大菱鲆脂质代谢相关基因PPARs的组织表达及其对高温胁迫的响应

为研究大菱鲆过氧化物酶体增殖物激活受体家族(PPARs)的组织分布和高温胁迫下PPARs在肾脏中的表达情况。实验采用荧光定量PCR(qPCR)技术检测PPARs基因3种亚型在大菱鲆不同组织中的表达情况以及高温胁迫下大菱鲆肾脏中PPARs的表达情况。qPCR结果显示,大菱鲆PPARs 3种亚型的组织分布存在显著差异。PPARα1和PPARα2在心脏中的表达量显著高于其他组织;PPARβ在大菱鲆的各个组织中普遍表达;PPARγ在大菱鲆的鳃中出现了显著性的高表达。大菱鲆肾脏中PPARs的mRNA水平随着温度升高呈现出不同的响应模式。PPARα随温度升高表达水平先显著降低,后有所升高;PPARβ的表达量在14、20、23和25℃时差异不显著,当温度升高至大菱鲆的致死温度28℃时,表达量出现了显著性的升高;PPARγ在14℃时表达水平很低,但随着温度的升高不断增加。研究表明,大菱鲆中存在PPARα、PPARβ和PPARγ3种亚型,而且三者可能以组织特异性的方式参与脂质代谢的调节,首次指出PPARs 3种亚型在温度胁迫中的表达变化,对PPARs的研究将推动鱼类脂代谢研究,揭示鱼类PPARs在脂质代谢调控以及响应逆境胁迫中的重要作用。     

虹鳟PPARα基因克隆、序列分析及其组织表达分布

克隆了虹鳟(Oncorhynchus mykiss)过氧化物酶体增殖物激活受体(peroxisome proliferator activated receptor,PPAR)αmRNA全序列。对其序列分析发现,虹鳟PPARα与其他脊椎动物PPARα有较高的同源性,其中mRNA序列有66.4%~97.5%相同,氨基酸序列有69.2%~98.9%相同。DNA结合结构域和配体结合结构域从鱼类到人类高度保守,其中DNA结合结构域有88.0%~98.8%的氨基酸序列相同;配体结合结构域有74.6%~99.6%的氨基酸序列相同。系统发生树分析表明,虹鳟的PPARα基因与同属鲑科鱼类的大西洋鲑(Salmo salar)属于同一分支。实时定量RT-PCR方法分析虹鳟不同组织中的表达发现,PPARα基因在脂肪、肌肉、卵巢、肾脏和肠中表达量较高。     

水中Mn(Ⅱ)对大菱鲆幼鱼生长及碱性磷酸酶和超氧化物歧化酶活性的影响

水温14~16℃下,将初始体质量为(5.0±1.0)g的大菱鲆养殖在40L水体的水族箱中,研究在不同Mn(Ⅱ)质量浓度(0.04、0.08、0.16、0.32、0.64、1.28、2.56、5.12、10.24mg/L)下大菱鲆的生长及碱性磷酸酶和超氧化物歧化酶的活性。结果表明,当水体中Mn(Ⅱ)质量浓度低于2.56mg/L时,鱼的生长较快,其中Mn(Ⅱ)质量浓度为0.64mg/L时生长最快;当Mn(Ⅱ)质量浓度低于0.64mg/L时,大菱鲆肝脏的碱性磷酸酶和超氧化物歧化酶活性随着Mn(Ⅱ)质量浓度的提高而增加,当Mn(Ⅱ)质量浓度超过0.64mg/L时,碱性磷酸酶和超氧化物歧化酶的活性急剧下降;0.64mg/L组的碱性磷酸酶和超氧化物歧化酶的活力均较高,随着Mn(Ⅱ)质量浓度增加,碱性磷酸酶和超氧化物歧化酶的活力先增后减;据剂量效应推测,0.64mg/L为Mn(Ⅱ)表现出毒性作用的临界质量浓度。     

