尼罗罗非鱼无乳链球菌基因缺失株I>cpsE和I>neuA的构建及其生物学特性

为探究尼罗罗非鱼无乳链球菌(GBS)荚膜多糖合成基因cpsE和neuA对菌株生物学特性的影响,本研究利用同源重组的方法,构建了GBS的cpsE与neuA的单基因缺失突变株.具体方法为:用Infusion-PCR的方法分别构建带有氯霉素抗性基因的cpsE与neuA基因敲除重组质粒pSET4s-cpsE和pSET4s-neuA.将构建好的质粒电转化入GBS感受态细胞中,通过改变培养温度实现双交换和质粒丢失,最后经氯霉素抗性筛选获得疑似敲除株.通过菌落PCR、RT-PCR及DNA测序等方法对疑似敲除株进行验证.结果显示GBS的两个突变株△cpsE和△neuA被成功构建.在此基础上,通过生物学功能分析比较基因缺失突变株△cpsE、△neuA与野生株在菌株生长速率、荚膜多糖厚度、唾液酸含量和毒力方面的差异.结果发现缺失突变株△cpsE和△neuA的生长速度与野生株无显著差异,但荚膜多糖厚度、唾液酸含量和菌株毒力均显著低于野生株.进一步研究显示,cpsE是鱼源GBS荚膜多糖合成的关键基因,neuA基因则是荚膜多糖唾液酸化的关键基因,它们的缺失导致了GBS荚膜唾液酸含量的降低,且显著降低了菌株的毒力.     

精·新·实·易·活——优化课堂教学初探

为了实现由应试教育向素质教育的转变 ,适应现代社会对高素质人才的需要 ,必须加强素质教育 ,而实现素质教育的核心问题是提高课堂教学质量 ,因此 ,创设最优化课堂教学、促进学生个性健康发展和素质的全面提高是极其必要的。本文拟就这一问题 ,举《淡水生物学》教学中之例 ,谈谈粗浅的“五字法”。一、精指授课“精”和板书“精”。1、授课“精”。教师讲课时照本宣科 ,会使学生无所适从 ,觉得枯燥乏味 ,甚至于“丢了西瓜 ,拣了芝麻”。因此 ,教师应深入钻研教材 ,挖掘课程体系 ,找出每章、每节课的难点和重点 ,分清轻重、强弱 ,强化重点和…     

罗非鱼 MC4R 基因克隆及与其生长相关的 SNPs 位点

黑素皮质素受体-4(Melanocortin receptor-4,MC4R)是5种已发现的黑素皮质素受体家族成员之一,属于跨膜G-蛋白耦联受体超级家族,对于调节动物的采食量、体质量及能最稳态有着重要的作用.本研究采用基因组步移技术获得了尼罗罗非鱼(Oreochromis niloticus)MC4R基因组DNA序列.该基因全长2547 hp,其中5'侧翼序列长为1481 bp,3'侧翼序列长为82 bp,编码区序列长为984 bp,只有1个外显子.采用PCR产物直接测序法、PCR-RFL和CRS-PCR技术对尼罗罗非鱼MC4R基因启动子和外显子区域进行了SNPs位点的检测和分犁,结果共检测到5个SNPs位点(-G1000C、-T974A、C276T、C561T和C708T),其中外显子上的3个SNPs位点均为同义突变.利用一般线性模型分析5个SNPs位点与尼罗罗非鱼生长性状的关系,结果表明,5个SNPs位点不同基因型之间体质量均值差异最大值分别为9.5%、23.2%、14.4%、18.6%和13.5%,没有达到显著水平(P＞0.05).将5个SNPs位点不同基因型组合成7种双倍型(去掉频率小于3%的组合),关联分析表明,在体质量和体长这2个主要生长性状方面,双倍型D4与D1、D3、D6存在显著性差异(P＜0.05),双倍型D2与D3、D6也存在显著性差异(P＜0.05).因而可以考虑将MC4R基因作为影响罗非鱼体质量等生长性状的候选基因,应用于罗非鱼分子标记辅助育种.     

