基于转录组学技术对斑马鱼肝脏组织低氧胁迫影响的研究

为探究鱼类响应低氧胁迫的调控机制,将1月龄的野生型斑马鱼(Danio rerio)在1.5 mg·L-1的低氧浓度下胁迫2个月后,对其肝脏组织进行转录组测序比较分析。对常氧与低氧组的3 270个差异基因进行KEGG分析,主要富集于细胞增殖、脂质代谢、糖类代谢和氨基酸代谢等通路。其中,上调的1 864个基因主要与细胞增殖相关,下调的1 406个基因主要参与脂质代谢。对差异基因进行GO富集分析,发现铁离子束(Iron ion banding)功能差异显著。对铁代谢相关基因的表达量进行分析,发现铁离子储存相关基因fthl28和fthl31变化显著,提示在低氧胁迫下斑马鱼肝脏(Zebrafish liver, ZFL)组织中铁离子含量发生显著变化。利用ZFL细胞进行体外验证实验,将ZFL细胞进行0.1%(体积分数) O2低氧胁迫,发现随着胁迫时间的延长,ZFL细胞的成活率降低,且细胞中与铁代谢相关基因和铁蛋白(Ferritin)的表达均显著降低。综上所述,铁代谢调节是低氧胁迫下的重要响应过程,低氧会导致细胞内铁代谢紊乱,延长低氧时间会形成新的铁稳态。研究结果为探究鱼类的低氧适应机制提供了理论基...     

NaHCO3生境胁迫下方正银鲫鳃靶器官代谢组学研究

为探究方正银鲫(Carassius auratus gibelio)鳃靶器官在细胞层面应对盐碱生境胁迫时的代谢响应机制, 实验设置了淡水对照组(Con)和 3 个碱胁迫组: 20 mmol/L NaHCO₃ (CA20)、40 mmol/L NaHCO₃ (CA40)和 60 mmol/L NaHCO₃ (CA60), 通过非靶向代谢组学方法, 解析暴露于不同浓度盐碱环境中 30 d 后的方正银鲫鳃靶器官中的差异显著代谢物及代谢通路。结果显示, 鳃组织在 ESI 正、负离子数据采集模式下共筛选出 89 个差异显著代谢物, 其中 50 个上调, 39 个下调, 其主要富集于甘油磷脂代谢、鞘脂代谢、花生四烯酸代谢、苯丙氨酸代谢以及苯丙氨酸、 酪氨酸和色氨酸的生物合成等 12 条代谢通路。本研究结果表明, 盐碱生境胁迫后, 方正银鲫体内甘油磷脂调控了细胞膜内外物质转运, 导致合成鞘磷脂、鞘氨醇的途径被抑制。而随着前列腺素、白三烯和 L-苯丙氨酸等代谢物的浓度上升, 鲫鳃的解毒功能增加, 炎症创伤获得逐渐修复, 该代谢机制可能是方正银鲫应对盐碱胁迫耐受策略的重要原因之一。本研究所探究的方正银鲫对盐碱生境胁迫的耐受策略及代谢调控机制, 为后期的耐盐碱优良鱼种培育及增养殖研究提供了科学理论依据。     

高温胁迫下大黄鱼肝脏的蛋白质组学

为探究大黄鱼在高温胁迫条件下的蛋白表达情况,实验应用串联质谱标签tan-dem mass tag(TMT)标记结合液相色谱-串联质谱(LC-MS/MS)技术对大黄鱼耐高温组和不耐高温组的肝脏组织进行蛋白组学分析.共鉴定到3 369个蛋白,其中有687个差异表达蛋白,包括281个上调蛋白和406个下调蛋白.用平行反应监测...     

