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1.
磁珠富集法分离草鱼微卫星分子标记 总被引：26，自引：0，他引：26
磁珠富集法是一种快速、高效的分离微卫星分子标记的方法。本研究通过该方法分离草鱼的微卫星分子标记。将草鱼（Ctenopharyngodon idella）基因组DNA经Sau3AI酶切,同收纯化400～900bp片段,连上接头,构建“基因组PCR文库”。用生物素标记的简单重复序列（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,经一系列的洗涤过程,去除磁珠表面不含有微卫星的片段。将吸附在磁珠上的片段洗脱,PCR扩增放大,再进行克隆和测序,根据微卫星两端的保守序列设计引物,即可得到微卫星分子标记。本研究义通过同位素标记的探针（CA）15进行二次杂交筛选,获得阳性克隆132个,所得到的阳性克隆经测序,86．36％含有微卫星序列,共获得130个微卫星DNA序列。用引物设计软件Primer Premier5．0没计引物83对。 相似文献
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采用磁珠富集法与放射性杂交相结合开发团头鲂（Megalobrama amblycephala）基因组微卫星资源。以团头鲂基因组DNA为材料，经Sau 3AⅠ限制性内切酶消化后，选取400～900bp的片段进行PCR全基因组扩增，并利用生物素标记的（CA）15探针进行微卫星片段的富集。将得到的片段与T载体连接后转入DH5α大肠杆菌中，然后利用γ-32P标记的放射性同位素探针进行第二轮杂交。结果表明，共获得微卫星基因组文库2000个菌，杂交前菌落PCR检测阳性克隆率为50％；杂交后得到的阳性克隆为230个，占11．5％。从得到的230个阳性克隆中挑出120个进行测序，有94个克隆含有重复次数大于5次的微卫星序列，其中46个（48．94％）有随机侧翼区，可以设计引物；14个缺乏足够的侧翼序列。在得到的微卫星序列中，重复单元除CA／GT外，还观察到CT、AG、CG、CAA、CTCA等重复单元。在单一型标记中，完美础占53．15％，非完美型为37．84％；混合型标记占9．01％。另外，微卫星重复次数主要集中在5-30次，占75．68％。本研究旨在对团头鲂基因组资源的开发利用起到一定的促进作用，并为团头鲂养殖品系的优化、遗传多样性的检测及遗传图谱的构建等奠定基础。 相似文献
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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
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合浦珠母贝微卫星DNA标记分离与分析 总被引：1，自引：0，他引：1
采用生物素标记的（CA）15、（AG）12、（ACA）15、（GATA）8、（GATT）75个探针和磁珠富集法构建马氏珠母贝（Pinctada martensii Dunker）基因组微卫星富集文库。随机挑选500个进行PCR筛选,得到138（27．6％）个候选克隆,测序分析发现130个克隆含有微卫星基重复单元。进一步通过序列比对,最终获得109个具有特异微卫星序列的阳性克隆,其中包含179个微卫星DNA结构域。获得的微卫星序列中,属于完全型序列的有154条,不完全型重复型序列有19条,复合型重复序列有6条。序列长度为70～490bp,平均295bp。微卫星核心序列两碱基重复3到39次,绝大多数序列重复次数大于10。基于微卫星两端的侧翼序列设计并获得了13对能够在马氏珠母贝基因组有效扩增的微卫星引物。本研究旨为进一步开展马氏珠母贝分子育种及资源评价分析提供基础资料。 相似文献
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根据生物素与链霉亲和素的亲和原理，采用生物素-磁珠富集微卫星，与传统放射性同位素杂交法相结合，构建筛选大黄鱼的微卫星文库。用生物素-微卫星捕捉单链限制性酶切片段（含有接头和大黄鱼微卫星序列），经PCR扩增单链目的片段形成双链，然后连接至T载体上，转化感受态细胞。将移至硝酸纤维素膜的重组菌用^32P标记的放射性同位素探针50[γ-^32P]ATP（CA）15筛选出阳性克隆菌。测序结果发现，阳性克隆率为71.9％，105个微卫星位点。其中选取设计合成30对并筛选出22对可用引物。说明所建大黄鱼微卫星文库是一个高质量的文库，可为大黄鱼基因组结构分析、大黄鱼精密微卫星连锁图谱构建、分子进化和系统发育研究、分子标记辅助育种以及经济性状的QTL定位提供大量的微卫星标记。 相似文献
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日本蟳微卫星富集文库的建立与多态性标记的筛选 总被引：2，自引：1，他引：1
采用磁珠富集法筛选日本蟳微卫星分子标记。日本蟳基因组DNA经Sau3 AⅠ酶切后,收集400~1 200 bp大小的片段并纯化,利用生物素标记的寡核苷酸探针(AC)15从中筛选出含有微卫星序列的DNA片段,连接到pMD18-T载体中,构建富集微卫星序列的基因组文库,经PCR检测筛选出阳性克隆进行测序。从随机挑选的970个菌落中筛选出369个阳性克隆进行测序,结果86.99%(321个)含有微卫星序列,其中完美型占80.54%,非完美型占15.95%,混合型占4.28%。除使用的探针AC重复外,还得到GA、CT等重复序列。共设计出102对微卫星引物,其中65对能扩增出清晰条带,27对具有多态性。同时筛选出的微卫星标记可为今后研究日本蟳的分子遗传育种提供有效的遗传标记。 相似文献
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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
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采用生物素-磁珠吸附微卫星富集法，筛选虾夷扇贝（Patinopecten yessoensis）微卫星分子标记，并用同位素法进行二次筛选。结果在筛选的192个菌落中获得136个阳性克隆，经测序分析，获得微卫星序列179个，其中完美型占50.8％，非完美型43.0％，混合型占6.1％。除探针中使用的CA重复外，还得到TC、AG、ACA、CTAT的重复序列。用引物设计软件Primer Premier5.0设计引物85对，挑选其中的40对合成并进行筛选。 相似文献
