翘嘴鳜铜锌超氧化物歧化酶重组蛋白表达、纯化及特性分析

超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的一种重要抗氧化酶。根据翘嘴鳜(Siniperca chuatsi)Cu/Zn-SOD基因序列(Gen Bank登录号:KJ558392.1)设计表达引物,扩增获得截去信号肽后的一段460 bp的序列,序列经过鉴定后,构建了重组表达质粒p ET-30a+Sc Cu/Zn-SOD,并将该质粒转入到BL21(DE3)中,用IPTG诱导进行表达。经过优化表达条件得到可溶的Sc Cu/Zn-SOD重组蛋白(rScCu/Zn-SOD),纯化重组蛋白后测定rScCu/Zn-SOD的浓度和酶活性。结果发现在20℃和37℃条件下均能够诱导Sc Cu/ZnSOD的表达。37℃时重组蛋白主要以包涵体形式存在。降低诱导温度和补充Cu~(2+)/Zn~(2+)可提高rScCu/Zn-SOD的表达量。在20℃、0.5 mmol/L IPTG条件下,添加0.5 mmol/L CuSO_4和0.1 mmol/L ZnCl_2于培养基中,重组蛋白的表达量明显升高。纯化后的重组蛋白浓度为0.14 mg/m L,酶活力为108.5 U/mg。rScCu/Zn-SOD最适温度为37℃,最适pH为7.0,可耐受5%浓度的SDS蛋白质变性剂。     

日本沼虾cytMnSOD和mtMnSOD基因全长cDNA克隆及表达

利用RACE技术从日本沼虾肝胰腺中克隆了cytMnSOD和mtMnSOD基因cDNA全长序列。cytMnSOD基因cDNA全长1 233 bp,开放阅读框为858 bp,编码286个氨基酸,N端含有60个氨基酸残基组成的延伸区;mtMnSOD基因cDNA全长1 113 bp,开放阅读框为654 bp,编码218个氨基酸,N端含有20个氨基酸残基组成的信号肽;cytMnSOD和mtMnSOD预测蛋白分子量及等电点分别为31.33、24.05 ku和5.62、7.12。日本沼虾cytMnSOD推导的氨基酸序列与其mtMnSOD的相似性为40%,二者均含有MnSOD的特征肽段(DVWEHAYY)、4个Mn2+结合位点和2个N-糖基化位点。Real-time PCR结果表明,cytMnSOD和mtMnSOD在日本沼虾肝胰腺、肌肉、血细胞、大颚器官、卵巢和鳃等组织均有表达,其中肝胰腺表达量最高;肝胰腺cytMnSOD和mtMnSOD基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低。嗜水气单胞菌刺激后3 h,肝胰腺cytMnSOD和mtMnSOD的表达量显著增加,推测MnSOD是参与机体免疫防御反应的一种重要分子。     

Effect of dietary vitamin C supplementation on the oxygen consumption, ammonia-N excretion and Na/K ATPase of Macrobrachium nipponense exposed to ambient ammonia

The 96-h LC₅₀ of ammonia-N and the effects of dietary vitamin C on oxygen consumption, ammonia-N excretion and Na⁺/K⁺ ATPase activity of Macrobrachium nipponense exposed to ambient ammonia were investigated. The results showed that the 96-h LC₅₀ of ammonia-N was 36.6 mg l⁻¹ for the freshwater prawn, M. nipponense, at pH 8.0. When prawns were exposed to high ambient ammonia-N concentrations, the oxygen consumption rate increased and ammonia excretion decreased. Dietary vitamin C supplementation led to higher oxygen consumption and lower ammonia excretion. Na⁺/K⁺ ATPase activity increased with increased ambient ammonia-N exposure in the range of 0–18.3 mg l⁻¹, and then was reduced at ambient ammonia-N 36.6 mg l⁻¹. Na⁺/K⁺ ATPase activities of prawns fed a vitamin C-supplemented diet were significantly lower than those of prawns fed a diet which was not supplemented with vitamin C.     