碳酸盐碱度胁迫下凡纳滨对虾基因的差异表达

以广盐性养殖的凡纳滨对虾(Litopenaeus vannamei)为研究对象,采用抑制消减杂交(suppression subtractive hybridization,SSH)和定量PCR的方法,研究其在高碳酸盐碱度胁迫下转录组水平的基因表达差异,以期从基因组水平研究对虾对高碳酸盐碱度胁迫的适应机制。结果表明,以高碳酸盐碱度(20 mmol/L)胁迫第4天凡纳滨对虾鳃组织和低碱度(2 mmol/L)对照组鳃组织为材料,通过斑点杂交筛选,发现鳃组织中有158个克隆子表达上调,291个克隆子表达下调。挑选150个高表达差异的克隆子进行测序,获得100个序列,其中50个得到成功注释。经过GO分析,这些注释的差异基因主要分为8大类群,其中碳酸酐酶基因(CA)、Na+-K+-ATPase基因(NKA-α)等与离子调控相关的基因表达量上调,而溶菌酶基因等与先天免疫相关的基因表达量下调,这些结果表明高碳酸盐碱度胁迫下,凡纳滨对虾以增加离子调控的方式进行酸碱平衡的调控,同时其免疫功能受到抑制。此外,还对CA和NKA-α两个基因在鳃和触角腺中的时空表达规律进行了研究,发现高碳酸盐碱度胁迫9 d过程中,两个基因在鳃组织中的表达均呈现先高表达后回落的现象,而在触角腺中NKA-α基因则一直维持较高表达水平,说明CA和NKA-α基因在凡纳滨对虾高碳酸盐碱度适应离子调控中起着关键作用,同时还发现除了鳃组织之外,触角腺同样参与了调控。本研究从转录水平初步筛选了高碳酸盐碱度胁迫下凡纳滨对虾的表达差异基因,探索了凡纳滨对虾的耐盐碱机制,对培育适于盐碱地水产养殖的优良品种有着重要的意义。     

Liposome‐mediated messenger RNA: An alternative for fish cell transfection in culture

Liposome‐mediated plasmid DNA (pDNA) transfection is a relatively efficient transfection method that depends highly on actively dividing cells. However, numerous fish cells are mitotically inactive under in vitro culture conditions, thus limiting the application of liposome‐mediated pDNA transfection. Liposome‐mediated mRNA transfection is an attractive alternative in post‐mitotic cells because it eliminates the transfer of genes into the nucleus and therefore is independent of cell proliferation. In this study, pDNA is compared with mRNA encoding biomarker proteins (enhanced green fluorescent protein [eGFP] and luciferase) mediated by cationic liposome in zebrafish embryo fibroblast cell line (ZF4), with or without proliferation restriction, and in mitotically inactive turbot liver primary cells. The results indicated that mRNA encoding eGFP exerted much higher transfection efficiency than pDNA in ZF4 cells. Moreover, mRNA encoding eGFP was detected and disappeared sooner than pDNA. Chemiluminescence of the biomarker protein luciferase showed that liposome‐mediated mRNA transfection translated about threefold more protein without proliferation restriction and about tenfold more protein with proliferation restriction than pDNA transfection. In turbot liver cells, mRNA induced higher transfection efficiency and greater luciferase protein expression than pDNA. Greater PPARα expression and expression of downstream target genes were induced by mRNA rather than by pDNA transfection. In conclusion, cationic liposome‐mediated mRNA is an alternative for fish cell transfection due to higher transfection efficiency and protein expression levels and faster translation onset.     

Molecular characterization and expression analysis of AMPK α subunit isoform genes from <Emphasis Type="Italic">Scophthalmus maximus</Emphasis> responding to salinity stress

AMP-activated protein kinase (AMPK) is a highly conserved and multi-functional protein kinase that plays important roles in both intracellular energy balance and cellular stress response. In the present study, molecular characterization, tissue distribution and gene expression levels of the AMPK α1 and α2 genes from turbot (Scophthalmus maximus) under salinity stress are described. The complete coding regions of the AMPK α1 and α2 genes were isolated from turbot through degenerate primers in combination with RACE using muscle cDNA. The complete coding regions of AMPK α1 (1722 bp) and α2 (1674 bp) encoded 573 and 557 amino acids peptides, respectively. Multiple alignments, structural analysis and phylogenetic tree construction indicated that S. maximus AMPK α1 and α2 shared a high amino acid identity with other species, especially fish. AMPK α1 and α2 genes could be detected in all tested tissues, indicating that they are constitutively expressed. Salinity challenges significantly altered the gene expression levels of AMPK α1 and α2 mRNA in a salinity- and time-dependent manners in S. maximus gill tissues, suggesting that AMPK α1 and α2 played important roles in mediating the salinity stress in S. maximus. The expression levels of AMPK α1 and α2 mRNA were a positive correlation with gill Na⁺, K⁺-ATPase activities. These findings will aid our understanding of the molecular mechanism of juvenile turbot in response to environmental salinity changes.     