高温胁迫下大黄鱼肝脏的蛋白组学

为探究大黄鱼在高温胁迫条件下的蛋白表达情况,实验应用串联质谱标签tandem mass tag (TMT)标记结合液相色谱-串联质谱(LC-MS/MS)技术对大黄鱼耐高温组和不耐高温组的肝脏组织进行蛋白组学分析。共鉴定到3 369个蛋白,其中有687个差异表达蛋白,包括281个上调蛋白和406个下调蛋白。用平行反应监测(parallel reaction monitoring, PRM)对随机挑选的13个蛋白进行验证,其结果与TMT标记的蛋白组学分析结果一致。随后,通过生物信息学分析发现,高温胁迫对大黄鱼的蛋白质折叠、能量代谢有显著影响,其中热休克蛋白70 (heat shock proteins 70, HSP70)、钙网蛋白(calreticulin, CRT)和葡萄糖调节蛋白(glucose-regulated protein, GRP)参与调节蛋白质的正确折叠,丙酮酸激酶(pyruvate kinase, PK)参与高温条件下的能量供给。由此推测HSP70、CRT、GRP和PK在大黄鱼应对高温胁迫时发挥着重要的作用。     

美洲大蠊粉替代鱼粉对虹鳟幼鱼生化指标、抗病力及代谢组学的影响

美洲大蠊(Periplaneta americana)是一种富含蛋白质、脂肪和生物活性成分的昆虫蛋白来源,具有成为鱼粉替代源的潜力。选取初始体质量约3 g的虹鳟(Oncorhynchus mykiss)幼鱼为研究对象,采用美洲大蠊粉分别替代0%、50%鱼粉配制的2种等氮等能饲料,持续投喂18周,探究了美洲大蠊粉替代鱼粉对虹鳟幼鱼生长、生化指标、抗病力及代谢组学的影响。养殖实验结束后,采用嗜水气单胞菌(Aeromonas hydrophila)对剩余虹鳟幼鱼进行攻毒实验。结果表明：实验组幼鱼生长性能及攻毒后成活率均显著高于对照组(P<0.05);实验组幼鱼血清中免疫球蛋白(IgM)、总抗氧化能力(T-AOC)、肝脏中溶菌酶(LZM)和头肾中过氧化氢酶(CAT)活性均显著高于对照组(P<0.05);代谢组学分析结果显示,两组血清中的差异代谢物主要参与甘油磷脂代谢、鞘脂代谢、缬氨酸、亮氨酸和异亮氨酸生物合成等8条生化代谢途径,其中参与甘油磷脂代谢的溶血磷脂酰胆碱(LysoPC)和磷脂酰胆碱(PC)含量均在实验组显著上调。综上,采用美洲大蠊粉替代饲料中50%的鱼粉可显著提高虹鳟幼鱼...     

高温胁迫条件下大菱鲆肾脏转录组研究

为探索大菱鲆(Scophthalmus maximus)耐高温的分子机制,筛选耐高温相关基因,采用高通量测序平台(IlluminaHiSeq-2500)分别对5个不同高温处理组的大菱鲆肾脏组织开展转录组测序,进行生物信息学分析,包括GO(基因功能注释)、SSR(简单重复序列)分析等。结果显示,通过组装得到Unigenes总数目为68525,长度范围为201~23456 bp,平均长度为1124 bp,N50长度为2316 bp。将Unigenes分别在Nr、Swissprot、KEGG、KOG、GO数据库进行序列比对及功能注释,共注释25498条,其中,Nr数据库注释到的Unigenes最多;按GO功能分类,共分为细胞组分、分子功能及生物学过程3类,包括56个功能组,其中,大量Unigenes与细胞进程、代谢过程、催化活性、生物调节、应激反应相关。将Unigenes进行pathway注释,归属于218条代谢通路,分为5类KEGG途径：代谢途径、遗传信息处理、细胞过程、环境信息和生物系统。进行转录因子分析,共检测到65类转录因子,其中,C2H2锌指蛋白家族的基因数目最多。通过对不同温度胁迫下基因表达谱结果进行分析,不同温度组之间存在着显著差异,在不同温度胁迫组中,20℃组与28℃组存在差异最大,差异基因达到4734个,其中,上调基因3386个,下调基因1348个。本研究建立了大菱鲆热应激肾脏转录组数据库,为大菱鲆高温胁迫分子机理研究提供了参考数据。     

急性高温胁迫对虹鳟和硬头鳟幼鱼抗氧化酶活性的影响

虹鳟(Oncorhynchus mykiss)与硬头鳟(O.mykiss)为同一种的不同生态型,都属冷水性鱼类.为探讨急性高温胁迫对虹鳟和硬头鳟抗氧化酶活性的影响,选取虹鳟[体重(22.76±2.89)g]和硬头鳟[体重(23.2±1.22)g]幼鱼,分别在不同温度梯度(16℃、20℃、22℃和24℃;16℃、19℃、...     

