尼罗罗非鱼肌肉蛋白质双向电泳体系的建立

为建立1种尼罗罗非鱼肌肉组织蛋白质的双向电泳体系,实验将尼罗罗非鱼肌肉组织蛋白质提取后,用双向电泳(2-DE)分离蛋白质,分别对蛋白质样品的制备方法、IPG胶条长度及p H范围、SDS-PAGE凝胶浓度及染色方法4个关键因素进行了探索和优化。结果显示,采用液氮研磨-丙酮沉淀法制备样品蛋白质,使用24 cm p H 4~7的IPG胶条进行第一向等电聚焦电泳,第二向SDS-PAGE电泳采用浓度为12.5%的凝胶进行,双向电泳后的凝胶采用硝酸银染色法进行染色处理,该条件下扫描所得尼罗罗非鱼肌肉蛋白质二维电泳图谱具有蛋白质分离程度好、斑点清晰、分辨率高及横纹少等优点。研究表明,技术体系适用于尼罗罗非鱼肌肉蛋白质的分离,可用于后续罗非鱼肌肉蛋白质组学研究。     

鳜幼鱼配合饲料最适蛋白质含量初步研究

采用精制饲料蛋白质梯度法，以鱼粉蛋白和酪蛋白为蛋白源，测定鳜幼鱼配合饲料最适蛋白质含量。试验期平均水温２１℃；试验鱼为平均尾重１５９±２２ｇ幼鳜；试验饲料蛋白质含量梯度为０％—４９９％；养殖３０天后采用直线回归和抛物线回归计算初步确定，鳜幼鱼配合饲料蛋白质最适含量为４４７％—４５８％。对饲料蛋白质含量与饲料蛋白质利用率、饲料系数和投饲率的相关关系进行了讨论     

摄食不同饵料对翘嘴鳜生长、体成分和消化酶活性的影响

为探究三种不同饵料对翘嘴鳜(Siniperca chuatsi)生长性能、体成分和消化酶的影响,实验选取初始体重为(113.56±11.82)g的翘嘴鳜900尾,随机分成3组,分别投喂活饵料鱼、冰鲜饵料鱼和商品饲料,进行60 d的养殖试验。结果显示:(1)冰鲜组、活饵组和饲料组翘嘴鳜的成活率、增重率、特定生长率和蛋白效率均无显著性差异。饲料组翘嘴鳜肥满度显著小于其他两组,肝体比和脏体比均显著大于其他两组。(2)三组翘嘴鳜体成分中的水分、蛋白质、粗脂肪和粗灰分均没有显著性差异。(3)翘嘴鳜肌肉各种氨基酸含量和总氨基酸量在三组之间的差异均不显著。饲料组翘嘴鳜肌肉中不饱和脂肪酸中的棕榈油酸、油酸、亚油酸、花生烯酸、EPA、神经酸、DHA的含量及总饱和脂肪酸、总单不饱和脂肪酸、总多不饱和脂肪酸均显著高于其他两组。(4)肝脏脂肪酶、肠道淀粉酶的活性在三组翘嘴鳜之间均没有显著性差异。冰鲜组翘嘴鳜肝脏淀粉酶、肠道脂肪酶活性均显著高于其他两组。结果表明:翘嘴鳜经过驯化可以摄食配合饲料,且不影响其生长性能。翘嘴鳜摄食饲料后,其肌肉氨基酸没有发生显著性的变化,不饱和脂肪酸的含量显著升高。     

