盐藻钙依赖蛋白激酶基因DsCDPK的表达分析

以盐藻总RNA为模板,利用RT-PCR技术扩增了盐藻DsCDPK基因的cDNA序列,将其克隆到pMD^TM19-T simple载体上,经测序获得的克隆片段全长1650 bp,与已发表的盐藻DsCDPK(GenBank:JQ964113)的编码区序列同源性达100%。将DsCDPK基因的开放阅读框与质粒pET-32a(+)连接,构建原核表达载体pET-32a-DsCDPK。将该重组质粒转化到大肠杆菌BL21中,经IPTG诱导融合,蛋白在大肠杆菌BL21中得到成功表达。通过SDS-PAGE检测发现,融合蛋白为部分可溶性表达,将上清蛋白经过His柱纯化后获得了纯度较高的可溶性融合蛋白,Western杂交检测显示,融合蛋白能被抗His单克隆抗体特异性识别,初步证明该融合蛋白就是带有His标签的DsCDPK蛋白。采用实时荧光定量PCR方法分析了高盐胁迫下DsCDPK基因的表达模式。试验结果表明,盐藻DsCDPK基因为盐胁迫上调基因,在高盐(3.0 mol/L NaCl)胁迫下,DsCDPK基因表达量显著增加,盐胁迫1 h时表达量达到最高,为正常生长状况(1.0 mol/L NaCl)下的3倍,差异达到极显著水平(P<0.01)。该研究成果为进一步阐明盐藻钙依赖蛋白激酶基因的功能及作用机制奠定了基础。     

盐藻醛缩酶的盐适应性及其与物质积累的关系

在不同N aC l质量浓度下培养盐藻,分析盐藻醛缩酶的盐适应性及其与物质积累的关系。试验结果表明,在NaCl质量浓度为40～120 g/L时,盐藻醛缩酶活性与盐度具有正相关性;在NaCl质量浓度为120～200 g/L时,盐藻醛缩酶活性与盐度具有负相关性;在NaCl质量浓度为120 g/L时,盐藻醛缩酶活性最高。盐藻醛缩酶是一种高盐适应酶。盐藻醛缩酶活性与盐藻细胞密度、叶绿素形成和蛋白质积累均呈正相关关系。在盐藻醛缩酶活性最高时,盐藻物质积累量最多。     

锰对盐藻生长与物质积累的调控作用

试验结果表明,培养液中锰质量浓度过高或过低都不利于盐藻细胞的生长与物质积累。培养基中锰质量浓度为4.0 mg/L时,盐藻细胞生长、蛋白质合成与β-胡萝卜素积累最多。当培养液中锰质量浓度较高(8.0 mg/L)或较低(2.0 mg/L)时,单个盐藻细胞中的蛋白质与β-胡萝卜素含量较高。但此时,因培养液中细胞密度较低,盐藻细胞积累的物质总量仍然较少。在锰质量浓度较高或较低的逆境条件下,盐藻可能通过适应性反应形成了逆境蛋白质与胡萝卜素等。     

盐藻过氧化氢酶对盐的适应性及其与物质积累的关系

将盐藻分别接于NaCl质量浓度为40、80、120、160、200g/L的培养液中,置光照培养箱中培养,分析盐藻过氧化氢酶对盐的适应性及其与物质积累的关系。结果表明,在NaCl质量浓度为40～120g/L和120～200g/L的范围内,盐藻过氧化氢酶活性与NaCl质量浓度分别具有正负相关性;在盐度为120g/L时,盐藻过氧化氢酶活性最高。盐藻过氧化氢酶是一种高盐适应酶,活性与盐藻细胞密度、叶绿素形成和蛋白质积累呈正相关关系。在盐藻过氧化氢酶活性最高时,盐藻物质积累量最多。     

6-BA与盐藻生长的关系

试验结果表明,在6-BA质量浓度较低时,6-BA与盐藻生长正相关;在6-BA质量浓度较高时,6-BA与盐藻生长负相关;培养液中1.5 mg/L的6-BA是盐藻的最佳促生长质量浓度,且6-BA对盐藻生长的促进效果可被萘乙酸(NAA)提高.     

磷浓度对盐生杜氏藻和纤细角毛藻叶绿素荧光特性及生长的影响

在温度(23±1)℃,盐度31,光照度5000 lx的条件下,用含有不同磷浓度(0、2.27、18.15、36.3、72.6、290.4μmol/L)的培养基对盐生杜氏藻和纤细角毛藻进行培养,研究2种微藻在一次性培养过程中,不同磷浓度对其PSII最大光能转化效率(Fv/Fm)、叶绿素含量以及细胞密度的影响。单因子方差分析结果表明,在整个培养周期中,磷浓度对两种微藻的光合作用及生长均有显著影响(P<0.05)。盐生杜氏藻的PSII最大光能转化效率、叶绿素含量以及细胞密度均随着起始磷浓度的增加而增加,在290.4μmol/L处达到最大值;纤细角毛藻的PSII最大光能转化效率、叶绿素含量以及细胞密度在18.15μmol/L处达到最大值,而后随着磷浓度的继续升高,其上述指标反而降低。所以盐生杜氏藻和纤细角毛藻进行光合作用和生长的最适磷浓度分别为290.4μmol/L和18.15μmol/L。     

