鱼生长激素基因在大肠杆菌中表达水平的研究

采用聚合酶链反应（ＰＣＲ）技术对鲤和鲑生长激素ｃＤＮＡ的５‘端和３’端进行定向改造。将改造后的５种基因分别克隆到大肠杆菌表达质粒ｐＢＶ２２０进行原核表达,以研究鱼生长激素基因在大肠杆菌中的表达水平。实验结果表明：（１）核糖核蛋白体结合位点（ＳＤ）与起始密码子ＡＵＧ之间的距离对鱼生长激素的表达水平有影响;（２）鲑鱼生长激素基因的终止密码子ＵＡＧ可能不会有效终止该基因在大肠杆菌中翻译的进行而造成部分通     

中华绒螯蟹线粒体DNA 12S rRNA基因片段的序列研究

首次进行了中华绒螯蟹（Ｅｒｉｏｃｈｅｉｒ ｓｉｎｅｎｓｉｓ）线粒体ＤＮＡ１２ＳｒＲＮＡ的序列分析。参考果蝇、蚤状序列对中华绒螯蟹线粒体ＤＮＡ１２ＳｒＲＮＡ基因片段做了引物设计、ＰＣＲ扩增及序列测定,结果得到４５７ｂｐ的碱基序列,其Ａ、Ｔ、Ｇ、Ｃ含量分别为１５９ｂｐ（３４．７９％）、１７７ｂｐ（３８．７３％）、５１ｂｐ（１１．１６％）、７０ｂｐ（１５．３２％）、与果蝇、蚤状的相同基因片段的序列含量基     

中华绒螯蟹线粒体DNA l2S rRNA基因片段的序列研究

首次进行了中华绒螯蟹（ Ｅｒｉｏｃｈｅｉｒ ｓｉｎｅｎｓｉｓ）线粒体 ＤＮＡ １２ＳｒＲＮＡ的序列分析。参考果蝇、蚤状溞序列对中华绒螯蟹线粒体ＤＮＡ １２ＳｒＲＮＡ基因片段做了引物设计、ＰＣＲ扩增及序列测定,结果得到 ４５７ ｂｐ的碱基序列,其Ａ、Ｔ、Ｇ、Ｃ含量分别为 １５９ｂｐ（３４．７９％）、１７７ｂｐ（３８．７３％）、５１ｂｐ（１１．１６％）、７０ｂｐ（ １５． ３２％）,与果蝇、蚤状溞的相同基因片段的序列含量基本相似。     

鱼类基因分子生物学及转基因鱼研究进展↑（*）

真核基因分子克隆技术的建立，提供了从核苷酸水平了解鱼类基因结构的手段。鱼类基因的分子克隆始于八十年代初、中期对虹鳟（Ｓａｌｍｏｇａｉｒｄｎｅｒｉ）鱼精蛋白基因［５２］和美洲黄盖鲽（Ｐｓｅｎｄｏｐｌｅｕｒｏｎｅｃｔｅｓａｍｅｒｉｃａｎｕｓ）抗冻蛋白（ＡＦＰ）基因［２２］的分子克隆，随后扩展到其他极地鱼类ＡＦＰ基因，接着扩展到鱼类生长激素（ＧＨ）基因、催乳激素（ＰＲＬ）基因、生长催乳素（ｓｏｍａｔｏｌａｃｔｉｎＳＬ）基因等数十个基因。对这些控制鱼类重要生理机能的基因结构的认识，无论在理论上还是在渔业…     

鲇细胞 b基因序列变异及遗传多样性分析

从４个种群的鲇（Ｓｉｌｕｒｕｓａｓｏｔｕｓ）线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ,ｍｔＤＮＡ）扩增出约５００ｂｐ的细胞色素ｂ基因,经ＣｌｕｓｔａｌＸ同源排序后得４００ｂｐ序列。用ＰｏｐＧｅｎ３２软件计算遗传距离和遗传一致度。１７个个体间的遗传 距离的变化范围为０～０．０２１１,遗传一致度的变化范围为１～０．９７９１。结果显示,在长江上游的支流中,雅砻江种群在该基因片段上基本不存在变异,在被分析的４个个体中,其细胞色素ｂ基因的序列完全一致;岷江种群和乌江种群在该基因片段上有一定的变异。沅江上游的贘阳河种群在该基因片段上基本不存在变异,在被分析的５个个体中,其细胞色素ｂ基因的序列完全一致。但各个种群间在该基因片段上差异较大。在４个种群的鲇Ｃｙｔｂ基因比较分析的基础上,构建了遗传关系聚类图,雅砻江、岷江、乌江、贘阳河４个种群各聚为一支后,雅砻江和岷江２个种群再聚为一支,乌江和贘阳河２个种群再聚为另一支。     

