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1.
牙鲆基因组 (CAG)n微卫星 DNA 特征分析   总被引:1,自引:0,他引:1       下载免费PDF全文
通过磁珠富集法筛选牙鲆(Paralichthys olivaceus)的微卫星分子标记,采用限制性内切酶Sau 3A Ⅰ对牙鲆完整基因组DNA进行酶切;通过蔗糖溶液梯度离心,收集400~900 bp大小的片段,连接Brown接头,构建牙鲆基因组文库.用生物素标记的微卫星探针(CAG)15,对基因组文库进行杂交,利用磁珠富集含有微卫星的DNA单链序列,并对其进行PCR扩增;将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星序列文库.利用大量质粒检测法进行二次筛选,成功地从牙鲆基因组中分离出含有CAG重复的微卫星序列,测序其中的3000个单菌落,获得2805个(占93.5%)含有微卫星序列的克隆,其中含有微卫星座位3120个,完美型1808个,占57.97%;非完美型226个,占7.25%;混合型1085个,占34.78%.从中选出186个微卫星序列设计120对引物并合成,经过筛选,74对引物可扩增清晰条带,其中68对呈多态性.  相似文献   

2.
将牙鲆基因组DNA经限制性内切酶RsaⅠ和BstUI双酶切后采用FIASCO法构建酶切片段基因组文库。共挑取269个克隆,177个为阳性克隆,阳性克隆率为65.79%。经测序后获得191条微卫星序列,其中完美型占74.35%;非完美型占14.66%;混合型占10.99%。用引物设计软件Primer Premier 5.0设计引物153对,挑选其中的50对合成并在32个野生牙鲆个体中进行扩增,共31个位点具有多态性,统计结果后使用POPGENE软件进行分析,平均等位基因个数为3.939 4,平均有效等位基因数为3.052 2,平均观测杂合度为0.650 1,平均期望杂合度为0.586 6,各引物的Hardy-Weinberg平衡指数在0.012 587~0.984 917变动。这些筛选出的多态性微卫星标记可应用于进一步的牙鲆遗传多样性分析、家系分析及遗传图谱的构建等工作中。  相似文献   

3.
剑尾鱼微卫星DNA的筛选   总被引:19,自引:4,他引:19       下载免费PDF全文
以剑尾鱼(Xiphophorus helleri)为材料,经MboⅠ限制性内切酶消化基因组DNA后,选取500~2 000 bp的片段连接到经BamHⅠ酶切的pUC18载体上,转化大肠杆菌DH5α构建部分基因组文库.采用设计合成的(AC)7、(GT)7重复序列为引物,PCR筛选部分基因组文库,对其中9个重组阳性克隆进行测序,结果共获得24个微卫星序列,其中Perfect(完美型)13个,占54.2%;Imperfect(非完美型)3个,占12.5%;Compound(混合型)8个,占33.3%.表明(AC/GT)n在剑尾鱼的基因组DNA中含量非常丰富.同时,根据其中3个克隆微卫星的侧翼序列设计引物,PCR扩增剑尾鱼基因组DNA,结果均扩增到目的片段.而且,这3对引物扩增出来的微卫星片段在非选育的剑尾鱼中显示出多态性,而在近交系19代则表现为单态,为剑尾鱼的实验动物化遗传研究提供了理论依据.  相似文献   

4.
青石斑鱼微卫星DNA 标记的筛选及群体遗传多样性分析   总被引:5,自引:0,他引:5  
以中国南海海域青石斑鱼(Epinephelus awoara)为材料,构建青石斑鱼小片段部分基因组DNA文库。以M13通用引物和设计合成的微卫星核心序列引物(CA)15,用PCR法对文库进行筛选,共获得96个微卫星序列,分别分布于28个阳性重组克隆中,其中perfect(完美型)共39个(占40.6%),imperfect(非完美型)30个(占31.3%),compound perfect(混合完美型)7个(占7.3%),compound imperfect(混合非完美型)20个(占20.8%)。同时发现(CA/GT)。序列在青石斑鱼的基因组DNA中含量非常丰富。根据微卫星侧翼序列设计28对引物扩增青石斑鱼基因组DNA,有26对引物能扩增出目的片段,选用其中13对多态性稳定的引物对19尾青石斑鱼进行遗传多样性分析。结果显示,13个位点共检测到48个等位基因,平均观测杂合度(Ho)为0.5982,平均期望杂合度(He)为0.5080,平均多态信息含量(PIC)为0.4722,平均Hardy-Weinberg遗传偏离指数(D)为0.1503。实验初步表明中国南海海域青石斑鱼的遗传多样性较为丰富,但在某种程度上受到了人为的干扰。  相似文献   

5.
应用磁珠富集法构建兰州鲇( Silurus lanzhouensis) CAG重复和GATA重复的微卫星文库,并分析其序列特征。兰州鲇基因组DNA经MseI酶切,选取200~800 bp的片段与生物素标记的探针(CAG)8和(GATA)6杂交,捕获到含有微卫星序列的目的DNA片段连接到pMD19-T载体,转化到大肠杆菌DH5α菌株中构建微卫星富集文库,经PCR检测筛选出阳性克隆进行测序。从126个阳性克隆中随机选取96个进行测序,获得59个微卫星序列( GenBank登录号: KJ545973~KJ545998, KJ598088~KJ598120)。其中完美型31个(52.54%)、非完美型20个(33.9%)、混合型为8个(13.56%)。根据侧翼序列,成功设计48对引物,选取25对微卫星引物在10个个体进行扩增与多态性筛选,共获得10对多态性引物。结果表明,经优化的磁珠富集法能够高效地获得兰州鲇微卫星标记,这些标记将为兰州鲇种质资源保护、微卫星连锁图谱构建、经济性状的QTL定位及分子标记辅助选育奠定基础。  相似文献   

