Quanqi Zhang

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.

Polymorphism in a serine protease inhibitor gene and its association with disease resistance in the eastern oyster (Crassostrea virginica Gmelin)

Serine protease inhibitors (SPIs) are a superfamily of structurally related but functionally diverse proteins found in almost all organisms ranging from viruses to humans. Some of them play important roles in host defense. A recently identified SPI from the eastern oyster (Crassostrea virginica), cvSI-1, has been shown to inhibit the proliferation of the Dermo pathogen Perkinsus marinus in vitro, although direct evidence linking it to disease resistance is lacking. In this study, we identified polymorphism in the cvSI-1 gene and studied its association with improved survival after disease-caused mortalities and in disease-resistant eastern oyster strains. Full-cDNA sequence of cvSI-1 was sequenced in a diverse panel of oysters, revealing 12 single-nucleotide polymorphisms (SNPs) in the 273 bp coding region: five were synonymous and seven non-synonymous. The Dn/Ds ratio, 1.4, suggests that cvSI-1 is under positive selection. Selected SNPs were genotyped in families before and after disease-caused mortalities as well as in disease-resistant and susceptible strains. At SNP198, the C allele consistently increased in frequency after mortalities that are caused primarily by Dermo and possibly also by MSX. Its frequency in the disease-resistant strain is significantly higher than that in the susceptible strains and the base population from which the selected strains were derived. These results indicate that polymorphism at cvSI-1 is associated with Dermo (possibly also MSX) resistance in the eastern oyster. SNP198 is a synonymous mutation, and its association with disease resistance may be due to its close linkage to a functional polymorphism nearby.

Mutation in promoter region of a serine protease inhibitor confers Perkinsus marinus resistance in the eastern oyster (Crassostrea virginica).

Protease inhibitors from the host may inhibit proteases from invading pathogens and confer resistance. We have previously shown that a single-nucleotide polymorphism (SNP198C) in a serine protease inhibitor gene (cvSI-1) is associated with Perkinsus marinus resistance in the eastern oyster. As SNP198 is synonymous, we studied whether its linkage to polymorphism at the promoter region could explain the resistance. A 631 bp fragment of the promoter region was cloned by genome-walking and resequenced, revealing 22 SNPs and 3 insertion/deletions (indels). A 25 bp indel at position -404 was genotyped along with SNP198 for association analysis using before- and after-mortality samples. After mortalities that were primarily caused by P. marinus, the frequency of deletion allele at -404indel increased by 15.6% (p = 0.0437), while that of SNP198C increased by only 3.4% (p = 0.5756). The resistance alleles at the two loci were coupled in 79.6% of the oysters. Oysters with the deletion allele at -404indel showed significant (p = 0.0189) up-regulation of cvSI-1 expression under P. marinus challenge. Our results suggest that mutation at the promoter region causes increased transcription of cvSI-1, which in turn confers P. marinus resistance in the eastern oyster likely through inhibiting pathogenic proteases from the parasite.

Identification and characterization of a hepcidin from half-smooth tongue sole Cynoglossus semilaevis.

Hepcidin, an antimicrobial peptide, has a dual function including innate immunity and iron regulation. Here, based on the sequence of an EST database, we have isolated and characterized a hepcidin gene (referred to as CsHepcidin) from half-smooth tongue sole (Cynoglossus semilaevis). Analysis of the coding regions indicated CsHepcidin gene comprised 3 exons and 2 introns. The putative CsHepcidin showed a great similarity to other hepcidin orthologues, particularly with respect to its 24 aa signal peptide, typical RX(K/R)R motif and eight conserved cysteine residues in the mature cationic peptide. Phylogenic analysis indicated that CsHepcidin was a hepcidin 1-type peptide of acanthopterygians, with highly homologous with Solea senegalensis hepcidin. In C. semilaevis ontogeny, CsHepcidin mRNA was detected at a low level in unfertilized eggs, increased on 6 d after hatching, and decreased remarkably at metamorphic stage. CsHepcidin transcripts showed a constitutive basal expression in most of the tissues, especially in liver. Challenge with formalin-inactivated Vibrio anguillarum led to significantly up-regulations of CsHepcidin gene in liver, head kidney and spleen in time-dependent manners. Biological activity analysis showed that recombinant CsHEP exhibited direct antimicrobial activity against bacterial pathogens in vitro, particularly showed strong activity against the principal fish pathogens, V. anguillarum and Edwardsiella tarda. All these results suggest that CsHepcidin may be involved in the initial response to invasion of microbial pathogens. Further exploration to elucidate the role of CsHepcidin in iron regulation and embryogenesis in C. semilaevis are needed.

Intragenomic variability and pseudogenes of ribosomal DNA in Stone flounder Kareius bicoloratus

Molecular study of Stone flounder Kareius bicoloratus revealed a high level of intra-individual polymorphism of ribosomal DNA (18S rDNA, ITS1 and 5.8S rDNA). This polymorphism includes not only multiple functional genes but also putative pseudogenes and recombinants. Three major types (Type I, II and III) of 18S rDNA differing in lengths coexisted in the genome of each studied individual. Type II and Type III have a 15-bp-deletion and Type III has an additional 7-bp-deletion relative to Type I. Expression analyses gave clear evidence that Type I 18S rDNA was abundantly expressed. In contrast, transcription of Type II and III 18S rDNA were not detected. Further sequence analysis revealed that Type I was the expected and functional 18S rDNA in K. bicoloratus, while Type II and Type III were pseudogenes. Phylogeny of the ITS1 sequences formed three groups (Group I, II and III), of which Group I linking with Type I 18S rDNA were functional, whereas the remaining two Groups (II and III) linking with Type II and Type III 18S rDNA were pseudogenes. Groups II and III ITS1 sequences were shorter (685-717bp in length) than Group I ITS1 (762-776bp), but had much higher sequence divergence (9.8%) than the later ones did (4.9%). Group II sequences were recombinants between Group I and Group III sequences, providing the possibility that the rDNA of K. bicoloratus is in a transition stage of concerted evolution. In addition, by comparing standard PCR and high-stringency PCR, we concluded that DMSO might prevent functional ITS1 sequences from being amplified instead of enhancing its amplification efficiency. The analysis suggests a non-concerted evolution of rDNA in K. bicoloratus. Features of rDNA functional genes and pseudogenes are described. Possible causes for the lack of homogenization of rDNA paralogues within individuals are discussed.

