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1

Molecular cloning, expression, and in silico analysis of a long type peptidoglycan recognition protein from half-smooth tongue sole, Cynoglossus semilaevis (Actinopterygii: Pleuronectiformes: Cynoglossidae)

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Qi Z. T., Zhang Q. H., Wang Z. S., Qiu M., Huang Y., Gao Q.
Acta Ichthyologica et Piscatoria
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2014
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tom 44
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nr 3
Background. Half-smooth tongue sole, Cynoglossus semilaevis Günther, 1873, a marine teleost, is an important aquaculture species of great economic value. In recent years, its farm production increase coincided, however, with the number of reported cases of bacterial diseases. Further understanding of its immune response to bacterial pathogens can provide more information on pathogenesis and how to prevent disease using immune-related strategy. Peptidoglycan (PGN) recognition proteins (PGRPs) play important roles in the innate immunity against bacterial infection. In the presently reported study, a long type PGRP in half-smooth tongue sole (csPGRP-L) was cloned, and its sequence features, PGN binding ability, and mRNA expressions in different tissues after bacterial infection were also analyzed. Materials and Methods. The full length of csPGRP-L cDNA was obtained by RT-PCR and RACE-PCR method, and its sequence features were analyzed by multiple sequence alignment and phylogenetic tree. Meanwhile, its 3-D structure and PGN binding ability were analyzed by comparative modelling and molecular docking methods. Furthermore, the expressions of csPGRP-L in different tissues of healthy fish and fish infected with Streptococcus dysgalactiae were examined using quantitative real-time PCR method. Results. The full length of csPGRP-L cDNA was 1509 bp (GenBank accession No. HQ909441), with a 1446 bp of open reading frame (ORF) encoding 481 amino acids (aa), which possessed several conserved PGRP family features, e.g., a typical PGRP domain at its C-terminal, 3-D structure. Molecular docking showed that the csPGRP-L also possessed the PGN-binding ability. csPGRP-L was constitutive expressed in all the selected tissues from healthy fish and following S. dysgalactiae infection its expression was up-regulated in a tissue-specific expression pattern. Conclusion. The gene we cloned was exactly the homologue of vertebrates’ long type PGRP in half-smooth tongue sole which was confirmed by several analyses and the up-regulation of csPGRP-L after bacterial infection suggest that csPGRP-L plays important role in antibacterial and anti-infective action.
2

Changes of globins expression in tongue sole, Cynoglossus semilaevis (Actinopterygii: Pleuronectiformes: Cynoglossidae) in response to short-term hypoxia

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Wang Z. S., Qi Z. T., Qiu M., Zhao W. H., Wang A. M., Huang J. T., Guo X. J.
Acta Ichthyologica et Piscatoria
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2011
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tom 41
|
nr 3
Background. Fishes live in the aquatic systems which are first affected by low or temporally changing O2 availability. Under hypoxia, the fish possess varieties of mechanism to ensure the uptake of an adequate amount of O2. Tongue sole, Cynoglossus semilaevis Günther, 1873, lives on the bottom of water where contain low O2 concentration, indicating that this species possess specific feature against hypoxia. In this study, we examined the mRNA expression of three globins in tongue sole under short-term hypoxia. Materials and methods. Using bioinformatics analysis, three globin genes including hemoglobin-α1 (Hb-α1), myoglobin (Mb), and cytoglobin (Cygb) were first obtained from tongue sole EST database. Then, their expression levels in different tissues of tongue sole under short-term hypoxia were detected using quantitative real-time PCR method. Results. Under short-term hypoxia, Hb-α1 significantly increased in all the tissues except intestine and stomach. The Cygb expression level was up-regulated from 5 min to 120 min, whilst changed to normal level at 36 h. Mb was only up-regulated in blood and heart at 120 min. But, as the hypoxia prolonged, there was a mild increase of Mb in blood, heart, spleen, and liver. Conclusion. The three globins of tongue sole were up-regulated in some tissues, but their rolemay be different in different stage of hypoxia. Hb and Cygb might play role in the earlier stage, while Mb might play role in the late stage of hypoxia.
3

Molecular cloning and expression analysis of hypoxia inducible factor 1α in tongue sole, Cynoglossus semilaevis (Actinopterygii: Pleuronectiformes: Cynoglossidae), subjected to acute hypoxia

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Qi Z. T., Wang Z. S., Zhang Q. H., Zhao W. H., Qiu M., Peng J. Q.
Acta Ichthyologica et Piscatoria
|
2013
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tom 43
|
nr 3
Background. Hypoxia-inducible factor-1α (HIF-1α), a subunit of the HIF-1 protein, plays a key role in the regulation of genes involved in hypoxia physiological response. Tongue sole, Cynoglossus semilaevis Günther, 1873, a marine teleost, had been proved to be a hypoxia-tolerant species. In this study, the HIF-1α in tongue sole was cloned and its expression under acute hypoxia was examined to provide further basis for understanding the molecular response of tongue sole under hypoxia. Materials and Methods. The full length of HIF-1α cDNA sequence was cloned from the liver of tongue sole by RT-PCR and RACE-PCR method. Then, the expression pattern of tongue sole HIF-1α under acute hypoxic conditions were detected using quantitative real-time PCR method. Results. The open reading frame of tongue sole HIF-1α is 2208 bp, encoding 735 amino acids. The amino acid sequence of tongue sole HIF-1α shared high identities (52.7%–81.8%) with HIF-1α from other vertebrates, and possessed six typical domains of the HIF-1 family (bHLH, PAS-A, PAS-B, PAC, N-TAD, and C-TAD). In adult fish, HIF-1α mRNAs were highly expressed in the liver, moderately in the heart, spleen, kidney, stomach, blood and gills, and low in intestine. Under acute hypoxia stress, expression of HIF-1α mRNAs were significantly up-regulated in many tissues, including the liver, spleen, stomach, blood, heart and gills. Conclusion. Tongue sole HIF-1α possessed the similar sequence length, shared higher identities and clustered well with other known HIF-1α, thus revealing a high degree conservation of HIF-1α during evolution. Tongue sole HIF-1α began to be up-regulated from 5 min to 120 min after hypoxia, indicating that it might play a significant role at the early stage of hypoxia.
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