Expressed sequence tag analysis of phyllosomas and hemocytes of Japanese spiny lobster Panulirus japonicus

Two complementary DNA (cDNA) libraries were constructed from phyllosomas and hemocytes of adult Japanese spiny lobster Panulirus japonicus and a total of 2,673 expressed sequence tags (ESTs) were obtained. After assembly and clustering, 450 and 458 unique sequences were found from the phyllosoma and hemocyte cDNA libraries, respectively. Of these, 114 and 220 ESTs showed significant homologies with known genes in the National Centre for Biotechnology Information (NCBI) database. The remaining sequences were of unknown function. Immune-related genes found in this study include lectin, proteinase inhibitor, prophenoloxidase, heat-shock protein, antimicrobial peptide, and a few putative defense-related proteins.     

First application of PCR-Luminex system for molecular diagnosis of fungicide resistance and species identification of fungal pathogens

Fungicide resistance in plant pathogens is often caused by a single point mutation in a gene encoding fungicide target proteins. Such is the case for resistance to MBI-D (inhibitors of scytalone dehydratase in melanin biosynthesis) fungicides in rice blast fungus (Magnaporthe oryzae), which is caused by a mutation in the scytalone dehydratase gene that results in a replacement of valine with methionine at codon 75 of the fungicide target protein. PCR-Luminex, a novel system developed for high-throughput analysis of single nucleotide polymorphisms (SNPs) was successfully introduced to diagnose MBI-D resistance using specific oligonucleotide probes coupled with fluorescent beads. The PCR-Luminex system was further tested for its potential in identifying species causing Fusarium head blight on wheat. Four major pathogens, Fusarium graminearum (=F. asiaticum), F. culmorum, F. avenaceum, and Microdochium nivale, known to cause the disease, were tested, and the species were identified using the PCR-Luminex method. So far, this report is the first on the application of the DNA-based PCR-Luminex system in the area of crop protection and/or agricultural sciences.     

Identification and characterization of Japanese flounder, Paralichthys olivaceus interferon-stimulated gene 15 (Jf-ISG15)

Previously, using cDNA microarray analysis, we demonstrated that an EST clone of Japanese flounder (Paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (ISG15) was strongly induced by treatment with DNA vaccine encoding the glycoprotein gene of Hirame rhabdovirus (HIRRV). In this study, we conducted molecular cloning and expression analysis of the Japanese flounder ISG15 (Jf-ISG15). Jf-ISG15 encoded two exons. The first exon was non-coding, while the second exon encoded a protein of 158 amino acids. The coded protein has two tandem ubiquitin-like domains with a carboxyl-terminus conjugation motif “LRLRGG”. Phylogenetic analysis revealed an evolutionary relationship among Jf-ISG15, mammalian and fish ISG15 orthologues. The interferon-stimulated response element (ISRE) sites were conserved among DNA sequences of Jf-ISG15 and mammalian ISG15 promoter regions. An RT-PCR analysis of healthy tissues showed that Jf-ISG15 mRNA was notably strongly expressed in gills, PBLs and spleen. Expression of Jf-ISG15 was strongly induced by poly-I:C treatment in head-kidney cells, peripheral blood leukocytes (PBLs) and spleen cells, and by HIRRV infection in kidney of juvenile fish suggesting that Jf-ISG15 plays a role in fish antiviral response.     

Analysis of plasmids encoding the tyrosine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to elucidate mechanisms of tyramine accumulation during fish sauce production, two tyramine-producing bacterial strains, referred to as TyrA and TyrB, were isolated from fish sauce mash accumulating over 141 mg of tyramine per 100 g of sample. Both strains were identified as Tetragenococcus halophilus based on phenotypic characterization and a 16S rRNA gene sequence analysis. Molecular analysis of the tyramine-producing gene in the two strains confirmed the presence of a ~30-kb plasmid encoding a single copy of the pyridoxal phosphate-dependent tyrosine decarboxylase gene (tdcA) along with three other genes related to tyramine synthesis (tdc cluster). The complete nucleotide sequences of plasmids extracted from the two strains indicated that both plasmids were almost identical, except for a 1.6-kb transposon sequence in the plasmid from the strain TyrB. Both plasmids had a replication region, a plasmid maintenance region, and two putative mobile genetic elements located upstream and downstream of the tdc cluster. This structure was identical to that of tetragenococcal plasmids encoding histidine decarboxylase (hdcA), which were sequenced previously. These results suggest a common origin for plasmids encoding hdcA and tdcA. In addition, the genes for both these biogenic amines are distributed among tetragenococcal species via this plasmid.     

Locus-specific EPIC-PCR primers for four distinct calmodulin genes of the Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844)

International Aquatic Research - Nucleotide sequences of four distinct calmodulin genes (designated as CaM-A to -D) of the Pacific bluefin tuna (Thunnus orientalis) were compared. Nucleotide...     

Gene expression profile of HIRRV G and N protein gene vaccinated Japanese flounder, Paralichthys olivaceus during HIRRV infection

The glycoprotein (G protein) gene, but not the nucleocapsid protein (N protein) gene, of the hirame rhabdovirus (HIRRV) was previously shown to be highly effective in inducing a protective immune response in Japanese flounder (Paralichthys olivaceus) when used as a DNA vaccine. Our previous cDNA microarray analysis demonstrated that interferon-stimulated genes (ISGs) were strongly induced by the HIRRV G protein gene (pHRV-G) but not by the N protein gene (pHRV-N). However, the molecular basis for the difference in protective immunity between pHRV-G- and pHRV-N-vaccinated fish during HIRRV infection is still unclear. In this study, we use a DNA microarray to analyze differences of gene expression in pHRV-G- and pHRV-N-vaccinated fish during HIRRV infection. Microarray analyses showed substantial difference in gene expression patterns during HIRRV infection between fish vaccinated with pHRV-G and pHRV-N. In addition, genes having homology to mammalian T cell activation-related genes were up-regulated in the HIRRV G protein-vaccinated group.     

Genotyping‐by‐sequencing for construction of a new genetic linkage map and QTL analysis of growth‐related traits in Pacific bluefin tuna

Pacific bluefin tuna (Thunnus orientalis) has high market value, but its wild populations have decreased in recent years. The broodstock of Pacific bluefin tuna that were hatched artificially and reared under aquaculture conditions is beginning to be used for production. The creation of broodstock with commercially valuable traits, such as rapid growth, is therefore of great interest. Genetic linkage map‐based identification of markers associated with quantitative trait loci (QTLs) facilitates marker‐assisted selection (MAS) breeding and allows efficient genetic improvement of broodstock. Single nucleotide polymorphism (SNP)‐based genetic linkage map construction using the genotyping‐by‐sequencing method can expand the number of mapped markers and help identify growth‐related QTLs. In this study, we constructed sex‐specific maps for 24 linkage groups consisting of 677 SNP and 651 microsatellite markers. The total lengths of 93 progenies in the mapping population followed normal distribution, with an average length of 9.4 mm. We performed composite interval mapping in the mapping population. QTL analysis revealed one significant QTL in LG10 on the female linkage map. The genetic linkage map—the second such map generated for Pacific bluefin tuna—and the growth‐related QTLs detected in this study will be useful for tuna aquaculture MAS programs.