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Quinolone-resistant strains of the fish-pathogenic bacterium, Photobacterium damselae subsp. piscicida are distributed widely in cultured yellowtail, Seriola quinqueradiata (Temminck & Schlegel), in Japan. The quinolone resistance-determining region (QRDR) was amplified with degenerate primers, followed by cassette ligation-mediated PCR. Open reading frames encoding proteins of 875 and 755 amino acid residues were detected in the gyrA and parC genes, respectively. Resistant strains of P. damselae subsp. piscicida carried a point mutation only in the gyrA QRDR leading to a Ser-to-Ile substitution at residue position 83. No amino acid alterations were discovered in the ParC sequence. A mutation in the gyrA gene was also detected in nalidixic acid-resistant mutants of strain SP96002 obtained from agar medium containing increased levels of quinolone. These results suggest that GyrA, as in other Gram-negative bacteria, is a target of quinolone in P. damselae subsp. piscicida. Furthermore, we attempted to detect a point mutation using targeting-induced local lesions in genomes (TILLING), which is a general strategy used for the detection of a variety of induced point mutations and naturally occurring polymorphisms. We developed a new detection method for the rapid and large-scale identification of quinolone-resistant strains of P. damselae subsp. piscicida using TILLING. 相似文献
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Duangjai Pisuttharachai Motoshige Yasuike Hideaki Aono Keisuke Murakami Hidehiro Kondo Takashi Aoki Ikuo Hirono 《Fisheries Science》2009,75(1):195-206
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. 相似文献
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Rodkhum C Hirono I Stork M Di Lorenzo M Crosa JH Aoki T 《Journal of fish diseases》2006,29(3):157-166
The genome of Vibrio anguillarum strain H775-3 was partially determined by a random sequencing procedure. A total of 2,300 clones, 2,100 from a plasmid library and 200 from a cosmid library, were sequenced and subjected to homology search by the BLAST algorithm. The total length of the sequenced clones is 1.5 Mbp. The nucleotide sequences were classified into 17 broad functional categories. Forty putative virulence-related genes were identified, 36 of which are novel in V. anguillarum, including a repeat in toxin gene cluster, haemolysin genes, enterobactin gene, protease genes, lipopolysaccharide biosynthesis genes, capsule biosynthesis gene, flagellar genes and pilus genes. 相似文献
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S D Hwang N Midorikawa P Punnarak Y Kikuchi H Kondo I Hirono T Aoki 《Journal of fish diseases》2012,35(12):927-934
RNA aptamers are artificial nucleic acids that specifically bind to a wide variety of targets. They are an effective tool for pharmaceutical research and development of antiviral agents. Here, we describe four Hirame rhabdovirus (HIRRV)‐RNA aptamers (H1, H2, H3 and H4) that we obtained from an in vitro process called the systematic evolution of ligands by exponential enrichment (SELEX). The HIRRV‐RNA aptamers specifically bind to HIRRV. Hirame natural embryo (HINAE) cells treated with virus and the RNA aptamer showed a decrease in appearance of cytopathic effect when compared with control (treated only with virus). Rhodovulum sulfidophilum was transformed with genes for the RNA aptamers, and the aptamers were detected in the culture medium, indicating that they were secreted from the cells. Thus, the recombinant R. sulfidophilum might be a powerful tool for the prevention of HIRRV in aquaculture. 相似文献
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Pitchaya Chainark Shuichi Satoh Ikuo Hirono Takashi Aoki Makoto Endo 《Fisheries Science》2008,74(2):380-390
ABSTRACT: Foreign DNA fragments from genetically modified defatted soybean meal (GM SBM) in rainbow trout was traced by nested polymerase chain reaction (PCR) and located by in situ hybridization. Either a GM or non-GM SBM formulated diet (42% protein) was fed to fish (average weight 50.5 g) for 2 weeks. The degradation results showed that the cauliflower mosaic virus 35S promoter (220 bp) fragment was detected in the contents of digestive system only in fish fed the GM SBM diet, and it was not detected on the third day after changing the diet to the non-GM SBM diet. For the possible transferal results, the promoter fragment was detected in the leukocyte, head kidney and muscle only of fish fed the GM SBM diet; it was not detected on the fifth day after changing the diet to the non-GM SBM diet. These results suggest that a foreign DNA fragment was not completely degraded and might be taken up into organs through the gastrointestinal tract. However, foreign DNA was not detected after the withdrawal period. Thus, the data show that uptake of DNA from GM SBM might not remain in the tissues of fish fed GM SBM diet. 相似文献
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Ryosuke?Yazawa Ikuo?Hirono Ehchi?Yamamoto Takashi?AokiEmail author 《Fisheries Science》2005,71(4):869-874
ABSTRACT: Fish transgenesis has progressed considerably. However, the technique of gene transfer for most marine aquaculture species has not been established. A method to introduce foreign genes into fertilized eggs of Japanese flounder Paralichthys olivaceus by particle gun bombardment was developed by the authors. A recombinant plasmid which contains the Japanese flounder keratin gene promoter linked to the green fluorescence protein (GFP) gene was introduced into fertilized eggs by particle gun bombardments twice at 250 psi. In each experimental group, 25 000–35 000 eggs were treated. However, the survival rate was 12.8% which is lower than that of the control groups (56.8%). Of 2606–5205 the embryos survived for 24 h, 43–61 GFP positive embryos were obtained in one experiment, giving a final gene transfer efficiency of 1.4%. All GFP-positive embryos developed and hatched normally. GFP expression was observed in epithelial tissue throughout the developmental stages. At 3 months after gene transfer, foreign DNA was detected by genome polymerase chain reaction analysis in 37 of 69 fry (53.6%). These results suggest that the particle gun method is an effective method to use with fertilized Japanese flounder eggs. 相似文献
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AbstractThe aim of this study was to assess the mitigating effects of lime nitrogen (calcium cyanamide) and dicyandiamide (DCD) application on nitrous oxide (N2O) emissions from fields of green tea [Camellia sinensis (L.) Kuntze]. The study was conducted in experimental tea fields in which the fertilizer application rate was 544 kg nitrogen (N) ha?1 yr?1 for 2 years. The mean cumulative N2O flux from the soil between the canopies of tea plants for 2 years was 7.1 ± 0.9 kg N ha?1 yr?1 in control plots. The cumulative N2O flux in the plots supplemented with lime nitrogen was 3.5 ± 0.1 kgN ha?1, approximately 51% lower than that in control plots. This reduction was due to the inhibition of nitrification by DCD, which was produced from the lime nitrogen. In addition, the increase in soil pH by lime in the lime nitrogen may also be another reason for the decreased N2O emissions from soil in LN plots. Meanwhile, the cumulative N2O flux in DCD plots was not significantly different from that in control plots. The seasonal variability in N2O emissions in DCD plots differed from that in control plots and application of DCD sometimes increased N2O emissions from tea field soil. The nitrification inhibition effect of lime nitrogen and DCD helped to delay nitrification of ammonium-nitrogen (NH4+-N), leading to high NH4+-N concentrations and a high ratio of NH4+-N /nitrate-nitrogen (NO3–-N) in the soil. The inhibitors delayed the formation of NO3–-N in soil. N uptake by tea plants was almost the same among all three treatments. 相似文献