基于UPLC-Q-TOF/MS代谢组学技术探究α-酮戊二酸对碳酸盐碱胁迫下鲫血清代谢的影响

为探究饲料中添加α-酮戊二酸(α-ketoglutarate,AKG)对碳酸盐碱生境胁迫下鲫(Carassius auratus)血清代谢变化的影响机理,设置淡水对照组(Con组)、NaHCO₃暴露组(CA组)以及NaHCO₃暴露AKG饲料添加组(AKG组),采用UPLC-Q-TOF/MS代谢组学技术,结合主成分分析(PCA)和正交偏最小二乘判别分析(OPLS-DA)等多元模式识别技术分析鲫血清代谢物组成及含量变化特征,鉴定显著差异代谢物并进行相关代谢通路分析。结果显示,与对照组相比, CA组有27个差异代谢物,富集到了甘油磷脂代谢、亚油酸代谢、色氨酸代谢等12条代谢通路;与CA组相比, AKG组有38个差异代谢物,富集到了甘油磷脂代谢、亚油酸代谢、不饱和脂肪酸生物合成等14条代谢通路。研究结果表明,碳酸盐碱胁迫会造成鱼类氧化应激损伤,发生甘油磷脂代谢、鞘脂代谢和能量紊乱,而饲料中添加AKG能够促进TCA循环提供稳定的碳源和能量供应,通过正向调节甘油磷脂代谢、鞘脂代谢和氨基酸代谢等代谢途径来提高盐碱胁迫下鱼类的抗氧化能力和免疫能力,从而缓解盐...     

基于转录组学解析铜驯化缓解高温胁迫对大黄鱼的氧化损伤

为探讨铜驯化对高温胁迫下大黄鱼(Larimichthys crocea)氧化损伤和基因表达水平的影响,将体质量为(48.92±3.62) g的大黄鱼暴露在铜离子浓度为0和10μg·L^-1的水体中14 d,再暴露在温度为32℃的水体中24 h。结果显示,高温胁迫显著增加了大黄鱼肝脏中活性氧(ROS)和脂质过氧化物(LPO)含量(P<0.05)。铜驯化对常温下ROS和LPO含量不产生影响,而显著降低了高温胁迫下大黄鱼ROS和LPO含量(P<0.05),表明铜驯化缓解了高温胁迫对大黄鱼的氧化损伤。从高温胁迫组vs对照组、铜驯化组vs对照组和(铜驯化+高温胁迫)组vs高温胁迫组中分别筛选到828个、2 311个和1 084个差异基因。GO和KEGG分析发现,差异基因主要富集在与TCA循环、氧化磷酸化、谷胱甘肽代谢、内质网蛋白加工、吞噬体和MAPK信号通路等相关功能上。聚类分析表明,高温胁迫下调了TCA循环、氧化磷酸化、谷胱甘肽代谢、吞噬体和MAPK信号等通路中的大部分基因表达,上调了内质网蛋白加工通路中的大部分基因表达,而铜驯化则对高温胁迫下大黄鱼的这些基因表...     

