Assessment of internal controls for data normalization of gene expression after different bacterial stimulation by quantitative real-time PCR in golden pompano Trachinotus blochii

Trachinotus blochii is one of the important commercial fish species.In this study,we aim to confirm the reliability reference genes in T.blochii during different bacterial challenge through quantitative real-time PCR(qRT-PCR).The expression of the seven selected genes in four immune organs(i.e.,spleen,kidney,intestine,and gill) stimulated with Vibrio harveyi,Edwardsiella tarda,and Streptococcus agalactiae were determined by qRT-PCR.The PCR data was analyzed using the geNorm and NormFinder algorithms.The results showed the selection of the internal controls should be tissue specific when studying gene expression in response to bacterial stimulation.After 48 h of stimulation with V.harveyi,geNorm ranked EF1 A/Actin,18 S rRNA/B2M,UBCE/B2M,and 18 S rRNA/B2M,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with E.tarda,geNorm ranked 18 S rRNA/EF1 A,18 S rRNA/B2M,B2M/RPL13,and 18 S rRNA/EF1 A,as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.After 48 h of stimulation with S.agalactiae,18 S rRNA/EF1 A,18 S rRNA/B2 M,B2 M/Actin,and 18 S rRNA/B2M were ranked as the most stably expressed genes in spleen,kidney,intestine,and gill,respectively.Compared to the results analyzed by geNorm,reference genes received similar rankings when using NormFinder software.The results showed that the reference genes appeared to be not only tissue specific,but also specific to the infecting species of bacteria.If one gene is preferred when T.blochii were infected by bacteria,18 S rRNA,B2M,B2M,18 S rRNA may be used in spleen,kidney,intestine,and gill,respectively.     

Cloning and stage-specific expression of CK-M1 gene during metamorphosis of Japanese flounder, Paralichthys olivaceus

The symmetrical body of flatfish larvae changes dramatically into an asymmetrical form after metamorphosis. The molecular mechanisms responsible for this change are poorly understood. As an initial step to clarify these mechanisms, we used representational difference analysis of cDNA for the identification of genes active during metamorphosis in the Japanese flounder, Paralichthys olicaceus. One of the up-regulated genes was identified as creatine kinase muscle type 1 (CK-M1). Sequence analysis of CK-M1 revealed that it spanned 1 708 bp and encoded a protein of 382 amino acids. The overall amino acid sequence of the CK-M1 was highly conserved with those of other organisms. CK-M1 was expressed in adult fish tissues, including skeletal muscle, intestine and gill. Whole mount in-situ hybridization showed that the enhanced expression of CK-M1 expanded from the head to the whole body of larvae as metamorphosis progressed. Quantitative analysis revealed stage-specific high expression of CK-M1 during metamorphosis. The expression level of CK-M1 increased initially and peaked at metamorphosis, decreased afterward, and finally returned to the pre-metamorphosis level. This stage-specific expression pattern suggested strongly that CK-M1 was related to metamorphosis in the Japanese flounder. Its specific role in metamorphosis requires further study.     

Screening of stable internal reference genes by quantitative real-time PCR in humpback grouper Cromileptes altivelis

Humpback grouper Cromileptes altivelis is one commercial fish with considerable economic value.To determine the expression stabilities of six commonly used internal reference genes in C.altivelis challenged by Vibrio harveyi and viral nervous necrosis virus(VNNV) through quantitative real-time PCR(qRT-PCR),the expression levels of selected genes in five immune organs stimulated with pathogenic infection were carefully evaluated using algorithms of geNorm,NormFinder,and BestKeeper.The results show that the expre ssion stabilities of the six candidate inte rnal reference genes were diffe re nt.Under no rmal physiological conditions,RPL13 were identified as the most stably expressed genes among five different immune organs(liver,spleen,kidney,intestine,and gill).After V.harveyi stimulation,RPL13,RPL13,EF1 A,RPL13,and EF1 A were identified by geNorm,NormFinder,and BestKeeper as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.Combining these three algorithms suggested that under stimulation of VNNV,RPL13,EF1 A,Actin,RPL13,and Actin were as the most stable genes in liver,spleen,kidney,intestine,and gill,respectively.These results suggest that specific experiment conditions and tissue types shall be considered when selecting the reference genes in qRT-PCR analysis.This study provided a solid foundation for future studies on gene expression of C.altivelis under different conditions.     