Biochemical properties of the sensitivity to GABA<Subscript>A</Subscript>ergic ligands,Cl<Superscript>−</Superscript>/HCO<Subscript>3</Subscript><Superscript>−</Superscript>-ATPase isolated from fish (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>) olfactory mucosa and brain

This paper presents a comparative study of the roles of Cl^? and HCO₃ ^? in the functioning of the GABA_AR-associated Cl^?/HCO₃ ^?-ATPase of the plasma membranes of the olfactory sensory neurons (OSNs) and mature brain neurons (MBNs) of fish. The ATPase activity of OSNs and its dephosphorylation were increased twofold by Cl^?(15–30 mmol l^?1), whereas the enzyme from MBNs was not significantly affected by Cl^?. By contrast, HCO₃ ^?(15–30 mmol l^?1) significantly activated the MBN enzyme and its dephosphorylation, but had no effect on the OSN ATPase. The maximum ATPase activity and protein dephosphorylation was observed in the presence of both Cl^?(15 mmol l^?1)/HCO₃ ^?(27 mmol l^?1) and these activities were inhibited in the presence of picrotoxin (100 μmol l^?1), bumetanide (150 μmol l^?1), and DIDS (1000 μmol l^?1). SDS-PAGE revealed that ATPases purified from the neuronal membrane have a subunit with molecular mass of ~?56 kDa that binds [³H]muscimol and [³H]flunitrazepam. Direct phosphorylation of the enzymes in the presence of ATP-γ-³²P and Mg²⁺, as well as Cl^?/HCO₃ ^? sensitive dephosphorylation, is also associated with this 56 kDa peptide. Both preparations also showed one subunit with molecular mass 56 kDa that was immunoreactive with GABA_AR β3 subunit. The use of a fluorescent dye for Cl^? demonstrated that HCO₃ ^?(27 mmol l^?1) causes a twofold increase in Cl^? influx into proteoliposomes containing reconstituted ATPases from MBNs, but HCO₃ ^? had no effect on the reconstituted enzyme from OSNs. These data are the first to demonstrate a differential effect of Cl^? and HCO₃ ^? in the regulation of the Cl^?/HCO₃ ^?-ATPases functioning in neurons with different specializations.     

团头鲂 HSP70 cDNA 的克隆、序列分析以及热应激对其 mRNA 表达的影响

采用RT-PCR和cDNA末端快速扩增法(RACE)分离、克隆出团头鲂(Megalobrama amblycephala)肝脏热休克蛋白70(HSP70)基因全长cDNA序列,共2 224 bp,包括135 bp5'非翻译区、1 932 bp阅读框以及157 bp 3'非翻泽区[不包括Poly(A)].阅读框共编码643个氨基酸,分子量计算值为70.53 kD,理论等电点为5.25.该HSP70基因在翻译区不含内含子,符合诱导型HSP70的特征.团头鲂HSP70氨基酸序列中含有HSP70家族的3个签名序列以及细胞质特异性调控基序EEVD等.同源性分析表明,团头鲂HSP70氨基酸序列与斑马鱼等脊椎动物的相似性高达84.0%以上,与无脊椎动物果蝇的相似性也高达73.4%.生物信息学分析显示,团头鲂HSPTO基因所编码的蛋白质以亲水性区域为主,具有丰富的B细胞抗原位点,无信号肽,无跨膜区域,为非分泌型蛋白;同时还含有众多的蛋白激酶C磷酸化位点、N-肉豆蔻酰化位点、酪蛋白激酶Ⅱ磷酸化位点和N-糖基化位点,推测其可能在细胞信号转导与调控中发挥重要作用.该蛋白含有2个部分重叠的双向核定位序列,二级结构以α-螺旋和无规卷曲为主,空间结构包括N端ATPase功能域和C端多肽结合功能域.应用实时定量PCR研究高温(34℃)应激对团头鲂HSP70 mRNA表达的影响.结果表明,在热应激过程巾肝脏HSP70 mRNA水平呈先升高后下降的变化趋势,说明该基因的表达受热应激调控,本实验克隆的序列为诱导型HSP70 cDNA.     

