不同添加方式植酸酶处理豆粕对牙鲆生长和饲料利用率的影响

以初始平均体重(2.02±0.02)g的牙鲆(Paralichthys olivaceus)为实验对象,进行为期70d的摄食生长实验,研究不同添加方式的植酸酶对牙鲆生长和饲料利用的影响。在5000.0g豆粕中添加2.5g植酸酶,然后用产朊假丝酵母(Candidautilis)进行发酵预处理,得到植酸酶预处理豆粕。共制作4种等氮等能(粗蛋白49.7%、总能20.9kJ/g)饲料,对照饲料主要以鱼粉为蛋白源;在对照饲料的基础上,用豆粕蛋白替代45%的鱼粉蛋白配制成豆粕组饲料;在每千克豆粕组饲料中添加1000IU植酸酶,配制成植酸酶组饲料;用植酸酶预处理豆粕蛋白替代45%的鱼粉蛋白配制成植酸酶预处理豆粕组饲料。结果表明,与对照组相比较,用豆粕蛋白替代饲料中45%的鱼粉蛋白,若不添加植酸酶则显著降低牙鲆的特定生长率(P0.01)、饲料效率、蛋白质效率和氮贮积率(P0.05);直接添加植酸酶组、植酸酶预处理豆粕组牙鲆的特定生长率、饲料效率、蛋白质效率和氮贮积率与鱼粉对照组相比较没有出现显著差异(P0.05);与不添加植酸酶的豆粕组相比较,在含豆粕饲料中添加1000IU/kg饲料的植酸酶显著提高牙鲆的特定生长率(P0.01)、氮贮积率(P0.05)和磷贮积率(P0.01),显著降低氮排放率(P0.05)和磷排放率(P0.01),但饲料效率和蛋白质效率没有显著变化(P0.05);在豆粕中添加植酸酶进行发酵预处理,降低了豆粕中植酸含量,在饲料中添加植酸酶预处理豆粕显著提高牙鲆的特定生长率(P0.01)、饲料效率、蛋白质效率和氮贮积率(P0.05),显著降低氮(P0.05)、磷和钙的排放率(P0.01)。       

Molecular analysis of complement component C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus

 The amino acid sequences of the human terminal complement components show extensive structural similarity to each other. In this study the C8β and C9 cDNAs of Japanese flounder, Paralichthys olivaceus, were cloned and analyzed. The derived deduced amino acid sequences of the two terminal components were homologous to those of humans, in that the sequences of both species contained LDL receptor, EGF precursor, and two thrombospondin domains. Japanese flounder C9 was found to have a second thrombospondin region in the C-terminus, similar to that reported for rainbow trout and pufferfish. Moreover, these two complement component cDNAs of Japanese flounder had partial similarity to human perforin. These findings show that Japanese flounder C8β and C9 have similar structures, which supports the hypothesis that the terminal complement genes originated from the same ancestral gene. Collectively, these features emphasize the strong similarity among the members of the terminal complement family. Received: 23 March 1999 / Revised: 1 June 1999     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Molecular cloning, expression analysis and enzymatic characterization of cathepsin K from olive flounder (Paralichthys olivaceus)

We assessed the putative physiological roles of cathepsin K from a flatfish, olive flounder. We cloned a cDNA encoding for cathepsin K (PoCtK), a cysteine protease of the papain family from olive flounder, Paralichthys olivaceus. The tissue-specific expression pattern of PoCtK, determined via real-time PCR analysis, revealed ubiquitous expression in normal tissues with high levels of expression in the spleen and bone marrow. However, PoCtK expression was significantly increased in the muscle and gill at 3–24 h post-injection with bacterial lipopolysaccharide (LPS). The cDNA encoding for the mature enzyme of PoCtK was expressed in Escherichia coli using the pGEX-4T-1 expression vector system. Its activity was quantified via the cleavage of the synthetic peptide Z-Gly-Pro-Arg-MCA, zymography, and the collagen degradation assay. The optimum pH for the protease activity was 8, and the recombinant PoCtK enzyme degraded collagen types I, II, III, IV, and VI and acid-soluble collagen from olive flounder muscle in the presence of chondroitin 4-sulphate (C-4S). Therefore, our data indicate that cathepsin K may play a role in the immune system of fish skin and muscle, in addition to its principal bone-specific function as a collagenolytic enzyme.     