2月龄花鲈形态性状与体质量相关性及通径分析

【目的】探明花鲈(Lateolabrax maculatus)早期主要形态性状对体质量的影响程度,为其良种选育提供参考。【方法】采用数理统计方法对500尾2月龄花鲈的全长(X₁)、体长(X₂)、体高(X₃)、头长(X₄)、头高(X₅)、躯干长(X₆)、吻长(X₇)、眼径(X₈)、尾柄长(X₉)、尾柄高(X₁₀)等10个形态性状和体质量(Y)进行测量分析。【结果】相关性分析结果表明,10个形态性状与体质量的相关性均达到了极显著水平(P<0.01)。通径分析结果发现,体高、全长、眼径、躯干长、吻长、体长等6个性状是影响2月龄花鲈体质量的主要形态性状,其中仅体高对体质量的直接作用大于间接作用,为影响体质量的核心变量。决定系数显示,体高对体质量的单独决定系数最大(0.324),全长和体高对体质量的共同决定系数最大(0.287),6个形态性状对体质量的总决定系数为0.947,表明本研究已将影响2月龄花鲈体质量的主要形态性状全部纳入。经逐步回归分析,拟合得到最优线性回归方程为Y=-1.468+1.314X₃+0.160X₁-0.307X₈+0.149X₆+0.305X₇+0.046X₂。【结论】除体质量外,还须将体高作为花鲈早期选育的目标性状。     

尼罗罗非鱼pik3r1基因的克隆和mRNA表达分析

【目的】哺乳动物pik3r1基因参与多种免疫途径,探索pik3r1基因在罗非鱼(Oreochromis)中的作用。【方法】克隆尼罗罗非鱼(Oreochromis niloticus)pik3r1(命名为On-pik3r1)cDNA全长,对该基因进行生物信息学分析,并运用荧光定量PCR方法分析无乳链球菌(Streptococcus agalactiae)刺激后On-pik3r1 mRNA在各组织种的表达模式。【结果与结论】On-pik3r1基因编码区2190 bp,编码729个氨基酸,5′端非编码区(UTR)为542 bp,3′UTR为2248 bp,理论分子质量为83.99 ku,等电点为5.73。On-pik3r1与斑马拟丽鱼(Maylandia zebra)相似性最高(98.53%),与其他物种的同源性在70%以上,表明pik3r1在物种进化过程中高度保守。On-pik3r1在健康尼罗罗非鱼各组织中均有表达,在肌肉中表达量最高,其次是鳃、皮肤,在胸腺中表达量最低。经灭活无乳链球菌刺激后,On-pik3r1表达量在肠道、鳃、脾脏、头肾、脑部等5个组织中均极显著下调(P<0.01),在胸腺中表现为4 h时极显著下调(P<0.01),24、48、72 h时为显著下调(P<0.05)。On-pik3r1参与了罗非鱼的对无乳链球菌的免疫应答过程。     

花染色体组型分析及细胞核DNA含量的测定

采用PHA、秋水仙碱腹腔或背部肌肉注射,活体培养法,取花肾细胞制成悬浮液,用空气干燥法制片。经Giemsa染色,镜检细胞分裂相,统计结果为2n=50,核型公式为:2n=20m+12sm+10st+8t,NF=82。同时,取花肌肉和肝脏为材料,以鸡血细胞为DNA标准(2.30pg/2c),使用EPICS-XL型流式细胞仪测定了花细胞DNA含量,分别为2.219±0.055pg/2c和2.214±0.083pg/2c。     

脂多糖、苯酚、硫酸铜对红笛鲷非特异性细胞毒性细胞受体基因表达的影响

应用Real-time PCR技术,研究脂多糖(lipopolysaccharide,LPS)、苯酚、硫酸铜刺激红笛鲷(Lutjanussanguineus)后非特异性细胞毒性细胞受体(NCCRP-1)基因在不同组织里的表达差异。结果发现,LPS刺激红笛鲷24 h后NCCRP-1在红笛鲷头肾、脾脏、胸腺、肝脏、心脏、脑、肌肉和肠组织中均有表达,其中头肾表达量最高,脾脏次之,然后依次是肝脏、脑、肌肉、胸腺和肠,心脏表达量最少。LPS、苯酚和CuSO4刺激红笛鲷后,随着刺激时间的增长,NCCRP-1表达量在各组织达到峰值的时间不同。以头肾为模式组织,RT-PCR的结果显示,红笛鲷NCCRP-1在LPS、苯酚和CuSO4的刺激下的表达模式相似,随着时间的增加NCCRP-1表达量逐渐增加,分别在24、9、12 h处达到最高,达到对照组的52、30、24倍左右,之后表达量开始下降。免疫组织化学表明,NCCRP-1只在头肾、脾脏和胸腺的特定细胞中表达。     

Expression Pattern of Peptide and Amino Acid Genes in Digestive Tract of Transporter Juvenile Turbot(Scophthalmus maximus L.)