广东"十一五"水产科技成果荟萃

“十一五”期间，广东水产科技硕果累累。经遴选，本刊现将部分成效显著的成果分为6类刊登如下，排名不分先后。     

"南海1号"斑节对虾

“南海1号”斑节对虾是中国水产科学院南海水产研究所以我国海南三亚、临高、文昌和泰国普吉岛4个海域野生斑节对虾群体为基础群体，通过群体选育方式，经过连续五代选育得到的有较明显抗逆优势、生长速度较快的斑节对虾品种。经第四届全国水产原种和良种审定委员会第三次会议审定通过，“南海1号”斑节对虾被农业部确定为水产新品种（2011年4月第1563号公告，品种登记证号为GS－01—009—2010）。     

广东谋划淡水渔业"十二五"

2010年11月22日召开的广东省淡水渔业作会议提出了“十二五”发展目标：力争到2015年，全省淡水渔业综合实力显著提升，总产量达到360万吨；总产值达450亿元，产业结构得到优化，区域布局更趋合理，     

斜带石斑鱼肝细胞分离及原代培养方法的建立

本研究以斜带石斑鱼肝细胞为实验对象,在不同培养条件下进行原代培养,旨在探讨稳定可靠的斜带石斑鱼肝细胞分离及原代培养方法。采用组织块分离法和胰蛋白酶(含EDTA)消化法分离肝细胞,并通过密度梯度离心法分离纯化肝细胞,细胞悬液于DMEM/F-12、M199和L-15培养液中培养;细胞活力及数量采用血球计数板计数,并通过MTT法测定细胞增殖率;同时,测定不同时间培养上清液中乳酸脱氢酶(LDH)活性、白蛋白(ALB)和尿素氮(BUN)的含量,以分析肝细胞生长状态。结果表明,组织块方法不适于斜带石斑鱼肝细胞的培养,未见细胞从组织块中迁出,而胰蛋白酶消化法获得良好稳定的培养效果,细胞产量达到1.6×108个/g肝重,活细胞数达到95%;L-15培养基细胞生长明显优于DMEM/F-12和M199培养基;启动原代培养的48~72 h阶段肝细胞生长代谢旺盛,培养上清液中LDH活性显著降低,ALB和BUN含量显著升高。结果显示,0.25%的胰蛋白酶常温消化法适合斜带石斑鱼肝细胞的分离,斜带石斑鱼肝细胞原代培养的最适培养基为L-15培养基,肝细胞在启动原代培养的48~72 h生长代谢旺盛。     

Maintenance of steelhead trout (Oncorhynchus mykiss) sperm at different in vitro oxygen tensions alters ATP levels and cell functional characteristics

Adenosine triphosphate (ATP) levels in sperm from steelhead trout (Oncorhynchus mykiss) were found to be 12.0 ± 1.4 pmol ATP per 10⁶ sperm cells. Sperm were stored at 0–2 °C for up to 72 h under 100 and 21% O₂, and 100% N₂. The sperm ATP content of samples maintained under 100 and 21% O₂ both decreased to about 70 and 50% of initial values after 24 and 72 h, respectively. ATP levels of sperm stored under 100% N₂ decreased to 20% of initial values after only 4 h and to less than 10% of initial values after 24 h; they remained unchanged throughout the following 48 h. These low levels, however, were reversible. Following 4 h incubation under 100% N₂, sperm ATP levels rose upon exposure to and maintenance under 100% O₂, increasing to 50% of initial values after a total of 72 h. ATP levels in sperm maintained with cyanide for 4 h decreased to less than 5% of initial values. The magnitude of the change in sperm ATP levels after 24 h in samples maintained under 100% O₂ correlated positively with the initial sperm ATP levels. Sperm ATP levels, motility, and fertility were all correlated positively in a subset of samples examined. The initial percent of living sperm (sperm viability) was 97.5 ± 0.5% and was unaltered through 24 h under all O₂ tensions. Sperm viability remained unchanged through 72 h under 21% O₂ and decreased by 10 and 30% by 72 h under 100% O₂ and 100% N₂, respectively. Therefore, reductions in sperm ATP levels could not be attributed to cell death as viability decreased only modestly over these durations. Spermatocrit values were unaltered throughout the 72 h incubation for samples maintained under 100 and 21% O₂, however, a 95% increase in spermatocrit occurred after 72 h in samples maintained under 100% N₂. These data indicate that intracellular ATP in steelhead trout sperm can be modulated by ambient oxygen without significant loss of viability and that this ATP has a probable role in cell motility and fertility, and sperm packing and cell flexibility.     