鲇鱼线粒体DNA的酶切图谱

采用差速离心法及ＤＮａｓｅⅠ、ＲＮａｓｅ消化法制备并纯化了鲇（Ｓｉｌｕｒｕｓａｓｏｔｕｓ）肝脏线粒体ＤＮＡ（ｍｉｔｏｃｈｏｎｄｒｉａｌＤＮＡ，ｍｔＤＮＡ）。用８种限制性内切酶对ｍｔＤＮＡ进行了分析。ＢｇⅡ、ＥｃｏｒⅠ、ＰｓｔⅠ、ＢｇｌⅠ、ＢａｍＨⅠ、ＸｂａⅠ、ＨｉｎｄⅢ、ＸｈｏⅠ在鲇ｍｔＤＮＡ分子上分别具１、１、１、２、２、７、７和０个切点。ｍｔＤＮＡ分子量约１０．８４×１０￣６道尔顿，大小为１７．５４ｋｉｌｏｂａｓｅｐａｉｒｓ。根据单酶和双酶解片段的数目和分子量，建立了鲇ｍｔＤＮＡ的限制性酶切图谱。     

斑节对虾白斑综合症病毒部分基因组文库及核酸探针检测法

通过分离纯化白斑综合症病毒（ＷＳＳＶ）粒子,抽提病毒ＤＮＡ。用限制性内切酶ＥｃｏＲⅠ或ＳａｌⅠ酶切后,克隆入质粒ｐＢｌｕｅｓｃｒｉｐｔⅡＫＳ中,从而建立了ＷＳＳＶ部分基因组文库。估计ＷＳＳＶ基因组ＤＮＡ在１６５ｋｂ以上。将ＷＳＳＶ ＥｃｏＲⅠ克隆片段标记制备为探针。进行Ｓｏｕｔｈｅｒｎ杂交、打点杂交和原位杂交,其结果证明了克隆片段对ＷＳＳＶ特异,并为检测ＷＳＳＶ提供了方法。通过对部分基因组文库序列     

对虾暴发性白斑病毒病综述

到目前为止，国内虾病工作者 达成了基本的共识，即造成对虾暴 发性流行病的病原体是一种无包　涵体病毒。但由于不同学者研究方 法及侧重点不同，对此病毒的叫法 不一、分别为：“白斑杆状病毒 （Ｗｈｉｔｅ　ｓｐｏｔ　ｂａｃｕｌｏｖｉｔｕｓ，ＷＳＢＶ）” “皮下造血组织坏死杆状病毒 （Ｈｙｐｏｄｅｒｍａｌ　ａｎｄ　Ｈｅｍａｔｏｐｏｉｔｅｔｉｃ Ｎｅｃｒｏｓｉｓ　Ｂａｃｕｌｏｖｉｒｕｓ，　ＨＨＮＢＶ）” “对虾白斑综合病（ｗｈｉｔｅ　ｓｐｏｔ ｓｙｎｄｒｏｍｅ　ｖｉｒｕｓ　ＷＳＳＶ）”、“对虾 杆状ＤＮＡ病毒（ＰＲＤ…     

华东地区中华鳖地方群体mtDNA多态分析

应用多聚酶链式反应（ＰＣＲ）技术和限制性长度多态（ＲＥＬＰ）方法，对我国华东地区中华鳖三个地方群体的线粒体ＤＮＡ（ｍｔＤＮＡ）的细胞色素（ｃｙｔ）ｂ基因进行了分析。６种限制性内切酶共获得１８种酶谱和１４种基因型。ｃｙｔｂ基因大小的估算值为１０８０±６０ｂｐ。基因型多样性和核苷酸序列多样性指数分别为０．６８７９±０．０９７８和０．０２６６±０．０９７８。浙江绍兴、江苏南京和山东青岛中华鳖群体间存在显著的遗传差异（Ｐ＜０．０５），其中南京群体与绍兴群体的遗传距离最近，青岛群体与绍兴群体的遗传距离最远。三群体的遗传关系与地理距离相一致，暗示地理分隔可能是造成中华鳖群体遗传分化的主要原因。还发现内切酶ＨｅⅢ和ＨｈａⅠ可作为中华鳖种群鉴定的标志酶。     

牙鲆和大菱鲆养殖群体的分子标记和遗传变异

用１６个１０碱基随机引物对养殖牙鲆（Ｐａｒａｌｉｃｈｔｈｙｓ ｏｌｉｖａｃｅｕｓ）和大菱鲆（Ａｃｏｐｈｔｈａｌｍｕｓ ｍａｘｉｍｕｓ）进行基因组随机扩增多态性ＤＮＡ（ＲＡＰＤ）遗传分析。发现：（１）多个引物具有种的特异性扩增带。其中引物Ｓ７扩增的６９０和１１６０ｂｐ带,引物Ｓ２扩增的６８０ｂｐ带为牙鲆的种的特异性谱带;而引物Ｓ７扩增的６５３和８８９ｂｐ带,引物Ｓ２扩增的６５０、８７０、９９０及１１     

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.     

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.     

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.     

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).     

Protein and amino acid composition from the mantle of juvenile O ctopus vulgaris exposed to prolonged starvation

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.     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.     

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.