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运用生物素与链霉亲和素的强亲和性原理,用生物素-磁珠吸附微卫星富集法,筛选企鹅珍珠贝(Pteria penguin)的微卫星分子标记序列,进而对南海区企鹅珍珠贝的遗传多样性进行分析。对136个菌落中的85个阳性克隆进行微卫星测序,获得64个序列,达到85.93%。所得到的55个重复6次以上的微卫星序列中,除生物素探针中使用的CA重复外,还得到TG、TC、GA的重复序列。重复序列中完美型36个,占65.5%;不完美型14个,占25.5%;混合型5个,占9.1%。利用Primer Premier5.0设计引物40对,合成其中的20对并进行PCR筛选,15对可扩增出特异性条带。研究表明,筛选出的企鹅珍珠贝微卫星分子标记可用于进一步的遗传多样性分析。 相似文献
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采用磁珠富集法构建哲罗鱼（Hucho taimen Pallas）荩因组微卫星文库。哲罗鱼基因组DNA经MboⅠ限制性内切酶消化后，选取400-900bp的片段，用生物素标记的简单重复序列（ACA）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，获得目的片段，连接T载体克隆，构建基因组微卫旱富集文库。再用同位素标记的（ACA）15探针进行二次筛选，筛选出686个阳性克隆，阳性克隆率为35．94％。对其中140个阳性克隆进行测序，共获得149个微卫星序列，4个小卫星序列，其GenBank Accession Number为DQ110955～DQ121108。其中perfect（完美型）62个，占41．61％；imperfect（非完美型）92个，占61．74％；compound（混合型）5个，占3．36％，重复次数主要分布于6～45（81．21％），平均重复次数为32．5，这表明（ACA／TGT）。在哲罗鱼基因组DNA中含最非常丰富。本研究旨为从DNA水平上研究哲岁鱼种群结构、遗传多样性及目前群体现状等方面提供有效工具。 相似文献
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Association between the interannual variation in the oceanic environment and catch rates of bigeye tuna (Thunnus obesus) in the Atlantic Ocean 
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下载免费PDF全文 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
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Liping Li Rui Wang Wanwen Liang Xi Gan Ting Huang Yan Huang Jian Li Yunliang Shi Honglin Luo Ming Chen 《广西水产科技》2014,(1)
Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China. 相似文献
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Iciar Martinez Amaya Velasco Ricardo Pérez-Martín Eskil Forås Michiaki Yamashita Carmen G. Sotelo 《Journal Of Aquatic Food Product Technology》2013,22(4):289-297
The purpose of this work was to evaluate the suitability of a method based on the presence of two restriction sites (for Hae III and Hindf I) in the mitochondrial NADH dehydrogenase subunit 5 (mt ND5) gene to identify Scomber species. The evaluation was performed on 144 reference and market samples by sequencing of the entire 505-bp fragment of the mt ND5 gene and of a 464-bp fragment of the Kocher fragment of the cytochrome b gene (mt Cytb). Sequence analysis of any of the two fragments allows the identification of each of the four Scomber species, but S. japonicus and S. colias had the same restriction sites at the ND5 amplicon and would not have been differentiated by this analysis. Similarly, loss of the Hae III site in some S. scombrus individuals would have misidentified them as not being Scomber. All the market products were correctly labeled except one acquired in Spain labeled as originating in the Atlantic and containing S. japonicus. 相似文献