ATP levels of chinook salmon (Oncorhynchus tshawytscha) sperm following in vitro exposure to various oxygen tensions

Adenosine triphosphate (ATP) levels in sperm from chinook salmon (Oncorhynchus tshawytscha) were found to be 12.1±1.9 pmol ATP per 10⁶ sperm cells (mean±SEM, n=18). Sperm were stored at 0–2 °C for up to 72 h under 100, 21, and 0% O₂. Changes in sperm ATP content of samples maintained under 100 and 21% O₂ were indistinguishable, decreasing to 50% of initial values after 72 h. ATP levels of sperm stored under 0% O₂ decreased to 10% of initial values within 8 h and remained unchanged throughout the following 64 h. The initial percent of living sperm (sperm viability) was 94.7±1.7% (mean±SEM, n=12) and was unaltered at 24 h under all O₂ tensions. At 72 h, sperm viability remained unchanged under 21% O₂ and had decreased by 20% under 100% O₂ and by 30% under 0% O₂. Therefore, reductions in sperm ATP levels could not be attributed to cell lysis, as viability decreased only modestly over these durations. Fertilizing ability correlated positively with sperm ATP levels. Spermatocrit values increased by 20% for the samples maintained under 100 and 21% O₂ after 72 h. However, a 20% increase in spermatocrit occurred within 24 h in samples maintained under 0% O₂, rising 70% after 72 h under 0% O₂. These data indicate that chinook sperm ATP (1) can be altered and manipulated without significant loss of viability, (2) is positively associated with fertilization, and (3) probably plays a role in sperm packing and cell flexibility.     

内刺盘管虫耗氧率的研究

通过实验生态学方法研究内刺盘管虫在饥饿状态下,其耗氧率在不同温度、盐度和pH值下的变化。结果表明,温度、盐度、pH对内刺盘管虫的耗氧率有极显著影响（F〉F0.01）,在12～24℃范围下,随温度升高,内刺盘管虫的耗氧率明显增大,在24℃达到最高,随着温度进一步升高,耗氧率反而下降;在盐度S16～36范围内,内刺盘管虫耗氧率与盐度呈正相关;pH值8时内刺盘管虫代谢最为旺盛。     

Co‐viability of a Mediterranean demersal fishery affected by the discard ban under alternative management scenarios

The joint viability, or co‐viability, of a Mediterranean Sea mixed demersal fishery was examined by applying a bioeconomic fisheries model to the main seven target stocks of the fishery under biological, social and economic constraints. The stocks of interest were hake, black‐bellied angler, deep‐water rose shrimp, red mullet, blue and red shrimp, Norway lobster and blue whiting, all of which were assessed as overexploited. Their combined production corresponds to 34% by volume and 52% by value of the landings of the demersal fishery studied. Simulation scenarios based on effort reduction, changes in the selectivity pattern and implementing a fisheries restricted area were compared over the simulation horizon of 2015–2030. The latter two scenarios were also examined in terms of full or partial compliance. The effect of a discard ban on the economy of the fleet was also analysed. The results show that the fishery is not co‐viable because some stocks are not viable in the projection period, but social and economic viability was ensured in all cases. As is often the case for mixed fisheries, a unique management measure is unlikely to improve the situation of all stocks simultaneously, but all the management measures tested are better than the status quo.     

