Cloning and Analysis of Calmodulin Gene from Porphyra yezoensis Ueda （Bangiales, Rhodophyta）

In order to understand the mechanisms of signal transduction and anti-desiccation mechanisms of Porphyra yezoensis, cDNA and its genomic sequence of Calmodulin gene (CaM) was cloned by the technique of polymerase chain reaction (PCR) based on the analysis of P. yezoensis ESTs from dbEST database. The result shows that the full-length cDNA of CaM consists of 603 bps including an ORF encoding for 151 amino acids and a terminate codon UGA, while the length of genomic sequence is 1231 bps including 2 exons and 1 intron. The average GC content of the coding region is 58.77%, while the GC content of the third position of this gene is as high as 82.23%. Four Ca²⁺ binding sites (EF-hand) are found in this gene. The predicted molecular mass of the deduced peptide is 16688.72 Da and the pI is 4.222. By aligning with known CaM genes, the similarity of CaM gene sequence with homologous genes in Chlamydomonas incerta and Chlamydomonas reinhardtii is 72.7% and 72.2% respectively, and the similarity of the deduced amino acid sequence of CaM gene with homologous genes in C. incerta and C. reinhardtii are both 71.5%. This is the first report on CaM from a species of Rhodophyta.     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)     

Isolation and Characterization of Pathogenic Listonella anguillarum of Diseased Haft-Smooth Tongue Sole （Cynoglossus semilaevis Günther）

Studies were conducted to determine the cause of the acute mortality of half-smooth tongue sole Cynoglossus semilaevis Günther juveniles in a fish farm in Jimo, Shandong Province, China, in June 2006. Gross signs of the diseased tongue sole included several petechiae and ecchymoses on the body and fin necrosis and hemorrhagic lesion at the base of the fin. Bacteria were isolated from kidney, liver and hemorrhagic lesions of the diseased tongue sole. Among 14 strains, SJ060621 was proved to be highly virulent to juvenile tongue sole with LD50 value of 〈1.0×10^5 colony forming units （CFU）mL^-1, while the remaining 13 were avirulent. Among the 16 antibiotics tested, SJ060621 was sensitive to gentamicin and nitrofurantoin. It was identified as Listonella anguillantm with conventional plate and tube tests in combination with API 20E analysis. 16S rRNA gene and partial HSP60 gene sequenceing analysis revealed that the strain was highly homologous with L. anguillarum. Examination of the infected musculature by electron microscopy indicated numerous bacteria and lots of macrophages containing phagocytosed bacteria. Histopathological investigations revealed severe necrotic degenerative changes in the infected organs. Indirect immunofluorescence assay （IFA） was employed to detect the location of occurrence of bacteria, and bacteria were found in aggregations in the inflammatory areas in musculature.     

The adaptation of Arctic phytoplankton to low light and salinity in Kongsfjorden （Spitsbergen）

The basic environmental variables and adaptability ofphytoplankton communities to low light and salinity were stud- ied using incubation experiments in Kongsfjorden, a high Arctic fjord of Spitsbergen, in late summer 2006. Chlorophyll a concentrations were steady or decreased slightly in darkness after one day or one week incubation. Chlorophyll a concentrations showed an initial decline when exposed to natural light after one week incubation in darkness, and then increased significantly. In a salinity experiment, the maximal growth rate was observed at a dilution ratio of 10%, however, higher dilution ratios （≥0%） had an obvious negative effect on phytoplankton growth. We suggest that the phytoplankton communities in fjords in late summer are dark- ness adapted, and the inflow of glacial melt water is favorable for phytoplankton growth in the outer fiords where the influence of freshwater is limited.     

Efficient expression of green fluorescent protein （GFP） mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii,a high efficient expression vector was constructed.Green fluorescent protein(GFP) was expressed in C.reinhardtii under the control of promoters:RBCS2 and HSP70A-RBCS2.Efficiency of transformation and expression were compared between two transgenic algae:RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ.Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2,and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ.In addition,a threefold increase of GFP in Tran-Ⅱwas induced by heat shock at 40°C.All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C.reinhardtii.     

Estimation of natural mortality coefficient from fish abundance and catch data using Virtual Population Analysis (VPA)

1 Introduction Natural mortality coefficient (M) is one of the key population parameters common to most analyses in fish stock assessment. Many mathematical models of fish stock dynamics include M directly or indirectly. However, until now M is the least known among the four factors affecting fish stock variation: recruitment, growth, natural death and fishing death (Quinn and Deriso, 1999). There is an extensive literature concerning the impact of errors in M on stock assessment (Mertz and…     