Turbot(Scophthalmus maximus L.),a carnivorous fish species with high dietary protein requirement,was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach,pyloric caeca,rectum,and three equal parts of the remainder of the intestine.The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns.Peptide transporter 1(Pep T1) was rich in proximal intestine while peptide transporter 2(PepT2) was abundant in distal intestine.A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B~0-type amino acid transporter 1(B~0AT1),L-type amino acid transporter 2(LAT2),T-type amino acid transporter 1(TAT1),proton-coupled amino acid transporter 1(PAT1),y~+L-type amino acid transporter 1(y~+LAT1),and cationic amino acid transporter 2(CAT2) while ASC amino acid transporter 2(ASCT2),sodium-coupled neutral amino acid transporter 2(SNAT2),and y~+L-type amino acid transporter 2(y~+LAT2) abundantly expressed in stomach.In addition,system b~(0,+) transporters(rBAT and b~(0,+)AT) existed richly in distal intestine.These findings comprehensively characterized the distribution of solute carrier family proteins,which revealed the relative importance of peptide and amino acid absorption through luminal membrane.Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.     

斑节对虾细菌性烂鳃病的病理组织学观察

对自然发生的斑节对虾烂鳃病进行了病理组织切片观察,发现病虾由杆状细菌感染造成鳃丝萎缩、坏死,鳃小片呼吸上皮脱落,鳃血中有菌团存在,而且病虾的肝胰脏、胃、肠、心脏等器官组织受毒素影响而发生组织变性坏死。证实斑节对虾烂鳃病由杆状细菌感染引起。本文对细菌引起虾体的损害及对虾的防御机制进行了探讨,认为斑节对虾是由于细菌感染造成鳃呼吸功能障碍和细菌毒血症造成肝胰脏功能障碍而死亡。     

Expression pattern of peptide and amino acid genes in digestive tract of transporter juvenile turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis> L.)

Turbot (Scophthalmus maximus L.), a carnivorous fish species with high dietary protein requirement, was chosen to examine the expression pattern of peptide and amino acid transporter genes along its digestive tract which was divided into six segments including stomach, pyloric caeca, rectum, and three equal parts of the remainder of the intestine. The results showed that the expression of two peptide and eleven amino acid transporters genes exhibited distinct patterns. Peptide transporter 1 (PepT1) was rich in proximal intestine while peptide transporter 2 (PepT2) was abundant in distal intestine. A number of neutral and cationic amino acid transporters expressed richly in whole intestine including B⁰-type amino acid transporter 1 (B⁰AT1), L-type amino acid transporter 2 (LAT2), T-type amino acid transporter 1 (TAT1), proton-coupled amino acid transporter 1 (PAT1), y⁺L-type amino acid transporter 1 (y⁺LAT1), and cationic amino acid transporter 2 (CAT2) while ASC amino acid transporter 2 (ASCT2), sodium-coupled neutral amino acid transporter 2 (SNAT2), and y⁺L-type amino acid transporter 2 (y⁺LAT2) abundantly expressed in stomach. In addition, system b^0,+ transporters (rBAT and b^0,+AT) existed richly in distal intestine. These findings comprehensively characterized the distribution of solute carrier family proteins, which revealed the relative importance of peptide and amino acid absorption through luminal membrane. Our findings are helpful to understand the mechanism of the utilization of dietary protein in fish with a short digestive tract.     