台盼蓝活体染色法观察瓦氏黄颡鱼血脾屏障研究

为探讨瓦氏黄颡鱼(Pelteobagrus vachelli)脾脏组织结构和血脾屏障位置,该研究对其脾脏进行了形态大小、组织学和活体染色观察。结果显示,瓦氏黄颡鱼的脾脏位于腹腔中部,呈暗红色,脾体指数为0.120%~0.273%;对健康脾脏制备石蜡切片,分别进行HE染色和改良James染色,显微镜观察发现其脾组织主要由不发达的被膜系统、分界不清的红髓和白髓及由网状纤维和胶原纤维组成的纤维支架构成。其中网状纤维主要位于脾索和椭球体周围,胶原纤维主要位于脾索;使用台盼蓝活体染色法对健康瓦氏黄颡鱼的血脾屏障部位和功能探索发现,台盼蓝主要位于脾脏的椭球体内皮细胞中,最早出现在注射后的第4小时,随时间推移在椭球体内逐渐累积,24 h后数量显著增加,并持续增加至第72小时。结果表明椭球体内皮细胞是血脾屏障的重要组成成分。     

Adenosine triphosphate levels of chinook salmon (Oncorhynchus tshawytscha) eggs following in vitro maintenance and activation/fertilization

To further examine the concept of egg quality and the physiology of stored salmonid eggs, we investigated the effects of different oxygen tensions on the adenosine triphosphate (ATP) levels of unfertilized, activated, and fertilized chinook salmon (Oncorhynchus tshawytscha) eggs. The ATP levels of unfertilized chinook salmon eggs were 2.61±0.14 nmol ATP per egg (17.6±0.9 mol l^–1 relative to cell water) and ranged from 1.98 to 3.63 nmol ATP per egg. The ATP content of unfertilized eggs maintained at 10 °C under 100% O₂, 21% O₂, and 100% N₂ remained unaltered throughout a 120 h storage period. Storing eggs under identical conditions at 20 °C (in an effort to speed egg metabolism and ATP turnover) resulted in significant O₂-independent decreases in ATP levels. However, ATP levels of unfertilized eggs exposed to 1 mmol l^–1 potassium cyanide (a potent inhibitor of oxidative phosphorylation) at 10 °C were significantly decreased after 24 h and continued to decline throughout the 120 h maintenance period to about 30% of time=0 values. Maintenance with exogenous nutrients (5 mmol l^–1 acetate plus 5 mmol l^–1 pyruvate) over 120 h at 10 °C did not alter the ATP content of unfertilized eggs. Eggs activated by exposing them to 10 °C water for a few minutes showed a rapid decrease in ATP values, regardless of whether the eggs were fertilized or not. Following an initial 25% drop after fertilization, the ATP levels remained stable for the remainder (5 d) of the incubation period in eggs maintained in 10 °C water. Therefore, unfertilized chinook salmon egg ATP levels appear to be relatively stable and maintained by a low, cyanide-inhibitable metabolism. The stability of egg ATP levels may be one reason that salmonid eggs can be stored for several days while eggs from other fishes cannot.     

口虾蛄耗氧量、耗氧率及窒息点初步研究

<正> 前言口虾蛄(Oratosquilla oratoria(DeHaan)系口足类甲壳动物,又名东方虾蛄、虾蛄,俗称琵琶虾。广布于中国、日本、韩国、东南亚地区。我国沿海均有分布,浙江沿海资源量颇大,为虾蛄类的优势种。随着海洋水产资源的衰退,早先不太被重视的口虾蛄也因其肉质鲜美、营养丰富而日益受消费者的青睐,但如何合理开发利用     

Cortisol-induced changes in oxygen consumption and ionic regulation in coastal cutthroat trout (Oncorhynchus clarki clarki) parr

