大菱鲆两种转基因方法的比较研究

对外源基因导入大菱鲆(Scophthat musmaximus)的两种方法进行了研究,采用经过改进的电脉冲方法以及哺乳动物细胞中常用的转染法导入外源基因,并对两种方法进行比较。电脉冲最佳导入条件为:脉冲电压为300V/cm,脉冲次数为5,外源DNA质量浓度为20mg/L;转染法的最佳操作时间为受精后50min。应用上述两种方法进行批量转基因鱼实验,得到外源基因的导入率分别为20%～28%和60%～70%。实验表明利用转染试剂jetPEI处理单细胞期的大菱鲆受精卵能得到较高的孵化率及较高的转基因效率。     

用基因枪将GUS基因导入褐藻细胞中表达

于１９９３年１１月－１９９４年２月，用高压氦气式基因枪，将ＰＨ１２２１质粒装有ＧａＭＶ ３５Ｓ启动子，ＧＵＳ基因以及ｎｏｓ的３＇调控区导入海带和裙带菜组织切块中。４８ｈ后，在海带假根细胞和裙带菜中肋部叶片细胞中检测到ＧＵＳ基因的表达。实验表明，微粒子轰击法是外源基因导入大型褐藻的一个有效途径。ＧａＭＶ ３５Ｓ启动子能够驱动外源基因在海洋藻类中的表达，可作为藻类基因工程的启动元件。     

大菱鲆与耐高温性状相关的微卫星示记筛选

采用微卫星分子标记(SSR)技术分析大菱鲆(Scophthalmus maximus L.)耐高温相关特性,为耐高温大菱鲆的分子辅助育种提供合适的分子标记.将大菱鲆经过高温实验处理,区分为耐高温组与高温敏感组,用于实验分析.采用Salah.M法抽提大菱鲆肌肉组织的DNA,根据已知的30个大菱鲆微卫星位点的侧翼保守序列设...     

真鲷精子在外源DNA 下的生理特性和转染效果

为研究真鲷(Pagrus major)精子在外源DNA下的生理特性和转染效果,探索真鲷精子介导转基因技术,作者通过计算机辅助分析外源DNA环境下(不同孵育时间、DNA长度、DNA浓度)真鲷精子活率和运动速度的变化,进一步采用PCR和荧光探针的方法检测外源DNA与真鲷精子的结合情况,并通过人工授精来评价外源DNA能否转染精子并传递至F0代。结果表明:外源DNA浓度、孵育时间及DNA长度对精子活率无显著影响。在5μg/106个精子的高浓度10 kb DNA下孵育12 h,真鲷精子仍具有(75.89±5.55)%的精子活率,平均直线速度(70.97±6.37)μm/s,与对照组无显著差异,但精子平均曲线速度显著下降,比对照组低21.85μm/s。真鲷精子与带Fluorescein荧光素的DNA片段共孵育后,部分精子在荧光显微镜下观察到绿光。将DNA孵育后精子进行人工授精表明经过1μg DNA/106个精子孵育后的受精率和孵化率无显著下降,通过PCR法并没有在外源DNA处理的精子和F0代中检测到目的基因,表明外源DNA虽然能够吸附在真鲷精子表面,但并不足以携带进入卵子产生转基因后代。     

星斑川鲽、大菱鲆及其杂交后代的线粒体DNA 序列比较分析

为了揭示星斑川鲽(Platichthys stellatus)♀×大菱鲆(Scophthalmus maximus)♂杂交后代的种质遗传属性,作者采用线粒体(mt)DNA COI基因片段和控制区(D-loop)序列对星斑川鲽、大菱鲆及两种间的杂交后代(星斑川鲽♀×大菱鲆♂)遗传特性进行研究。研究结果显示,基于mtDNA COI和D-loop序列结果显示星斑川鲽和大菱鲆的种间遗传距离分别为22.9%和41.2%,相似的研究结果显示星斑川鲽♀×大菱鲆♂杂交后代与大菱鲆的遗传距离分别为22.9%和41.2%,遗传差异极其显著。而基于mtDNA D-loop序列结果显示星斑川鲽♀×大菱鲆♂杂交后代与星斑川鲽的遗传距离仅为0.4%,并且在mtDNA COI基因片段上两者序列完全一致,杂交后代在线粒体DNA上呈现明显的偏母遗传。     