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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
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Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL?1 (1.25 ng well?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation. 相似文献
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Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection 下载免费PDF全文
下载免费PDF全文 G Sharon M Pimenta‐Leibowitz M C L Vilchis N Isakov D Zilberg 《Journal of fish diseases》2015,38(1):67-74
Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality). 相似文献
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Qin-Cheng Huang Di‐Gen Qin Bei‐Ping Tan Tao Du Yuan-Zhi Yang Qi-hui Yang Shu‐Yan Chi Xiao-hui Dong 《Aquaculture Research》2020,51(2):816-827
A 5 × 3 factorial growth trial was conducted to evaluate optimal dietary protein and lipid levels (dietary protein level, DP; dietary lipid level, DL) for juvenile Sillago sihama (S. sihama) (2.0 ± 0.02 g, initial weight). Fish were fed 15 diets containing 5 DPs (350, 400, 450, 500 and 550 g/kg) and 3 DLs (60, 90 and 120 g/kg) for 8 weeks. The interaction between proteins and lipids significantly influenced the feed conversion ratio, condition factor, body composition, antioxidant indices and lipase activity (p < .05). DP 450 g/kg showed the highest average final body weight. DPs 500 and 550 g/kg significantly decreased the protein efficiency ratio (p < .05). DL 120 g/kg showed the highest percentage weight gain. The low feed conversion ratio was found in diets P45L12, P55L9 and P55L12. Diet P45L12 showed high superoxide dismutase activities. DP 450 g/kg showed the lowest average malondialdehyde content. Lipase activity was increased by increasing DP (p < .05) with a fall at DP 550 g/kg. Under the present experimental conditions, the optimal DP for S. sihama was 450 g/kg under the DL 120 g/kg. 相似文献
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Cholecalciferol (Vitamin D3) increased plasma inorganic phosphate concentration in American eels,Anguilla rostrata, in a dose-dependent fashion. This response was more marked in phosphate loaded fish. In control as well as phosphate loaded
eels the hyperphosphatemic response to D3 was associated with a sharp reduction in renal phosphate clearance relative to14C-polyethelene glycol (PEG) clearance. Glomerular filtration and urine flow rates were not affected by D3. As renal phosphate clearance, even in phosphate loaded eels, never significantly exceeded that of PEG, it is suggested that
D3 reduced the relative clearance rate of phosphate by increasing renal phosphate reabsorption rather than by reducing the tubular
secretion of phosphate. 相似文献