The influence of cortisol on oxygen consumption and osmoregulatory variables was examined in coastal cutthroat trout (Oncorhynchus clarki clarki) parr kept in fresh water (FW) and transferred to seawater (SW). Intraperitoneal implants containing cortisol (50 g g^–1) in vegetable oil resulted in elevated plasma cortisol titres similar to those observed in fish following a 24h SW exposure. Cortisol treatment significantly increased the oxygen consumption and plasma glucose levels of trout in FW, consistent with the glucocorticoid role of cortisol. Cortisol treatment did not cause any changes in plasma ion concentrations or gill Na⁺,K⁺-ATPase activity in FW after 10 days. Cortisol-implanted fish exposed to SW for 24h showed slightly improved ion regulatory ability compare to non-implanted controls. The results of this study suggest that during SW transfer in juvenile salmonids, increases in cortisol may act as both a mineralocorticoid and a glucocorticoid, depending on the developmental state of the fish (e.g., smolt versus parr). Furthermore, the relative energetic costs of osmoregulation and that of the stress associated SW transfer cannot be discerned using whole-animal oxygen consumption rates.     

光照周期对褐牙鲆幼鱼生长、能量分配及生化指标的影响

在循环水实验系统中,设置1 L∶23 D、9 L∶15 D、12 L∶12 D、15 L∶9 D、24 L∶0 D(光L∶暗D)共5个光照周期处理组,研究其对褐牙鲆幼鱼生长及部分生化指标的影响。实验结束时褐牙鲆幼鱼的体质量为41.10~43.98 g,不同处理组间无显著差异,体质量未表现出与日光照周期长短的相关性。日生长系数只在21~30 d阶段内出现显著差异,整个实验期间的平均日生长系数为1.86~2.02,不同处理组间不存在显著差异,日生长系数随日光照周期的延长略呈下降趋势。不同阶段的摄食率在31~40 d阶段出现显著差异,12 L∶12 D处理组摄食率显著高于9 L∶15 D处理组。在整个实验期间,1 L∶23 D的褐牙鲆幼鱼摄食率为1.64,显著低于12 L∶12 D的褐牙鲆幼鱼的摄食率(1.79)。整个实验期间,1 L∶23 D的褐牙鲆幼鱼平均饲料转化效率为113.49,显著高于12 L∶12 D、15 L∶9 D、24 L∶0 D的褐牙鲆幼鱼,并略高于9 L∶15 D的褐牙鲆幼鱼,饲料转化效率随光照时间的延长呈下降趋势。实验期间摄食能的分配比例仅在排泄能上出现显著差异,15 L∶9 D最高,12 L∶12 D最低,其他各项能量分配均无显著差异。不同光照周期对耗氧率影响显著,15 L∶9 D最高,为0.252 mg/(g·h),24 L∶0 D最低,为0.173 mg/(g·h)。耗氧率随着光照时间的延长呈先上升后下降的趋势。血浆生长激素和类胰岛素生长因子I含量在不同处理组间也不存在显著差异,随光照时间延长也未表现出明显变动趋势。血浆褪黑素含量随光照时间的增加呈先下降后上升的趋势,12 L∶12 D处理组MT含量最低,但与其他处理组差异不显著。实验结束时肌肉RNA/DNA比值随光照周期延长呈先上升后下降变动趋势,其中15 L∶9 D和24 L∶0 D处理组的肌肉RNA/DNA比值显著高于1 L∶23 D处理。肝脏RNA/DNA比值以12 L∶12 D最高,15 L∶9 D最低。实验结果表明光照周期对褐牙鲆幼鱼摄食量和饲料转化效率产生显著影响,但摄食量减少的处理组由于耗氧率降低因而饲料转化效率得到提高,因此光照周期对生长没有显著影响。光照周期明显影响血浆褪黑素、肌肉和肝脏RNA/DNA比值,但生化指标的差异与生长无明显相关性。