应用高通量测序技术分析大菱鲆幼鱼肠道及其养殖环境的微生物群落结构

采用基于Illumina测序平台的高通量测序技术,对大菱鲆(Scophthalmus maximus)幼鱼肠道及其养殖水体、生物饵料中细菌种类及丰度进行研究。测序结果显示,养殖水体、生物饵料和大菱鲆幼鱼肠道等19个样品共获得有效序列547621条,可聚类于3771个可分类操作单元(OTUs),归属于养殖水体、生物饵料、健康幼鱼和发病幼鱼的操作分类单元(OTU)个数分别为3038、1090、87和777,其中,健康幼鱼与生物饵料、健康幼鱼与养殖水体特有的OTU个数分别为57和0,发病幼鱼与生物饵料、发病幼鱼与养殖水体特有的OTU个数分别为481和31。表明幼鱼肠道微生物多样性与生物饵料密切相关。根据细菌注释结果,拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和变形菌门(Proteobacteria)在大菱鲆幼鱼肠道中占优势地位,其中,健康幼鱼肠道微生物共聚类为8个门,发病幼鱼的肠道微生物可聚类为19个门。与健康幼鱼相比,发病幼鱼肠道门水平上的3种主要优势菌群落结构出现失衡。此外,对各样品中丰度最高的100位OTU分析显示,幼鱼肠道优势菌种类与生物饵料中的优势菌种类密切相关,而每个发病幼鱼肠道优势菌种类具有一定的独立性。本研究旨在为大菱鲆健康养殖和微生态调控提供实验依据。     

温度对大菱鲆幼鱼生长及免疫相关酶活性的影响

将体质量(31.13±1.67)g的大菱鲆幼鱼饲养在容积100L的水桶中,每个水桶40尾,采用OKE-6710HF微电脑温控仪控制温度为15、18、21、24℃和27℃,在第0、15、30、45、60d进行采样分析血清中免疫相关酶活性及幼鱼全长、体质量及存活率。结果显示,在15~27℃条件下,大菱鲆幼鱼生长速度随温度升高先升后降;水温为18℃时,幼鱼存活率为100%,生长速度最快,为最佳生长水温。大菱鲆幼鱼血清中酸性磷酸酶和碱性磷酸酶活力,随温度及饲养时间的变化而呈波动性,但没有表现出显著的规律性;大菱鲆幼鱼血清中超氧化物歧化酶的活力随温度的升高先升后降;刚达到设定温度(即第0d)时,各温度组之间溶菌酶活力并无显著差异(P0.05),随着饲养时间的加长及温度的升高,溶菌酶活力不断升高,表明温度对大菱鲆幼鱼生长及存活率具有显著的影响。     

Effect of waterborne zinc exposure on lipid deposition and metabolism in hepatopancreas and muscle of grass carp <Emphasis Type="Italic">Ctenopharyngodon idella</Emphasis>

The aim of the present study was to explore the effect of waterborne zinc (control, 0.85, 2.20, 3.10 mg/l, respectively) exposure on lipid deposition and metabolism in the hepatopancreas and muscle of grass carp Ctenopharyngodon idella. The lipid content, Zn accumulation, and the activities and expression levels of several enzymes involved in lipid metabolism were determined in hepatopancreas and muscle. Waterborne Zn exposure reduced growth performance and increased Zn accumulation in both tested tissues. In hepatopancreas, Zn exposure increased lipid content, the activities of lipogenic enzymes, such as 6PGD, G6PD, ME, ICDH and FAS, as well as the mRNA expression level of G6PD, 6PGD, ICDH, FAS and SREBP-1. But the activity of CPT I and the mRNA expression of HSL, CPT Iα1a, CPT Iα2a and PPARα were down-regulated by Zn exposure. In contrast, in muscle, waterborne Zn exposure decreased lipid deposition, activities of 6GPD, ICDH and ME, as well as the mRNA expression level of G6PD, ICDH, ME, FAS and SREBP-1. However, the activity of CPT I as well as the mRNA expression level of PPARα, HSL, CPT Iα2a, CPT Iα1b and CPT Iβ were up-regulated by Zn exposure. Our results indicate that waterborne Zn increases lipid content by up-regulating lipogenesis and down-regulating lipolysis in hepatopancreas. But, in muscle, waterborne Zn reduces lipid accumulation by up-regulating lipolysis and down-regulating lipogenesis. Differential patterns of lipid deposition, enzymatic activities and genes’ expression indicate the tissue-specific regulatory mechanism in fish.     