6.
贾舒雯  刘萍  韩智科  李健  潘鲁青 《水产学报》2011,35(12):1787-1794
采用人工合成的生物素标记(AG)15探针及磁珠富集法构建了脊尾白虾基因组微卫星富集文库.将脊尾白虾基因组DNA经HaeⅢ酶切后,收集400 ~1 200 bp片段,连接特定接头,与生物素标记(AG)15探针杂交并捕获含有微卫星的DNA片段,然后连接到pMD18-T载体中,构建脊尾白虾微卫星富集文库.随机挑取947个克隆,经菌落PCR检验,其中667个为阳性克隆.从阳性克隆中随机选取184个克隆进行测序,有150个克隆含有微卫星序列,共获得199个微卫星序列,其中完美型158个(79.4%),非完美型27个(13.6%),复合型14个(7.0%).根据微卫星序列,设计了119对微卫星引物,64对扩增出稳定的具有特异性的条带,经30个脊尾白虾个体进行多样性评价后,有26个位点表现出多态性.26个微卫星位点获得的等位基因数从3 ~ 16个不等,平均每个位点扩增得到6.5个等位基因.观测杂合度(Ho)、期望杂合度(4)及多态性信息含量值(PIC)的范围分别为0.111 ~ 0.929、0.246 ~0.932、0.231 ~0.909.本研究开发的微卫星位点将为脊尾白虾种群多样性研究、家系识别和种质鉴定提供有效的工具.  相似文献   

7.
青蟹微卫星DNA的筛选及特征分析   总被引:1,自引:0,他引:1  
利用磁珠富集法,结合生物素标记的(CA)16寡核苷酸探针从青蟹基因组MboI酶切片段中筛选微卫星DNA序列.将得到的片段与pGEM-T Easy载体连接后转化克隆构建微卫星富集文库.经PCR筛选检测,对89个阳性克隆进行测序,其中52个克隆中含有64个微卫星,完全型占87.50%,重复次数超过11次的占78.12%.根据所获微卫星的侧翼序列设计并合成了30对引物,通过优化PCR反应条件,并在35只青蟹个体中进行PCR扩增检测,最终获得了10对具有多态性的引物,为进一步开展青蟹遗传多样性分析、遗传图谱构建等研究提供了基础资料.  相似文献   

8.
磁珠富集法制备大口鲶的微卫星分子标记   总被引:10,自引:0,他引:10  
通过磁珠富集的方法分离大口鲶(Silurus meriaionalis)的微卫星分子标记。将基因组DNA酶切,梯度离心收集400~900bp片段并纯化,连接Brown接头,用生物素标记的寡核苷酸(CA)15作探针与其杂交,杂交复合物结合到包被有链霉亲和素的磁珠上,然后将这些片段洗脱,PCR扩增,进行克隆构建“基因组文库”,再通过同位素标记的探针(CA)15进行2次杂交筛选,所得到的阳性克隆测序。从所获得593个阳性克隆中选取178个经测序,97.19%(173个)含有微卫星序列,90.60%重复数在10以上,75.98%为完美型。除探针中使用的CA重复单元外,还观察到Cr、GA、ATG的重复序列。设计获得120对微卫星引物,合成40对经PCR筛选,结果31对引物扩增出多态性条带。显示出,磁珠富集法是获得微卫星分子标记的一种有效的方法,可成为今后发展该标记的主要方法。同时,制备出的微卫星标记可为今后研究大口鲶的分子遗传育种提供有用的遗传标记。  相似文献   

9.
采用磁珠富集法,以生物素标记的(CA)10寡核苷酸为探针,构建了三角帆蚌(Hyriopsis cumingii)基因组微卫星富集文库。根据微卫星位点的侧翼序列设计引物,随机挑选扩增出与预期大小相符的32对引物,引物荧光标记后对24个三角帆蚌个体分别进行PCR扩增,共筛选出20对多态性较好的引物。结果表明,20个微卫星位点的等位基因数为5~20,有效等位基因数2.321 3~13.260 3。观察杂合度和期望杂合度分别为0.318 2~1.000 0和0.582 5~0.946 1;PIC值0.547 2~0.919 9;其中14个位点符合Hardy-Weinberg平衡(P>0.05)。本研究筛选的20个微卫星标记可作为三角帆蚌遗传多样性、种群遗传结构等研究的理想分子标记。  相似文献   

10.
漠斑牙鲆引进群体子一代遗传多样性的RAPD分析   总被引:1,自引:1,他引:0       下载免费PDF全文
运用随机扩增多态性DNA(RAPD)技术对引进群体漠斑牙鲆Paralichthys lethostigma 子一代48个个体的遗传多样性进行了分析,筛选的40个10bp的随机引物中,25个引物产生了稳定性好,可重复性强的谱带。共检测到154个位点,其中多态位点有122个,多态位点的比例为79.22%,有效等位基因数为1.5629±0.3631,Nei's基因多样性指数为0.3184±0.1843,Shannon信息指数是0.4651±0.2574。结果表明,漠斑牙鲆处于较高的遗传多样性水平。  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.
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   

14.
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   

15.
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   

16.
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   

17.
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   

18.
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   

19.
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   

20.
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.  相似文献   

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