动植物蛋白源替代鱼粉研究进展

With the fast development of aquaculture, fish meal needs increased in recent years, however the quantity of fish catching decreases gradually. Fishmeal is a limited feed resource, and serious concem exists on the future availability of this feedstuff for incorporation in fish diets. Undoubtedly, fish meal is well recognized as the best dietary protein source for most marine carnivorous fishes which required high dietary protein levels compared to omnivorous or herbivorous fish. Fishmeal is known for their high content of essential amino acids and fatty acids, low carbohydrates, high digestibility, low levels of anti-nutritional factors (for fresh fish meal) and is a very good source of minerals and is highly palatable. Thus fish meal is in high demand as the protein source for many formulated diets. However, production of fish meal consumes approximately 35 % of the total global fish catch, and the increasing price and potentially unstable supply in the market could be limiting factors for marine fish culture. There have been strong efforts to define and develop cost-effective protein sources that can, at least partly, substitute for expensive high-quality fish meals in least-cost feed formulations. The search for fish meal substitutes and altemative dietary protein sources is an international research priority that could be of considerable economic advantages. Therefore it is urgent task to find animal and plant protein sources in place of fish meal. Among these, plant feedstuffs have received most attention in recent years, but due to their amino acid unbalances, .presence of anti-nutritional factors and low palatability, a high level of replacement of fish meal with plant feedstuffs in omnivorous fish is generally not well accepted. This paper reviews the research status for other protein sources replacing fish meal based on available information in the literature. Animal and plant protein sources nutrient values are evaluated from the aspect of digestibility, antinutrients, physiological status and suitable supplementation.     

鳗源嗜水气单胞菌主要外膜蛋白基因克隆及其表达

A pair of primers were designed according to the published nucleotide sequence of a putative outer membrane protein gene (omp) of Aeromonas hydrophila . With the specific primers, a target fragment about 1.1 kb was amplified from Aeromonas hydrophila ML316 via PCR .The target fragment was inserted into the linearized pGEM-T easy vector. After enzyme restriction and sequencing analysis,the nucleotide data had been further analyzed by DNAman and ClutalW software. The analysis results showed that the cloned DNA fragment had a longest open reading frame (ORF) of 1035 nt,it predicted to be encoded a 344 aa protein with the molecular weight of 36 kD. Hydrophobicity analysis suggested that the protein was highly hydrophilic, especialy at the first 24 aminoacid, this region could function as signal peptide. The homologious comparison proved the cloned gene had 96% homology to the sequence of the omp gene, and the alignment of the amino acid sequence was 98% . The recombinant plasmid was constructed with the target gene and the expressing vector pGEX-4T-1 and then was transformed into E. coli BL21（DE3）by BamH and Sal I .The fusion protein was expressed under the IPTG inducing condition,and exhibited about 62 kD in size,very close to the predicted molecular weight of GSTMOMP, furthermore,the fusion protein was specifically recognized by antiserum which raised against the major outer membrane protein of AHML316. Considering all these together, it proved that the cloned gene represented the major outer membrane protein gene of AHML316, and the expressed gene products shared identical antigenicity with the natural main outer membrane protein,and also provided technical support for developing an advanced gene engineering vaccine against Aeromonas hydrophila.     

鱼类白细胞介素1β基因的研究概况

Interleukin-1β(IL-1β) is one of the most pleiotropic cytokines and a central regulator of the immune and inflammatory responses. Interleukin-1β was initially discovered within mice and humans and over the last 10 years has been characterized within a wide variety of animals. The IL-1β plays a key role in the inflammatory process, enhancing cell-mediated immunity by inducing the growth and proliferation of lymphocytes, connective tissue cells, and by stimulating immune and inflammatory response effector cells.As an immunoregulatory cytokine, IL-1β has the potential to enhance the immune response induced by a vaccine and/or to modulate the immune response leading to different effector mechanisms. It is produced by many cell types, including monocytes, macrophages,T and B lymphocytes, fibroblasts, and endothelial and epithelial cells. Expression is induced by a diverse range of stimuli, including mitogens, cytokines, and microbial products. There have been considerable evidences provided by biological cross reaction that fish produce IL-1β during immune responses, and the bioactivity of IL-1 in fish has been known for over a decade. But only. since 1999,IL-1β gene has been cloned from the rainbow trout. And from then on, IL-1β gene has been cloned and expressed in many fish confirming that fish produces IL-1β gene during immune responses. In mammals it is produced as an inactive precursor that is processed by interleukin converting enzyme (ICE) to give a biologically active ‘mature‘ peptide. There is no signal peptide in IL-1β and its mechanism is unknown. This is the special part of the gene structure of the IL-1β. And through the program analyzing, we found this special structure in fish IL-1β gene. This paper reviews the functions and structure of gene IL-1β and the studies of the gene IL-1β in fish recently.     

