Qiwang Zhong

Expression Profiling and Validation of Potential Reference Genes During Paralichthys olivaceus Embryogenesis

Differential expression of genes is crucial to embryogenesis. The analysis of gene expression requires appropriate references that should be minimally regulated during the embryonic development. To select the most stable genes for gene normalization, the expression profiles of eight commonly used reference genes (ACTB, GAPDH, rpL17, α-Tub, EF1-α, UbcE, B2M, and 18S rRNA) were examined during Japanese flounder (Paralichthys olivaceus) embryonic development using quantitative real-time polymerase chain reaction. It was found that all seven mRNA genes appeared to be developmentally regulated and exhibited significant variation of expression. However, further analyses revealed the stage-specific expression stability. Hence when normalization using these mRNA genes, the differential and stage-related expression should be considered. 18S rRNA gene, on the other hand, showed the most stable expression and could be recommended as a suitable reference gene during all embryonic developmental stages in P. olivaceus. In summary, our results provided not only the appropriate reference gene for embryonic development research in P. olivaceus, but also possible guidance to reference gene selection for embryonic gene expression analyses in other fish species.

Full-length sequence and expression analysis of a myeloid differentiation factor 88 (MyD88) in half-smooth tongue sole Cynoglossus semilaevis.

Myeloid differentiation factor 88 (MyD88) is a universal and crucial adaptor protein, which plays an essential role in the intracellular signalling elicited by IL‐1R/TLR superfamily. In the present study, we report the full‐length sequence of MyD88 gene in half‐smooth tongue sole (Cynoglossus semilaevis). In the 2855 bp genomic sequence, five exons and four introns were identified. The cloned cDNA exhibited 110 bp of 5′ UTR, 576 bp of 3′ UTR and 858 bp of the entire open‐reading frame encoding a polypeptide of 285 amino acids. The protein sequence included a typical conserved cytosolic Toll/interleukin‐1 receptor (TIR) domain, an intermediate domain (ID) and a death domain (DD), and shared greater than 70% identity with Japanese flounder Paralichthys olivaceu ortholog. Real‐time polymerase chain reaction (RT‐PCR) analysis indicated a broad expression of csMyD88, especially in ovary and spleen. Quantitative RT‐PCR analysis indicated that the csMyD88 mRNA levels were significantly increased in the spleen and head kidney after inactive Vibrio anguillarum challenge and the expression of csMyD88 appeared to be developmentally regulated during C. semilaevis ontogeny. Although, species‐specific differences were present, the similarity between mammalian and piscine MyD88s suggested that the main function of MyD88 might be conserved across vertebrates.

Identification of differential genes in the ovary relative to the testis and their expression patterns in half-smooth tongue sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a rare marine flatfish whose mature ovary and testis greatly differ in volume and weight. The length and weight of mature females are over twice greater than those of mature males. To obtain sufficient information on gonad differentiation and the relationship between gonad development and growth in the fish, we compared gene expression between the ovary and testis using suppression subtractive hybridization (SSH). Testis cDNAs are subtracted from ovary cDNAs and are used to establish an ovary testis-subtracted cDNA library. A total of 41 nonredundant clones are identified, including 20 known cDNAs, 9 uncharacterized cDNAs (EST clones), and 12 novel sequences. For selected genes such as ZPC, RacGAP, survivin, aquaporin, CPEB, O5, O15, and O18, gene expression patterns are analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). The results confirm that these genes are only expressed in the ovary and not in the testis, or at higher levels in the ovary than in the testis. At the same time, expressions of certain genes such as ZPC, survivin, aquaporin, CPEB, and O15 are demonstrated to possess sexual dimorphism in the kidney or muscle, and spleen. The results suggest that these genes could play key roles not only in the ovary but in other female tissues as well.

Isolation, polymorphism and expression study of two distinct major histocompatibility complex class II B genes from half-smooth tongue sole (Cynoglossus semilaevis)

