated as future hypoallergen vaccine therapeutic target.

From EST to novel spider silk gene identification for production of spidroin-based biomaterials

A cDNA library from a pool of all the seven silk glands from a tropical spider species was constructed. More than 1000 expressed sequence tag (EST) clones were created. Almost 65% of the EST clones were identified and around 50% were annotated. The cellular and functional distribution of the EST clones indicated high protein synthesis activity in spider silk glands. Novel clones with repetitive amino acid sequences, which is one of the most important characteristics of spider silk genes, were isolated. One of these clones, namely TuSp2 in current research, contains two almost identical fragments with one short C-terminal domain. Reverse transcription (RT) PCR and expression analysis showed that it is expressed in the tubuliform gland and involved in eggcase silk formation. Furthermore, its single repetitive domain can be induced to form various types of materials, including macroscopic fibers, transparent film and translucent hydrogel. This study implies promising potentials for future identification of novel spidroins and development of new spidroin-based biomaterials.

Functional analysis of Pacific oyster (Crassostrea gigas) β-thymosin: Focus on antimicrobial activity

An antimicrobial peptide, ∼5 kDa in size, was isolated and purified in its active form from the mantle of the Pacific oyster Crassostrea gigas by C18 reversed-phase high-performance liquid chromatography. Matrix-assisted laser desorption ionisation time-of-flight analysis revealed 4656.4 Da of the purified and unreduced peptide. A comparison of the N-terminal amino acid sequence of oyster antimicrobial peptide with deduced amino acid sequences in our local expressed sequence tag (EST) database of C. gigas (unpublished data) revealed that the oyster antimicrobial peptide sequence entirely matched the deduced amino acid sequence of an EST clone (HM-8_A04), which was highly homologous with the β-thymosin of other species. The cDNA possessed a 126-bp open reading frame that encoded a protein of 41 amino acids. To confirm the antimicrobial activity of C. gigas β-thymosin, we overexpressed a recombinant β-thymosin (rcgTβ) using a pET22 expression plasmid in an Escherichia coli system. The antimicrobial activity of rcgTβ was evaluated and demonstrated using a bacterial growth inhibition test in both liquid and solid cultures.

Stearoyl‐CoA Desaturase mRNA in Channel Catfish: A Potential Molecular Marker to Investigate Development of Obesity Using Non‐model Fish Species

Objectives of the present study were to characterize SCD‐1 gene in channel catfish and examine its mRNA expression in various tissues. Genetic selection toward increased growth results in development of obesity‐like phenotype in channel catfish, suggesting that channel catfish may serve as an alternative animal model to study human obesity development. However, the underlying mechanism(s) that results in the development of obesity‐like phenotype, as well as the consequence of developing such a phenotype, is unclear. In mammals, development of obesity leads to changes in expression and function of various enzymes involved in lipid metabolism such as stearoyl‐CoA desaturase‐1 (SCD‐1). Currently, little is known about the role of SCD‐1 in channel catfish physiology. Channel catfish expressed sequence tag clones that correspond to the zebrafish SCD‐1 sequence were identified and used as templates to generate primers for the RT‐PCR. The PCR amplicon (599 bp) was generated from channel catfish liver cDNA and shared high sequence similarity (around 70%) with SCD‐1 mRNA of various fish species. Expression of SCD‐1 mRNA was detected in brain, liver, and kidney. Currently, changes in expression of SCD‐1 mRNA in relation to changes in food intake and genetic selection toward growth are being investigated. This project is supported by the Kansas IDeA Network of Biomedical Research Excellence.

Identification and comparison of gonadal transcripts of testis and ovary of adult common carp Cyprinus carpio using suppression subtractive hybridization