蓝太阳鱼生长激素全长cDNA的克隆与序列分析

The full length cDNA encoding growth hormone of a freshwater fish, Lepomis cyanellus, (LcGH) was cloned from pituitary RNA with RT-PCR, 3‘ and 5‘ RACE (rapid amplification of cDNA ends). The LcGH cDNA (Genbank No. AY530822), about 989nt (nucleotide) long, consisted of a open reading frame with 615nt long, 5‘and 3‘untranslated regions with 93nt and 224nt long respectively, and a 57nt poly (A) tail. The DNA sequence analysis showed that there are typical Kozak sequence and polyadenylation signal. The pregrowth hormone peptide of 204aa deduced from LcGH cDNA included a putative signal peptide (17aa) locating in its Nterminal. There exist a Asn-Cys-Thr glycosylation site at amino acid 201, and 4 cysteine residues (No. 69, 177, 194, 202) that are essential to construct two S-S bonds in this pregrowth hormone peptide. Homological comparision among LcGH and other species growth hormones showed that There is high homology (more than 85%) between growth hormone of Lepomis cyanellus and that of most perciformes fish, but low homology (less than 70%) in comparison with other species such as Siluriformes and Cypriniformes fish.     

草鱼FGF8蛋白的理化特性分析

Physico-chemical properties of FGF8 in Ctenopharyngodon idellus were analyzed by bioinformatics. The full - length grass carp FGF8 pro-polypeptide of 210 amino acids including 40 basic amino acids and 16 acidic amino acids. The signal peptide sequence is MRLIPSRLSYLFLHLFAFCYYA, and its cleavage site is located between Ala22 and Gln23. Grass carp FGF8 protein is a secreted protein since the typical character of secreted protein is that its N-domain contains a signal peptide. There are two N-glycosylation in the sites of Asn37 and Asn136, respectively, which were Asn-Phe-Thr and Asn-Tyr-Thr. The molecular weight （Mw） of grass carp FGF8 protein is 24.68 kDa. Its isoelectnic point （pl） is 10.93. The protein contains four main secondary structures： α-helix, β- sheet, β-turn and random coil.     

鱼粉在水产饲料中的应用研究

As a main protein source in aquafeeds, fish meal has been extensively studied. Fish sources, freshness, processing temperature, lipid quality and microbiological index are five main aspects of the evaluation of fish meal quality. This paper reviewed the researches on fish meal including the evaluation of fish meal quality, the use of fishmeal and the environmental problems. Biogenic amine is the main potential toxin in decomposed fish meal including mainly histamine, cadaverine, putrescine and tyramine and most studies showed that they could affect the fish growth performance and health. The determination of protein digestibility of fish meal includes pepsin-digestion method, animal test, capillary electrophoresis, etc. The content of phosphorus in fish meal and its utilization can introduce pollution to water bodies and the use of alternative protein and improvement of utilization of fish meal can help to reduce the pollution from fish meal.     

美洲黑石斑鱼营养成分分析与营养价值评价

In this experiment, the contents of the main nutrients such as protein, fat, moisture, ash, calcium and phosphorus were analysed, and the contents of the trace elements, amino acids and fatty acids in its muscle were determined for Centropristis striata. Moreover, its nutritional value was evaluated and compared with other fishes,livestocks and poultries. The results showed that the content of dry matter and protein are higher in its muscle. Then, all of 17 common amino acids（AA） contents in muscle was 17.51% in the fresh sample, and 7 of them are human essential amino acids（EAA）41.58% in the total amino acids（TAA）. The ratio of essential amino acids to nonessential amino acids（EAA/NEAA） was 84.46% ,and the essential amino acids index（EAAI） was 57.71. According to AAS and CS,which basically accorded with the issued standard value by FAO and WHO. The content of 5 delicious amino acids was 44.37% in the TAA, and the ratio of branched chain amino acids to aromatic amino acids was 2.83. The content of polyunsaturated fatty acids was 22.3% rich in its muscle also, in particular EPA and DHA. In conclusion, Centropristis striata is a species of delicious and high nutritional value,which well deserves exploitation and utilization. At the same time, this experiment was preparing for the next study to provide reference data for artificial feed and the expansion of aquaculture.     

