EST analysis and identification of gonad-related genes from the normalized cDNA library of large yellow croaker,Larimichthys crocea

On grounds of the especially limited numbers of identified gonad-specific or gonad-related genes of large yellow croaker Larimichthys crocea which may represent a major obstacle for the study of gonad development and sex differentiation, we initiated a sequencing program of Expressed Sequence Tags (ESTs) in large yellow croaker. In this study, we firstly constructed a normalized gonad cDNA library using the combination of SMART technique and DSN treatment. The titer of amplified cDNA library was 4.8 × 10¹¹ and the percentage of unique cDNA sequences of the library was 82.49%. 2916 unique cDNAs were clustered from the 3535 high quality ESTs. Among the 1785 ESTs which had significant homology with known genes in the NCBI database, about 64 significant gonad-related genes were found, accounting for 3.59% of the total unique cDNAs. Specifically, the testis-specific LRR gene and testis-specific chromodomain Y-like protein gene were identified from fish for the first time. Six gonad-related microsatellite-containing ESTs were identified from the 129 ESTs containing 149 microsatellites. Expression patterns of 10 of these gonad-related gene homologues in ovaries and testes were examined by qRT-PCR. The results will be powerful resources for our further investigation to establish the molecular mechanisms of gonad development and sex differentiation in large yellow croaker.     

Cloning and molecular characterization of lycC1INH genes in large yellow croaker (Pseudosciaena crocea)

Complement component 1 inhibitor (C1INH) is a crucial protein in controlling activation of many plasma mediator pathways and can directly interact with Gram negative bacteria. The full-length cDNA of lycC1INH gene was identified from the large yellow croaker. It is of 2046 nucleotides (nt) encoding a protein of 599 amino acids, with a 5′-untranslated region of 99 nt and a 3′-untranslated region of 147 nt including the poly (A) tail. The deduced protein contains a C-terminal serpin (serine protease inhibitor) domain, and two N-terminus immunoglobulin domains without significant homology to other species. Western blot analysis of the protein expression showed that the expression of lycC1INH was obviously up-regulated in liver, spleen and head kidney of the fish challenged by attenuated live Vibrio anguillarum strain. This indicated that lycC1INH might be involved in the immune response of large yellow croaker to bacterial challenge.     

Beta2-microglobulin is involved in the immune response of large yellow croaker to Aeromonas hydrophila: A proteomic based study

Outbreaks of infectious diseases in cultured large yellow croaker have resulted in great economic losses. However, information regarding its immune defense is limited. In the present study, an approach by the combination of differential proteomics with EST resource was applied for investigation of a profile of serum immune response by large yellow croaker to Aeromonas hydrophila challenge after immunization and for characterizing of one of the targeted immune molecules. Of the twelve altered proteins involved in the response, eight were identified by MS, in which three were randomly selected for antiserum preparation and were further confirmed by Western blotting. Furthermore, three β₂m clones, one of the altered molecules, were obtained from a previously constructed Kidney Smart cDNA library of this fish, and were compared for their identity, which contributed to the identification of β₂m cDNA diversity. Meanwhile, the up-regulated β₂m in response to the bacterial immunization and challenge was further confirmed by Western blotting. Our results indicate that β₂m is involved in the immune response of large yellow croaker to the challenge by A. hydrophila after immunization, which suggests an efficient approach for characterizing of targeted molecules at both the gene and protein levels.