Rapid Amplification Of cDNA Ends-polymerase Chain Reaction Research Articles

Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin.

The paired-box 3 (Pax3) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new Pax3 gene was identified from Pteria penguin (Röding, 1798) (P. penguin) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpPax3 was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed PpPax3 shared the highest (69.2%) identity with Pax3 of Mizuhopecten yessoensis. Tissue expression profile showed that PpPax3 had the highest expression in mantle, a nacre-formation related tissue. The PpPax3 silencing significantly inhibited the expression of PpPax3, PpMitf, PpTyr and PpCdk2, genes involved in Tyr-mediated melanin synthesis, but had no effect on PpCreb2 and an increase effect on PpBcl2. Furthermore, the PpPax3 knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (Tyr) knockdown. These data indicated that PpPax3 played an important regulating role in melanin synthesis by Tyr pathway in P. penguin.

Cloning and expression analysis of the nuclear factor erythroid 2-related factor 2 (Nrf2) gene of grass carp (Ctenopharyngodon idellus) and the dietary effect of Eucommia ulmoides on gene expression

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor in the regulation of redox homeostasis, which plays a vital role in metabolism, the cell cycle, and cell protection. In this study, the complete cDNA sequence of Nrf2 gene of grass carp was obtained by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR), and the characteristics of Nrf2 proteins predicted from gene sequencing were analyzed by bioinformatics methods. Tissue differential expression patterns were also detected by Real-time Fluorescence quantitative PCR (qRT-PCR). Then, Eucommia ulmoides (EU), a traditional Chinese medicine, was added into diet with an inclusion of 2% to feed grass carp for 3 months to investigate the effects on the expression of Nrf2 gene in tissues. The results showed that the full-length of Nrf2 gene of grass carp is 2760 bp, encoding 593 amino acids, and the predicted Nrf2 protein is a hydrophilic and non-secretory protein. Grass carp Nrf2 gene showed higher expression in kidney, gill, liver, spleen, and blood tissues than in other tissues. In the feeding trial, dietary EU significantly promoted the Nrf2 mRNA expression in kidney, gill, and liver tissues after three months feeding. The results suggest that Nrf2 plays an important role in the anti-oxidative capacity of grass carp and its expression is enhanced by dietary EU.

Molecular Cloning and Expression of Major Histocompatibility Complex Class Iα Gene of Swamp Eel (Monopterus albus)

Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. In this study, we isolated MHC class Iα gene from swamp eel (Monopterus albus) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of MHC Iα in swamp eel, tissue distribution and immune response of the MHC Iα after bacterial challenge were analyzed. All the characteristic features of MHC Iα chain structure were identified by the deduced amino acid sequence, including the leader peptide, α1, α2, and α3 domains, connecting peptide, transmembrane and cytoplasmic regions, conserved cysteines, and N-glycosylation site. The deduced amino acid sequence of the MHC Iα shares 27.3% to 69.6% similarity with those of other vertebrates. Quantitative real-time PCR (qRT-PCR) demonstrated that MHC Iα gene was ubiquitously expressed in ten tissues, comparatively higher in immune related tissues, including the liver and intestine. Challenge of M. albus with the extracellular pathogen, Aeromonas hydrophila, resulted in a significant increase in the expression of MHC Iα within 72 h after infection and these high levels were maintained for 72 h in kidneys, spleen, and skin, whereas in the intestine and liver, it was found first in the form of up-regulated and then down-regulated gene. These results demonstrated that MHC Iα gene played an important role in response to bacterial infection in M.albus.