The limited number of gonad-specific and gonad-related genes that have been identified in fish represents a major obstacle in the study of fish gonad development and sex differentiation. In common carp Cyprinus carpio from China's Yellow River, the ovary and testis differ in volume and weight in adult fish of the same age. Comparing sperm, egg, and somatic cell transcripts in this carp may provide insight into the mechanisms of its gonad development and sex differentiation. In the present work, gene expression patterns in the carp ovary and testis were compared using suppression subtractive hybridization. Two bidirectional subtracted complementary DNA (cDNA) libraries were analyzed in parallel using testis or ovary as testers. Eighteen nonredundant clones were identified in the male library, including 15 known cDNAs. The expression patterns of selected genes in testis and ovary were analyzed using reverse transcriptase polymerase chain reaction. Tektin-1, GAPDS, FGFIBP, IGFBP-5, and an unknown gene from the Ccmg4 clone were observed to be expressed only in testis. GSDF, BMI1b, Wt1a, and an unknown gene from the Ccme2 clone were expressed at higher levels in testis than in ovary at sexual maturity. Thirty functional expressed sequence tags (ESTs) were identified in 43 sequenced clones in the female library, including 28 known cDNAs, one uncharacterized cDNA (EST clone), and one novel sequence. Eight identified ESTs showed significant differences in expression between the testis and the ovary. ZP3C and Psmb2 were expressed exclusively in ovary, whereas the expression levels of IFIPGL-1, Setd6, ATP-6, CDC45, AIF-1, and an unknown gene from the Ccfh2 clone were more strongly expressed in ovary than in testis. In addition, the expression of ZP3C, Wt1a, and Setd6 was analyzed in male and female gonads, heart, liver, kidney, and brain. ZP3C was expressed only in ovary. Setd6 expression was significantly stronger in female tissues than that in the male, except in the liver, and Wt1a expression showed sexual dimorphism in the kidney and liver. Results suggest that these genes could play key roles during carp growth, both in the gonad and other tissues. The results provide a resource for further investigation of molecular mechanisms responsible for gonad development and sex differentiation in Yellow River common carp.

Gene discovery in the finger leather coral Sinularia notanda by construction and sequencing of a normalized cDNA libary

The transplantation of coral fragments is one of methods that restore coral communities. To form coral colonies, the fragmented corals initiated skeletal extension from the cut-edge of fragment then success the settlement. In order to understand the molecular events underlying fragment adhesion and settlement, we constructed a normalized cDNA library and generated and annotated expressed sequence tags (ESTs) from the fragmented adult polyps of soft coral Sinularia notanda. We generated 3251 high-quality ESTs with an average length of 580bp and the EST cluster and assembly analyses produced 2796 unigenes, including 2487 singletons and 309 contigs. Of the known genes, 55 genes were selected to be involved in polyp fragment adhesion and settlement based on Gene Ontology (GO) classification. Notably, two EST clones were identified to show homology with galaxin gene which was demonstrated as coral specific calcifying protein of organic matrix. These EST sequences can provide utility as molecular markers in molecular and genetic studies of S. notanda and other soft coral.

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss)

BackgroundOocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes.MethodsThrough analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene (named fbxoo) was obtained by assembling sequences from an EST clone and a 5′RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening.Resultsfbxoo mRNA is specifically expressed in mature oocytes as revealed by tissue distribution analysis. The fbxoo cDNA sequence is 1,996 bp in length containing an open reading frame, which encodes a predicted protein of 514 amino acids. The novel protein sequence does not match any known protein sequences in the NCBI database. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that Fbxoo is a new member of the F-box protein family. The expression of fbxoo mRNA and protein is high in ovaries at early pre-vitellogenesis stage, and both fbxoo mRNA and protein are predominantly expressed in early pre-vitellogenic oocytes. Several proteins including tissue inhibitor of metalloproteinase 2 (Timp2) were identified as potential Fbxoo protein binding partners.ConclusionsResults suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.

독성 Alexandrium tamarense 의 EST 분석 및 삭시톡신 생합성 유전자의 확인

Expressed sequence tag (EST) library was constructed from A. tamarense. Base sequences of EST clones were analyzed and saxitoxin biosynthesis-related genes were cloned. Sequences of 827 clones were analyzed and 564 EST were functionally clustered using Blast searches against GenBank. Main genes in the EST had functions on cellular organization, cell metabolism, energy, cell cycle and DNA processing, cellular transport and transport, cell rescue, defense, death and aging, and transcription. Moreover, expression of S-adenosylmethionine synthetase and H2A histone family genes were increased in the toxic A. tamarense. These results show that two genes could be a good biomarkers for the detection of saxitoxin biosynthesis in the A. tamarense.