动植物蛋白对日本沼虾生长、肌肉组成、抗氧化、TOR信号通路及食欲调节相关基因表达的影响

为探讨动植物蛋白对日本沼虾生长、肌肉组成、抗氧化、TOR信号通路及食欲调节相关基因表达的影响,实验配制了3组等氮等能饲料,分别为鱼粉组(FM)、混合植物蛋白组(豆粕,菜粕,玉米蛋白粉)(PPB)和混合植物蛋白添加氨基酸组(PPB/Aa),投喂饲养日本沼虾8周。结果发现,PPB和PPB/Aa组日本沼虾增重率和摄食率显著低于FM组,而饲料系数显著高于FM组,各组之间成活率和肌肉必需氨基酸组成无显著差异;FM组肝胰腺丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)活性显著低于PPB组和PPB/Aa组,各组谷草转氨酶(AST)和谷丙转氨酶(ALT)无显著差异,PPB与PPB/Aa组血清碱性磷酸酶(AKP)、酸性磷酸酶(ACP)和ALT的活性均显著高于FM组;与FM组相比,PPB与PPB/Aa组显著降低了雷帕霉素靶蛋白(TOR)、核糖体蛋白S6激酶(S6K)及神经肽(NPY)基因的表达,但各组瘦素(leptin)基因的表达无显著差异。研究表明,鱼粉与混合植物蛋白对日本沼虾肌肉中氨基酸组成无显著影响,但混合植物蛋白源降低了虾的生长和抗氧化能力及TOR信号通路与NP...     

Induced thermotolerance and stress resistance in larvae of the freshwater prawn, Macrobrachium rosenbergii (de Man, 1879)

Optimal conditions for heat shock (HS) were used to demonstrate induced thermotolerance (ITT) in larvae of the prawn, Macrobrachium rosenbergii. Larvae from three different broodstock nutritional regimes exhibited comparable degrees of ITT, which remained high for about 4 days. Survival and growth of larvae given the standard HS treatment (37 °C for 30 min) were not statistically different from those of controls, so the cost of mounting a heat shock response was not sufficient to reduce those parameters. However, the percentage of heat-shocked larvae undergoing metamorphosis appeared to be slightly lower than that of controls. Previously heat-shocked larvae withstood hypersalinity exposures much better than control larvae, but showed the same survival level when both were challenged with ammonia toxicity. We suggest that the use of induced thermotolerance might provide a means to improve the performance of larvae during transport and/or initial inoculation into grow-out ponds. The present paper provides the basis upon which that suggestion might be examined.     

Classification of differentiating oocytes during ovarian cycle in the giant freshwater prawn, Macrobrachium rosenbergii de man

Based on the light microscopic observations of cells' sizes, chromatin patterns, amount of lipid droplets and yolk granules, the female germ cells could be classified into four different phases, which include 1) oogonia (Oog), 2) primary oocytes (pOc), 3) secondary oocytes (sOc), and 4) mature oocyte (mOc). Oog are small oval-shaped cells with irregular-shaped nuclei sizing 4–6 μm in diameter. They rest on the connective tissue germinal cord at the tip of each ovarian pouch (lobule). Oogonia increase their number through mitotic division, and the daughter cells move into ovarian pouch where they undergo first meiotic division to become primary oocytes, which have various steps of 1st meiotic prophase accumulating at the innermost zone of the ovarian pouch. The primary oocytes are small oval-shaped cells (8.5–10 μm in diameter) with large nuclei containing chromatin in various states of condensation that finally transform into chromatids. Their nuclei are surrounded by thin rim of faint blue-stained cytoplasm. The secondary oocytes derived from 2nd meiosis and comprise five steps: Oc1 and Oc2, classified as previtellogenic oocytes, Oc3 and Oc4, classified as vitellogenic oocytes, and mature oocyte (mOc) The zones of ovarian pouch are defined based on the accumulation of various steps of developing oocytes, namely, oogenic, previtellogenic, vitellogenic and mature zones, respectively. The ovarian cycle is divided into five stages based on the number and types of oocytes present in each stage. Stage 0 and I are spawn and spent stages. Stage II and III are proliferative and premature stages, while stage IV is mature stage. During ovarian stage I, each ovarian pouch contains primarily oogonia, primary oocytes, Oc1 and a few Oc2. In stage II, the pouch contains mainly Oc2 and Oc3, while in stage III the predominant cells are Oc4. Mature oocytes appear synchronously, in stage IV. The ovulating mature oocytes pass through the thin disrupted wall of ovarian pouch into subcapsular space, that leads into the oviduct situated on the ventro-lateral side of the ovarian lobe. At spawning, the ovarian pouches break down and only connective sheaths and hemolymph sinuses remain. The germinal cords and islets of oogonia remain in the central area of stage 0 ovary. The ovarian capsule, including the muscular layer, becomes attenuated as the ovary progresses from stage 0 to IV. The hemolymph vessels become highly convoluted in the central area of the ovary, and they branch radially into smaller hemolymph sinuses around each oogenic pouch.     

