仿刺参cDNA文库构建以及序列分析

应用Gubler-Hoffman cDNA Library Construction技术,构建仿刺参(Apostichopus japonicus)cDNA文库.测试结果表明,库容量为2.0×106 pfu/mL,利用PCR方法检测cDNA文库插入DNA片段的大小,其长度范围在0.5～4.1 kb,插入片段平均长度为1.3kb,表明该文库达到建库要求,可用来筛选低丰度mRNA的基因克隆.随机挑选100个1kb以上的cDNA克隆进行测序分析,发现有25条EST序列和网上公布的其他物种的序列相似性较高,这些基因与刺参能量代谢和蛋白水解相关.其中发现的几个重要基因,如编码遍在蛋白以及缬酪肽蛋白等的基因,可能与仿刺参自溶过程的蛋白酶水解和细胞凋亡事件相关,进一步的研究还在进行.另有49条EST序列相似性较低,推测可能是功能未知的新基因.仿刺参cDNA文库的成功构建,为下一步大量EST序列分析和功能性新基因的发现,以及从基因水平认识仿刺参这一特殊棘皮动物的生长、发育和代谢规律奠定了基础.     

鲤脑组织cDNA文库的构建及脑室管膜素基因鉴定

用Gubler-Hoffman法构建了低温下鲤(Cyprinus carpio Linnaeus)脑组织cDNA文库,经测定库容量为5.7×105 CFU/mL,文库的重组率接近95%,平均插入片段长度约为1.5 kb,符合文库保存和筛选实验的要求.同时,根据鲤与低温相关消减cDNA文库中低温下特异表达基因片段序列,设计PCR引物在此cDNA文库中进行PCR检测和序列测定,使用BLAST软件将测序的10个cDNA序列同GenBank等数据库进行比对,结果显示8个阳性克隆有相关同源性,2个克隆为功能未知的基因,全长率近75%;以上结果说明本实验所构建的cDNA文库质量较高,可作为筛选与鲤低温性状相关的功能基因的重要资源.同时对其中一个与低温相关的基因脑室管膜素基因构建了表达载体,为进一步验证该基因在鱼类低温适应中所发挥的作用及其作用机理奠定了基础.     

败血症鲢肝与肾cDNA文库构建及MHC classⅠ的克隆与分析

采用CloneMinerTM cDNA文库构建试剂盒构建了患败血症的鲢(Hypophthalmichthys molitrix)肝和肾的cDNA文库.经检验,文库的滴度为1.34×107 cfu/mL,总库容为2.68×107 cfu,阳性克隆率为97.5％,平均插入片段大于1.2 kb.对80个随机挑选的克隆进行两端测序,结果显示有74个测序成功.经在GenBank上BLAST比对,其中57个为已知功能基因,属于45个不同基因,另外17个没有明显的同源序列.在已知功能的57个克隆中,含有全长编码区序列的克隆共有49个,全长cDNA比率为61.25％.采用PCR法对文库进行筛选,获得一个含MHC classⅠ完整编码区的序列,该序列具有典型的脊椎动物MHC class Ⅰ的序列特征.本研究完成鲢肝和肾较高质量的cDNA文库的构建,旨在为今后开展鲢功能基因研究奠定基础.     

施氏鲟精巢和卵巢差减cDNA文库的构建和差异表达EST的筛选

应用抑制差减杂交（SSH）技术构建了施氏鲟（Acipenser schrenckii）精巢和卵巢组织的SMART cDNA文库及其cDNA差减文库，从两个差减文库中随机挑取200个克隆进行PCR检测，随机挑选其中的123个cDNA克隆测序。将所测序列经GenBank检索和生物信息学比较，发现有55个cDNA片段序列在GenBank上无明显的同源性，68个片段与已报道的基因有较高的同源性，其中17个cDNA片断为精巢中特异表达的基因，而51个为卵巢中特异表达的基因。精巢中大量表达的主要为延长因子（EF-1）和脂肪酸连接蛋白等与精子生成发育有关的基因，而卵巢主要为ZPC、组蛋白和周期蛋白等与卵细胞发育成熟相关的基因。这些EST数据为进一步筛选和克隆鲟鱼性腺组织特异性表达基因提供了材料平台，为鲟鱼基因组数据增补了大量信息。     

牙鲆变态早期cDNA文库的构建和parvalbumin基因的克隆

在前期的研究中,克隆到牙鲆变态早期和变态前差异表达的parvalbumin基因片断cDNA。为了克隆parvalbumin基因的全长cDNA,构建了变态早期(孵化后23d仔鱼)牙鲆头部的全长cDNA文库,并对文库的滴度和插入片段大小进行了评估。同时采用锚定PCR法从cDNA文库筛选出parvalbumin基因的全长cDNA。结果表明,所构建的牙鲆变态早期头部cDNA文库的滴度为1.2×106PFU·mL-1,插入片段的平均长度为1000bp左右。parvalbumin基因的全长cDNA的长度为603bp,编码109个氨基酸,含有两个EF手型钙离子结合域:DQDGSGFIEEEEL(52～64)和DSDGDGKIGVEEF(91～103),以及一个ATP/GTP结合位点模序:ACGLAGKS(33～40),是一种典型的钙离子结合蛋白。通过parvalbumin基因氨基酸序列的同源比较分析表明,牙鲆parvalbumin基因是比较保守的蛋白,与其它鱼类享有61.5%～68.8%序列同源。系统分析表明,牙鲆的parvalbumin基因与狭鳕(Theragrachalcogramma)最为接近。     