Identification of a drought- and cold-stress inducible WRKY gene in the cold-hardy Citrus relative Poncirus trifoliata

WRKY transcription factors (TFs) are involved in regulation of abiotic and biotic stress responsive genes and play a role in abiotic and biotic stress tolerance in plants. Although commercial Citrus species are sensitive to cold and drought stresses, an interfertile Citrus relative, Poncirus trifoliata, is resistant to cold and more drought tolerant than most commercial Citrus cultivars. An expressed sequence tag clone sharing homology with WRKY-type TFs was previously isolated from a cDNA library constructed using two-day cold-acclimated P. trifoliata cv. Rubidoux seedlings. In this study, a full-length sequence of this WRKY gene was cloned and isolated from 2-day cold-acclimated P. trifoliata plants using the rapid amplification of cDNA ends (RACE) method. The full-length PtrWRKY1 cDNA is 1807 bp and encodes a polypeptide of 405 amino acids containing a single WRKY domain with a Cys2His2 motif, which is a signature of WRKY TFs. A homologous open reading frame (ORF) sequence was isolated from Citrus grandis, CgWRKY1, sharing 98% identity at the nucleotide level with PtrWRKY1. The expression of PtrWRKY1 and CgWRKY1 was investigated in response to cold and drought stresses by northern blot analysis. While the expression of both genes was induced in response to drought, only PtrWRKY1 expression was induced in response to cold. This differential expression in cold-hardy Poncirus and cold-sensitive pummelo suggests that PtrWRKY1 gene may play a role in adaptation and tolerance to cold stress in Poncirus.

Identification of MV-generated ROS responsive EST clones in floral buds of Litchi chinensis Sonn.

A suppression subtractive hybridization library was constructed using inflorescence primordia of 'Nuomici' litchi to identify EST clones responsive to MV-generated ROS. 93 ESTs could be aligned as unique gene sequences in the inflorescence primordia of litchi. Litchi is an evergreen woody tree widely cultivated in subtropical and tropical regions. However, defective flowering is a pending problem of litchi production. Our previous study indicated that reactive oxygen species (ROS) induced by methyl viologen dichloride hydrate (MV) promotes flowering in litchi. In the present study, a suppression subtractive hybridization (SSH) library was constructed using inflorescence primordia of 'Nuomici' with the aim to find out ROS responsive clones during floral differentiation. 1856 Expressed sequence tag (EST) clones were randomly selected. Clones carrying single exogenous fragments were screened by reverse northern analysis to identify those responsive to MV-generated ROS. A total of 783 differentially expressed EST clones were identified as MV responsive cDNA and were subjected to sequencing. Among them, 26 clones were represented more than three times. 783 clones were aligned to 93 unique gene sequences. The unique genes were classified into 9 categories. 16% of them were involved in transport facilitation, 11% in transcription regulation, 4% in stress response, 9% in carbohydrate metabolism, 1% in secondary metabolism, 14% in intracellular signaling, and 25% in other metabolism, while 9% were genes with unknown functions and 11% were genes with no match in the database.

Coupling Deep Transcriptome Analysis with Untargeted Metabolic Profiling in Ophiorrhiza pumila to Further the Understanding of the Biosynthesis of the Anti-Cancer Alkaloid Camptothecin and Anthraquinones

The Rubiaceae species, Ophiorrhiza pumila, accumulates camptothecin, an anti-cancer alkaloid with a potent DNA topoisomerase I inhibitory activity, as well as anthraquinones that are derived from the combination of the isochorismate and hemiterpenoid pathways. The biosynthesis of these secondary products is active in O. pumila hairy roots yet very low in cell suspension culture. Deep transcriptome analysis was conducted in O. pumila hairy roots and cell suspension cultures using the Illumina platform, yielding a total of 2 Gb of sequence for each sample. We generated a hybrid transcriptome assembly of O. pumila using the Illumina-derived short read sequences and conventional Sanger-derived expressed sequence tag clones derived from a full-length cDNA library constructed using RNA from hairy roots. Among 35,608 non-redundant unigenes, 3,649 were preferentially expressed in hairy roots compared with cell suspension culture. Candidate genes involved in the biosynthetic pathway for the monoterpenoid indole alkaloid camptothecin were identified; specifically, genes involved in post-strictosamide biosynthetic events and genes involved in the biosynthesis of anthraquinones and chlorogenic acid. Untargeted metabolomic analysis by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) indicated that most of the proposed intermediates in the camptothecin biosynthetic pathway accumulated in hairy roots in a preferential manner compared with cell suspension culture. In addition, a number of anthraquinones and chlorogenic acid preferentially accumulated in hairy roots compared with cell suspension culture. These results suggest that deep transcriptome and metabolome data sets can facilitate the identification of genes and intermediates involved in the biosynthesis of secondary products including camptothecin in O. pumila.

