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1.
本研究将100~300 pg含有肌肉特异表达启动子和绿色荧光蛋白(Green fluorescent protein,GFP)基因的重组质粒(smyd1:gfp)显微注射到大菱鲆(Scophthalmus maximus)受精卵动物极细胞中,通过细心培育,成功孵化出鱼苗约120尾。统计分析显示,显微注射后,大菱鲆胚胎存活率为4.8%。利用荧光显微镜观察大菱鲆胚胎及仔鱼,只在注射smyd1:gfp质粒的胚胎及仔鱼的肌肉中发现有绿色荧光。通过进一步PCR扩增检测,在注射的大菱鲆胚胎及仔鱼DNA中扩增出了GFP特异片段,大小约为340 bp。研究表明,本研究成功建立了大菱鲆显微注射技术,可为大菱鲆基因功能研究和遗传育种奠定基础。  相似文献   

2.
显微注射技术在制备鱼类嵌合体和转基因海水鱼上的应用   总被引:1,自引:0,他引:1  
以鱼类胚胎细胞和胚胎干细胞为核供体进行细胞移植构建鱼类嵌合体研究方面,现有的成功报道均采用显微注射方法;在转基因海水鱼类研究中,显微注射也是最为常用的技术,本实验室在花鲈胚胎干细胞嵌合体构建和外源基因向花鲈胚胎的转移研究中取得的结果也充分证实,显微注射技术是开展海水鱼类细胞移植和转基因研究的首选技术。  相似文献   

3.
为研究半滑舌鳎(Cynoglossus semilaevis)vasa(Csvasa)基因调控区的功能,在已克隆的Csvasa基因编码序列的基础上,采用基因组步移和PCR扩增的方法克隆得到Csvasa调控区,通过生物信息学方法分析vasa基因5′区,并构建了含Csvasa基因调控区的绿色荧光蛋白(GFP)表达载体(p Csvasa-GFP-T),进一步通过显微注射技术初步验证调控区的驱动活性。结果表明,通过基因组步移和PCR扩增获得Csvasa 5′区5 166 bp和3′区1 655 bp,利用在线生物信息学软件对5′区序列进行分析,发现在转录起始点上游26 bp处存在保守的TATA框,以及潜在的转录因子结合点如SRY、Oct-1、Sox-5、CREB、GATA、AP-1、C/EBP、Sp-1、c-Myc、HNF、NKX2-5、V-Myb等。通过显微注射技术,将所构建的p Csvasa-GFP-T表达载体注射于青鳉(Oryzias latipes)受精卵并进行培养观测,发现Csvasa调控区能够驱动GFP在青鳉胚胎内表达,荧光表达率为81%。将有荧光的胚胎培养为成鱼,检测外源基因的整合率为11.5%。这些结果为进一步研究半滑舌鳎原始生殖细胞(PGCs)的标记、追踪和操作研究以及半滑舌鳎的性别控制等奠定了基础。  相似文献   

4.
对花鲈(Lateolabrax japonicas)成鱼腹腔注射促黄体激素释放激素类似物(LHRH-A3)和人体绒毛膜促性腺激素(HCG),利用放射性免疫激素测定方法(RIA)检测了花鲈血清中类胰岛素生长因子(Insulin-like growth factor 1,IGF-1)在注射后6、12、24、48 h内的变化情况,同时还利用荧光实时定量PCR技术检测了花鲈肝脏中IGF-1和类胰岛素生长因子结合蛋白1(Insulin-like growth factor-binding protein 1,IGFBP-1)mRNA的相对表达量的变化情况。结果表明,注射LHRH-A3的花鲈血清中IGF-1的含量在24 h后出现了明显的降低(P0.05),而花鲈肝脏中IGF-1 mRNA的相对表达量在注射6 h后较对照组明显升高(P0.05),而肝脏中IGFBP-1 mRNA的相对表达量则呈现下降趋势。注射HCG的花鲈血清中IGF-1的含量在注射后12 h出现了明显降低(P0.05),24 h后血清中IGF-1含量持续降低。肝脏中IGF-1 mRNA的相对表达量在注射后的24 h内均保持稳定,而在处理后的48 h出现了显著上升(P0.05)。肝脏中IGFBP-1 mRNA的相对表达量在注射后12h检测到显著升高(P0.05)。研究结果表明,LHRH-A3和HCG均可以通过直接或间接的刺激作用来触发花鲈IGF系统的应答反应,但是其中具体机制尚不明了。  相似文献   

