甘肃金鳟RAPD反应体系构建及优化

以甘肃金鳟尾鳍为试验材料,提取基因组DNA。对影响甘肃金鳟RAPD扩增的重要参数进行了优化试验,包括模板DNA浓度、dNTP浓度、引物浓度、Taq酶、变性时间、退火温度等,建立了甘肃金鳟RAPD反应的最适体系。即在25μl反应体系中:模板DNA用量为4.0 ng/μl;Mg2 浓度为2.5mmol/L;dNTP浓度为0.5 mmol/L;引物浓度为0.5μmol/L;Taq酶用量为1 U;扩增程序:94℃预变性4 min,94℃变性45 s,36℃退火50 s,72℃延伸1 min,共35个循环,最后延伸10 min。     

鳡鱼ISSR-PCR反应体系的正交优化

利用正交试验设计的方法,对鳡鱼ISSR-PCR反应体系中的Mg2+浓度、引物浓度、dNTPs浓度、DNA模板浓度及Taq DNA聚合酶浓度进行优化分析。建立了最佳ISSR-PCR反应体系,即25μL反应体系中含Mg2+2mmol/L、引物0.4μmol/L、dNTPs 0.2mmol/L、模板DNA 100ng、Taq DNA聚合酶1.0U。并在此基础上对退火温度和循环次数进行梯度试验,得到最佳退火温度为52℃,最佳循环次数35次。     

扩增香螺SSR指纹研究的试验条件的优化

利用SSR技术对香螺进行了PCR扩增,探索香螺基因组DNA的提取方法。从香螺23对近缘种引物中筛选出8对可以扩增的引物,对引物浓度、Taq酶浓度、循环次数、模板浓度等方面进行了研究,从而探索出一个适合香螺SSR分析的反应体系和反应条件并对微卫星引物通用性进行初步分析。     

西施舌基因组DNA的提取及其RAPD反应条件的优化

通过实验比较获得了一种高效、简便的西施舌基因组DNA的提取方法,以该法提取的基因组DNA为模板,对西施舌RAPD分析中的DNA模板浓度、镁离子浓度、dNTPs浓度、引物浓度和Taq酶浓度进行了研究,初步确定了适于西施舌RAPD分析的最佳反应体系:25μL反应体系中,含模板DNA 25 ng,10×PCR缓冲液2.5μL,dNTPs 0.3 mmol/L,MgCl22.5 mmol/L,引物15 ng,Taq DNA聚合酶1.5U。     

双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)

根据Genbank中对虾白斑病由白斑综合征病毒（WSSV）和传染性皮下及造血器官坏死病毒（IHHNV）的基因序列,设计了两对能分别检测WSSV和IHHNV保守片段基因的特异性引物,而且这两对引物在同一反应体系中可以同时对WSSV和IHHNV的DNA模板进行多重PCR扩增,得到大小分别为110bp（WSSV）和356bp（IHHNV）的扩增条带。对影响PCR反应的主要因素Mg“浓度和退火温度进行了优化,证明当Mg^2＋浓度为2．0～4．0mmd,退火温度为57～59℃时可获得最佳的扩增和检测效果。特异性试验结果表明,这两对引物检测WSSV和IHHNV具有很好的特异性,对其它对虾常见病原的PCR扩增结果均为阴性。敏感性测定结果表明,该反应体系最低能检测100pg的WSSV和IHHNV的DNA模板。临床检测表明,所建立的双重PCR方法可以适用于WSSV和IHHNV的同时检测和鉴别。     

中国对虾SRAP分子标记体系正交优化及在遗传多样性分析中的应用

利用正交设计L16(45)对影响中国对虾SRAP分子标记分析的5个因素(Taq酶,Mg2+,模板DNA,dNTP和引物浓度)在4个水平上进行了优化,筛选出各反应因素的最佳水平为:20μl反应体系中包含1.0UTaq酶,2.0mmol/L的Mg2+,40.0ng的模板DNA,0.125mmol/L的dNTP以及0.4μmol/L的引物,退火温度为53.5℃。研究表明,各因素的不同水平对扩增结果有显著影响,其中Mg2+影响最大,影响大小顺序依次为Mg2+,Taq酶,引物,dNTP和模板DNA。利用优化的SRAP分子标记体系对中国对虾"黄海1号"第12世代选育群体进行了遗传多样性分析,实验结果表明,此标记技术可作为中国对虾遗传分析的良好技术工具。遗传多样性分析共筛选出8对可以扩增出清晰稳定条带的引物组合,共获得171个位点,其中多态性位点154个,占90.08%;有效等位基因数为1.7741,期望杂合度均值为0.4219,Shannon多样性指数为0.6055。上述参数值均高于应用AFLP技术对前几代选育群体的遗传多样性分析结果。     

刀鲚RAPD分析条件的优化

以刀鲚基因组DNA为模板,对刀鲚RAPD分析中的DNA模板浓度、镁离子浓度,dNTPs浓度、引物浓度进行了研究。研究结果初步确定了适用于刀鲚遗传多样性分析的RAPD反应体系为:25μl反应体系中,含10×PCR缓冲液2.5μl,模板DNA20ng, dNTPs0.1m mol/L,Mg~(2+)2.5m mol/L,引物0.4μ mol/L,Taq DNA聚合酶1U。     

红尾仙女虾养殖群体ISSR反应体系建立及遗传多样性分析

针对24尾养殖红尾仙女虾Branchinella thailandensis样本的25 μL PCR体系,进行了 Mg2+、引物浓度、Taq DNA聚合酶3因素3水平L9(34)正交实验;退火温度范围设置为48～59℃(间隔1℃),优化了 ISSR反应体系及筛选的6个多态性和重复性较好的引物,分析了红尾仙女虾养殖群体的...     

拟穴青蟹ISSR-PCR条件的优化

分析DNA模板、Mg2+、dNTPs、引物和Taq DNA聚合酶及甲酰胺、二甲基亚砜等对拟穴青蟹ISSR-PCR反应的影响.研究结果确定了获得清晰条带的模板DNA、Mg2+、dNTPs、引物、Taq DNA聚合酶的浓度范围分别为:模板DNA 8～50 ng/μl ,Taq DNA聚合酶0.75～1.0 U, Mg2+1.0～2.0 mmol/L(与引物密切相关),dNTP 0.15～0.20 mmol/L,引物浓度0.6～0.8 μmol/L.甲酰胺的添加并不能优化拟穴青蟹ISSR-PCR反应结果,而低浓度的二甲基亚砜可提高扩增产物的清晰度,但与引物相关.     

中华绒螯蟹RAPD扩增条件及个体间遗传差异

该项研究优化了河蟹ＲＡＰＤ反应体系,初步确定：Ｔａｑ酶用量为ＩＵ,引物浓度０．３５μｍｏｌ／Ｌ,ｄＮＴＰｓ浓度１００μｍｏｌ／Ｌ模板量５０－１００ｎｇ,ＭｇＣｌ２浓度２．５ｍｍｏｌ／Ｌ,退火温度３８℃,得出同一养殖群中不同个体河蟹遗传差异在２０％以下。     

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.     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

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.     

Evaluation of a Fast Method Based on the Presence of Two Restriction Sites in the Mitochondrial ND5 (mt ND5) Gene for the Identification of Scomber Species

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.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

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.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

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).