低值鱼蛋白多肽-铁(Ⅱ)螯合物的酶解制备及其抗氧化、抗菌活性

以南海低值鱼蛋白为原料,采用木瓜蛋白酶和风味酶的复合酶水解得到多肽水解物(HP),并制备多肽-铁(Ⅱ)螯合物,对螯合工艺及螯合产物的结果及抗氧化和抗菌功能特性进行了初步研究。结果表明:1)水解度为5%的多肽与铁(Ⅱ)螯合效果最佳,螯合率为94.15%;2)化学分析及红外光谱表明活性肽与亚铁离子螯合并形成稳定结构的螯合物;(3)经过50%乙醇(体积分数)沉淀的螯合铁具有明显的抗氧化活性,其抗氧化作用相当于维生素E的90%,经过80%乙醇(体积分数)沉淀的螯合物具有显著的抗枯草牙孢杆菌和金黄色葡萄球菌活性,其抑菌圈均直径为13mm。     

低值鱼蛋白多肽-铁（Ⅱ）螯合物的酶解制备及其抗氧化、抗菌活性

以南海低值鱼蛋白为原料，采用木瓜蛋白酶和风味酶的复合酶水解得到多肽水解物（HP），并制备多肽．铁（Ⅱ）螯合物，对螯合工艺及螯合产物的结果及抗氧化和抗菌功能特性进行了初步研究。结果表明：1）水解度为5％的多肽与铁（Ⅱ）螯合效果最佳，螯合率为94．15％；2）化学分析及红外光谱表明活性肽与亚铁离子螯合并形成稳定结构的螯合物；（3）经过50％乙醇（体积分数）沉淀的螯合铁具有明显的抗氧化活性，其抗氧化作用相当于维生素E的90％，经过80％乙醇（体积分数）沉淀的螯合物具有显著的抗枯草牙孢杆菌和金黄色葡萄球菌活性，其抑菌圈均直径为13mm。     

Purification and identification of a clotting protein from the hemolymph of Chinese shrimp (Fenneropenaeus chinensis)

The clotting protein (CP) plays important and diverse roles in crustaceans, such as coagulation and lipid transportation. A clotting protein was purified from the hemolymph of Chinese shrimp Fenneropenaeus chinensis (named as Fc-CP) with Q sepharose HP anion-exchange chromatography and phenyl sepharose HP hydrophobic interaction chromatography. Fc-CP was able to form stable clots in vitro in the presence of hemocyte lysate and Ca²⁺, suggesting that the clotting reaction is catalyzed by a Ca²⁺-dependent transglutaminase in shrimp hemocytes. The molecular mass of Fc-CP was 380 kDa under non-reducing conditions and 190 kDa under reducing conditions as was determined with SDS-PAGE. CP exists as disulfide-linked homodimers and oligomers. The N-terminal amino acid sequence of Fc-CP was identical to that of shrimps including Penaeus monodon, Farfantepenaeus paulensis and Litopenaeus vannamei; and similar to that of other decapods. The purified Fc-CP was digested with trypsin and verified on an ABI 4700 matrix-assisted laser desorption/ionization tandem time-of-flight (MALDI-TOF/TOF) mass spectrometry. Our results will aid to better understanding the coagulation mechanism of shrimp hemolymph.     

Replacement of Dietary Fish Oil with Vegetable Oils Improves the Growth and Flesh Quality of Large Yellow Croaker （Larmichthys crocea）

We investigated the effect of the replacement of dietary fish oil with vegetable oils on the growth and flesh quality of large yellow croaker(Larmichthys crocea). The basal diet(FO) was formulated to contain 66.5% fish meal and 6.4% menhaden fish oil; whereas the other 3 experimental diets were formulated by replacing the fish oil with 50% soybean oil(SO50), 100% soybean oil(SO100) and 100% palm oil(PO100), respectively. The 4 diets were randomly assigned to 4 floating sea cages(3.0 m × 3.0 m × 3.0 m), and each was stocked with 250 fish individuals with an initial average weight of 245.29 g ± 7.45 g. The fish were fed to apparent satiation twice a day at 5:00 and 17:00, respectively, for 12 weeks. Experimental analysis showed that the specific growth rate of fish fed SO50 or PO100 were significantly higher than that of fish fed FO or SO100(P0.05), and crude lipid contents of ventral muscle and viscera were significantly lower in fish fed FO than in those fed the other 3 diets(P0.05). No significant differences in condition factor, viscerosomatic index, hepatosomatic index, gutted yield and colorimetric values of fish among the dietary treatments were observed(P0.05). Compared to FO diet, SO50, SO100 and PO100 diets led to substantial decreases in the liquid loss and water loss from fresh fillets(1 d, 4℃)(P0.05). Similarly, thiobarbituric acid reactive substance(TBARS) values of fillets under different storage conditions(1 d, 4℃; 7 d, 4℃; 4 weeks,-20℃; 8 weeks,-20℃) decreased significantly after partial or complete replacement of fish oil with vegetable oils. These findings indicated that the growth performance and selected flesh quality properties(liquid holding capacity and TBARS value) of large yellow croaker were substantially improved by replacing dietary fish oil with vegetable oils.     