5.
对花鲈(Lateolabrax japonicas)成鱼腹腔注射促黄体激素释放激素类似物(LHRH-A3)和人体绒毛膜促性腺激素(HCG),利用放射性免疫激素测定方法(RIA)检测了花鲈血清中类胰岛素生长因子(Insulin-like growth factor 1,IGF-1)在注射后6、12、24、48 h内的变化情况,同时还利用荧光实时定量PCR技术检测了花鲈肝脏中IGF-1和类胰岛素生长因子结合蛋白1(Insulin-like growth factor-binding protein 1,IGFBP-1)mRNA的相对表达量的变化情况。结果表明,注射LHRH-A3的花鲈血清中IGF-1的含量在24 h后出现了明显的降低(P0.05),而花鲈肝脏中IGF-1 mRNA的相对表达量在注射6 h后较对照组明显升高(P0.05),而肝脏中IGFBP-1 mRNA的相对表达量则呈现下降趋势。注射HCG的花鲈血清中IGF-1的含量在注射后12 h出现了明显降低(P0.05),24 h后血清中IGF-1含量持续降低。肝脏中IGF-1 mRNA的相对表达量在注射后的24 h内均保持稳定,而在处理后的48 h出现了显著上升(P0.05)。肝脏中IGFBP-1 mRNA的相对表达量在注射后12h检测到显著升高(P0.05)。研究结果表明,LHRH-A3和HCG均可以通过直接或间接的刺激作用来触发花鲈IGF系统的应答反应,但是其中具体机制尚不明了。  相似文献   

6.
7.
为探究花鲈(Lateolabrax maculatus)干扰素刺激基因 15 (Interferon-stimulated gene 15, ISG15)在抵抗病毒过程的免疫应答反应, 本研究克隆、鉴定了花鲈 ISG15 基因(LmISG15), 并对其进行生物信息学分析, 获得了 LmISG15 在各组织中的表达特征及其在受到传染性脾肾坏死病毒(infectious spleen and kidney necrosis virus, ISKNV)感染后的时空表达特征。结果显示, 该基因全长 1625 bp, ORF 为 480 bp, 编码 160 个氨基酸, 分子质量为 17.5 kD, 理论等电点为 9.41。LmISG15 的氨基酸序列较为保守, 与眼斑拟石首鱼(Sciaenops ocellatus)和条石鲷(Oplegnathus fasciatus)相似度分别为 74.07%和 71.88%。LmISG15 在花鲈 10 种组织中均有表达, 在心脏中表达量最高, 其次在脑、中肾和鳃中的表达量较高, 在肌肉中表达量最低。注射 ISKNV 后, 花鲈头肾组织中病毒拷贝数升高, 同时 LmISG15 在花鲈肝脏、头肾和鳃中的表达量显著上调(P<0.05)。研究表明, LmISG15 参与了抵抗病毒刺激的免疫应答过程。  相似文献   

8.
王志远  李金库  李昀  王灵钰  齐鑫  李吉方  温海深 《水产学报》2023,47(8):089104-089104
为探究ncc、nkcc基因在花鲈渗透调节中发挥的作用,实验通过全基因组鉴定、多重序列比对、系统进化树构建以及蛋白结构预测对花鲈ncc进行了鉴定及序列分析,利用实时荧光定量PCR (qRT-PCR)检测ncc和nkcc在海水、淡水花鲈鳃组织中的表达水平,利用原位杂交技术确定ncc2和nkcc1a在海水及淡水花鲈鳃中的表达位置。结果显示,从花鲈中鉴定出2个ncc基因,即ncc1和ncc2,其编码序列(CDS)长度分别为2 691和3 120bp,编码896和1 039个氨基酸,在进化上具有保守性。ncc2在淡水花鲈鳃组织中的表达量显著高于海水,而nkcc1a在海水花鲈鳃组织中的表达量显著高于淡水,ncc1、nkcc1b、nkcc2在海淡水中的表达量则无显著差异。淡水适应过程中花鲈鳃组织中的ncc2的表达量逐渐上调,而nkcc1a的表达量逐渐下调;海水适应过程则呈现相反的表达趋势。此外,原位杂交结果显示,ncc2和nkcc1a基因分别位于淡水与海水中鳃组织的相邻鳃小片间的鳃丝上皮。以上结果表明,ncc2和nkcc1a基因分别编码淡水及海水花鲈鳃中重要的Na+及Cl  相似文献   