建鲤与异育银鲫生长、生化组成和饲料利用的比较

In order to investigate the difference in utilization of diets with different quality in Jian carp（Cyprinus carpio var. Jian）and allogynogenetic silver curcian carp(Carassius auratus gibelio), a 55 d growth trial was conducted and low quality diet (LQ-diet) and high quality diet (HQ diet) were tested. LQ-diet contained 33.91% dietary protein which is mainly from soybean meal while HQ-diet contained 45.59% dietary protein which is mainly from fish meal. The initial average body weights were from 5.58 g to 5.82 g for two fish strains. The trial was carried out in a system consisting of 12 self circulation 320 L tanks. During the experiment, the fish were fed to satiation twice a day (at 9: 00 and 15:00), and uneaten feed was collected 1 h after feeding and dried. Feces were collected twice a day (at 11:00 and 16:45) from the fecal traps and dried at 70 ℃. The results show that feed intake was higher in Jian carp than in allogynogenetic silver curcian carp when fed LQ diet, while there was no significant difference between weight gain, feed conversion efficiency, protein efficiency rate and apparent digestibility. When fed HQ diet, Jian carp showed a lower feed intake, but higher feed conversion efficiency and protein efficiency rate than allogynogenetic silver curcian carp while there was no significant difference in the weight gain and apparent digestibility of both species. For Jian carp, feed intake and protein efficiency rate for the fish fed HQ diet and LQ diet were not significantly different, while the fish fed HQ diet showed higher weight gain, feed conve rsion efficiency and apparent digestibility. For allogynogenetic silver curcian carp, the fish fed HQ diet showed significantly higher feed intake, apparent digestibility, weight gain, feed conversion efficiency and lower protein efficiency rate. For Jian carp, body contents of dry matter, protein, lipid and energy for the fish fed HQ diet and LQ diet were significantly higher. For allogynogenetic silver curcian carp, body contents of dry matter and protein was significantly higher, while body contents of lipid and energy were affected by diet qualities. Compared to Jian carp, allogynogenetic silver curcian carp showed better utilization when fed LQ diet while poorer utilization when fed HQ diet.     

鱼类几种新型免疫因子的研究进展

Cytokines are low molecular weight proteins that serve as chemical messengers within the innate and adaptive immune systems. To date, great progresses have been made in fish cytokine researches. A number of cytokine genes have been cloned and sequenced in fish. This review will focus on a number of novel immune-related cytokines including interleukin, interferon, interferon regulatory factors, Myxovirus resistance proteins, transforming growth factor, tumor necrosis factor, chemokines (CC and CXC chemokines), NK dell enhancement factor, MHCⅠ, MHCⅡ and some of their receptors, which have been identified in many fish species recently. Their genes and molecular structures are clarified. These cytokines are evolutionary well conserved. They share high identities at both the nucleotide and amino acid levels with the high vertebrate cytokines, and maintain characteristic structural motifs of those higher vertebrates. The function of some cytokine genes are analyzed in conventional manner by production of recombinant molecules. Several fish cytokines have been identified based on functional similarity to, or cross-reactivity with, mammalian cytokines. Moreover, molecular techniques, such as suppression subtractive hybridization, PCR and cDNA library screening, have recently enabled the identification of fish cytokine genes. Because of fish phylogenetic position and the fact that their immune systems have not been elaborated to the extent seen in mammals, progresses in this field will deepen our understanding of the molecular origins of cytokine genes and extend our knowledge on their mechanisms conferring disease resistance and the recombinant cytokines to control fish diseases.     