Major histocompatibility complex (MHC) class II antigens are important in vertebrate immune system, which present peptides to CD4+ T cells. In the present study, cDNAs encoding MHC class II B gene were isolated from the cDNA library of half‐smooth tongue sole (Cynoglossus semilaevis), and the full length cDNA sequences were got by rapid amplification of cDNA ends polymerase chain reaction. The polymorphism of its open reading frame, 3′ untranslated region and intron 1 was studied. Nineteen class II B alleles were identified from nine individuals and clustered into two groups, designated as Cyse‐DAB and Cyse‐DBB. The deduced amino acid sequences among Cyse‐DAB and Cyse‐DBB alleles shared identities from 94.0% to 99.6% and 92.4% to 99.6%, respectively, while the identities between Cyse‐DAB and Cyse‐DBB genes varied from 85.1% to 92.0%. Three Cyse‐DAB alleles and one Cyse‐DBB allele were observed in each of two individuals, and three Cyse‐DBB alleles and one Cyse‐DAB allele in another individual, which indicated at least two loci existed in each gene. Two different 3′ UTR sequences were identified and one belonged to Cyse‐DAB, the other belonged to Cyse‐DBB. Both five Cyse‐DAB and five Cyse‐DBB intron 1 sequences were identified from genomic sequences, among which two sequences of each gene were identified in a single individual, which suggested the existence of at least two Cyse‐DAB and two Cyse‐DBB loci. Both the two genes’‐specific tissue and developmental stage expression were studied by reverse‐transcription polymerase chain reaction, which showed that the two genes had similar expression patterns in tissue study with high expression in spleen and kidney, low expression in liver, gill and ovary, moderate expression in brain, heart, intestine and testis. While in developmental analysis, Cyse‐DBB had higher expression than Cyse‐DAB in early developmental stages, which indicated that the two genes might have different functions in those stages. Therefore, in half‐smooth tongue sole, two different MHC class II B genes exist and could differentiate from each other whether by sequence analysis or by developmental stage expression study.

Isolation and characterization of twenty novel microsatellite markers for half-smooth tongue sole (Cynoglossus semilaevis)

Twenty novel microsatellite markers for half-smooth tongue sole (Cynoglossus semilaevis) were isolated and characterized from a simple sequence repeat-enriched library constructed with (GA)15 and (CA)15. The polymorphism was assessed using 32 individuals collected from one locality along the Chinese coast. The result showed the number of alleles ranged from 2 to 14, with an average of 8.7 alleles/locus. The values of observed and expected heterozygosities ranged from 0.2609 to 1.0000 and from 0.2367 to 0.9716, respectively. Six microsatellite loci departed significantly from Hardy–Weinberg equilibrium. No linkage disequilibrium was found. These markers will be useful for the conservation studies and population structure assessment for this species.

Identification, structural characterization, and expression analysis of toll-like receptors 2 and 3 from gibel carp (Carassius auratus gibelio)

ABSTRACT Toll‐like receptors (TLRs) are important components of innate immunity. TLRs recognize pathogen‐associated molecular patterns (PAMPs) and initiate downstream signaling pathways in response. In present study, we report the identification of two TLRs from gibel carp (Carassius auratus gibelio), TLR2 and TLR3 (designated CagTLR2 and CagTLR3, respectively). We report on the genomic structures and mRNA expression patterns of CagTLR2 and CagTLR3. Five exons and four introns were identified from the genomic DNA sequence of CagTLR3 (4749 bp in total length); this genomic organization is similar to that of TLR3 in zebrafish and human. However, only one intron was identified from the CagTLR2 genomic locus (3166 bp in total length); this unique genomic organization of CagTLR2 is different from that of TLR2 in fish and humans. The cDNAs of CagTLR2 and CagTLR3 encoded 791 and 904 amino acid residues, respectively. CagTLR2 and CagTLR3 contained two distinct structural/functional motifs of the TLR family: a leucine‐rich repeat (LRR) domain in the extracellular portion and a toll/interleukin‐1 receptor (TIR) domain in the intracellular portion. The positions of critical amino acid residues involed in PAMP recognition and signaling pathway transduction in mammalian TLRs were conserved in CagTLR2 and CagTLR3. Phylogenetic analysis revealed a closer clustering of CagTLR2 and CagTLR3 with TLRs from freshwater fish than with marine fish species. In healthy gibel carp, transcripts of these genes were detected in all examined tissues, and high expression levels of CagTLR2 and CagTLR3 were observed in liver and brain, respectively. Following injection with CyHV‐2, expression levels of CagTLR2 and CagTLR3 were significantly upregulated in the spleens of gibel carp after three days, and CagTLR3 transcript levels were rapidly increased in head kidney after 12 h. These results suggest that CagTLR2 and CagTLR3 are functionally involved in the induction of antiviral immune response. HighlightsMolecular cloning and identification of TLR2 and TLR3 in gibel carp.Unique genomic organization of TLR2 in gibel carp was carefully characterized and discussed.TLR2 and TLR3 showed different expression patterns in response to DNA virus (CyHV‐2) infection in gibel carp.