低氧胁迫下中华圆田螺的肝脏转录组学分析

为了探究低氧胁迫下中华圆田螺(Cipangopaludina cathayensis)肝脏组织基因的差异表达,本研究通过高通量测序技术,分析中华园田螺低氧胁迫组(2.5 mg/L)和常氧组(6.9 mg/L)某些基因的差异表达,并对差异基因进行生物信息学分析,进一步采用实时荧光定量PCR (RT-qPCR)对关键差异表达基因进行验证。结果显示,测序共获得232 379条基因(unigenes),与对照组比较,低氧胁迫组筛到176个差异基因,包含64个上调基因和112个下调基因。GO功能注释分析显示,差异基因主要富集在生物学过程中的几丁质代谢过程和含氨基葡萄糖的复合代谢过程,细胞组分中的胶原三聚体成分,分子功能中的几丁质结合功能和糖衍生物结合功能。KEGG通路富集分析显示,差异基因主要集中于环境信息处理、遗传信息处理、代谢和生物系统这4大类通路。6个关键差异基因的RT-qPCR结果显示,热休克蛋白70B2和热休克蛋白β-6基因表达量上调,几丁质酶蛋白4、α-1胶原蛋白(XIV)、α-4胶原蛋白(XIV)、5-磷酸酶蛋白基因表达量下调,证实了转录组测序结果的可靠性。本研究发现,低氧胁迫激活了中华圆田螺适应缺氧的生理活动,并获得了低氧胁迫下中华圆田螺肝脏组织中相关功能基因的表达信息,为深入研究中华圆田螺响应低氧胁迫的调控机制提供了基础数据和理论依据。     

Hsp60/10 and sHsp families of heat shock protein genes in rainbow trout (Oncorhynchus mykiss) and their expression under heat stress

Heat shock proteins (Hsps) are highly conserved proteins whose expression can be induced by high temperature and play an important role in a variety of biological processes. However, systematic identification of the Hsp60/10 and small Hsp (sHsp) gene family in rainbow trout has not yet been reported, and there is little available information about its roles in evolution in rainbow trout, a typical economical cold-water fish. In this study, we performed a comprehensive analysis of the rainbow trout Hsp60/10 and sHsp gene family and to investigate their expression profiles. A total of one Hsp60 gene, one Hsp10 gene, and ten sHsp genes were identified. According to RNA-seq analysis of rainbow trout liver and head kidney under heat stress, a total of six out of ten sHsp genes were significantly upregulated in liver and head kidney. Real-time RT-PCR (RT-qPCR) was used to quantitatively analyze the expression levels of these genes in different tissues of rainbow trout. Results showed that the expression of hspe1 and hspd1 was lowest in liver and gill, respectively, and highest in brain. In sHsp gene family, all genes are highly expressed in the liver and head kidney, but relatively low in the heart, spleen, brain, gills, and muscles. This systematic analysis provided valuable information about the diverse roles of Hsp60/10 and sHsp in the evolution of teleost fish, which will contribute to the functional characterization of Hsp60/10 and sHsp genes in further research.

     

Glucose-stimulated lipolysis in rainbow trout,Oncorhynchus mykiss,liver

Rainbow trout, Oncorhynchus mykiss, were used to characterize further the influence of glucose on hepatic lipolysis. Liver was removed from fed fish, cut into 1 mm³ pieces and incubated for up to 5 h in Hanks medium containing either 2 mM, 5.5 mM, 10 mM, or 25 mM glucose. Glucose-stimulated lipolysis was indicated by tissue triacylglycerol (TG) lipase activity and by medium concentrations of glycerol and fatty acids (FA). Triacylglycerol lipase activity in liver pieces incubated in the presence of higher concentrations (25 mM) of glucose was significantly higher than that in liver pieces incubated in lower concentrations (2 mM) of glucose, rising from 0.075 ± 0.002 (mean ± SEM) nmol FA released/h/mg protein to 0.092 ± 0.004 units. Similarly, higher concentrations of glucose stimulated significantly more FA release and glycerol release from liver pieces than that stimulated by lower concentrations of glucose. Glycerol release from liver pieces incubated in the presence of 10 mM glucose and 25 mM glucose was ca. 2-fold to 2.8-fold, respectively, higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Fatty acid release from liver pieces incubated in the presence of 10 mM or 25 mM glucose was ca. 1.8-fold higher than that from liver pieces incubated in the presence of either 2 mM or 5.5 mM glucose. Notably, increased glycerol release was not accompanied by a parallel increase in FA. Fatty acid reesterification was more pronounced in liver pieces exposed to higher glucose (10 mM and 25 mM) than in liver pieces exposed to lower glucose (2 mM and 5.5 mM). ¹⁴C-incorporation studies indicated that glucose serves as a carbon source for reesterified FA in trout liver. The route of reesterification appears to be from glucose to glycerophosphate to phosphatidic acid to diacylglycerol to TG. Increasing concentrations of glucose did not affect glycerol kinase activity, indicating that glucose-stimulated lipolysis was not accompanied by increased glycerol recycling within the liver. These results suggest that glucose stimulates fatty acid reesterification and directly enhances net lipolysis in trout liver incubated in vitro.A part of this study was presented at the Annual Meeting of the American Society of Zoologists, December 26–30, 1991, Atlanta, GA.     