Computational analysis of common bean (Phaseolus vulgaris L., genotype BAT93) lycopene β-cyclase and β-carotene hydroxylase gene's cDNA.

The identification of genes and understanding of genes' expression and regulation in common bean (Phaseolus vulgaris L.) is necessary in order to strategize its improvement using genetic engineering techniques. Generation of expressed sequence tags (ESTs) is useful in rapid isolation, identification and characterization of the genes. To study the gene expression in P. vulgaris pods tissue, ESTs generation work was initiated. Early stage and late stage bean-pod-tissues cDNA libraries were constructed using CloneMiner cDNA library construction kit. In total, 5972 EST clones were isolated using random method of gene isolation. While processing ESTs, we found lycopene β-cyclase (PvLCY-β) and β-carotene hydroxylase (PvCHY-β) gene's cDNA. In carotenoid biosynthesis pathway, PvLCY-β catalyzes the production of carotene; and PvCHY-β is known to function as a catalyst in the production of lutein and zeaxanthin. To understand more about PvLCY-β and PvCHY-β, both strands of both cDNA clones were sequenced using M13 forward and reverse primers. Nucleotide and deduced protein sequences were analyzed and annotated using online bioinformatics tools. Results showed that PvLCY-β and PvCHY-β cDNAs are 1639 and 1107 bp in length, respectively. Analysis results showed that PvLCY-β and PvCHY-β gene's cDNA contains an open reading frame (ORF) that encodes for 502 and 305 amino acid residues, respectively. The deduced protein sequence analysis results also showed the presence of conserved domains needed for PvLCY-β and PvCHY-β functions. The phylogenetic analysis of both PvLCY-β and PvCHY-β proteins showed it's closeness with the LCY-β and CHY-β proteins from Glycine max, respectively. The nucleotide sequence of PvLCY-β and PvCHY-β gene's cDNA and it's annotation is reported in this paper.

Dynamic Nucleotide-dependent Interactions of Cysteine- and Histidine-rich Domain (CHORD)-containing Hsp90 Cochaperones Chp-1 and Melusin with Cochaperones PP5 and Sgt1

Mammals have two cysteine- and histidine-rich domain (CHORD)-containing Hsp90 cochaperones, Chp-1 and melusin, which are homologs of plant Rar1. It has been shown previously that Rar1 CHORD directly interacts with ADP bound to the nucleotide pocket of Hsp90. Here, we report that ADP and ATP can bind to Hsp90 cochaperones Chp-1 and PP5, inducing their conformational changes. Furthermore, we demonstrate that Chp-1 and melusin can interact with cochaperones PP5 and Sgt1 and with each other in an ATP-dependent manner. Based on the known structure of the Rar1-Hsp90 complex, His-186 has been identified as an important residue of Chp-1 for ADP/ATP binding. His-186 is necessary for the nucleotide-dependent interaction of Chp-1 not only with Hsp90 but also with Sgt1. In addition, Ca(2+), which is known to bind to melusin, enhances the interactions of melusin with Hsp90 and Sgt1. Furthermore, melusin acquires the ADP preference for Hsp90 binding in the presence of Ca(2+). Our newly discovered nucleotide-dependent interactions between cochaperones might provide additional complexity to the dynamics of the Hsp90 chaperone system, also suggesting potential Hsp90-independent roles for these cochaperones.

Olfactomedin 1 Interacts with the Nogo A Receptor Complex to Regulate Axon Growth

Olfm1, a secreted highly conserved glycoprotein, is detected in peripheral and central nervous tissues and participates in neural progenitor maintenance, cell death in brain, and optic nerve arborization. In this study, we identified Olfm1 as a molecule promoting axon growth through interaction with the Nogo A receptor (NgR1) complex. Olfm1 is coexpressed with NgR1 in dorsal root ganglia and retinal ganglion cells in embryonic and postnatal mice. Olfm1 specifically binds to NgR1, as judged by alkaline phosphatase assay and coimmunoprecipitation. The addition of Olfm1 inhibited the growth cone collapse of dorsal root ganglia neurons induced by myelin-associated inhibitors, indicating that Olfm1 attenuates the NgR1 receptor functions. Olfm1 caused the inhibition of NgR1 signaling by interfering with interaction between NgR1 and its coreceptors p75NTR or LINGO-1. In zebrafish, inhibition of optic nerve extension by olfm1 morpholino oligonucleotides was partially rescued by dominant negative ngr1 or lingo-1. These data introduce Olfm1 as a novel NgR1 ligand that may modulate the functions of the NgR1 complex in axonal growth.