9.
红色荧光蛋白(RFP)基因在转基因青Jian中的表达   总被引:5,自引:1,他引:5  
  相似文献   

10.
为获得快速生长的泥鳅(Misgurnus anguillicaudatus),研究了泥鳅β-actin基因启动子介导的革胡子鲶(Clarias gariepinus)生长激素(growth hormone,GH)重组基因。采用PCR和RT-PCR技术克隆泥鳅β-actin基因近端启动子、3′-UTR及革胡子鲶GH基因编码区,构建一个长为2 418bp的GH重组基因DPRK。首先,构建了以增强型绿色荧光蛋白(EGFP)基因作为报告基因的重组表达载体p AF(pβ-actin promoter-EGFP),然后转染DF1真核细胞,同时经酶切线性化后显微注射到斑马鱼(Danio rerio)和泥鳅的受精卵中,以评价泥鳅β-actin启动子的启动活性。其次,将重组基因DPRK显微注射到泥鳅受精卵中。结果显示:泥鳅β-actin启动子能在DF1细胞、斑马鱼和泥鳅的受精卵中启动绿色荧光蛋白的表达;泥鳅的荧光表达率(78.44%)显著高于斑马鱼(29.62%);泥鳅受精卵显微注射DPRK 20d后,在m RNA水平检测到GH基因的表达。这表明泥鳅β-actin基因近端启动子具有显著的启动活性,且重组基因DPRK能在泥鳅体内表达,为下一步泥鳅的基因工程育种奠定了理论基础。  相似文献   

11.
The environmental processes associated with variability in the catch rates of bigeye tuna in the Atlantic Ocean are largely unexplored. This study used generalized additive models (GAMs) fitted to Taiwanese longline fishery data from 1990 to 2009 and investigated the association between environmental variables and catch rates to identify the processes influencing bigeye tuna distribution in the Atlantic Ocean. The present findings reveal that the year (temporal factor), latitude and longitude (spatial factors), and major regular longline target species of albacore catches are significant for the standardization of bigeye tuna catch rates in the Atlantic Ocean. The standardized catch rates and distribution of bigeye tuna were found to be related to environmental and climatic variation. The model selection processes showed that the selected GAMs explained 70% of the cumulative deviance in the entire Atlantic Ocean. Regarding environmental factors, the depth of the 20 degree isotherm (D20) substantially contributed to the explained deviance; other important factors were sea surface temperature (SST) and sea surface height deviation (SSHD). The potential fishing grounds were observed with SSTs of 22–28°C, a D20 shallower than 150 m and negative SSHDs in the Atlantic Ocean. The higher predicted catch rates were increased in the positive northern tropical Atlantic and negative North Atlantic Oscillation events with a higher SST and shallow D20, suggesting that climatic oscillations affect the population abundance and distribution of bigeye tuna.  相似文献   