三角帆蚌肝脏瘟病感染期抑制性消减cDNA文库的构建与分析

采用抑制性消减杂交技术构建了三角帆蚌肝脏瘟病感染期抑制性消减cDNA文库。经检验，差异表达基因均被富集了 2¹⁰倍左右，证明构建的 cDNA 消减文库具有很强的消减效率。PCR 鉴定发现，在随机挑取的阳性克隆中，95% 的克隆均含有 0.2～1.0 kb 的插入片段，这些片段可能是三角帆蚌瘟病病毒感染后差异表达基因的cDNA片段。测序共获得 214 个有效 cDNA 序列，分别属于8 大类，共 98 个基因。其中细胞分裂基因 2个、细胞结构与运动基因 9 个、代谢基因 10 个、信号传导基因 7 个、细胞防疫基因 10 个、基因与蛋白表达基因 20 个、未知功能蛋白基因 26 个，GenBank中找不到任何同源序列的基因 14 个。结果说明，构建的差异表达cDNA文库，可较好地反映三角帆蚌瘟病病毒对三角帆蚌影响的基因信息。     

海带雄性配子体差异表达基因片段的克隆及筛选

利用抑制消减杂交法（suppression subtractive hybridization，SSH）获得了海带雌、雄配子体差异表达的cDNA片段，将雄配子体差异表达的cDNA片段克隆于pGEM-T载体并转化大肠杆菌（Escherichia coli）JM109，构建雄配子体差异表达的cDNA消减文库。自750个阳性克隆中随机挑选100个进行PCR扩增鉴定，电泳图谱表明插入片段大小在200～1000bp之间。进一步经过Southern斑点杂交筛选后，选取10个阳性克隆进行测序和序列分析，有7个片段与GenBank数据库中已知基因序列无明显同源性，初步判断为新的基因序列，3个片段与已知脚基因和Lhcf6基因的同源性分别达到88％和98％。     

条斑紫菜基因组Fosmid文库构建

高分子量基因组文库是开展条斑紫菜基因组学研究的必备工具。构建了由23 040个Fosmid克隆组成的条斑紫菜孢子体无偏倚基因组文库。检测分析表明：文库克隆重组率为100%；插入DNA片段长度为28~40 kb，平均长度为35 kb，文库覆盖率约为条斑紫菜基因组的2.78倍；从超级池中筛查条斑紫菜18S rRNA、atpA、h2A、rbcL基因，至少能得到一个阳性克隆，印证了计算所推测的文库覆盖率；随机选取6个Fosmid克隆经100代传代后插入DNA片段未发生丢失或长度变化，表明克隆传代的稳定性。该文库的构建为开展条斑紫菜基因组特性分析和基因克隆奠定了基础。     

酵母双杂交系统用于WSSV粘附蛋白VP37相关蛋白基因的研究

为研究WSSV的粘附蛋白VP37相关蛋白的基因，应用酵母双杂交系统Ⅲ筛选中国明对虾(Fenneropenaeus chinensis)鳃细胞全长cDNA文库。构建VP37基因的重组载体PGBKT7-VP37，把它转化酵母Y187后与含文库质粒的酵母AH109配合，在营养缺陷培养基上进行双杂交筛选。经过PCR鉴定确定了1个阳性克隆。测定该段cDNA序列并进行生物信息学分析，结果显示，此cDNA片段编码的蛋白与蛋白酶的同源性较高，该蛋白在WSSV识别并结合宿主的过程中所起的作用还有待于进一步研究。成功筛选了VP37相关蛋白的cDNA片段，为研究WSSV入侵以及感染致病的机制等方面提供了新线索。     

Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean

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.     

Growth performance,digestive enzyme activity and immune response of Japanese sea bass,Lateolabrax japonicus fed with fructooligosaccharide

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.     

Involvement of gonadal steroids in final oocyte maturation of white perch (Morone americana) and white bass (M. chrysops): in vivo and in vitro studies

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.     

Cardiac,ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide

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.     

An overview of stress physiology of fish transport: changes in water quality as a function of transport duration

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.     

Are hatchery‐reared abalone naïve of predators? Comparing the behaviours of wild and hatchery‐reared northern abalone,Haliotis kamtschatkana (Jonas, 1845)

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.     

Effects of cadmium on the kinetics of calcium uptake in developing tilapia larvae, Oreochromis mossambicus

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 K_m 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.     

Migratory dynamics of stream-spawning longnose gar (Lepisosteus osseus)

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.     

Nutritional regulation of hepatocyte fatty acid desaturation and polyunsaturated fatty acid composition in zebrafish (Danio rerio) and tilapia (Oreochromis niloticus)

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.     

Production of diatom–bacteria biofilm isolated from Seriola lalandi cultures for aquaculture application

Controlled incorporation of selected microalgae and bacteria in aquaculture systems can be beneficial because they can act as microbiological control. That is why the characteristics of biofilm generated naturally in Seriola lalandi culture cages were analysed, their potential benefit to the growth of larvae was studied, and their controlled use for improving the larval viability and as a vector to improve incorporation of previously studied probiotic bacteria was tested. According to biodiversity results, these biofilms are composed of a diatom–bacteria mix showing a decrease in biodiversity in laboratory culture conditions being dominated by Navicula phyllepta and bacteria of the family Rhodobacteraceae. This can be produced on mesh substrates incorporated in bioreactors with rapid growth rate and adhesiveness. Preliminary results from the addition of substrates with this specific biofilm in larvae culture systems showed that it is consumed by the larvae without negative effects, while positive effects on the viability of larvae in combination with probiotics were observed. Considering preliminary results, the addition of these specific substrates with diatom–bacteria biofilms could be a good improvement for aquaculture systems and together with the use of probiotics can contribute to maintaining a stable and controlled system improving the viability of the larval fish culture in its early stages.