不同光周期条件下日本牙鲆尾部神经分泌系统转录组分析

为发掘日本牙鲆响应光周期变化的重要功能基因,采用新一代高通量测序RNA-seq技术分析8L∶16D、12L∶12D和16L∶8D等3个光周期条件下日本牙鲆尾部神经分泌系统(caudal neurosecretory system,CNSS)的基因表达变化。转录组测序结果显示,3个样品分别产生了5 807 622、6 147 140和6 116 872个Clean reads。分别对8L∶16D、12L∶12D和16L∶8D条件下的文库进行两两比较,共获得200个差异表达基因。GO分类分析表明,差异表达基因属于生物学过程、细胞定位和分子功能的42个类别。KEGG Pathway显著性富集分析差异表达基因共涉及29条代谢途径,包括糖酵解/糖异生、钙离子信号转导、血管平滑肌收缩和光信号转导等通路。上述结果不仅加深了对鱼类尾部神经分泌系统功能的认识,也为进一步探索硬骨鱼类光信号转导机制提供了多方面多层次的信息。

日本牙鲆尾部神经分泌系统CRH、UⅠ和UⅡ基因表达及荷尔蒙分泌日节律变化

尾部神经分泌系统（CNSS）是硬骨鱼类特有的神经分泌系统,其主要分泌物为促肾上腺皮质激素释放因子（CRH）、尾紧张素Ⅰ和尾紧张素Ⅱ（U Ⅰ、UⅡ）.选取平均体重为600 g的日本牙鲆（Paralichthys olivaceus）,系统研究了日本牙鲆尾部神经分泌系统日节律的变化.荧光定量PCR分析表明,昼夜变化过程中,UⅠ和UⅡmRNA表达量没有显著变化;CRH mRNA表达量在6：00～12：00显著升高,12：00后降低.余弦法分析表明,CRH、UⅠ和UⅡmRNA表达峰值依次出现在12：00、18：00、12：00.免疫组织化学染色结果显示,CNSS分泌细胞——Dahlgren细胞中CRH、UⅠ和UⅡ抗血清免疫反应阳性细胞总数目均无显著性差异,均无日节律现象.酶联免疫反应及放射免疫反应检测血浆中激素含量表明,血浆中CRH、UⅠ、UⅡ、促肾上腺皮质激素（ACTH）和皮质醇（Cortisol）含量在光照-黑暗循环中均没有显著性差异,但UⅠ、UⅡ和ACTH含量随昼夜交替呈先升高后降低的趋势.上述结果表明,日本牙鲆尾部神经分泌系统各基因存在转录振荡,有一定的日节律变化,激素分泌量亦随日周期发生改变.这证明CNSS在牙鲆适应环境变化过程中发挥重要调节作用.

Distribution and expression in vitro and in vivo of DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

Lymphocystis disease,caused by the lymphocystis disease virus (LCDV),is a significant worldwide problem in fish industry causing substantial economic losses.In this study,we aimed to develop the DNA vaccine against LCDV,using DNA vaccination technology.We evaluated plasmid pEGFP-N2-LCDV1.3 kb as a DNA vaccine candidate.The plasmid DNA was transiently expressed after liposome transfection into the eukaryotic COS 7 cell line.The distribution and expression of the DNA vaccine (pEGFP-N2-LCDV1.3kb) were also analyzed in tissues of the vaccinated Japanese flounder by PCR,RT-PCR and fluorescent microscopy.Results from PCR analysis indicated that the vaccine-containing plasmids were distributed in injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver,6 and 25 days after vaccination.The vaccine plasmids disappeared 100 d post-vaccination.Fluorescent microscopy revealed green fluorescence in the injected muscle,the muscle opposite the injection site,the hind intestine,gill,spleen,head,kidney and liver of fish 48 h post-vaccination,green fluorescence did not appear in the control treated tissue.Green fluorescence became weak at 60 days post-vaccination.RT-PCR analysis indicated that the mcp gene was expressed in all tested tissues of vaccinated fish 6-50 days post-vaccination.These results demonstrate that the antigen encoded by the DNA vaccine is distributed and expressed in all of the tissues analyzed in the vaccinated fish.The antigen would therefore potentially initiate a specific immune response.The plasmid DNA was injected into Japanese flounder (Paralichthys olivaceus) intramuscularly and antibodies against LCDV were evaluated.The results indicate that the plasmid encoded DNA vaccine could induce an immune response to LCDV and would therefore offer immune protection against LCD.Further studies are required for the development and application of this promising DNA vaccine.