半滑舌鳎(Cynoglossus semilaevis) Nramp基因克隆与表达分析及SNP筛选

天然抗性相关巨噬细胞蛋白(Natural resistance-associated macrophage protein, Nramp)属于膜整合转运蛋白,具有抑制胞内寄生菌侵染、调节巨噬细胞的抗菌活性等作用。本研究对半滑舌鳎(Cynoglossus semilaevis)Nramp基因进行了克隆和表达分析,并对其与抗鳗弧菌感染相关的单核苷酸多态性(Single Nucleotide Polymorphism, SNP)位点进行了筛选。该基因cDNA序列全长3717 bp,其中开放阅读框(Open reading frame, ORF)1677 bp,所编码蛋白含有558个氨基酸,该蛋白具有Nramp家族的典型特征,包括10个跨膜区(Transmembrane, TM)、1个由20个氨基酸残基组成的胞质内转运蛋白特征结构域(Consensus Transport Motif, CTM)。半滑舌鳎Nramp的ORF末端有1个类似于脊椎动物Nramp2中的铁反应控制蛋白结合位点(Iron-responsive regulatory protein-binding site, IRE)。半滑舌鳎Nramp与其他14个物种的Nramp氨基酸序列同源性在63%?91%之间,系统进化分析表明,半滑舌鳎Nramp和所有鱼类Nramp聚集为一簇,与其他物种Nramp2的亲缘关系较近。实时荧光定量PCR分析显示,Nramp基因在半滑舌鳎脾脏和肾脏中的表达量最高,而在肌肉和性腺中的表达量最低;在哈维氏弧菌感染的半滑舌鳎肾脏、脾脏和肝脏中表达量呈升高趋势,而在鳃中则表现为下调趋势。利用直接测序法检测感染鳗弧菌后同一家系的233个个体(抗病个体165个,感病个体68个),共检测到15个SNP位点,对其中3个SNP 位点即 SNP-g.3113(T→C)、SNP-g.3125(A→G)和 SNP-g.3164(A→T)进行测序分型后发现,SNP- g.3125(A→G)的等位基因(G)频率和基因型(GG)频率与半滑舌鳎抗鳗弧菌疾病呈极显著相关(P<0.01)。研究结果表明,Nramp 基因不同基因型对半滑舌鳎的抗病能力有着极其重要的影响,SNP-g.3125(A→G)可作为潜在的抗性遗传标记位点。本研究将为半滑舌鳎抗性品系培育提供技术支持。     

RACE法分离团头鲂生长抑素全长cDNA及其序列测定

生长抑素具有抑制脑垂体GH释放的作用，是调控鱼类生长的主要激素之一。本研究采用RT和RACE(rapid amplification of cDNA ends)法，分离和测定了团头鲂脑中生长抑素PSSI cDNA的全长核苷酸序列，并对该基因进行了结构和系统进化分析。3’RACE扩增得到700bp左右的片段，5’RACE分离得到500bp左右的片段，把3’片段与5’片段拼接得到全长cDNA。cDNA全长735bp[不含poly(A)]，5’端非翻译区有100nt。3’端290bp[不包含poly(A)]，阅读框(open reading frame，ORF)345bp。该序列与金鱼PSSI cDNA序列同源性为90％，主要差异在5’端非翻译区。团头鲂生长抑素mRNA阅读框编码114个氨基酸，包括一些酶切位点，产生26个氨基酸的大分子态生长抑素，进一步加工成与人等结构相似的14个氨基酸的生长抑素；团头鲂生长抑素前体氨基酸序列与金鱼、虹鳟、鲶鱼、鲅鱇、蛙、牛、鼠、鸡、猴、人等的比较发现，它与金鱼的同源性最高达95％，与鮟鱇最低50％，与人68％。这说明生长抑素基因在长期的进化中相当保守，同时，也因为鱼类生活环境多样导致基因变异较大。     

奥利亚罗非鱼DMO cDNA的分离和克隆

采用RTPCR和RACE法分离和测定了奥利亚罗非鱼DMOcDNA的全序列。得到1571bp[不含poly(A)]的全长cDNA,包括148bp5’非翻译区,1230bp阅读框以及含Poly(A)信号AATAAA的193bp3’非翻译区[不包括Poly(A)]。阅读框共编码409氨基酸,与尼罗罗非鱼DMO编码的氨基酸序列进行比较,同源性为96.3%,表明DMO在同一物种中差别较小。而与尼罗罗非鱼,红鳍东方豚,虹鳟,青鱼将,鼠,人等动物的DMRT1编码的氨基酸序列进行比较,同源性分别为:25.7%,25.8%,24.3%,29.7%,22.5%,22.0%,这说明DMO和DMRT1可能是两个不同的基因。     