Structural comparison of promoter and coding sequence of type I collagen alpha 1 chain gene duplicates between zebrafish and flounder/fugu lineages

Alpha 1 chain (Colα1(I)) and alpha 2 chain (Colα2(I)) are universal components of type I collagen in tetrapods, but rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) have a third: alpha 3 chain (Colα3(I)). This study tests whether Colα3(I) is a duplicate of Colα1(I) by whole-genome duplication (WGD) that occurred early in the ray-fin fish lineage. We also examine how their promoter sequence was modified after WGD. We cloned Colα1(I), Colα2(I) and Colα3(I) cDNAs and their promoters from flounder (Paralichthys olivaceus) and obtained corresponding sequences from the genome databanks of two pufferfishes Takifugu rubripes and Tetraodon nigroviridis, by BLAST-Search using flounder sequences. Phylogenetic analysis of N-terminal sequences of ca. 100 amino acids, including signal peptide and N-propeptide sequences before short triple helical domain, indicates that Colα3(I), found only in teleosts, is a duplicate of Colα1(1) by WGD. Colα1(I) and Colα3(I) genes begin to be transcribed at different stages of Takifugu embryogenesis, suggesting that their structure of promoter is modified differently after WGD. In flounder, Takifugu and Tetraodon, the structure of proximal region of promoter is highly conserved within Colα1(I) and within Colα3(I); no homology is apparent except for the TATA element motif between Colα1(I) and Colα3(I) of each species. Unexpectedly, zebrafish Colα1(I) promoter is more homologous to Colα3(I) of flounder and fugu than Colα1(I) is. These results suggest that each duplicated Colα1(I) gene promoter inherited a unique structure after WGD, but the manner of modification differed between the phylogenetically separated zebrafish and flounder/pufferfish lineages.     

Five new microsatellite loci for Oliver flounder (Paralichthys olivaceus) from an expressed sequence tag (EST) library and cross‐species amplification

Five microsatellite loci have been isolated and characterized from a cDNA library of Oliver flounder, Paralichthys olivaceus. All loci were found to be polymorphic and had between four and 10 alleles. Observed and expected heterozygosities varied from 0.70 to 0.90 and from 0.52 to 0.80, respectively. Five additional fish species assessed for cross‐species amplification revealed between zero and three positive amplifications and between zero and two polymorphic loci per species.     

Salinity effects on osmoregulation and growth of the euryhaline flounder Paralichthys orbignyanus

The flounder, Paralichthys orbignyanus, is found in coastal and estuarine waters of the Western South Atlantic Ocean. It is being considered for aquaculture due to its high market price and wide tolerance to environmental factors such as salinity, pH, and nitrogenous compounds. The objective of this study was to characterize the ionic and osmotic regulation of P. orbignyanus over the range of its tolerated ambient salinities (0-40‰) and to evaluate the survival and growth in freshwater (0‰) and seawater (30‰) over 90 days. After 15 days of exposure to different salinities (0‰, 10‰, 20‰, 30‰ and 40‰), plasma osmolality and ionic (Na⁺, Cl⁻, K⁺ and Ca²⁺) concentrations slightly increased with salinity. The isosmotic point was estimated as 328.6 mOsm kg⁻¹ H₂O and corresponded to 10.9‰ salinity. After 90 days, survival was similar in freshwater and seawater, but osmo- and ionoregulation was significantly affected in freshwater and flounders reared in this medium showed a lower growth rate than those reared in seawater. Based on the results from this study, P. orbignyanus can be characterized as a marine/estuarine euryhaline teleost capable of hyper/hypo iono- and osmoregulation over the fluctuating salinity regime faced by this species in the environment. Furthermore, results suggest that the lower growth rate exhibited by P. orbignyanus in freshwater could be due, at least partially, to a higher energy expenditure associated to a higher branchial Na⁺, K⁺-ATPase activity in this environment.     

A set of polymorphic trinucleotide and tetranucleotide microsatellite markers for the Japanese flounder (Paralichthys olivaceus)

We have developed the first set of trinucleotide and tetranucleotide markers for the Japanese flounder, Paralichthys olivaceus. One hundred and sixty-seven polymorphic trinucleotide and tetranucleotide microsatellites were isolated using clones derived from two libraries. Of almost 200,000 clones analysed, 0.5% presented trinucleotide or tetranucleotide repeat regions. Among the trinucleotide repeats analysed in this study, the most frequent one was (CAG)(n) and the most common tetranucleotide repeat was (GATA)(n). The position of the new markers in the genetic linkage map was determined. Markers were evenly distributed along the P. olivaceus linkage groups, without distinction between the kinds of repeats and library of origin. The markers isolated in this study contribute significantly to the genetic linkage map of the Japanese flounder.     

Purification of flounder (Paralichthys olivaceus) fibronectin and its localization during re-epithelialization at a fin wound

Plasma fibronectin (FN) of flounder Paralichthys olivacem was purified and characterized. Flounder FN was purified by a combination of affinity chromatography using Sepharose coupled with flounder gelatin and gel filtration on Superose 6. Flounder FN was found to be a disulphide-linked heterodimer of 220 and 230 kDa polypeptides. It had cell-spreading activity for baby hamster kidney (BHK) cells, which was inhibited by the synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro. In flounder explants, anti-flounder FN antiserum distinguished fibroblast-like cells from epithelial cells; indirect immunofluorescence showed that the fibroblast-like cells exhibited a fibrous staining to the antiserum, but that epithelial cells did not. These results suggest that flounder FN is a homologue of mammalian FN.
The localization of FN during re-epithelialization at the site of a severed fin was investigated. When the top of the fin was cut, epidermal cells covered the surface of the wound within 1 h. FN is detected at the wound site during epidermal cell migration, suggesting that it serves as a cell-adhesion factor for prompt re-epithelialization at wound sites.