12.
In this experiment, a feeding trial was performed to determine the effects of fructooligosaccharide (FOS) on growth performance, digestive enzyme activity and immune response of Japanese sea bass, Lateolabrax japonicus juveniles (initial weight 38.3 ± 0.5 g), and the fish were examined following feeding with six levels of FOS (0, 0.5, 1, 2, 4 and 6 g/kg) for 28 days. Significant enhancement of weight gain (WG) and specific growth rate (SGR) was found in fish fed 1 g/kg FOS incorporated diets (p < .05), while the feed conversion ratio (FCR) in the 1, 2 g/kg FOS groups reduced significantly compared with the control (p < .05). Besides, the crude lipid in the 4, 6 g/kg FOS groups increased significantly compared with the control (p < .05). On the other hand, the erepsin and lipase activities significantly elevated in intestine of fish fed 2 g/kg FOS (p < .05) and the lysozyme activity in serum of fish fed 2 g/kg FOS were significantly higher than that in the control (p < .05). Moreover, the alkaline phosphatase activities in serum of fish fed 0.5, 1, 2 g/kg FOS were significantly higher than in control (p < .05). Regression analysis showed that the relationships between dietary FOS levels and either SGR, FCR, erepsin or lysozyme activities were best expressed by regression equations, and the optimal inclusion levels are 1.37, 1.80, 3.06, 3.11, 1.93 and 1.80 g/kg for SGR, FCR, erepsin, lipase, lysozyme and total superoxide dismutase activities, respectively. Overall, this study revealed that FOS incorporated diets could beneficial for L. japonicus culture in terms of increasing the growth, digestion and immune activities. Under the present experimental condition, the optimal supplementary level of FOS in the diet of L. japonicus is 1–3 g/kg.  相似文献   

13.
Protein and amino acid composition of the mantle of juvenile O ctopus vulgaris (Cuvier, 1797) during fasting for 27 days were determined. Average protein content of octopus mantle was of 711.19 ± 46.80 g kg?1 DW, and it decreased with increasing fasting days. The non‐essential amino acids content was higher (486.18 ± 11.08 g kg?1 protein) than essential amino acids (425.82 ± 9.15 g kg?1 protein) at the start of the experiment (unstarved animals). The results suggest that the amino acid profile of the mantle where the most abundant amino acids are Arg, His, Lys, Gly, Leu and Pro could indicate a prolonged fasting condition (>20 days) or poor nutrition of O . vulgaris. This study supports the idea of using mantle for metabolic needs of starved O . vulgaris suggesting that the degradation pathway of amino acids to pyruvate and tricarboxylic acid cycle intermediates was favoured contrary to the degradation pathway of ketogenic amino acids. Special considerations should be taken concerning Thr, Ile, Ser, Ala, Asx (Asp, Asn), Glx (Glu, Gln) (because of their fast intake) and Lys and His (due to their stable contents) during a prolonged period of fasting.  相似文献   

14.
Plasma estradiol-17 (E2), testosterone (T), 17,20-dihydroxy-4-pregnen-3-one (DHP) and 17,20,21-tri-hydroxy-4-pregnen-3-one (20-S) levels were measured by radioimmunoassay (RIA) in white perch (Morone americana) and white bass (M. chrysops) that were induced to undergo final oocyte maturation (FOM) with human chorionic gonadotropin (hCG). Plasma DHP levels increased in females of both species in association with oocyte germinal vesicle migration (GVM) and germinal vesicle breakdown (GVBD) and decreased thereafter. Plasma 20-S levels also increased with oocyte GVM in white bass, but were several-fold lower than DHP levels. Circulating E2 and T levels were greatest during GVM and GVBD in both species and decreased to low levels during oocyte hydration and ovulation. Follicles from white perch and white bass which received a priming injection of hCG in vivo, produced both DHP and 20-S in vitro after exposure to hCG and their oocytes underwent GVBD. Ovarian incubates from unprimed fish of either species produced only E2 and T and their oocytes did not complete GVBD. Oocytes from unprimed bass, but not perch, matured when follicles were exposed to hCG in vitro. Both trilostane and cycloheximide blocked in vitro production of DHP and 20-S and oocyte GVBD by white perch follices. DHP and 20-S were equipotent inducers of FOM in the GVBD bioassay. None of several other structurally-related steroids tested were effective within a physiological range of concentrations. These results indicate a role for DHP and 20-S in the control of FOM in white perch and white bass.  相似文献   