Studies on the optimum protein content and the ratio of animal to plant protein in the diet of the black sea bream (Sparus macrocephalus)

An experiment on the optimum dietary protein content for the black sea bream using semi-purified diets showed the optimum is 50.19% by weight as inferred from the growth rate and food consumption of the fish. Another experiment on the proper ratio of animal to plant protein in its diet using sandeel as the animal protein source and peanut cake meal as plant protein source showed that the ratio had great effect on its growth and should be 1.00: 0.89–0.96 as judged from the growth rate and food consumption of the fish. Contribution No. 1973 from the Institute of Oceanology, Chinese Academy of Sciences.     

Effects of Dietary Protein Level on Growth and Utilization of Protein and Energy by Juvenile Mangrove Red Snapper （ Lutjanus argentimaculatus ）

A feeding trial was conducted in a recirculating water system to investigate the effects of dietary protein levels on growth, feed utilization, hepatosomatic index and liver lipid deposition of juvenile red snapper, Lutjanus argentimaculatus (average initial wet weight 8.0 ± 0.39 g and total length 3.14 ± 0.3 cm). In the experiment, six fishmeal-based diets were formulated to contain various protein levels (20% to 45% in 5% increments), with dietary energy ranging from 2210.7kJ lOOg to 2250.2kJlOOg dry matter. The protein to energy ratios of diets ranged from 8.58 mg protein kJ⁻¹ to 20.03 mg protein kJ⁻¹. Diets were fed for 90d to triplicate groups of fish stocked in 0.128m³ seawater tanks, 25 individuals each. The daily ration of 2% wet body weight was offered to the fish thrice a day. The fish at the end of the study had more than ten-fold (77.0g) increase in weight compared to the initial (8.0g). Fish fed diets of 40% and 45% protein produced significantly (P<0.05) higher weight gain of 77.2g and 76.5g, and specific growth rate (SGR) of 2.65% and 2.62% than those of 67.0 g and 68.3g, and 2.49% and 2.51% of the other diets. The broken-line regression of SGR against dietary protein level yielded an optimum dietary protein requirement of 42.6% (Y=−1.6295 + 0.1114 X ²,P<0.05). Survival remained 100% among groups. Feed conversion ratio decreased from 0.45 for fish fed 20% dietary protein to 0.35 for fish fed 45% dietary protein. Nitrogen intake increased with an increase in dietary protein, which in turn resulted in an increase in nitrogen gain of fish whole body. Fish fed 40% and 45% protein diets showed higher (P<0.05) nitrogen gain (0.27g and 0.26g) than those (0.23g and 025g) fed all other diets. Gross energy intake (GEI) in fish fed 45% protein was lower (600.67kJ) than that (607.97 kJ) of 40% protein diet, though the differences were not statistically significant (P>0.05); GEI ranging from 677.31 kJ to 663.20 kJ at remaining four diets (20% to 35% protein) did not appear to differ significantly (P>0.05). The highest energy gain of 518.33 kJ was obtained with fish fed 40% protein, resulting in the highest energy retention efficiency of 85.26%. The hepatosomatic index of fish fed diets of 20%, 25%, 30% and 35% protein were significantly (P<0.05) higher (2.09% to 2.57%) than those (1.44% and 1.41%) of fish fed diets containing 40% and 45% protein. Liver lipid contents decreased from 8.72% to 7.0% in fish fed dietary protein of 20% to 45% in 5% increments. Results suggest that the diet containing 40% to 42.6% protein with a P/E ratio of 17.6 mg protein kJ⁻¹ is required for good growth of L. argentimaculatus weighing between 8.0 g and 85.2 g under the culture conditions of the present study.     

Cloning, Expression and Characterization of Translationally Controlled Tumor Protein （TCTP） Gene from Flatfish Turbot （Scophthalmus maximus）

A full-length cDNA encoding translationally controlled tumor protein of marine flatfish turbot (Scophthalmus maximus), SmTCTP, was isolated with rapid amplification of cDNA Ends (RACE). SmTCTP consisted of a 5' untranslated region (UTR) of 84 bp, a 3' UTR of 451 bp and an open reading flame (ORF) of 513 bp, encoding a protein of 170 amino acid residues, which contained two signature sequences of TCTP family. The 5'UTR of SmTCTP started with a 5'-terminal oligopyrimidine tract (5'-TOP), a typical feature for translationaily controlled mRNAs. The deduced amino acid sequence of SmTCTP was similar to the other known verte-brate TCTPs in a range of 58.8% to 64.1%. The length offish TCTPs was diverse among species, e.g., TCTP of turbot and sea perch (Lateolabrax japonicus) is 170 aa in length, while that of zebrafish (Danio rer/o) and rohu (Labeo rohita) is 171 aa in length. North-ern blot analysis revealed that SmTCTP has only one type of mRNA. Its expression level in albino skin was slightly higher than that in normal skin. We constructed the pET3Oa-SmTCTP expression plasmid. The recombinant protein of His-tag SmTCTP was over-expressed in E. coli, purified and identified with peptide mass fingerprinting. These results may pave the way of further inves-tigation of the biological function of TCTP in fish.