抑制性消减杂交技术在养殖鱼类免疫基因克隆中的应用

抑制性消减杂交技术(SSH)是近年来兴起的一种分离、克隆差异基因的新技术,它结合了消减杂交和抑制PCR的优点,具有操作简便、特异性强、背景低及重复性好的优点。目前已经用SSH技术鉴定出了鱼的许多免疫相关基因,如白细胞介素、趋化因子、肿瘤坏死因子、溶菌酶、NKEF、补体、干扰素及急性期蛋白基因等,对它们的结构和功能进行了较为深入的研究。本文对SSH技术在养殖鱼类中克隆的免疫基因进行了归纳与总结,旨为全面了解鱼类的抗病免疫基因提供基础资料。     

Physiological and morphological effects of severe hypoxia,hypoxia and hyperoxia in juvenile turbot (Scophthalmus maximus L.)

We studied the physiological status of juvenile turbot (Scophthalmus maximus L.) under severe hypoxia (1 and 2 mg/L dissolved oxygen, DO), hypoxia (3 and 5 mg/L DO), hyperoxia (11 and 14 mg/L DO) and normoxia (7 mg/L DO, control) conditions. The respiratory rates, haematology parameters, acid–base balance status and gill structure were analysed to find the effects of different DO concentration on turbot. Fish mortality was only observed under severe hypoxia conditions. Severe hypoxia caused an increase in respiratory rates and red blood cell counts, as well as an increase in haemoglobin and haematocrit levels in the fish. In fish exposed to hypoxia conditions, the respiratory rate increased overall as the DO concentration decreased. Lower pCO₂ and HCO₃^? levels led to a high blood pH, while the pO₂ remained stable. In hyperoxia groups, respiratory rate decreased as the DO concentration increased. The levels of pCO₂ and HCO₃^? significantly increased (P < 0.05), while the pO₂ level and blood pH did not change obviously. The gill structure was damaged after prolonged exposure to hyperoxia, but no obvious damage was found in hypoxia groups. The fish that survived the hypoxia or hyperoxia treatment were able to restore the structural integrity of the gills after 14 days' recovery. The results suggest that juvenile turbots can tolerate a wide range of DO concentrations. However, even mild hyperoxia condition (11 mg/L DO), which is widely used in fish culture, has adverse effects on juvenile turbot physiology.     

坛紫菜色素突变体色素基因的表达定量分析

以野生型和6种不同色泽的坛紫菜色素突变体为材料,通过实时荧光定量PCR技术分析了4种主要色素基因(Cpeα、Cpcα、Apcβ、Chl.a)在不同色泽突变体不同生长期(初期、盛期、末期)的相对表达水平。结果表明在不同色泽不同生长期的坛紫菜色素突变体中,各色素基因表达水平由高到低均为Apcβ>Cpcα>Chl.a>Cpeα,且Cpeα基因的表达水平最不稳定,会随着生长过程发生显著变化,而Cpcα、Apcβ和Chl.a基因表达水平则相对稳定;此外,对色素突变体和野生型藻体中色素基因表达水平和相应色素蛋白含量的线性相关回归分析结果表明二者间没有相关性,即各色素基因的表达水平与藻体最终显示的颜色无关,由此推测坛紫菜色素突变的可能机制是色素蛋白合成过程中一些相关调控基因突变所致。     

大菱鲆幼鱼对饲料中泛酸最适需求量的研究

为研究大菱鲆(Scophthalmus maximus)幼鱼对泛酸的最适需求量,在基础配方中添加不同梯度的泛酸钙,制成6组泛酸含量分别为6.24、10.64、15.02、23.81、41.40和76.57 mg/kg等氮等能实验饲料,投喂初始体重为(24.73±0.10) g的大菱鲆幼鱼80 d。结果显示：1)泛酸对幼鱼成活率(SR)无显著影响(P>0.05),10.64~76.57 mg/kg饲料组增重率(WGR)和特定生长率(SGR)显著提高(P< 0.05);泛酸含量超过23.81 mg/kg时,肝体比(HSI)显著降低(P<0.05);2)随着饲料中泛酸含量的提高,全鱼粗蛋白、粗脂肪和肌肉粗蛋白均呈先升后降趋势,肝脏脂肪含量显著下降(P<0.05);3)肠道消化酶、Na+, K+-ATPase和肝脏胆碱酯酶(ChE)活力随泛酸添加量的增加呈先上升后下降趋势,10.64~76.57 mg/kg饲料组肠道肌酸激酶(CK)活力显著提高(P<0.05);4)血清、肝脏中过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活力均呈先升后降趋势;6.24 mg/kg饲料组血清丙二醛(MDA)含量显著高于其他5组(P<0.05);5)随泛酸含量的增加,肝脏脂肪合成酶(FAS)基因表达量先升后降,脂蛋白酯酶(LPL)基因表达量显著上升(P<0.05)。研究表明,饲料中添加适宜的泛酸能显著增强大菱鲆幼鱼肠道消化、吸收能力和机体抗氧化能力,并可提高脂肪代谢相关基因的表达水平,从而提高其生长性能。以增重率为判据,经折线模型拟合得出,初体重为24.73 g的大菱鲆幼鱼对泛酸的需求量为16.08 mg/kg饲料。     