15.
Changes in heart rate, ventilatory activity and oxygen consumption were determined in trout (Salmo gairdneri) and brown bullhead catfish (Ictalurus nebulosus) during exposure to a steadily increasing concentration of waterborne cyanide selected to produce death in 8–9 hours for each species. The lethal cyanide concentration for the bullheads was an order of magnitude higher than for trout. Trout developed an immediate and gradually increasing bradycardia throughout the exposure period. Cyanide produced tachycardia in the bullhead followed by a gradual onset of bradycardia as the concentration of cyanide was raised. Pericardial injection of atropine (a muscarinic cholinergic antagonist) indicated that bradycardia in the trout was due initially to increased vagal tone but later due to the direct effect of cyanide on the heart. Hyperventilation in the trout persisted throughout the exposure period, although the rate and amplitude fluctuated and was variable between individual fish. During the last hour of exposure (highest cyanide concentration), ventilation was characterized by rapid, shallow breaths followed by a sudden respiratory arrest. The bullheads exhibited hyperventilation during the first 3 hours of exposure followed by a gradual, linear drop in ventilation rate and amplitude until death occurred. Cardiac and ventilatory responses in both species were attributed to stimulation of central and peripheral chemoreceptors by cyanide. Evidence is presented which suggests the initial response in the bullheads was due, at least in part, to gustatory stimulation by the cyanide. Oxygen consumption of the trout remained above pre-exposure levels for the majority of the test period. Oxygen consumption in the bullhead paralleled the changes in heart and ventilatory rates. Whole-body lactate levels of fingerlings of both species during cyanide exposure were measured to estimate the extent of anaerobiosis. Whole-body lactate levels were much greater in the bullheads than the trout, indicating a higher capacity for anaerobiosis, possibly due to a greater fuel supply. Overall, the trout responded to cyanide in a manner similar to that produced by environmental hypoxia whereas the bullheads experienced a gustatory stimulus which masked the hypoxia-like response.  相似文献   

16.
This study brings an integrated analysis about the relationship between water deterioration and its physiological consequences in live fish transport. The analysis was focused on the transport water and its deterioration, and physiological challenges imposed on the fish. Usual commercial handling procedures employed to mitigate fish stress during transport were discussed. Future topics of research for the establishment of safer fish transport protocols were proposed. Transport was classified into short (≤8 h) or long transport (>8 h). The main issue in short transports should be the prevention of water pH reduction, while in long transports it is the increase in ammonia. Plasma cortisol is the most employed marker for stress and is acutely elevated upon short episodes of transport, but remains elevated even in long‐transport events. Plasma glucose is perhaps a better marker for handling stress. Plasma lactate, pH, osmolality CO2 and ions should be more often evaluated. Plasma Na+ and Cl are very useful markers of acidosis, due to their respective exchange for H+ and , for acid–base regulation. The establishment of species‐specific transport protocols should be preceded by such combined analyses of water and physiological parameters.  相似文献   

17.
Abalone populations have declined worldwide, generating interest in enhancement using hatchery‐reared individuals. In many cases, such restoration efforts have met with limited success due to high predator‐induced mortality rates. Furthermore, the mortality rates of outplanted hatchery abalone are often considerably higher than for wild individuals. This study uses northern abalone (Haliotis kamtschatkana) as a case study to determine whether hatchery‐reared abalone behave differently than their wild counterparts. In the field, outplanted hatchery‐reared abalone were significantly less responsive than wild abalone, in terms of number of abalone responding and intensity of response, to nearby movement and to physical contact with an inert probe. Also, when encountering a cue to which all abalone responded (a seastar predator), hatchery‐reared individuals remained subdued. Anti‐predator behavioural deficits in hatchery‐reared abalone were more pronounced in 4‐year‐old individuals than in 1‐year‐old individuals, suggesting an influence of either age or amount of time spent in the hatchery environment. These behavioural differences are expected to increase the vulnerability of hatchery‐reared abalone to predators, and are likely a major cause of their elevated predator‐induced mortality when outplanted.  相似文献   