Intracellular transport of ovalbumin afterin vivo endocytosis in rainbow trout liver

The intracellular handing of a mannose-terminated glycoprotein taken up in rainbow trout liver cells by receptor-mediated endocytosis has been studied. The intracellular transport and degradation of ovalbumin (OA) were studied by means of subcellular fractionation in Nycodenz gradients and by differential centrifugation following intravenous injection of the ligand. By using OA labelled with¹²⁵I-tyramine cellobiose (¹²⁵I-TC), the subcellular distribution of labelled degradation products could be studied, since they are trapped intracellularly in the organelle where the degradation takes place. ¹²⁵I-TC-OA was shortly after injection (45 min) localized in a homogenous population of endosomes. Labelled degradation products firs appeared in an organelle with the same density distribution as the endosomes. In livers homogenized 2h after injection the degradation products appeared in organelles with increasing size and density. After 24h, the degradation products were recovered in at least two populations of lysosomes with a distribution profile which coincided with that of the lysosomal enzyme -acetylglucosaminidase.The heterogeneous distribution of the late degradation products seemed not to be due to uptake of ligand in different liver cell types as only the parenchymal liver cells took up labelled OA after intravenous injection of the ligand.     

Interaction of low density lipoproteins with liver cells in rainbow trout

Liver is the main catabolic tissue for low density lipoprotein in rainbow trout (Gjøen and Berg 1992). We have investigated the interaction of LDL with isolated trout liver cells and liver membranes. ¹²⁵I-TC labelled trout LDL bound to isolated trout liver cells in a time dependent and saturable manner with an apparant K_d of 20.1 g/ml, suggesting the existence of a specific binding site on the surface of these cells. The binding was Ca²⁺ dependent assessed by the 50% reduction obtained by 5 mM EDTA. Saturable binding to isolated trout liver membranes could also be demonstrated, but with lower affinity as compared to intact cells. Degradation of ¹²⁵I-TC-LDL in hepatocytes was also saturable as degradation could be inhibited about 60% by a 100 fold surplus of unlabelled LDL. The rate of degradation increased with temperature up to 20°C. Both cell association (binding + uptake) and degradation were reduced down to 20% of control in the presence of microtubular and lysosomal inhibitors. Hepatic catabolism of trout LDL therefore seems to depend on receptormediated endocytosis, followed by lysosomal degradation.Abbreviations TC tyramine cellobiose - LDL low density lipoproteins - MeLDL methylated low density lipoproteins - VLDL very low density lipoproteins - HDL high density lipoproteins - VTG vitellogenin - EDTA ethylenediamine tetraacetic acid - PBS phospate buffered saline - SDS-PAGE sodium dodecyl sulphatepolyacrylamide gel electrophoresis - DMPC L--phosphatidylcholine-dimyristoyl     

Comparison of different European rainbow trout populations under intensive production conditions

Growth and carcass quality of European rainbow trout populations were compared in a full sib family test over five experimental periods. In total, 131 full-sib families of 17 different populations, (i.e. 19 500 fish), were included. A special system for family testing in trout was developed. This consisted of separate hatching of single pair matings, separate rearing of families up to marketable size and common fattening under two stocking densities. Thus, growth was studied separately for the rearing and the fattening period. The observed differences in rearing and fattening weight between and within populations were quite distinct and of significance for production and breeding efficiency. Similar ranking of populations after fattening in different years and under different stocking densities indicated that the final weight was highly genetically controlled. Carcass characteristics determined at marketable size showed somewhat smaller differences between populations. Populations with the highest fattening weight did not generally produce the most valuable carcasses. The results are discussed with respect to their impact on breeding strategies.     