Analysis of Expressed Sequence Tags from the Antarctic Psychrophilic Green Algae, Pyramimonas gelidicola

Expressed sequence tags (ESTs) from the Antarctic green algae Pyramimonas gelidicola were analyzed to obtain molecular information on cold acclimation of psychrophilic microorganisms. A total of 2,112 EST clones were sequenced, generating 222 contigs and 219 singletons, and 200 contigs and 391 singletons from control (4 degrees C) and cold-shock conditions (-2 degrees C), respectively. The complete EST sequences were deposited to the DDBJ EST database (http:// www.ddbj.nig.ac.jp/index-e.html) and the nucleotide sequences reported in this study are available in the DDBJ/EMBL/ GenBank. These EST databases of Antarctic green algae can be used in a wide range of studies on psychrophilic genes expressed by polar microorganisms.

Structural and expression analysis of prolamin genes in Oryza sativa L.

Rice is a staple crop with a small genome of 389 Mb. Rice grain is a source of carbohydrates and proteins and has a relatively low protein content compared to other legume seeds. Glutelin and prolamin are the major storage proteins in rice. Prolamins are characterized by high glutamine and proline content and are generally soluble only in strong alcohol solutions. In this study, we obtained a total of 51,383 expressed sequence tags (ESTs) from Ilpumbyeo (Oryza sativa L.), of which 33,201 and 18,182 clones were obtained from immature and germinating seeds, respectively. From the EST clones, 15,148 unigenes were identified, and 2,590 genes were expressed in both immature and germinating seeds. Gene expression profiling of rice prolamins indicated that prolamin gene expression increased 5 days after heading and reached maximal expression after 30 days, suggesting a high demand for prolamins during seed development and germination. Phylogenetic analysis grouped 33 prolamin genes based on the abundance of sulfur-containing amino acids methionine and cysteine according to the deduced amino acid sequences. Our results enhance the understanding of the regulation of seed maturation and germination, which can result in improved agricultural traits for the seed industry.

Chemosensory protein genes of batocera horsfieldi (hope): identification and expression pattern

AbstractChemoreception is an essential feature for selection of host plants by insects. Chemosensory proteins (CSPs) are a group of small proteins (approximately 13 kDa) that are expressed widely in various insect sensory appendages. Batocera horsfieldi (Hope) is a major pest of Popolus and utilizes a variety of semiochemicals for its mating and oviposition. However, no CSP gene has been identified in B. horsfieldi. Here, to obtain the DNA sequences of B. horsfieldi CSPs, we used both bioinformatics and experimental approaches to analyse the antennal expressed sequence tags (ESTs). Among 649 EST clones, three unique CSP sequences (BhorCSP1, 2 and 3) were identified and characterized from B. horsfieldi (Hope) antennal cDNA libraries based on their four conserved cysteine residues, which have a characteristic spacing pattern. A phylogenetic tree of BhorCSPs and other insect CSPs showed that BhorCSP1 and BhorCSP2 are more similar to CSPs of Tribolium castaneum than BhorCSP3. BhorCSP3 is closely related to CSPs of the Dipteran, Culex quinquefasciatus. Using reverse transcription polymerase chain reaction (RT‐PCR) and real‐time Q‐PCR strategies, we examined the expression patterns of these three putative CSP genes in different tissues, sexes and developmental stages. Our analysis shows that all three genes were expressed in all tissues examined, including the antennae, labial palps, maxillary palps, legs, abdomen and wings but not in the head. Real‐time Q‐PCR results revealed that these CSP genes were expressed throughout the life of the adults, and the transcription levels of these genes depended on the sex, age and mating status of adults.

A Combination of Biochemical and Proteomic Analyses Reveals Bx-LEC-1 as an Antigenic Target for the Monoclonal Antibody 3-2A7-2H5-D9-F10 Specific to the Pine Wood Nematode

Pine wilt disease (PWD) is one of the most devastating forest diseases in Asia and Europe. The pine wood nematode, Bursaphelenchus xylophilus, has been identified as the pathogen underlying PWD, although the pathology is not completely understood. At present, diagnosis and confirmation of PWD are time consuming tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for B. xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically, but not those from other closely related nematodes. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions, or bands isolated from SDS-PAGE, two-dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two nonidentical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.