Phytoplankton,especially diatoms,in the gut contents and feces of two plantivorous cyprinids—Silver carp and bighead carp

In order to clarify whether the planktivorous silver carp and bighead carp can collect phytoplankton as small asCyclotella(<20 μm) in Donghu Lake, studies on phytoplankton in their gut contents and feces were made in 1990. The fish were cultured in both net cage in Donghu Lake and aquaria with the lake water. Past the intestine, the average valve diameter ofCyclotella changed little. The average ratio of empty frustule ofCyclotella to totalCyclotella in the foregut contents of the fishes were 1.8–1.9 times higher than that in the lake water, but changed little from foregut to feces. The aquarium experiment showed that both carps could collect particles as small as 8–10 μm, which was obviously narrower than the distance between their gill rakers. Probably, secretion of mucus plays an important role in collecting such small particles.     

Effects of Na^＋/K^＋ Ratio of Groundwaters on the Gill Ion-Transport Enzyme Activity, Plasma Osmolality and Growth of Cynoglossus semilaevis Juveniles

The effects of environmental Na /K ratio on the gill ion-transport enzyme activity,plasma osmolality and growth ofCynoglossus semi/aevis juveniles were investigated.The results showed that,plasma osmolality was similar among flounder adaptedto different Na /K ratios of saline groundwaters (P>0.05),while the growth,gill Na ,K -ATPase and HCO,3'--ATPase activities wereaffected by Na /K ratio significantly (P<0.05).The gill Na ,K -ATPase activity reached its maximum on day 3,then decreasedgradually from day 3 to day 9 and remained constant from day 9 to day 15.The peak values of gill Na ,K -ATPase activity weredetected on day 3 for all Na /K ratios of saline groundwaters,then the enzyme activities descended,and on day 9 the enzyme activi-ties achieved steady state,and the gill HCO,3--ATPase activity increased rapidly and achieved steady state after one day.Duringsteady state,the gill Na ,K -ATPase and HCO,3--ATPase activity of Na /K ratios 20 and 40 treatments were significantly higherthan those in the control group (Na /K ratio 27.5),while there were no significant differences between the Na /K ratio 30 treatmentand the control group; the gill Na ,K -ATPase activity of Na /K ratio 20 and 40 treatments were significantly higher than that forratio 30 treatment,but there were no significant differences of gill HCO3-ATPase activity among these treatments.At the end of the15-day experiment,the weight gain (%) and specific growth rate (SGR) of flounders maintained in seawater were significantly higherthan those in groundwaters; significant differences also occurred among the treatments; Na /K ratio 30 treatment had the highestvalues (33.7% and 1.94 respectively),which were significantly higher than those under Na /K ratios 20 and 40 treatments.There-fore,for the saline groundwater used in this experiment,it is suggested that the Na /K ratio be adjusted to approximately 30,I.e.,asclose to that of natural seawater as possible in the culture of flounder.     

CHARACTERIZATION OF LACTATE DEHYDROGENASE ISOZYME PATTERN AND MORPHOLOGY OF THREE MARINE FISH CELL LINES

Three continuous marine fish cell lines of FG (i. e., Hounder Gill) from flounder (Paralichthys olivaceus) gill, SPH (i. e. , Sea Perch Heart) from sea perch (Lateolabrax japonicus) heart and RSBF (i.e., Red Sea Bream Fin) from red sea bream (Pagrosomus major) fin, were characterized by lactate dehydrogenase (LDH) isozyme and morphological analysis. The LDH isozyme patterns of these three cell lines and their corresponding tissues of origin were investigated and compared. The results showed: (1) No difference was found in the LDH isozyme patterns of FG and flounder gill tissue. However, the LDH isozyme patterns of SPH and RSBF were significantly different from their corresponding tissues of origin; (2) LDH isozyme patterns of FG, SPH and RSBF were markedly different from each other and could serve as genetic markers for species identification and detection of cross contamination. Morphological change analysis of these three cell lines in comparison to their original tissues indicated that FG cells still appeared epithelioid without morphological transformation. However, morphological changes were found in SPH and RSBF compared to their original tissues. Therefore, the cellular morphology was still plastic in the relatively stable culture conditions, and it was possible that change of LDH patterns wasrelated to morphological changes of fish cells in vitro.     

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.     