鱼类转基因技术综述

基因转移的研究为鱼类遗传育种开辟了一条全新的途径。自1985年第1例转基因鱼问世至今已获得20～30种转基因鱼,在促进生长,提高鱼类抗逆性、抗病性等方面取得了巨大成绩。本文主要阐述了转基因鱼的构建机理、方法和检测方法以及各自的优缺点。外源基因的定点整合、稳定表达和遗传仍需要深入研究。构建“全鱼”基因则是今后发展的方向,受到越来越多的关注。     

大菱鲆免疫球蛋白轻链IgL全长cDNA的分离、鉴定及表达分析

实验采用末端快速扩增(RACE)技术从大菱鲆脾脏cDNA文库中筛选得到了免疫球蛋白轻链IgL的全长cD-NA片段。该序列包含47 bp的5′末端非编码区(5′UTR),738 bp的开放阅读框(ORF)和202 bp 3′UTR,整个开放阅读框编码246个氨基酸。系统发生分析表明大菱鲆IgL基因与五条鰤的IgL基因起源关系最近。RT-PCR分析表明,大菱鲆IgL基因只在正常脾脏、肾脏和头肾组织中表达;IgL在大菱鲆胚胎发育细胞期就已开始表达,在大菱鲆胚胎尾芽期和体节期表达持续增强;在鳗弧菌感染大菱鲆脾脏和头肾早期均检测到IgL基因强烈表达,后期表达逐渐减弱;大菱鲆胚胎细胞(TEC)在用鳗弧菌感染48h后,IgL开始表达。这些结果均表明IgL基因在大菱鲆免疫应答中起着重要作用。     

副溶血弧菌（Vibrioparahaemolyticus）tdh2基因和鳗弧菌（Kanguillarum）ompU基因二联DNA疫苗制备及其对大菱鲆（Scophthalmusmaximus）免疫保护作用

采用基因工程的方法,将副溶血弧菌的热稳定直接溶血素基因tdh2和鳗弧菌外膜蛋白基因ompU进行融合,在大肠杆菌中得到表达,并利用该融合基因构建二联DNA疫苗pEGFP．N1／tdh2-ompU。用DNA疫苗按10和50μg／尾的剂量通过肌肉注射免疫大菱鲆,对大菱鲆抵抗致病性副溶血弧菌和鳗弧菌的免疫效果进行研究。结果表明...     

溶藻弧菌的PCR快速检测方法

为建立溶藻弧菌(Vibrio alginolyticus)的PCR快速检测方法,本研究根据弧菌toxR基因的高变区序列设计1对扩增片段为161 bp的引物,进行了特异性和敏感性试验.结果表明该方法能特异地检测溶藻弧菌,每个PCR反应检测的敏感度为0.01 pg的DNA和3.7CFU的细菌.用溶藻弧菌人工感染大菱鲆,以建立的PCR方法检测感染鱼的肝、脾、肾阳性检出率为100%.该方法可用于对感染溶藻弧菌的水生动物疾病进行诊断.     

大菱鲆与耐高温性状相关的微卫星标记筛选

采用微卫星分子标记(SSR)技术分析大菱鲆(Scophthalmus maximus L.)耐高温相关特性,为耐高温大菱鲆的分子辅助育种提供合适的分子标记.将大菱鲆经过高温实验处理,区分为耐高温组与高温敏感组,用于实验分析.采用Salah.M法抽提大菱鲆肌肉组织的DNA,根据已知的30个大菱鲆微卫星位点的侧翼保守序列设计引物,进行微卫星引物PCR(SSR-PCR)扩增.对PCR扩增出的差异条带进行个体统计,最后进行微卫星位点与耐高温性状的相关性分析.结果表明,有1个微卫星位点与大菱鲆耐高温性状存在一定的负相关性;有3个微卫星位点与大菱鲆耐高温性状存在正相关性,其中位点Sma-USC27 286 bp的等位基因片段与耐高温性状的正相关性极显著,相关系数达到0.383(P＜0.01),其余2个位点为一般显著性相关.微卫星位点Sma-USC27所扩增出的差异等位基因片段可作为分子标记指导耐高温大菱鲆的辅助育种.     