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.

Detection of Alternative Splice and Gene Duplication by RNA Sequencing in Japanese Flounder, Paralichthys olivaceus

Japanese flounder (Paralichthys olivaceus) is one of the economic important fish in China. Sexual dimorphism, especially the different growth rates and body sizes between two sexes, makes this fish a good model to investigate mechanisms responsible for such dimorphism for both fundamental questions in evolution and applied topics in aquaculture. However, the lack of “omics” data has hindered the process. The recent advent of RNA-sequencing technology provides a robust tool to further study characteristics of genomes of nonmodel species. Here, we performed de novo transcriptome sequencing for a double haploid Japanese flounder individual using Illumina sequencing. A single lane of paired-end sequencing produced more than 27 million reads. These reads were assembled into 107,318 nonredundant transcripts, half of which (51,563; 48.1%) were annotated by blastx to public protein database. A total of 1051 genes that had potential alternative splicings were detected by Chrysalis implemented in Trinity software. Four of 10 randomly picked genes were verified truly containing alternative splicing by cloning and Sanger sequencing. Notably, using a doubled haploid Japanese flounder individual allow us to analyze gene duplicates. In total, 3940 “single-nucleotide polymorphisms” were detected form 1859 genes, which may have happened gene duplicates. This study lays the foundation for structural and functional genomics studies in Japanese flounder.

Isolation and characterization of 64 novel microsatellite markers from a fosmid library of female half‐smooth tongue sole (Cynoglossus semilaevis)

Microsatellite markers were developed from a fosmid library of female half‐smooth tongue sole, Cynoglossus semilaevis. Three hundred eighty‐four clones were randomly selected to sequence (double strand reading), and 168 sequences in 143 clones were found to contain microsatellites. Of the 101 primer pairs designed, 64 gave polymorphic polymerase chain reaction products. Based on characterization with 36 individuals, the number of alleles ranged from two to nine. The values of observed and expected heterozygosities varied from 0.06 to 1.00 and from 0.05 to 0.94, respectively. These markers have the potential as tools for population structure evaluation, ecological analyses and linkage map construction.

Reference gene selection for quantitative real-time RT-PCR normalization in the half-smooth tongue sole (Cynoglossus semilaevis) at different developmental stages, in various tissue types and on exposure to chemicals.

Quantitative real time RT-PCR has been described as the most sensitive method for the detection of low abundance mRNA. To date, no reference genes have been screened in the half-smooth tongue sole (Cynoglossus semilaevis). The aim of this study was to select the most stable genes for quantitative real-time RT-PCR. Eight housekeeping genes (18S, TUBA, B2M, ACTB, EF1A, GAPDH, RPL17 and UBCE) were tested at different developmental stages, in different tissues, and following exposure to the drug SB-431542. Using geNorm, BestKeeper and NormFinder software, GAPDH/B2M, GAPDH/18S and UBCE/GAPDH were identified as the most suitable genes from samples taken of different developmental stages while 18S/RPL17 were consistently ranked as the best reference genes for different tissue types. Furthermore, TUBA/B2M, TUBA/UBCE and B2M/TUBA were found to be the most suitable genes in samples treated with the drug, SB-431542 by geNorm, BestKeeper and NormFinder respectively. Across both different developmental stages and tissue types, the combination of 18S and GAPDH was the most stable reference gene analyzed by Ref-Finder. To test and verify the screened reference genes, the expression profiles of LEFTY-normalized to the combination of GAPDH/18S and ACTB were presented. These results will be useful for future gene-expression studies in the half-smooth tongue sole.

Characterisation of kisspeptin system genes in an ovoviviparous teleost: Sebastes schlegeli

Kisspeptins are neuropeptides that play important roles in the reproduction and the onset of puberty in vertebrate by activating their receptor, Kissr. In the present study, we first isolated kiss1 and kissr4 genes from an ovoviviparous fish, the black rockfish (Sebastes schlegeli) by homologue cloning. Phylogenetic analysis indicated that the kiss and kissr of S. schlegeli belonged to kiss1 and kissr4 respectively. Quantitative real-time PCR analysis showed that the kissr4 was expressed mainly in the brain and testis, while the kiss1 was expressed predominantly in the heart of both sexes. As for the different gonadal maturation stages the kiss1 showed different expression patterns in different tissues. During the early development stage, expression levels of the ligand and receptor genes showed similar increasing trends. The promoter region of kissr4 contained several putative transcription factor (TF) binding sites which may have the function of regulating kisspeptin system gene expression, providing potential targets for future in-depth investigation. These results together confirmed that the kisspeptin system in S. schlegeli may be involved in reproduction and other activities. Furthermore, our study laid the groundwork for further learning about the evolution and function of kisspeptin system in fish even vertebrate.