Diurnal expression of circadian clock genes period 1 and period 3 in Pelteobagrus vachellii

Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom,which entrain the rhythms of biochemical,physiological,and behavioural processes.To better understand the molecular oscillations of the circadian rhythms in darkbarbel catfish(Pelteobagrus vachellii),we isolated and characterized two circadian clock genes in P.vachellii,period 1(perl),and period3(per3).The circadian clock gene perl was found to encode a 1 428-amino acid polypeptide,including PER-ARNT-SIM(PAS) dimerisation domains,a PAS-associated C-terminal motif(PAC),a short mutable domain(S/M),and a nuclear export signal(NES).The 4 902-bp per3 cDNA includes an open reading frame encoding a 1 292-amino acid residue polypeptide with a PER-ARNT-SIM(PAS) domain,cytoplasmic localisation domain(CLD),interaction site(TIS),and a nuclear localisation signal(NLS).The perl and per3 gene was constitutively expressed in all examined tissues.Moreover,perl expression within a light/dark cycles showed rhythmic expression in the diencephalon,brain,liver and intestine,with,the acrophase at 15:15,12:52,7:51,and 12:55,respectively.Daily expression of per3 was rhythmic in the diencephalon,brain,liver and intestine,with the acrophase at 8:15,9:54,10:39,and 10:25 h,respectively.These findings expand our understanding of circadian mechanism at the molecular level in this species.     

3-甲基胆蒽对斑马鱼组织病理效应的初步研究

分别用25、50、100、200和400μg/L的3-甲基胆蒽(3-MC)对斑马鱼进行水浴染毒,于第7天和第14天采集各染毒组和对照组活鱼的肝、鳃、心、肠和肾,进行病理组织学检查。结果发现,一定剂量的3-MC能导致肝细胞变性,胞浆内出现脂肪滴,严重时肝细胞坏死解体,界限模糊;心肌细胞萎缩坏死;肠粘膜上皮细胞纹状缘不整齐,有的脱落;鳃丝结构异常,鳃小片表面粗糙,上皮细胞不完整;肾小管上皮细胞肿胀,细胞界限不清,管腔内均质红染。供试斑马鱼的病理组织变化与3-MC的作用剂量和作用时间存在一定的关系。     

Molecular Characterization and Expression Analysis of SKIV Infection of Interferon-Induced Protein with Tetratricopeptide Repeats 1(IFIT1) in Epinephelus lanceolatus

Interferon-induced protein with tetratricopeptide repeats 1(IFIT1), also known as interferon-induced protein 56(IFI56) or Interferon-stimulated protein 56(ISG56), was originally identified as a protein induced upon treatment with interferon and inhibited by viral replication and translational initiation. In this study, Epinephelus lanceolatus IFIT1(ELIFIT1) gene was cloned for the first time. The complete cDNA of El IFIT1 gene includes 2921 nucleotides, and encodes a 437-amino acid(AA) protein. The putative ELIFIT1 protein has 9 TRP domains and is highly similar with IFIT1 proteins in other teleosts. In healthy fish, ELIFIT1 gene was highly expressed in the blood, which indicate its specific function in the peripheral immune system. Its expression was also observed in various immunity-related tissues including spleen, intestine, and kidney, Inducted with spotted knifejaw iridovirus(SKIV), ELIFIT1 gene expression was upregulated in the spleen, kidney, and liver 24 h after induction and reached its peak at 72 h, indicating that ELIFIT1 may play an important role in antivirus. These findings contribute to the understanding of the antiviral regulation of ELIFIT1 gene in teleost.     

溶藻弧菌胞外产物注射赤点石斑鱼后的组织病理学观察

在注射溶藻弧菌胞外产物(ECP)48 h后,石斑鱼Epinephelus akaara出现死亡,临床症状表现为:运动迟缓,体色变深,腹部膨大,腹腔有明显的积水,肝脏出血有花斑,肾脏和脾脏肿大。病理组织切片观察发现:肝细胞坏死,解体,界限模糊,细胞核变形、裂解甚至消失;肝脏出血,肝索中有红细胞浸润,有的红细胞变形甚至破裂;肾小管上皮细胞脱落、坏死;肠粘膜上皮细胞脱落、坏死,微绒毛不整齐,模糊不清,脱落;脾窦和脾索不清晰,分界不清,且在脾窦和脾索中分布着大量的、形态不规则的红细胞。此外,还发现在肝脏、脾脏、肾脏、肠壁肌层、心肌和鳃等组织中存在一种相同结构,该结构有平滑肌环绕,管腔内壁衬以扁平上皮细胞,管腔中含有一团染成紫红色的物质,经推断为小动脉透明样变性,而在正常的组织切片中,均无此结构。