TmSR-C, scavenger receptor class C, plays a pivotal role in antifungal and antibacterial immunity in the coleopteran insect Tenebrio molitor

Scavenger receptors (SRs) constitute a family of membrane-bound receptors that bind to multiple ligands. The SR family of proteins is involved in removing cellular debris, oxidized low-density lipoproteins, and pathogens. Specifically, class C scavenger receptors (SR-C) have also been reported to be involved in phagocytosis of gram-positive and -negative bacteria in Drosophila and viruses in shrimp. However, reports are unavailable regarding the role of SR-C in antifungal immune mechanisms in insects. In this study, a full-length Tenebrio molitor SR-C (TmSR-C) sequence was obtained by 5′- and 3′-Rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The TmSR-C full-length cDNA comprised 1671 bp with 5′- and 3′-untranslated regions of 23- and 107-bp, respectively. TmSR-C encodes a putative protein of 556 amino acid residues that is constitutively expressed in all tissues of late instar larvae and 2-day-old adults, with the highest transcript levels observed in hemocytes of larvae and adults. TmSR-C mRNA showed a 2.5-fold and 3-fold increase at 24 and 6 h after infection with Candida albicans and β-glucan, respectively. Immunoassay with TmSR-C polyclonal antibody showed induction of the putative protein in the cytosols of hemocytes at 3 h after inoculation of C. albicans. RNA interference (RNAi)-based gene silencing and phagocytosis assays were used to understand the role of TmSR-C in antifungal immunity. Silencing of TmSR-C transcripts reduced the survivability of late instar larvae at 2 days post-inoculation of C. albicans, Escherichia coli, or Staphylococcus aureus. Furthermore, in TmSR-C-silenced larvae, there was a decline in the rate of microorganism phagocytosis. Taken together, results of this study suggest that TmSR-C plays a pivotal role in phagocytosing not only fungi but also gram-negative and -positive bacteria in T. molitor.

Expression analyses of salinity stress- associated ESTs in Aeluropus littoralis

Salinity is among the most important abiotic stresses affecting crop production throughout the earth. Halophyte plants can sustain high salinity levels, therefore elucidating molecular mechanisms underlying their salinity resistance is beneficial for crop improvement. Aeluropus littoralis, a halophyte weed, is a great genetic resource for this purpose. Isolated expressed sequence taq (EST) sequences from A. littoralis under salinity stress, have given us the chance to find and analyze transcripts of genes involved in response to salinity. Transcriptome analyses indicated the expression levels of mRNAs corresponding to 10 of sequences were increased under treatments. All mRNAs were significantly induced under salt treatment with the highest peaks observed at different hours of treatments. Moreover, the full-length cDNA of vacuolar H+-pyrophosphatase (VP) was isolated utilizing 3′ and 5′ rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) and characterized (GenBank accession number of KT253223.1). The extracted full-length of VP was 2732 bp, which contained ORF of 2292 bp encoding 763 amino acids.

A novel cecropin‐like peptide from black soldier fly, Hermetia illucens: Isolation, structural and functional characterization

AbstractCecropins are basic antibacterial peptides that have potent antimicrobial activities. We induced and purified a novel antimicrobial peptide exhibiting activity against Gram‐negative bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel cecropin‐like peptide 1 (CLP1) was purified using solid‐phase extraction and reverse‐phase chromatography. The purified CLP1 demonstrated a molecular weight of 4,840 Da, as determined by matrix‐assisted laser desorption/ionization‐time‐of‐flight (MALDI‐TOF). From analysis of CLP1 by N‐terminal amino acid sequencing using Edman degradation, combined with MALDI‐TOF and rapid amplification of cDNA ends‐polymerase chain reaction (RACE‐PCR), the amino acid sequence of the mature peptide was determined to be GWRKRVFKPVEKFGQRVRDAGVQGIAIAQQGANVLATARGGP PQQG. In NCBI BLAST, the amino acid sequence of CLP1 was found to be 60 % identical to the Drosophila melanogaster cecropin C. In silico analysis revealed that CLP1 was suggested to be part of the cecropin superfamily of AMPs characterized as cationic, linear, α‐helical, and amphipathic polypeptides. Analysis of the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) showed that CLP1 exerted antibacterial effects against Gram‐negative bacteria. The expression of CLP1 transcripts in several tissues after bacterial challenge was measured by quantitative real‐time PCR. CLP1 expression was negligible throughout the body before immunization, and was mostly evident in the fat body after immunization.