18.
The toxic effects of Cd2+ on Ca2+ influx kinetics in developing tilapia (Oreochromis mossambicus) larvae were evaluated. Addition of 20 µg l-1 of Cd2+ to the environment of 0 and 3 day-old larvae competitively inhibited the Ca2+ uptake within 4h resulting in a great increase in Km values for Ca2+ influx (19.3 and 17.4 fold, respectively) as compared with their respective controls. Consequently, the actual Ca2+ influx of larvae in solutions of 0.2 mM Ca2+ are suppressed by 32–45%. Also, 3 day-old larvae were more sensitive to internally accumulated Cd2+ than 0 day-old larvae. Although the Ca2+ influx in 0 and 3 day-old larvae may be restored to the levels of their respective controls with 24h of being transferred to a 20 µg l-1 Cd2+ solution, total body Ca2+ content was significantly reduced in 3 day-old larvae. Increased Ca2+ uptake efficiency ensures sufficient Ca2+ for normal growth. However, rapid increase in Ca2+ influx after hatching also leads to higher Cd2+ uptake. Exposure to Cd2+ will lead to a drop in body Ca2+ content resulting in retardation of larval growth. Therefore, we conclude that if Ca2+ uptake is interfered with at this critical stage of development, larvae will not be able to maintain normal levels of body Ca2+ and will show signs of Cd2+ poisoning.  相似文献   

19.
Migratory dynamics of stream-spawning longnose gar (Lepisosteus osseus)   总被引:1,自引:1,他引:0  
Abstract– Literature evidence suggests that lake-dwelling longnose gar (Lepisosteus osseus) enter tributary streams to spawn, Until the present study, the dynamics of this breeding migration had never been investigated quantitatively. During the summers of 1991 and 1992, longnose gar were captured as they entered Weaubleau Creek, Missouri, a tributary of Harry S. Truman Reservoir. The in-stream spawning migration began in early April and ended in late May, and was positively correlated with stream flow and water level, and negatively correlated with water temperature. In-stream residence times ranged from 15 to 94 days, with males exhibiting longer residence times than females. Once in-stream, longnose gar travelled as far as 10 km upstream and occupied certain pools at greater relative frequencies. Although the reason for this preferential utilization is not completely understood, it may relate to pool depth and riffle proximity. Longnose gar disperse from the spawning stream great distances, with gar captured in Weaubleau Creek being recaptured up to 48 km away. This information should provide fisheries biologists the means to consider the reproductive ecology of this species in their conservation and management decisions.  相似文献   

20.
The desaturation and elongation of [1-14C]18:3n-3 was investigated in hepatocytes of the tropical warm freshwater species, zebrafish (Danio rerio) and Nile tilapia (Oreochromis niloticus). The hepatocyte fatty acid desaturation/elongation pathway was assayed before and after the fish were fed two experimental diets, a control diet containing fish oil (FO) and a diet containing vegetable oil (VO; a blend of olive, linseed and high oleic acid sunflower oils) for 10 weeks. The VO diet was formulated to provide 1% each of 18:2n-6 and 18:3n-3, and so satisfy the possible EFA requirements of zebrafish and tilapia. At the end of the dietary trial, the lipid and fatty acid composition was determined in whole zebrafish, and liver, white muscle and brain of tilapia. Both zebrafish and tilapia expressed a hepatocyte fatty acid desaturation/elongation pattern consistent with them being freshwater and planktonivorous fish. The data also showed that hepatic fatty acid desaturation/elongation was nutritionally regulated with the activities being higher in fish fed the VO diet compared to fish fed the FO diet. In zebrafish, the main effect of the VO diet was increased fatty acid Δ6 desaturase activity resulting in the production of significantly more 18:4n-3 compared to fish fed the FO diet. In tilapia, all activities in the pathway were greater in fish fed the VO diet resulting in increased amounts of all fatty acids in the pathway, but primarily eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). However, the fatty acid compositional data indicated that despite increased activity, desaturation of 18:3n-3 was insufficient to maintain tissue proportions of EPA and DHA in fish fed the VO diet at the same level as in fish fed the FO diet. Practically, these results indicate that manipulation of tilapia diets in commercial culture in response to the declining global fish oil market would have important consequences for fish fatty acid composition and the health of consumers. Scientifically, zebrafish and tilapia, both the subject of active genome mapping projects, could be useful models for studies of lipid and fatty acid metabolism at a molecular biological and genetic level. This revised version was published online in August 2006 with corrections to the Cover Date.  相似文献   

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