大菱鲆趋化因子CCL34基因的鉴定及其响应细菌感染的表达特征

为了研究大菱鲆(Scophthalmus maximus)中趋化因子(Chenokine)的特征及其在大菱鲆免疫过程中发挥的作用,设计了本文中的实验。本实验从大菱鲆基因组和转录组数据库中鉴定了一个硬骨鱼特有的趋化因子CC亚家族成员—CCL34,并选取大菱鲆的8个健康组织以及2种细菌感染后的肠道和皮肤组织,采用实时荧光定量PCR(qRT-PCR)对其表达特征进行研究。序列分析结果显示该趋化因子全长mRNA包含1个327 bp的5’非编码区(UTR),1个246 bp的3’非编码区,和1个编码103个氨基酸残基长度为312 bp的开放阅读框(ORF)。此CCL34基因含有4个外显子和3个内含子。通过系统发育分析、共线性分析,将该大菱鲆趋化因子命名为CCL34。实时荧光定量PCR表明CCL34在大菱鲆健康组织中普遍表达,尤其在肾脏、肝脏、皮肤中有高水平表达。鳗弧菌(Vibrio anguillarum)和无乳链球菌(Streptococcus agalactiae)感染后,肠道组织CCL34基因表达量较对照组显著性上调(p<0.05),这表明CCL34可能在大菱鲆的肠道粘膜免疫中起重要作用。本研究为趋化因子家族功能的研究奠定了基础,为增强鱼类免疫力和抗病能力提供了理论依据。     

免疫增强剂紫锥菊(Echinacea purpurea)提取物对大菱鲆(Scophthalmus maximus)头肾内非特异性免疫基因表达量的影响

通过给大菱鲆(Scophthalmus maximus)注射不同浓度的紫锥菊(Echinacea purpurea)提取物,测定其非特异性免疫指标的变化水平,研究其对大菱鲆的头肾内非特异性免疫分子基因相对表达量的影响,进而为其在大菱鲆养殖生产过程中推广应用提供科学依据。试验选取体重(250±20)g的大菱鲆作为试验动物,分别注射10、20、40mg/m L浓度的紫锥菊提取物,试验共进行28d,注射试验结束后,分别从高、中、低剂量组及空白对照组选出6尾大菱鲆,分别取出大菱鲆的头肾并提取总RNA。采用?Ct法进行目标基因的荧光定量分析,再应用SPSS17.0软件对获得的实验数据进行单因素方差分析。研究结果表明,紫锥菊提取物能够不同程度地提高大菱鲆头肾内非特异性免疫分子基因相对表达量。注射紫锥菊提取物28d后对大菱鲆头肾中Lysozyme基因相对表达量的影响显著(P0.05),对C3补体基因相对表达量的影响极显著(P0.01),对Transferrin基因相对表达量的影响显著(P0.05),对TGF-β1基因相对表达量的影响极显著(P0.01),对IL-1h基因相对表达量的影响极显著(P0.01),本试验研究表明,紫锥菊提取物能够显著提高大菱鲆头肾内非特异性免疫分子基因的相对表达量。     

花鲈PPARα基因实时荧光定量PCR质粒标准品的构建

利用SYBR荧光定量PCR技术，制备用于花鲈（Lateolabrax japonicus）PPARα基因实时荧光定量PCR检测的质粒标准品．通过PCR扩增出目的片断344-bp，纯化后连接至pMDI8-T载体，转化宿主菌E．coli DH5α．通过PCR鉴定、双酶切鉴定和测序分析后抽提重组质粒，测定其拷贝浓度，10倍梯度稀释成标准品模板10^8～10^4 copy／mm^3，进行荧光定量PCR分析．结果显示，循环阈值（Ct）与起始模板量的对数值之间有着良好的线性关系．     

