RESUMO
Biofilm formation of pathogen bacterium is currently one of the most widely studied topics; however, little is known regarding pathogen bacteria biofilms in aquaculture. Aeromonas hydrophila is a representative species of the genus Aeromonas, which has been recognized as a common pathogen, is associated with many diseases in aquatic animals, and causes significant mortality. The objectives of this study are i) to confirm that A. hydrophila can form biofilms on abiotic substrates and construct a biofilm growth curve for this bacterium; ii) to identify the genes that play crucial roles in A. hydrophila biofilm formation. The biofilm growth curve of A. hydrophila was constructed using a crystal violet assay, which showed that biofilm formation for this bacterium is a dynamic process. Next, a mutant library of pathogenic A. hydrophila B11 was constructed using the mini-Tn10 transposon mutagenesis system. A total of 861 mutants were screened, and 5 mutants were stably deficient in biofilm formation. Molecular analysis of the mutant B112 revealed that the open reading frame that encodes the protein MshQ was disrupted. Comparison of biological characteristics including growth, motility, and adhesion between the mutant B112 and the wild-type strain B11 suggested that MshQ is necessary for mannose-sensitive hemagglutinin pilus biosynthesis of A. hydrophila, and that these pili play crucial roles in A.hydrophila adherence to a solid surface during the early stages of biofilm formation.
Assuntos
Aeromonas hydrophila/fisiologia , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Fímbrias Bacterianas/fisiologia , Aeromonas hydrophila/genética , Aeromonas hydrophila/metabolismo , Sequência de Aminoácidos , Aderência Bacteriana/genética , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Elementos de DNA Transponíveis/genética , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Fases de Leitura Aberta/genética , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
In order to investigate the immune role of ribosomal protein L10 (RPL10/QM-like gene) in marine fish, we challenged the large yellow croaker Pseudosciaena (= Larimichthys) crocea, the most important marine fish culture species in China, by injection with a mixture of the bacteria Vibrio harveyi and V. parahaemolyticus (3:1 in volume). Microarray analysis and real-time PCR were performed 24 and 48 h post-challenge to isolate and identify the QM-like gene from the gill P. crocea (designated PcQM). The expression level of the PcQM gene did not changed significantly at 24 h post-challenge, but was significantly downregulated at 48 h post-challenge, suggesting that the gene had an immune-modulatory effect in P. crocea. Full-length PcQM cDNA and genomic sequences were obtained by rapid amplification of cDNA ends (RACE)-PCR. The sequence of the PcQM gene clustered together with those of other QM-like genes from other aquatic organisms, indicating that the QM-like gene is highly conserved in teleosts.
Assuntos
Imunidade/genética , Perciformes/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Regulação da Expressão Gênica , Genoma/genética , Brânquias/metabolismo , Brânquias/microbiologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Perciformes/microbiologia , Filogenia , Proteína Ribossômica L10 , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Alinhamento de Sequência , Vibrio/fisiologia , Vibrioses/genética , Vibrioses/imunologiaRESUMO
We isolated and characterized 21 polymorphic microsatellite loci in Japanese Spanish mackerel (Scomberomorus niphonius) using a (GT)(13)-enriched genomic library. Forty individuals were collected from Qingdao, China. We found 3 to 24 alleles per locus, with a mean of 8.8. The observed and expected heterozygosities ranged from 0.263 to 0.975 and from 0.385 to 0.946, with means of 0.655 and 0.685, respectively. Deviation from Hardy-Weinberg proportions was detected at three loci. Two loci showed evidence for null alleles. These microsatellite markers will be useful for population genetic analysis of Japanese Spanish mackerel.
Assuntos
Repetições de Microssatélites/genética , Perciformes/genética , Polimorfismo Genético , Animais , Sequência de Bases , Primers do DNARESUMO
Growth hormone-releasing hormone (GHRH) and pituitary adenylate cyclase activating polypeptide (PACAP) regulate development and somatic growth in teleosts; they may be associated with sexual growth dimorphism in the half-smooth tongue sole (Cynoglossus semilaevis). We found that the full-length GHRH and PACAP gene sequences obtained from females and males consist of 4160, 4159, 2425, and 2446 bp, respectively, each of which includes four exons and three introns. When we analyzed normal females and males and extra-large male adults, GHRH and PACAP mRNA were found to be predominantly expressed in the brain; the expression levels were highest in normal males. The extra-large males exhibited the lowest mRNA levels of both GHRH and PACAP. Sex differences in GHRH and PACAP mRNA expression during development were also examined in a full-sib family; GHRH and PACAP mRNA were detected at all 27 times sampled from 10 to 410 days old. The GHRH expression levels in females were significantly higher than in males at most of the stages between 20 and 100 days old, while lower than those of males after 120 days old. Five microsatellite loci were identified in GHRH and PACAP genes. We used these five polymorphic markers to genotype 224 individuals, and no significant differences were found between females and males from the Bohai Sea, the Yellow Sea and hatchery samples.
Assuntos
Linguados/genética , Regulação da Expressão Gênica , Genoma/genética , Hormônio Liberador de Hormônio do Crescimento/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polimorfismo Genético , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Peso Corporal/genética , Diploide , Feminino , Linguados/anatomia & histologia , Linguados/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Genótipo , Hormônio Liberador de Hormônio do Crescimento/metabolismo , Masculino , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We isolated and characterized 14 polymorphic microsatellite loci in the Japanese anchovy (Engraulis japonicus) using a (GT)(13)-enriched genomic library. The numbers of alleles per locus ranged from 6 to 31, with a mean of 17.8. The observed and expected heterozygosities ranged from 0.180 to 0.949 and from 0.172 to 0.966, with means of 0.731 and 0.825, respectively. All 14 loci were in Hardy-Weinberg equilibrium and no significant linkage disequilibrium between loci pairs was detected. These microsatellite markers will be useful for analyzing the population genetic structure and gene flow of E. japonicus.