Demand-feeding behaviour of rainbow trout

Rainbow trout learned quickly to operate a trigger to deliver dry food and showed a peak of feeding at dusk, but also appreciable nocturnal feeding. Food deliveries were aggregated into feeding bouts with a preferred time between trigger presses of 4–8 min; the aggregation of feeds was more marked in groups of trout than with trout held singly. Total daily food intake varied with the reward level per trigger press, and could therefore be controlled at about the manufacturers recommended feeding levels. Territorial and hierarchical behaviour were observed and in groups of up to 20 trout only one member of the group pressed the trigger although all the fish took the food delivered, and there was no evidence that smaller fish in a group were deprived of food to the point at which they lost condition.     

虹鳟养殖技术之一利用水库底层水养殖虹鳟

虹鳟鱼属冷水性鱼类,生长快,抗病力强,是目前深受养殖户和消费者喜爱的品种之一.2002年,我们利用凌源菩萨庙水库底层水进行虹鳟商品鱼养殖试验,取得较好的经济效益.     

提高虹鳟、金鳟鱼苗成活率的关键技术

在虹鳟及金鳟鱼苗的养殖中,鱼苗的成活率是养鳟鱼场取得良好经济效益的关键,也是衡量鱼场技术水平的一个重要指标。根据多年的生产实践经验,总结出以下几点,供广大鳟鱼养殖户参考。     

Theoretical and practical analysis of waste heat recovery system in off-season rainbow trout production

The supply of heat for fish farms, especially during off-season production, is necessary for the establishment of commercial aquaculture. For this purpose, research was carried out on the supply of heating ener to a rainbow trout farm (cold water fish) using a waste heat recovery system in the city of Meshgin in Ardabil province. The study area has 20 indoor pools and 14 unguarded pools. The 14 pools that are in the indoor area, are used for this study. Seven pools of these 14 pools were at high altitude and other 7 pools were at low altitude. After collection of the temperature data for waste water ponds that had provided optimum temperature conditions in the off-season using geothermal fluid, three modes for waste heat recovery were designed according to the thermal, flow and physicochemical properties of the waste pond. In the first mode, the output of the first seven pools and the second seven pools were combined and the fluid flow rate reached to 24.92 L/sec. In the second mode, the output of the second seven pools (8.15 L/sec) was used to recover heat, and in the third case, the output of the first seven pools (at high-altitude pools and with the flow rate of 16.77 L/sec) was used to recover heat. After obtaining technical approval and determining the economic value of each component of the waste heat recovery circuit from specialized companies in Iran, the proposed systems were analyzed theoretically with COMSOL software. Because there was no equal rate of flow, velocity or geometry for comparison between the optimum state in the theoretical conditions of practice, the temperature differences between the input hot water and output cold water were used for all three modes of heat recovery. Technically, the results indicate that the second mode is best in both the theoretical and practical modes of the waste heat recovery circuit. The first mode recovery system had the smallest temperature difference between the hot water inlet and cold water outlet. Economically, the minimum cost of establishing a heat recovery system was for the second mode, with a decrease of 60.65% and 38.83% compared to the third and first modes, respectively. Considering technical and economic factors, the waste heat recovery system of the second mode was the most appropriate choice for the trout farm, both theoretically and practically. The first and third modes ranked next, respectively.     

虹鳟养殖技术 之三武陵山区养殖虹鳟应注意的技术环节

虹鳟是典型的冷水性鱼类,2005年北京市对口支援湖北省巴东县虹鳟鱼养殖项目,当年5月引进鱼苗1.5万尾后放入引水水渠中饲养,之后在水渠下方建新鱼池,并将鱼苗转入新池