Effect of dietary vitamin E on growth,immunity and regulation of hepatopancreas nutrition in male oriental river prawn,Macrobrachium nipponense

We evaluated the effects of dietary vitamin E on growth, immunity and regulation of the hepatopancreas in male oriental river prawn, Macrobrachium nipponense. Shrimps were fed 0, 40, 80, 160, 320 or 640 mg vitamin E/kg for 60 days. The 80 mg/kg group had the highest weight gain rate, specific growth rate and lowest feed conversion rate while there were no significant differences in survival rate and hepatosomatic index. The highest crude fat and lowest crude protein content were observed in the 160 mg/kg group. In the hepatopancreas, lysozyme, acid phosphatase and alkaline phosphatase activities were highest in the 160 mg/kg group, while superoxide dismutase, catalase and glutathione peroxidase activities decreased with increasing vitamin E levels. Malondialdehyde content initially decreased then increased with vitamin E levels, whereas the reverse was seen with total antioxidant capacity. Linoleic acid, DPA, DHA, total n‐3 polyunsaturated fatty acid and total polyunsaturated fatty acid first increased then decreased, while EPA and total saturated fatty acid rose with vitamin E levels. Total n‐6 polyunsaturated fatty acid content declined while there were no significant differences in linolenic and total monounsaturated fatty acid content. Following a toxicity test with Aeromonas hydrophila, hepatopancreas ultrastructure revealed that appropriate vitamin E levels promote an increase in mitochondria, endoplasmic reticulum and Golgi bodies, but excess vitamin E can damage cell structure. These results provide evidence that 80–160 mg/kg dietary vitamin E has a positive impact on growth, immunity and regulation of the hepatopancreas in male shrimp.     

Purification and characterization of a β–glucan binding protein from the haemolymph of freshwater prawn Macrobrachium rosenbergii

β‐glucan binding protein (βGBP), a pattern recognition protein was purified from the haemolymph of freshwater prawn Macrobrachium rosenbergii by heparin affinity chromatography that showed a single band in native gradient PAGE. The β‐glucan binding property of the purified protein was confirmed in a phenoloxidase (PO) assay, where addition of βGBP along with β‐glucan increased the specific PO activity compared with that of β‐glucan alone. The molecular weight of the βGBP was found to be ~316 kDa on gel filtration chromatography. In SDS‐PAGE, βGBP molecule was reduced to one polypeptide chain of molecular weight ~113 kDa. Thus the βGBP in M. rosenbergii is possibly a homotrimeric molecule. The purified sample run on unreduced condition in SDS‐PAGE also revealed a similar size band (~113 kDa) and hence, the polypeptide chains of βGBP are held by non‐covalent interactions. The purified βGBP samples run in native PAGE was stained positively with alcian blue for carbohydrates and Sudan black for lipids indicating the βGBP to be a glycolipoprotein. With rabbit polyclonal anti‐βGBP serum developed, an indirect ELISA was standardized and the normal βGBP concentration in adult M. rosenbergii serum was quantified to be ~2 mg mL^?1. Furthermore, the applicability of the developed ELISA is discussed.     

中国海洋红藻门新分类系统

蚣藻属(Grateloupia)种类多(中国已报道42种),可食用、药用(具有清热解毒、驱虫、抗肿瘤、抗病毒、抗凝血、抗氧化等作用),并可作为提取卡拉胶的工业原料,是有待于养殖和加工开发的经济红藻。文章综述了蜈蚣藻属分类变动、繁殖生物学、生理生态和养殖的研究现状及存在的问题,为蜈蚣藻属增养殖及研究开发利用提供参考。     

Acute effects of copper on superoxide dismutase, catalase and lipid peroxidation in the freshwater teleost fish, Esomus danricus

Static-renewal bioassays [Methods for acute toxicity tests with fish, macro-invertebrates and amphibians: USEPA, ERS, EPA 660/3 75-009 (1975)] were carried out on Esomus danricus exposed to sub-lethal (0.55 mg/l) and lethal (5.5 mg/l) concentrations of copper. The 96-h median lethal concentration (LC₅₀) was 5.5 mg/l. Biochemical stress responses, such as visceral superoxide dismutase (SOD) and catalase (CAT) activities, were measured during this 96-h period. Malondialdehyde, a product of lipid peroxidation, was present at elevated levels in the visceral tissue of copper-exposed fish. Copper was found to be highly toxic to the fish and induced significant declines (p < 0.05–0.001) in all of the biochemical profiles studied, demonstrating a linear and positive correlation with both the concentration and duration of exposure to copper. In E. danricus, CAT appeared to be more sensitive to copper exposure (p < 0.001) than SOD at both lethal and sub-lethal levels. These results indicate that antioxidant responses can be employed as biomarkers of oxidative stress for this species in aquatic environments contaminated with copper.     

Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

罗氏沼虾(Macrobrachium rosenbergii)幼体病原肠杆菌PCR检测技术的建立与应用

根据罗氏沼虾(Macrobrachium rosenbergii)幼体培育期主要细菌性病原阴沟肠杆菌omp A基因序列、产气肠杆菌gry B基因序列设计特异性引物,通过对PCR扩增产物进行测序鉴定与特异性和敏感性试验,建立了两种病原菌的PCR快速检测方法,并对发病样品进行了检测。结果显示,设计的阴沟肠杆菌与产气肠杆菌检测引物能分别扩增出与预计大小一致的385 bp和201 bp的特异性片段,与其余供试菌株无交叉反应。两种检测方法的灵敏度分别为103 CFU/ml和102 CFU/ml。罗氏沼虾幼体样品的检测结果与实际发病情况一致,建立的检测方法也可直接对样品进行PCR检测,而无需细菌分离培养。本研究建立的阴沟肠杆菌与产气肠杆菌PCR检测方法具有较高的特异性与灵敏度,可缩短检测时间,该方法的建立对罗氏沼虾幼体病原的快速诊断、分子流行病学的调查及无特定病原(SPF)群体的建立具有重要意义。     

Comparative performance of Gamma Amino Butyric Acid (GABA) and 5-Hydroxytryptamine (5-HT) in the diet of larvae and post larvae of giant freshwater prawn, Macrobrachium rosenbergii: Effect of dose and route of administration on growth and survival

Three experiments were conducted to evaluate the effect of different dose and route of administration of 5-Hydroxytryptamine (5-HT) and Gamma Amino Butyric Acid (GABA) on growth, survival and pigmentation of giant freshwater prawn, Macrobrachium rosenbergii, larvae (experiment 1) and post larvae (experiment 2 and 3). In experiment 1 larva were stocked at 100 /L in seven different treatments with each of three replicates. The treatments were T1 (control), T2 (100 μg/ml of 5-HT bath exposure for 2 days), T3 (1 μM of GABA bath exposure for 2 days), T4 (0.5% 5-HT in feed), T5 (0.25% 5-HT in feed), T6 (0.5% GABA in feed) and T7 (0.25% GABA in feed). Highest growth, transformation rate, pigmentation and survival of larvae were recorded in T2 group in experiment 1. In experiment 2, post larvae were stocked at 70 per tank with 200 L of water for 45 days. This experiment consisted of 5 treatment groups with each of 3 replication viz. T1 (control), T2 (0.5% 5-HT in feed), T3 (0.25% 5-HT in feed), T4 (0.5% GABA in feed) and T5 (0.25% GABA in feed). At the end of this experiment growth was found to be higher in the control than the other treatment group showing inhibitory effect of 5-HT and GABA on growth of post larvae. To confirm the result, a third experiment was conducted for 30 days. Thirty numbers of post larvae having similar size were segregated from the previous experimental tanks (experiment 2) and stocked in the tanks of 100 L of water for experiment 3. All treatments were fed with control diet. There was similar growth pattern in all the treatments, which were higher (P < 0.05) than control, confirms the inhibitory effect of neurotransmitter in the diet of PL. Therefore, the overall results of the present study suggest that the bath treatment of neurotransmitter is superior to the dietary addition with regard to the growth, survival and pigmentation of M. rosenbergii larvae. 5-HT is more effective than the GABA for larvae of M. rosenbergii.     

Molecular cloning,characterization and expression analysis of the fatty acid‐binding protein (MnFABP), involved in dietary lipid sources response in oriental river prawn,Macrobrachium nipponense