Isolation, expression and single nucleotide polymorphisms (SNPs) analysis of LACCASE gene (LkLAC8) from Japanese larch (Larix kaempferi)

Nucleotide diversity (π) and linkage disequilibrium (LD) analysis based on SNP marker could provide a sound basis for choosing an association analysis method. Japanese larch (Larix kaempferi) is an important timber coniferous tree species for pulping and papermaking, but its high lignin content has significantly restricted it application potential. In this study, the LACCASE gene, that plays an important regulatory role for lignin biosynthesis, was selected as research target. The full-length cDNA and genomic sequences of the encoding LkLAC8 gene were isolated from the LACCASE expressed sequence tags of the Japanese larch transcriptome database using the rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA was determined to be 1940 bp, with an open reading frame (ORF, 1734 bp) that encoded a protein of 577 AA. This protein contains four highly specific Cu2+ binding sites and 11 glycosylation sites, thus belonging to the LACCASE family. The deduced protein sequence shared an 89% identity with the PtaLAC from Pinus taeda. A real-time PCR analysis showed that the LkLAC8 transcript was expressed predominantly in mature xylem, with moderate levels in the immature xylem, cambium and mature leaves, the lowest in the roots. Lastly, the genomic sequences of LkLAC8 in 40 individuals from six naturally distributed populations of Japanese larch were amplified, and a total of 201 SNPs (103 and 98 mutation types of transition and transversion, respectively) were detected; the frequency of the SNPs was 1/19 bp. Nucleotide diversity among the six populations ranged from 0.0034 to 0.0053, which suggested that there were no significant differences among the populations. The LD analysis showed that the LD level decayed rapidly within the increasing length of the LkLAC8 gene. These results implied that LD mapping and association analysis based on candidate gene may be feasible for the marker-assisted breeding of new germplasms with low lignin in Japanese larch.

Overexpression of the mulberry latex gene MaMLX-Q1 enhances defense against Plutella xylostella in Arabidopsis thaliana

Purified mulberry latex chitinase (MLX) has a role in defense against some lepidopteran insects. In this study, a full length chitinase gene, MaMLX-Q1, of 1405 bp with a 1140 bp open reading frame, was obtained from mulberry leaves by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) procedure. The gene encoded a mature protein with the predicted molecular mass of 39.38 kDa and an isoelectric point (pI) of 6.43; it contained two chitin-binding domains and a hydrolase family 19 chitinase domain. Sequence alignment and phylogenetic analysis grouped it in the class I chitinase protein group. Heterogeneous expression of this MaMLX-Q1 in Arabidopsis showed non-visible alterations in growth phenotype, except for the higher transcriptional expression of MaMLX-Q1 when compared to that of wild-type Arabidopsis. This ectopic MaMLX-Q1 exhibited toxicity to the growth and development of Plutella xylostella larvae, causing significantly lower weight gain and higher mortality. These results indicate an application of MaMLX-Q1 as an insecticide for plant protection.

Molecular characterization and expression analysis of major histocompatibility complex class IIA and IIB genes of Odontobutis potamophila

The major histocompatibility complex (MHC) has a critical role in the adaptive immune system by presenting a foreign peptide to the T cell receptor. The full length of MHC class IIA and IIB cDNA was cloned from Odontobutis potamophila by expressed sequence tag and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). Tissue distribution and expression of MHC IIA and IIB were examined to study the functions of the MHC II gene in fish. The domain structure and antigen-binding motifs of other teleosts and mammals MHC II are conserved in O. potamophila as MHC IIA and IIB genes. The alignments of the deduced amino acid sequences from MHC IIA and MHC IIB in O. potamophila with those from other vertebrates demonstrated a 25–52 % similarity between other fish and mammalians. Real-time quantitative PCR demonstrated that MHC IIA and IIB genes were ubiquitously expressed in all tested tissues. The challenge of O. potamophila with Aeromonas hydrophila resulted in a significant change in the expression of MHC IIA and IIB mRNA in the liver, spleen and kidney. All these results suggest that both genes play crucial roles in immune responses of O. potamophila.