The effect of high ortho-phosphate water levels on growth,feed intake,nutrient utilization and health status of juvenile turbot (Psetta maxima) reared in intensive recirculating aquaculture systems (RAS)

In intensive recirculating aquaculture systems (RAS) ortho-phosphate (ortho-P) is one of the main accumulating substances, but effects of chronically elevated concentrations on fish health and production performance are still unknown. Therefore 120 juvenile turbot (Psetta maxima) were exposed to ortho-P concentrations of 3 mg/L (control – C), 26 mg/L (low – LP), 52 mg/L (medium – MP) and 82 mg/L (high – HP) for 56 days and fed until satiation with a commercial diet. Health status and feed conversion ratio (FCR) were not significantly affected by treatment (p > 0.05). Specific growth rates (SGR) and daily feed intake (DFI) of C were not considered significantly different from LP, MP and HP treatments, however LP showed significant higher DFI and SGR than HP (p < 0.05). Using non-linear regression between SGR and ortho-P concentrations, 27 mg/L ortho-P was found as the optimum for turbot growth. Although not reflected in blood plasma P levels (p > 0.05) a potential aqueous P uptake might result in metabolic benefits leading to the observed growth enhancement in the LP treatment.In a second experiment 114 juvenile turbot were exposed to ortho-P concentrations of 2 mg/L (C2) and 25 mg/L (LP2) for 63 days and fed until satiation with a low P diet (4.6 g digestible-P/kg diet). Overall production performance was low due to low voluntary feed intake. Whereas the FCR was unaffected by treatment (p > 0.05), significantly higher feed intake and biomass gain were observed for LP2 compared to C2 (p < 0.05). LP2 treatment showed a trend for higher protein retention efficiency and lower whole body lipid content (p < 0.1). The dry matter, ash, Phosphorus, Calcium and protein content in whole body did not significantly vary between treatments (p > 0.05).In conclusion the accumulation of ortho-P in RAS does not negatively affect health of turbot. Elevated ortho-P seems to have slight positive effects on production performance of juvenile turbot. Further research to quantify dietary P requirements for turbot in general, as well as for turbot raised under elevated ortho-P conditions in RAS is strongly required.     

Effects of size grading on growth and survival of juvenile turbot at two temperatures

Juvenile turbot were size graded into three size groups (mean initial size): Small (3.4 g), medium (7.0 g) and large (10.5 g), and additional fish were held in ungraded (6.6 g) groups. Subgroups (n = 396) of fish were tagged and individual growth rates and social interactions within different size categories were studied in fish reared at 13 and 19 °C. Size grading of juvenile turbot did not improve growth. Specific growth rates (SGR) were mainly size-related, and no differences in SGR or mortality between the experimental groups at both tem-peratures were found. A higher level of social interactions was indicated amongst medium-sized fish than amongst those in the smallest and largest categories. Excess feeding may have been important factors in reducing aggression, so that the growth of the smallest individuals was not suppressed by the larger individuals in the present study. Grading seems to be an unnecessary operation to improve the growth and survival of juvenile turbot (5- 150 g). However, as it was mainly the smallest individuals in each group that died, grading of very small turbot (2-5 g) can be recommended.     

三倍体虹鳟实时荧光定量PCR内参基因稳定性分析

采用实时荧光定量PCR(qRT-PCR)方法比较甘油醛-3-磷酸脱氢酶(gapdh)、核糖体大亚基蛋白基因(rplp2)、真核延伸因子基因(ef1-α)、β-肌动蛋白(β-actin)和18S核糖体核酸基因(18S rRNA)等5个候选内参基因在体质量约800 g三倍体虹鳟脑、眼、鳃、皮肤、心脏、肾脏(头肾、中肾、后肾...