A fatty acid‐binding protein (FABP) gene designated as MnFABP10 was cloned and characterized from the freshwater prawn Macrobrachium nipponense. The full‐length cDNA of MnFABP10 was 646 bp encoding a 130 amino acid. Real‐time quantitative RT‐PCR showed that the MnFABP10 gene was expressed in various tissues with the highest expression in the hepatopancreas. The MnFABP10 mRNA levels in the hepatopancreas and ovary of M. nipponense were dependent on the stages of ovarian development. Western blot results revealed a single immunoreactive band with an estimated molecular mass of approximate 14 kDa in the developmental ovary. Then, M. nipponense with an initial body weight of 0.090 ± 0.0010 g were fed with four isonitrogenous and isocaloric diets with different oils, that is, beef tallow (BT), soybean oil (SO), pollack fish oil (FO) and a mixture of fish oil and soybean oil (FO/SO 2 : 1 w/w) for 52 days. The mRNA levels of MnFABP10 in the hepatopancreas were influenced by different lipid sources, with a peak expression observed in prawns fed SO. This study suggests that MnFABP10 may have a putative function in ovary maturation, and its mRNA expression in the hepatopancreas can be regulated by the source of dietary lipids in M. nipponense.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

大口黑鲈生长性状相关标记的聚合效果分析

为摸清生态基养殖池塘系统的能量流动规律,以放置生态基的大口黑鲈 (Micropterus salmoides) 池塘为研究对象,采用原位实验方法,研究了生态基对大口黑鲈池塘养殖系统的水质及能量收支的影响。实验期间,生态基可显著降低池塘水体中氨氮、硝态氮、总氮及总磷含量(P<0.05),但对亚硝态氮、磷酸盐、底泥总氮和总磷含量无显著影响(P>0.05);饵料投入是系统能量输入的主要来源,分别占对照组和实验组总输入能的53.26%和55.02%,其次为浮游生物生产,两组分别为45.92%和44.22%;浮游生物呼吸是能量输出的主要途径,分别占对照组和实验组总输出能的60.01%和56.68%,其次为养殖生物收获,两组分别为28.78%和31.99%;生态基实验组生物净产出能、光合能转化效率、饲料能转化效率及总能量转化效率均显著高于对照组(P<0.05),而单位净产量耗饲料能和单位净产量耗总能则显著低于对照组(P<0.05)。结果表明,在大口黑鲈池塘放置生态基能改善池塘环境,有效提高系统产出量及能量利用效率。     

Amino acid-chelate: a better source of Zn, Mn and Cu for rainbow trout, Oncorhynchus mykiss

This study aimed to evaluate the amino acid-chelated trace elements as dietary supplement to rainbow trout. Three diets were formulated containing trace elements either from the inorganic salt (SF) or amino acid-chelate (AM). Diets 1 (SF) and 2 (AM) contained the same amount of trace elements from inorganic and amino acid-chelates, respectively. Diet 3 (AM-Hf) was added with trace elements from amino acid-chelatex at one-half of their levels in Diets 1 and 2. Each diet was fed for 15 weeks to three groups of 30 fish each, with an average weight of 1.52±0.21 g. Growth of fish was not affected by the treatment (P>0.05). However, bone (P<0.01) and liver (P<0.05) Cu contents were higher in the AM than the SF group. Similarly, hematocrit level (P<0.05) and alkaline phosphatase (ALP) activity (P<0.01) were higher in the chelate-fed fish. Further, DNA polymerase and CuZnSOD expression in the AM group was highly upregulated (P<0.05) compared to the SF fed fish as quantified by RT-PCR. Absorption and whole body retention of Mn and Zn from the AM were higher (P<0.05) than the inorganic salt. Half supplementation of those fed the elements from AM was at par with the full provision from the inorganic source tested.     

Cloning, expression, and characterization of a novel anti-HIV lectin from the cultured cyanobacterium, Oscillatoria agardhii

OAA, the potent anti-HIV protein from Oscillatoria agardhii NIES-204 belongs to a new lectin family, shows strict binding specificity for high-mannose N-glycans, and has an extremely high association constant in the picomolar range for recombinant gp120, an envelope protein of HIV. In this study we have cloned the gene encoding OAA from the genomic DNA of the cyanobacterium, and efficiently expressed the recombinant lectin (rOAA) in Escherichia coli. The rOAA expressed as a His-tagged fusion protein was recovered in a soluble form and purified in high yield (48 mg/1 l-culture) by metal chelate chromatography. The fusion protein was cleaved with factor Xa, and the resulting rOAA was isolated in a final yield of 14.8 mg/1 l-culture by reversed-phase HPLC. Both the N-terminal sequence and the molecular mass of rOAA were found to be identical with those of OAA. The rOAA was fully functional with the same properties as OAA, as evidenced by hemagglutination activity, hapten-inhibition test, and binding specificity for high-mannose-type N-glycans. This rOAA should be applicable as a specific probe for high-mannose N-glycans and should contribute to elucidation of the molecular basis of its strict carbohydrate-binding specificity and potent anti-HIV activity.