Genetic diversity in two Japanese flounder populations from China seas inferred using microsatellite markers and COI sequences

Japanese flounder is one of the most important commercial species in China; however, information on the genetic background of natural populations in China seas is scarce. The lack of genetic data has hampered fishery management and aquaculture development programs for this species. In the present study, we have analyzed the genetic diversity in natural populations of Japanese flounder sampled from the Yellow Sea （Qingdao population, QD） and East China Sea （Zhoushan population, ZS） using 10 polymorphic mierosatellite loci and cytochrome c oxidase subunit I （COI） sequencing data. A total of 68 different alleles were observed over 10 microsatellite loci. The total number of alleles per locus ranged from 2 to 9, and the number of genotypes per locus ranged from 3 to 45. The observed heterozygosity and expected heterozygosity in QD were 0.733 and 0.779, respectively, and in ZS the heterozygosity values were 0.708 and 0.783, respectively. Significant departures from Hardy-Weinberg equilibrium were observed in 7 of the 10 microsatellite loci in each of the two populations. The COI sequencing analysis revealed 25 polymorphic sites and 15 haplotypes in the two populations. The haplotype diversity and nucleotide diversity in the QD population were 0.746±0.072 8 and 0.003 34±0.001 03 respectively, and in ZS population the genetic diversity values were 0.712±0.047 0 and 0.003 18±0.000 49, respectively. The microsatellite data （Fst=0.048 7, P〈0.001） and mitochondrial DNA data （Fst=0.128, P〈0.001） both revealed significant genetic differentiation between the two populations. The information on the genetic variation and differentiation in Japanese flounder obtained in this study could be used to set up suitable guidelines for the management and conservation of this species, as well as for managing artificial selection programs. In future studies, more geographically diverse stocks should be used to obtain a deeper understanding of the population structure of Japanese flounder in the China seas and adjacent regions.

Analysis of New Microsatellite Markers Developed From Reported Sequences of Japanese Flounder Paralichthys olivaceus

The expressed sequence tags（ESTs）of Japanese flounder,Paralichthys olivaeeus,were selected from GenBank to identify simple sequence repeats（SSRs）or microsatellites.A bioinformatic analysis of 11111 ESTs identified 751 SSR-containing ESTs,including 440 dinucleotide,254 trinucleotide,53 tetranueleotide,95 pentanucleotide and 40 hexanucleotide microsatellites respectively.The CA/TG and GA/TC repeats were the most abundant microsatellites.AT-rich types were predominant among trinucleotide and tetranucleotide microsatellites.PCR primers were designed to amplify 10 identified microsatellites loci.The PCR results from eight pairs of primers showed polymorphisms in wild populations.In 30 wild individuals,the mean observed and expected heterozygosities of these 8 polymorphic SSRs were 0.71 and 0.83 respectively and the average PIC value was 0.8.These microsatellite markers should prove to be a useful addition to the microsatellite markers that are now available for this species.

Cloning of the cDNA encoding adenosine 5'-monophosphate deaminase 1 and its mRNA expression in Japanese flounder Paralichthys olivaceus

AMP deaminase catalyzes the conversion of AMP into IMP and ammonia. In the present study, a full-length cDNA of AMPD1 from skeletal muscle of Japanese flounder Paralichthys olivaceus was cloned and characterized. The 2 526 bp cDNA contains a 5'-UTR of 78 bp, a 3'-UTR of 237 bp and an open reading frame (ORF) of 2 211 bp, which encodes a protein of 736 amino acids. The predicted protein contains a highly conserved AMP deaminase motif (SLSTDDP) and an ATP-binding site sequence (EPLMEEYAIAAQVFK). Phylogenetic analysis showed that the AMPD1 and AMPD3 genes originate from the same branch, but are evolutionarily distant from the AMPD2 gene. RT-PCR showed that the flounder AMPD1 gene was expressed only in skeletal muscle. QRT-PCR analysis revealed a statistically significant 2.54 fold higher level of AMPD1 mRNA in adult muscle (750±40 g) compared with juvenile muscle (7.5±2 g) (P<0.05). HPLC analysis showed that the IMP content in adult muscle (3.35±0.21 mg/g) was also statistically significantly higher than in juvenile muscle (1.08±0.04 mg/g) (P<0.05). There is a direct relationship between the AMPD1 gene expression level and IMP content in the skeletal muscle of juvenile and adult flounders. These results may provide useful information for quality improvement and molecular breeding of aquatic animals.