Functional analysis of small ubiquitin-modifier (SUMO) in orange-spotted grouper (Epinephelus coioides)

Small ubiquitin-like modifiers (SUMOs), structurally similar to ubiquitin, ligated to lysine residues within sumoylation target proteins. Sumoylation and ubiquitination exhibit similar biological processes for post-translational modification regulation, apoptosis and protein stability which was participated in a number of cellular processes such as nuclear transport, transcriptional regulation, apoptosis and protein stability. In the present study, the cDNA of orange-spotted grouper SUMO, terms osgSUMO, was cloned by the combination of homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approaches. The SUMO1 and SUMO2-yellow fluorescent protein (YFP) were distributed in the cytoplasm. Our results show that SUMO1 was capable of decreasing coat protein (CP) of nucleus localization ratio, not SUMO2. Thus, we demonstrate here that SUMO1 was able to interact with CP by using analysis of fluorescence resonance energy transfer (FRET). Taken together, grouper SUMO can partially influence on localization of CP.

Molecular cloning of a coiled-coil-nucleotide-binding-site-leucine-rich repeat gene from pearl millet and its expression pattern in response to the downy mildew pathogen.

Downy mildew caused by Sclerospora graminicola is a devastating disease of pearl millet. Based on candidate gene approach, a set of 22 resistance gene analogues were identified. The clone RGPM 301 (AY117410) containing a partial sequence shared 83% similarity to rice R-proteins. A full-length R-gene RGA RGPM 301 of 3552 bp with 2979 bp open reading frame encoding 992 amino acids was isolated by the degenerate primers and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) approach. It had a molecular mass of 113.96 kDa and isoelectric point (pI) of 8.71. The sequence alignment and phylogenetic analysis grouped it to a non-TIR NBS LRR group. The quantitative real-time PCR (qRT-PCR) analysis revealed higher accumulation of the transcripts following inoculation with S. graminicola in the resistant cultivar (IP18296) compared to susceptible cultivar (7042S). Further, significant induction in the transcript levels were observed when treated with abiotic elicitor β-aminobutyric acid (BABA) and biotic elicitor Pseudomonas fluorescens. Exogenous application of phytohormones jasmonic acid or salicylic acid also up-regulated the expression levels of RGA RGPM 301. The treatment of cultivar IP18296 with mitogen-activated protein kinase (MPK) inhibitors (PD98059 and U0126) suppressed the levels of RGA RGPM 301. A 3.5 kb RGA RGPM 301 which is a non-TIR NBS-LRR protein was isolated from pearl millet and its up-regulation during downy mildew interaction was demonstrated by qRT-PCR. These studies indicate a role for this RGA in pearl millet downy mildew interaction.

Molecular cloning and characterization of NPR1 gene from Arachis hypogaea.

The NPR1 gene was an important regulator for a plant disease resistance. The cDNA of NPR1 gene was cloned from peanut cultivar Ri Hua 1 by rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full length cDNA of Arachis hypogaea NPR1 consisted of 2,078 base pairs with a 1,446 bp open-reading frame encoding 481 amino acids. The predicted NPR1 contained the highly conserved functional domains (BTB/POZ domain from M1 to D116), protein-protein interaction domains (three ankyrin repeats from K158 to L186; N187 to L217 and R221 to D250) and one NPR1-like domain (C262 to S469). The DNA sequence of the NPR1 gene was 2,332 or 2,223 bp. Both two sequences contained three introns and four exons. The NPR1 transcripts were expressed mainly in roots and leaves, while fewer signals were detected in the stems. Amount of the NPR1 transcript was significantly increased 1 h after salicylic acid challenge and was eventually 5.3 times greater than that in the control group. Both the DNA sequence and the coding sequence were obtained from eight cultivars and nine wild species of Arachis. Maximum likelihood analyses of d N/d S ratios for 25 sequences from different species showed that different selection pressures may have acted on different branches.