盐胁迫对雨生红球藻虾青素累积、虾青素合成相关酶基因表达和抗氧化指标的影响

为探讨盐胁迫对雨生红球藻(Haematococcus pluvialis)虾青素合成的影响与机理,以及雨生红球藻各抗氧化机制之间的关系,本研究采用生化和分子生物学方法研究了不同浓度(0.04 mol/L、0.08 mol/L、0.12 mol/L和0.16 mol/L)和不同时间(3 d、6 d和9 d)的盐(Na Cl)胁迫对雨生红球藻生长、虾青素积累、番茄红素β-环化酶(Lcy)、β-胡萝卜素羟化酶(Crt R-B)和β-胡萝卜素酮化酶(Bkt)基因表达、超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GSH-Px)的活性以及丙二醛(MDA)含量的影响。结果表明,各胁迫时间的雨生红球藻的密度均随着盐胁迫浓度的增加而不断下降,在盐胁迫的第9天,雨生红球藻的死亡率和孢子比例均随着盐胁迫浓度的增加而不断升高;雨生红球藻虾青素含量、Lcy、Crt R-B和Bkt基因表达量均随着盐胁迫浓度和时间的增加而不断提高;雨生红球藻SOD、CAT和GSH-Px活性以及MDA含量在不同浓度和不同时间的盐胁迫下与对照组(0.00 mol/L Na Cl)相比均升高,且在不同时间的0.12 mol/L Na Cl胁迫下与对照组相比均显著升高(P0.05);雨生红球藻虾青素含量、Lcy、Crt R-B和Bkt基因表达量在盐胁迫的早期(第3天)和中期(第6天)阶段较低,在盐胁迫的后期(第9天)阶段较高,而SOD、CAT和GSH-Px活性以及MDA含量在盐胁迫的早期和中期阶段较高,在盐胁迫的后期阶段较低。实验结果说明了适当浓度和时间的盐胁迫能促进雨生红球藻累积虾青素,雨生红球藻在盐胁迫下主要是通过提高虾青素合成相关酶基因的转录水平来促进虾青素的合成,其虾青素和抗氧化酶的抗氧化活性可能互为补充,共同保护雨生红球藻免受盐胁迫的氧化损伤。     

三十烷醇对铜藻生长及岩藻黄素含量的影响

为探讨三十烷醇对铜藻生长的影响,本研究采用不同浓度(0、0.1、 0.5、1.0 和2.0 mg/L)的三十烷醇连续施用20 d,或用三十烷醇浸泡处理24 h后,再恢复正常条件培养20 d,分别测定了铜藻的生长、生理生化指标(光合色素、可溶性蛋白、可溶性糖)以及岩藻黄素含量的变化情况。结果显示,连续施用5 d时,各组藻体的比生长速率(RGR)、叶绿素a(Chl.a)、类胡罗卜素(Car)、可溶性蛋白、可溶性糖的含量均显著高于对照组;15 d时,2.0 mg/L浓度组的铜藻生长受到抑制,各项生理指标均显著下降;20 d时,除了0.1 mg/L浓度组的铜藻生长仍有促进外,其他3个较高浓度的施用组均受到不同程度的抑制,且三十烷醇的浓度越高,抑制作用越明显。经三十烷醇浸泡处理的藻体恢复正常条件培养5 d时,各组藻体的RGR、Chl-a、Car、可溶性蛋白和可溶性糖的含量均显著上升;10 d时,0.5和1.0 mg/L浓度组的铜藻生长速率仍显著高于对照组,但15 d时已与对照组无显著差异,且综合各项生理生化指标,1.0 mg/L组的促进作用最为明显,这种促生长的作用能持续到处理后10 d左右。岩藻黄素的含量除连续施用2.0 mg/L组在实验20 d时低于对照组外,其余各实验组在培养20 d内均显著高于对照组,其中1.0 mg/L三十烷醇浸泡处理24 h恢复培养10 d,可使岩藻黄素的含量提高79.5%。研究表明,连续施用低浓度的三十烷醇(0.1 mg/L) 20 d或以1.0 mg/L三十烷醇每10 d浸泡处理24 h,对铜藻的生长和岩藻黄素的积累有显著的促进作用。     

一株野生型耐饱和盐度的盐藻

从江苏盐城射阳盐场自然海水中分离得到一株可在NaCl浓度为6.15 mol/L培养基中很好生活的野生型盐藻,是目前已报道的最高盐度.研究其在不同盐度下的生长适应性,发现该盐藻细胞可以生活在多个NaCl浓度培养基中;以纵裂方式增殖;盐藻细胞通过改变自身的形状和形成透明状膜来快速响应盐度突然改变.     

铜对盐藻生长与物质积累的调控作用

实验研究了不同浓度的铜对盐藻细胞生长与物质积累的调控作用。结果表明:培养液中供给铜过多或过少都不利于盐藻细胞的生长与物质积累。以培养基中125μg/L的铜浓度对盐藻细胞生长、蛋白质合成与β-胡萝卜素积累的促进作用最大。这一铜浓度可用于盐藻的生产性培养。当培养液中铜浓度较高(175μg/L)或较低(25μg/L)时,单个盐藻细胞中的蛋白质与β-胡萝卜素含量较高,但因培养液中细胞密度较低,盐藻细胞积累的物质总量仍然较少。在铜浓度较高或较低的逆境条件下,盐藻可能通过适应性反应形成了逆境蛋白质与胡萝卜素等。     

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 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).     

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.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

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.