Detection of lymphocystis disease virus in Japanese flounder Paralichthys olivaceus and other marine teleosts from northern China

We isolated a strain of lymphocystis disease virus （LCDV） from Japanese flounder （Paralichthys olivaceus） cultured in northern China. Based on published sequences of major capsid protein （MCP） gene of LCDV-cn （GenBank： AF126405）, we designed two primer sets P1/P2 and P3/P4. We then used one-step or nested PCR and in-situ hybridization （ISFI） to detect LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral supematant, skin nodules, gut, gill, kidney, spleen, stomach, heart, and liver of Japanese flounder. We compared the DNA sequence with 14 MCP nucleotide sequences from GenBank, including Megalocytivirus （OFIV and RSIV）, lridovirus （CzlV and W/V）, Ranavirus （TFV and FV3）, and Lymphocystivirus （8 LCDV）. Based on the alignment, we confirmed the PCR product was from Lymphocystivirus （GenBank accession number DQ279090 （LCDV-HD））. Using ISH, we noted the presence of LCDV in the skin nodules, gut, gill, spleen, stomach, and heart of spontaneously infected Japanese flounders. We successfully amplified LCDV fragments from Schlegel＇s black rockfish （Sebastes schlegeli Higendorf）, redwing sea robin （Lepidotrigla microptera Gtinther） and turbot （Scophthalmus maximus） using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method.

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV （lymphocystis disease virus）-infected Japanese flounder （Paralichthys olivaceus）, serum proteins from self-healing and sick Japanese flounder were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Protein spots demonstrating changes greater than two-fold in the expression level were digested and further identified in capillary liquid chromatography tandem mass spectrometry （LC-MS/MS）. Two immunityrelevant proteins were thus identified as transferrin and the complement component C3 of Japanese flounder. These findings suggest that the two proteins may play important roles in the self-healing of lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing in LCDV-infected Japanese flounder by improving their innate immunity.

Single Nucleotide Polymorphism of FSHβ Gene Associated with Reproductive Traits in Japanese Flounder (Paralichthys olivaceus)

Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development.This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder.We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals.We identified only an SNP (T/C) in the coding region of exon3 of FSHβ.The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene.Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P<0.05).Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P<0.05) than that of genotype CC.Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.

Histological change and heat shock protein 70 expression in different tissues of Japanese flounder Paralichthys olivaceus in response to elevated temperature

High temperature influences the homeostasis of fish. We investigated the effects of elevated temperature on tissues of Japanese flounder （Paralichthys olivaceus） by analyzing the histology and heat shock protein 70 （hsp70） expression of fish reared in warm conditions. In this study, temperature was increased at 1±0.5℃/day starting at 24±0.5℃, and was kept at that temperature for 5 days before the next rise. After raising temperature at the rate up to 32±0.5℃, tissue samples from midgut, spleen, stomach, liver, muscle, gill, heart, trunk kidney and brain were collected for histological analysis and mRNA assay. Almost all the tissues showed changes in morphological structure and hsp70 level at 32±0.5℃. Histological assessment of the tissues indicated that the gill had the most serious damage, including highly severe epithelial lifting and edema, curved tips and hyperemia at the ending of the lamellars, desquamation and necrosis. The next most severe damage was found in liver and kidney. The hsp70 levels in all the tissues first increased and then decreased. The gut, stomach, muscle, heart, and brain had the highest expressions in 6 h, whereas the spleen, liver, gill and kidney had the highest expressions in 2 h. Therefore, tissues with the most significant lesions （especially gill and liver） responded much earlier （2 h） in hsp70 expression than other tissues, and these tissues demonstrated the most marked histological disruption and elevated mRNA levels, making them ideal candidates for further studies on the thermal physiology of this species.