Isolation and Characterization of a Dominant Dwarf Gene, D-h, in Rice

Plant height is an important agronomic trait that affects grain yield. Previously, we reported a novel semi-dominant dwarfmutant, HD1, derived from chemical mutagenesis using N-methyl-N-nitrosourea (MNU) on a japonica rice cultivar, Hwacheong. In this study, we cloned the gene responsible for the dwarf mutant using a map-based approach. Fine mapping revealed that the mutant gene was located on the short arm of chromosome 1 in a 48 kb region. Sequencing of the candidate genes and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) analysis identified the gene, d-h, which encodes a protein of unknown function but whose sequence is conserved in other cereal crops. Real-time (RT)-PCR analysis and promoter activity assays showed that the d-h gene was primarily expressed in the nodes and the panicle. In the HD1 plant, the d-h gene was found to carry a 63-bp deletion in the ORF region that was subsequently confirmed by transgenic experiments to be directly responsible for the gain-of-function phenotype observed in the mutant. Since the mutant plants exhibit a defect in GA response, but not in the GA synthetic pathway, it appears that the d-h gene may be involved in a GA signaling pathway.

Molecular cloning, structural analysis, and expression of zona pellucida glycoprotein ZP3 gene from Chinese zokor, Myospalax fontanierii

The zona pellucida 3 (ZP3) plays a crucial role in reproductive immunology. We obtained a full-length cDNA encoding Chinese Zokor zp3, using rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The cDNA contains an open reading frame of 1269 nucleotides encoding a polypeptide of 422 amino acid residues. The amino acid sequence has a high degree of homology with hamster (78%), mouse (76%), and rat (74%). XhoI and SacI sites restricted 1158 bp fragment of zokor ZP3 cDNA, excluding the signal sequence and transmembrane-like domain was cloned under the phage T7 promoterlac operator control in the pET-28a(+) vector. Recombinant pET-zokorZP3 (r-ZP3) was expressed as a poly-histidine fusion protein in E. coli strain BL21 (DE3). Optimum expression of r-ZP3 was observed at 28 degrees C, 1 mM IPTG and 2 h of inducing. The purified protein was tested by Western blot.

Molecular cloning of four glutathione peroxidase (GPx) homologs and expression analysis during stress exposure of the marine teleost Sparus aurata

Glutathione peroxidase (GPx; EC 1.11.1.9) is an important family of enzymes that protects organisms from oxidative damage. Four full-length GPx cDNAs were cloned and characterized by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR) from the liver of gilthead sea bream (Sparus aurata), an economically important species for Mediterranean aquaculture. Structural and functional annotations were performed for all paralogs, which suggested possible differences in function and subcellular localization. The phylogenetic analysis, based on the amino acid sequences, revealed four groups corresponding to teleostean GPx1a, GPx1b, GPx4a, and GPx4b and three groups for mammalian GPx1, GPx2 and GPx4. The tree topology indicated past duplication events for fish genes, unlike their mammalian homologs. Transcriptional analysis in ten tissues by reverse transcription quantitative polymerase chain reaction (RT-qPCR) evidenced a tissue-specific pattern for each GPx homolog. Fish experimental groups were exposed to stress factors such as fasting and confinement. Relative expression analysis in fish liver demonstrated that GPx1 genes were not regulated by dietary restriction; GPx4b was differentially expressed opposed to regularly fed fish. On the other hand, both GPx1 and GPx4 genes were up-regulated in fish post exposed to confinement, considered as a response to acute stress. The results underline the role of GPx genes as indicators of stress and welfare status in gilthead sea bream aquaculture.

Molecular cloning and expression analysis of major histocompatibility complex class I, IIA and IIB genes of blunt snout bream (Megalobrama amblycephala)

