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《中国海洋湖沼学报》2020,(2)
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. 相似文献
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The appropriate reference gene is a prerequisite for accurate normalization of gene expression level, and research on suitable reference genes in clam Cyclina sinensis is scarce. To improve the situation, we selected five commonly used housekeeping genes, including β-actin, Elongation factor 1-α (EF1-α), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 40S ribosomal protein S18 (RPS18), and Tubulin α (TUB-α), then evaluated their expression stability in different adult tissues and under different experimental treatments (salinity stress and Vibrio parahaemolyticus infection). Their expression stability was analyzed by three frequently used programs, geNorm, NormFinder, and BestKeeper. This analysis indicated that multiple genes should be used for normalization, and we concluded that the reference gene combination GAPDH-RPS18-β-actin, should be used for qRT-PCR analysis in different tissues of C. sinensis under normal physiological conditions. For the clams under salinity stress and Vibrio infection, EF1-α-GAPDH-RPS18 was recommended as the gene combination for qRT-PCR normalization. TUB-α was generally poorly ranked by all programs, and should not be used in future studies. This study should provide fundamental support for accurate quantitative gene expression analysis of this species.
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为筛选能在马氏珠母贝外套膜边缘膜与中央膜区域稳定表达的内参基因,应用荧光定量PCR 技术,对3-磷酸脱氢酶（GAPDH）、β-肌动蛋白（β-actin）、胆色素原脱氨酶（PBGD）、微管蛋白（TUB）、TATA 盒结合蛋白（TBP）、磷脂酶A2（PLA-2）、葡萄糖苷酶（GUSB）、18S 核糖体rRNA（18SrRNA）等8 个常用的内参基因在马氏珠母贝外套膜边缘膜与中央膜区域的表达情况进行分析,并利用geNorm、NormFinder、BestKeeper 及RefFinder 软件对8 个内参基因的表达稳定性进行分析.结果表明：8 个内参基因均可获得特异性扩增产物,但稳定性各异, 其在外套膜边缘膜和中央膜区的表达稳定性顺序依次为PBGD/TBP〉TUB〉GUSB〉18SrRNA〉PLA-2〉GAPDH〉 β-actin;在荧光定量PCR 中,PBGD 和TBP 在马氏珠母贝外套膜边缘膜和中央膜区的表达比较稳定,为研究贝壳矿化机制中理想的内参基因. 相似文献
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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。 相似文献
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注射黄芪多糖对吉富罗非鱼c型溶菌酶基因表达量的影响 总被引：1，自引：0，他引：1
将黄芪多糖(APS)用无菌生理盐水配制成2 mg/mL和20 mg/mL针剂,腹腔注射吉富罗非鱼,以注射无菌生理盐水为对照。24 h后分别提取吉富罗非鱼鳃、头肾、肝脏、脾脏等组织中的总RNA并反转录成cDNA,利用Real-time PCR方法对不同组织中基因表达进行定量分析。结果表明:吉富罗非鱼腹腔注射20 mg/mL高剂量APS后,其鳃、头肾、肝脏等三个组织中的Lysozyme-c基因表达量显著高于对照组(P<0.05);注射2 mg/mL低剂量APS后,Lysozyme-c基因表达量仅在脾脏中出现显著上调(P<0.05)。APS可通过诱导Lysozyme-c基因在鳃、头肾、肝脏和脾脏等组织在的表达量,来提高吉富罗非鱼的机体免疫力。 相似文献
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通过同源引物从致病性哈维氏弧菌(Vibrio harveyi)ZJ0603基因组中克隆GST的开放阅读框(ORF),构建真核表达质粒pcDNA-GST。大量抽提重组质粒后,于背鳍基部肌肉注射重组质粒免疫斜带石斑鱼(Epinephelus coioides),分析重组质粒的免疫效果。通过核酸水平检测重组质粒在鱼体肝、肌肉、头肾和脾脏组织的分布;用ELISA法检测鱼体血清的抗体水平,用Western-blot检测目的蛋白的表达情况。结果表明:该序列全长615 bp;免疫7 d后,鱼体中均有质粒分布;斜带石斑鱼血清中产生抗GST的高效抗体(1∶4 096);相应的目的蛋白也在鱼体中成功表达。攻毒后,疫苗免疫保护率达80%,表明GST可作为防治哈维氏弧菌病有效候选抗原。 相似文献
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Histogenesis of the immune system and specific activity of superoxide dismutase(SOD) were studied in rock bream Oplegnathus fasciatus from fertilization to 50 days after hatching(DAH).The pronephric tubule primordium developed in the embryo,14 h 30 min post fertilization.The spleen anlage was observed between the swim bladder and the intestine at 5 DAH,and the thymus was formed as a paired structure under the pharyngeal epithelium above the gill arch at 10 DAH.The order of the immune organs becoming lymphoid was the pronephric kidney(10 DAH),thymus(15 DAH) and spleen(21 DAH).As the embryo developed,the specific activity of SOD gradually increased until hatching,but subsequently SOD activity continuously decreased to a minimum at 14 DAH.After the spleen became lymphoid,the specific activity of SOD was relatively stable.It is suggested that the immaturity of the lymphoid organs and low specific activity of SOD was the cause of the high mortality of fingerlings 12 to 16 DAH. 相似文献
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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. 相似文献
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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。 相似文献
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WANG Lei MA Teng XU Wenteng CHEN Zhangfan ZHOU Qian ZHENG Guiliang CHEN Songlin 《中国海洋大学学报(英文版)》2021,(2)
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. 相似文献
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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 (ISH) to detect
LCDV and identify the target tissues or cells in infected Japanese flounder. The PCR products were positive in purified viral
supernatant, 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), Iridovirus (CzIV and WIV), 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 Günther) and turbot (Scophthalmus maximus) using the one-step and nested PCR, suggesting the target genes can be widely detected in fish using this method. 相似文献