Diagnosis of iridovirus in large yellow croaker by PCR

A rapid and sensitive PCR-based method for the detection of the large yellow croaker iridovirus(LYCIV) is described,which involves the amplification of a 295 bp fragment of the LYCIV ATPase gene from DNA isolated from naturally infected fish spleen.Sequencing of LYCIV ATPase gene fragment showed it shared 100% nucleotide sequence homology with the corresponding region of the ATPase gene of red sea bream iridovirus(RSIV) and sea bass iridovirus(SBIV),suggesting that LYCIV was homologous with RSIV and SBIV at least in part of the gemone.The specificity and sensitivity of the PCR procedure were tested on the iridovirus-infected fishes,the expected fragment was detected from spleen DNA samples of infected fishes,whereas no fragments were amplified from healthy fish spleen DNA,white spot syndrome baculoviruses(WSBV) DNA and pseudorabies virus(PRV) DNA.Detection limit of this method was 10^-7 ng positive plasmid DNA containing target sequence,equal to about 100 virions.In the infected experiment,first positive detection(1/4) appeared at Day 3 post-infection,all fish(4/4) tested positive at Day 7,however obvious symptoms were observed at Day 8,so LYCIV infection could be detected prior to the appearance of obvious symptoms.These results indicate that this PCR method could be used for early,rapid and specific detection of LYCIV infection.     

Uptake of estradiol from sediment by hornyhead turbot (Pleuronichthys verticalis) and effects on oxidative DNA damage in male gonads

Male and female hornyhead turbot (Pleuronichthys verticalis) were exposed to four concentrations (0, 0.75, 14.7 and 46.5 ng/g dry weight) of E2-amended sediment for 7 days. Sediment-derived E2 was bioavailable to the flatfish, though the route of uptake was unclear. A concentration of 46.5 ng/g E2 in sediment led to a significant increase in vitellogenin concentrations in the plasma in both sexes after 7 days of exposure. Though plasma E2 concentrations increased significantly in males at sediment E2 concentrations of 0.75 ng/g dry weight and above, a dose-dependent increase was not observed. There was also no correlation between sediment E2 concentrations, plasma E2 concentrations, and oxidative DNA damage in male gonads. The results suggest that the DNA damage previously seen in the gonads of feral hornyhead turbot at a sewage outfall is likely not caused by acute exposure to exogenous E2 from sediments.     

大菱鲆亲子鉴定的微卫星多重PCR技术建立及应用

用8个微卫星标记组合建立了2个微卫星多重PCR体系,对大菱鲆7个人工选育家系进行了系谱认证、亲子鉴定和遗传多样性研究。2个多重PCR体系中8个微卫星位点共检测到等位基因49个,每个位点等位基因数为3~8个。根据已知亲本及子代基因型,成功推断出了3个缺失亲本的基因型。在双亲未知的情况下2个多重PCR的8个微卫星位点累积排除概率是96.58%,已知1个亲本时累积排除率为99.71%,亲子鉴定准确率为96.42%。采用70个个体进行双盲验证,利用UPGMA法对7个家系的70个体进行了聚类分析,同一家系95.71%的个体聚类分析结果与系谱来源一致。Cervus 2.0软件亲子鉴定结果表明亲子鉴定准确率为92.86%。2个多重PCR体系的建立为大菱鲆不同家系混养后的亲子鉴定、系谱分析和分子辅助家系管理提供了技术手段。     

Accumulation and effects of benzo(a)pyrene on cytochrome P450 1A in waterborne exposed and intraperitoneal injected juvenile turbot (Scophthalmus maximus)