加州鲈肌肉生长抑制素(MSTN)cDNA的克隆和序列分析

肌肉生长抑制素是抑制肌肉生长和发育的生长调控因子。对运用RT-PCR和RACE技术从加州鲈成鱼肌肉总RNA中扩增得到的MSTN cDNA全序列进行了序列分析。结果表明,加州鲈MSTN cDNA全长为1626bp,其开放阅读框为1 134bp,共编码377个氨基酸,前面的22个氨基酸为信号肽,中间有四个氨基酸(RARR)为蛋白水解加工位点;该基因总共有13个半胱氨酸残基,后面9个在蛋白水解加工位点之后的C端生物活性区,与其它脊椎动物比较,它们的位置完全一致,对该基因的结构和功能非常重要。与GenBank中已知的条纹狼鲈、金鲷、斑马鱼、虹鳟、斑点叉尾鮰、人、猪、鸡、鸽MSTN的ORF相比较,核苷酸序列同源性为63%~94.4%,氨基酸同源性为61.4%~96%,特别是在C端生物活性区氨基酸同源性为88.1%~100%,高度的保守性反映了该基因受到了高度的进化限制以及功能的重要性。加州鲈MSTN基因的克隆为研究该基因打靶和鱼类肌肉发育调控机理奠定了基础。     

虹鳟PPARα基因克隆、序列分析及其组织表达分布

克隆了虹鳟(Oncorhynchus mykiss)过氧化物酶体增殖物激活受体(peroxisome proliferator activated receptor,PPAR)αmRNA全序列。对其序列分析发现,虹鳟PPARα与其他脊椎动物PPARα有较高的同源性,其中mRNA序列有66.4%~97.5%相同,氨基酸序列有69.2%~98.9%相同。DNA结合结构域和配体结合结构域从鱼类到人类高度保守,其中DNA结合结构域有88.0%~98.8%的氨基酸序列相同;配体结合结构域有74.6%~99.6%的氨基酸序列相同。系统发生树分析表明,虹鳟的PPARα基因与同属鲑科鱼类的大西洋鲑(Salmo salar)属于同一分支。实时定量RT-PCR方法分析虹鳟不同组织中的表达发现,PPARα基因在脂肪、肌肉、卵巢、肾脏和肠中表达量较高。     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

Rainbow trout Oncorhynchus mykiss (Walbaum) type IV ice‐structuring protein LS‐12 in the acute‐phase response to Flavobacterium psychrophilum infection

A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 10⁶ or 10⁸ colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 10⁸ CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout.     

Isolation, cloning, sequencing of brain type aromatase and its expression in male and female Wrasse, Pseudolabrus sieboldi

Pseudolabrus sieboldi, wrasse being a diurnal spawner provides a good opportunity to study the endocrine mechanism of estrogen formation in brain and gonads. Moreover, an extremely large amount of E2 was produced in serum and testis of wrasse. It is assumed that the presence of E2 may play a major role in diurnal gametogenesis in male fish. In this study brain type aromatase have been isolated, cloned and sequenced from the brain of wrasse. Further, the expression pattern of brain type aromatase in gonads and adult tissue of male and female fish have been analyzed. In addition, the diurnal expression pattern of brain type aromatase in both male and female fish brain during spawning season have been analyzed. The P450arom (br) was isolated, cloned and sequenced from both male and female bamboleaf wrasse. The P450arom (br) gene (1877 sequenced nucleotide) contains an ORF of 1470 bp, a 5′-UTR of 18 bp and at least 407 bp in 3′-UTR. The amino acid sequence homology in the coding region of wrasse P450arom (br) is high compared to that of medaka, Oryzias latipes (80%), rainbow trout type 2, Oncorhynchu mykiss (78.2%), fugu, Takifugu ribripes (78%) rainbow trout type 1, (76%), goldfish, Carassius auratus (66.8%) and zebrafish, Danio rerio (66.2%). Expression study reveals that P450arom (br) mRNA were most abundant in brains of both male and female fish throughout the day during the spawning season. RT-PCR study revealed that P450arom (br) was expressed in skin, anal fin and tail fin of both male and female wrasse. P450arom (br) was not detected at any time of the spawning day in either ovary or testis of wrasse.