Major histocompatibility complex (MHC) plays an important role in the immune response of vertebrates. In this study, we isolated MHC class IIA and IIB genes from blunt snout bream (Megalobrama amblycephala) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of the MHC genes in M. amblycephala, tissue distribution and immune response of the MHC genes to bacterial challenge were analyzed. All the characteristic features of MHC class II chain structure could be identified in the deduced amino sequences of MHC IIA and IIB, including the leader peptide, α1/β1 and α2/β2 domains, connecting peptide and transmembrane and cytoplasmic regions, as well as conserved cysteines and N-glycosylation site. The deduced amino acid sequence of the MHC IIA and IIB molecules shared from 48% to 88% and from 65% to 77% similarity with those of other teleosts, respectively. Quantitative real-time PCR (qRT-PCR) demonstrated that MHC I and II genes were ubiquitously expressed in ten tissues, with high level in immune related tissues, including kidney, intestine, gill and spleen. Challenge of M. amblycephala with the extracellular pathogen, Aeromonas hydrophila, resulted in a significant increase in the expression of MHC I, MHC IIA and IIB mRNA within 72h after infection in gill, kidney, intestine and liver, followed by a recovery to normal level after 120h. The changes of expression levels for MHC IIA and IIB in most tissues were significantly higher than that of MHC I in the corresponding tissues at most time points (P<0.05). These results demonstrated the MHC genes played an important role in response to bacterial infection in M. amblycephala; however, MHC class I and II genes showed different functional activity, which need be further investigated in teleost.

A phase I dose-escalation study of TKM-080301, a RNAi therapeutic directed against polo-like kinase 1 (PLK1), in patients with advanced solid tumors: Expansion cohort evaluation of biopsy samples for evidence of pharmacodynamic effects of PLK1 inhibition.

TPS2621 Background: TKM-080301 is a lipid nanoparticle formulation of a small interfering RNA (siRNA) directed against PLK1, a serine/threonine kinase that regulates multiple critical aspects of cell cycle progression and mitosis. Anti-tumor activity, RNA interference and pharmacodynamic effects of PLK1 inhibition have been conclusively demonstrated in preclinical models. Demonstration of pharmacodynamic effects of PLK1 inhibition in patient biopsy samples is an exploratory objective of this first-in-human study. Methods: TKME080301 is being evaluated in an open-label, non-randomized, dose-escalation study in patients with advanced solid tumors or lymphoma. Sequential cohorts of 3 to 6 patients receive TKME080301 as a 30-minute intravenous infusion on Days 1, 8 and 15 of a 28-day cycle. Treatment can continue until disease progression, based on overall clinical benefit. Tumor response is determined according to RECIST criteria. Primary study objectives include determination of safety, maximum tolerated dose and dose limiting toxicities. Secondary objectives include characterization of pharmacokinetics and the preliminary assessment of anti-tumor activity. Five cohorts have been enrolled and a tentative Phase 2 dose has been identified. An expansion cohort of 10 patients began enrolling in February, 2013. The focus of the expansion cohort will be to collect additional safety and pharmacokinetic data at the tentative Phase 2 dose, as well as pharmacodynamic data from mandatory biopsy samples. Pre- and post-dose biopsy samples will be evaluated for potential evidence of PLK1 inhibition using 5’ RACE (rapid amplification of cDNA ends) polymerase chain reaction (to identify the predicted PLK1 mRNA cleavage product), histology (to assess for the presence of aberrant mitotic figures) and immunohistochemistry. An update on enrollment and pharmacodynamic evaluations will be presented. Clinical trial information: NCT01262235.

Molecular cloning, characterization and expression profiles of thioredoxin 1 and thioredoxin 2 genes in Mytilus galloprovincialis

Thioredoxin (Trx) proteins are involved in many biological processes especially the regulation of cellular redox homeostasis. In this study, two Trx cDNAs were cloned from the mussel Mytilus galloprovincialis using rapid amplifi cation of cDNA ends-polymerase chain reaction (RACE-PCR). The two cDNAs were named MgTrx1 and MgTrx2, respectively. The open reading frames of MgTrx1 and MgTrx2 were 318 and 507 base pairs (bp) and they encoded proteins of 105 and 168 amino acids with estimated molecular masses of 11.45 and 18.93 kDa, respectively. Sequence analysis revealed that both proteins possessed the conserved active site dithiol motif Cys-Gly-Pro-Cys. In addition, MgTrx2 also possessed a putative mitochondrial targeting signal suggesting that it is located in the mitochondria. Quantitative real-time polymerase chain reaction (qPCR) revealed that both MgTrx1 and MgTrx2 were constitutively expressed in all tissues examined. The MgTrx1 transcript was most abundant in hemocytes and gills, whereas the MgTrx2 transcript was most abundant in gonad, hepatopancreas, gill and hemocytes. Following Vibrio anguillarum challenge, the expression of MgTrx1 was up-regulated and reached its peak, at a value 10-fold the initial value, at 24 h. Subsequently, expression returned back to the original level. In contrast, the expression level of MgTrx2 was down-regulated following bacterial stimulation, with one fi fth of the control level evident at 12 h post challenge. These results suggest that MgTrx1 and MgTrx2 may play important roles in the response of M. galloprovincialis to bacterial challenge.