Juvenile turbot (Scophthalmus maximus) were exposed to benzo(a)pyrene (BaP) for 14 d using a glass bead generator flow-through system. Exposure was followed by a recovery period of 16 d. The highest BaP concentration in the edible portion of the fish, 16.5 ± 4.3 μg BaP/kg, was observed on the first day. Then concentrations dropped following first-order kinetics. BaP was below detection level at the end of the experiment. A statistically significant increase in bile fluorescence was observed from day 9 until the end of the experiment, suggesting the elimination of BaP metabolites by this route. No significant differences between control and exposed fish in EROD activity and CYP1A concentration, measured by immunodetection method, were observed. Intraperitoneal injection of 2.5 mg BaP/kg in juvenile turbot induced EROD activity. Under waterborne experimental conditions, bile fluorescence was observed to be a more sensitive biomarker of BaP exposure than EROD activity and CYP1A measurement.     

Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L

Hatchery-reared turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to sediment collected from polluted sites in Cork Harbour and a reference site at Ballymacoda, Co. Cork, Ireland. The potential of surficial sediment for inducing hepatic biomarkers was assessed at two levels of biological organisation: expression of cytochrome P450 [Western blotting analysis and 7-ethoxy-resorufin O-dealkylase (EROD), 7-benzoxy resorufin O-dealkylase (BROD), 7-methoxy resorufin O-dealkylase (MROD), 7-pentoxy-resorufin O-dealkylase (PROD) activities] and DNA integrity (Comet assay). Positive controls were generated, either by exposing turbot to cadmium chloride-spiked seawater (Comet assay) or to beta-naphthaflavone by intraperitoneal injection (cytochrome P450 induction). The induction of cytochrome P450 activity (EROD, MROD and PROD) in animals following a 7-day exposure to contaminated sediments was significantly higher than those exposed to reference site sediment and remained elevated thereafter; BROD was not induced. DNA single-strand breaks were also significantly higher following exposure to contaminated sediments throughout the experiment. Although no direct correlation between induction of alkoxyresorufin O-dealkylase activities and a particular chemical class was established, the induction of MROD and PROD activities in fish exposed to sediments containing complex contaminant mixtures, appeared to be more sensitive than conventional EROD activity assays. We conclude from the present laboratory study that S. maximus is a suitable sentinel species for the assessment of moderately contaminated sediments and therefore allows for the further development of this model for future, ecologically relevant, field studies.     

Correlative changes in metabolism and DNA damage in turbot (Scophthalmus maximus) exposed to benzo[a]pyrene

Juvenile turbot (Scophthalmus maximus) were injected intraperitoneally with either corn oil or 5 mg/kg benzo[a]pyrene (BaP) dissolved in corn oil and sampled I and 3 days after injection. After 1 day, no elevation of 7-ethoxyresorufin O-deethylase (EROD) activity was observed, however bile metabolites (BaP-7,8 dihydrodiol representing 70% of the total metabolites) and a single hepatic DNA adduct spot (0.47 adducts/10(8) nucleotides) identified by 32P-postlabelling were formed. No BaP metabolites or DNA adducts were observed in either control or carrier control fish. Fish sampled after 3 days reported 5-fold higher (P < 0.05) levels of EROD activity, a shift in the bile metabolite profile towards BaP phenol formation (1OH and 30H BaP comprising up to 60% of total metabolites detected) and the formation of two adduct spots (0.86 and 0.71 adducts/10(8) nucleotides). These results show that BaP can be metabolised and form hydrophobic DNA adducts in turbot without EROD elevation. Following EROD elevation, a shift in the profile of both BaP metabolites and BaP metabolite-DNA interactions occurs indicative of other oxidative processes.     

Studies on the edwardsiellosis and characterization of pathogenic bacteria from diseased flounder (Paralichthys olivaceus L.) and turbot (Scophthalmus maximus L.)

1IntroductionEdwardsiellahas been recognized as a world-wide-occurring pathogen of numerous marine fishspecies,and edwardsiellosis has been recorded in adiverse array of fish species,including chinooksalmonOncorhynchus tshawytschaL.(Amandi etal.,1982),cha…