Molecular cloning, genomic structure, polymorphism and expression analysis of major histocompatibility complex class II B gene of Nile tilapia (Oreochromis niloticus)

Major histocompatibility complex (MHC) class II molecules play important roles in the immune system of vertebrates. In this study, full-length MHC II B cDNA was obtained from Nile tilapia (Oreochromis niloticus) by rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). In order to study the function of the MHC II B gene in Nile tilapia, the genomic structure, three-dimensional model, molecular polymorphism, tissue distribution, and immune response of the MHC II B gene to bacterial challenge were analyzed. Six exons and five introns were identified in the Nile tilapia MHC II B gene, which is different from the general five-exon–four-intron structure of other teleosts. Interestingly, the β-2 domain of Nile tilapia MHC II B was encoded by two exons. Sequence comparison showed that the deduced amino acid sequence had 26.7–69.6% identity with those of other species. Ten alleles were observed among five healthy individuals. However, a putative N-linked glycosylation site located at theβ-1 domain was observed in all the sequences except in Orni-DAB*0301-0501. Five different alleles observed in a single individual may imply the existence of at least three MHC II B loci. Moreover, dN was significantly greater than dS at protein-binding region (PBR) positions (dN/dS=3.073, P=0.016) in Nile tilapia MHC II B, which may provide evidence of strong positive selection at β-1 PBR positions among Nile tilapia sequences. Semi-quantitative reverse transcriptase PCR demonstrated that the Nile tilapia MHC II B mRNA was expressed in all tested tissues. Significant changes were observed in the liver, gill and kidney after bacteria challenge, while no obvious change was found in the intestine and spleen.

Molecular cloning and pharmacological characterisation of a tyramine receptor from the rice stem borer, Chilo suppressalis (Walker)

Tyramine (TA) and octopamine (OA) are considered to be the invertebrate counterparts of the vertebrate adrenergic transmitters. Because these two phenolamines are the only biogenic amines whose physiological significance is presumably restricted to invertebrates, the attention of pharmacologists has been focused on the corresponding receptors, which are believed to represent promising targets for novel insecticides. For example, the formamidine pesticides, such as chlordimeform and amitraz, have been shown to activate OA receptors. A full-length cDNA (designated CsTyR1) from the rice stem borer, Chilo suppressalis (Walker), has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction (RACE-PCR). The mRNA of CsTyR1 is present in various tissues, including hemocytes, fat body, midgut, Malpighian tubules, nerve cord and epidermis, and it is found predominantly in the larval nerve cord with 16-80-fold enrichment compared with other tissues. The authors generated a HEK 293 cell line stably expressing CsTyR1 in order to examine functional and pharmacological properties of this receptor. Both TA and OA at 0.01-100 µM can reduce forskolin-stimulated intracellular cAMP levels in a dose-dependent manner (TA, EC(50) = 369 nM; OA, EC(50) = 978 nM). In agonist assays, activation of CsTyR1 by clonidine and amitraz but not by naphazoline and chlordimeform can also significantly inhibit forskolin-stimulated cAMP production. The inhibitory effect of TA at 10 µM is eliminated by coincubation with yohimbine, phentolamine or chlorpromazine (each 10 µM). This study represents a comprehensive molecular and pharmacological characterisation of a tyramine receptor in the rice stem borer.