中国明对虾肌肉组织蛋白质双向电泳技术体系的建立

为了探索并建立中国明对虾的肌肉蛋白双向电泳体系,实验将中国明对虾的肌肉组织溶解处理后,通过固相pH梯度胶条等电聚焦、SDS-PAGE垂直电泳对蛋白质进行分离,对不同的裂解液配方、IPG胶条的pH范围及其长度、等电聚焦程序、上样量等进行了优化,并分别利用银染和考染方法进行染色,应用PDQuest软件对图谱进行了初步分析.结果显示,中国明对虾肌肉蛋白的等电点主要位于4～7之间,裂解液中添加硫脲、CHAPS、DTT等,可以增加对虾肌肉蛋白的提取率;采用17 cm,pH 4～7的中型IPG胶条,主动水化上样120μg,适当延长除盐时间和等电聚焦时间,搭建盐桥,采用浓度为12.5％的二向分离胶和硝酸银染色,能有效提高双向电泳(2-DE)图谱中蛋白点的分离度和分辨率;建立的中国明对虾肌肉蛋白双向电泳体系具有良好的重复性和稳定性.     

鲢冻结过程中肌肉组织及蛋白质的变化

以鲜活鲢为研究对象，从肌肉组织冷冻切片，盐溶蛋白，Ca2 ,-ATP酶活性等角度研究在缓慢冻结条件下链的冷冻变性情况，并探讨链冷冻变性的要理，结果表明：冻结前后，链肌肉纤维组织 结构变化十分显著：链冻结至－35度后盐溶蛋白下降了14．8％；Ca2 -ATP酶活性变化则不明显。     

坛紫菜叶状体蛋白质双向电泳样品制备方法的比较

蛋白质样品的制备是双向电泳分析的关键。为探索适用于坛紫菜叶状体蛋白质双向电泳样品的制备方法,实验对3种常用植物蛋白质分离方法进行了优化和改进,并分别同3种蛋白质裂解液联用,比较了9种方法的总蛋白质得率及双向电泳图谱效果。结果发现,采用三氯乙酸/丙酮沉淀法与裂解液C(7 mol/L 尿素,2 mol/L 硫脲,2%CHAPS,2%TritonX-100,2% IPG Buffer pH 3~10,65 mmol/L DTT)联用的方法,坛紫菜叶状体的蛋白质得率最高,且双向电泳图谱显示此法获得的蛋白质点数最多,蛋白点形状规则,水平和垂直拖尾情况较轻,图像清晰。实验结果还发现,在进行双向电泳分析时,采用pH 4~7的胶条可以使坛紫菜叶状体蛋白质得到更为有效的分离。     

杂交鳜与鳜鱼、斑鳜肌肉营养成分和氨基酸含量比较

通过测定鳜鱼、斑鳜和杂交鳜的肌肉营养成分和18种氨基酸含量，结果表明：杂交鳜、斑鳜和鳜鱼的肌肉水分、脂肪、灰分的含量无显著差异（P〉0．05），杂交鳜与斑鳜的肌肉蛋白质含量无显著差异（P〉0．05），但均显著高于鳜鱼肌肉的蛋白质含量（P〈0．05）；鳜鱼、斑鳜和杂交鳜的18种氨基酸含量及其组成、18种氨基酸总量（TAA）、人体所需8种必须氨基酸总量（HEAA）及4种呈味氨基酸（天门冬氨孽、谷氨酸、甘氨酸和丙氨酸）的总量（FAA）均无显著差异（（P〉0．05）。     

基于水温的鳜与鲢配套催产模式研究

为解决汉江上游流域鳜饵料鱼的配套问题,建立鳜与鲢配套催产模式,分析了水温对鳜和鲢人工繁殖中效应时间、孵化时间、开口时间等的影响,了解鳜和鲢人工繁殖各时间段与水温的相关性。结果表明:鳜孵化时间和开口时间与水温(18~28℃)显著相关,受精卵孵化时间(y)与水温(x)的关系式为y=41 058x~(-2.142 4),R~2=0.995 2,仔鱼开口时间(y)与水温(x)的关系式为y=-0.099 6x~2-5.226 0x+266.02,R2=0.961 4;鲢的效应和孵化总时间(y)与水温(16℃x32℃)的关系式为y=0.051 4x~2-4.924 4x+129.91,R~2=0.978 2;鳜、鲢配套催产时间(y)与水温(x)的关系式为y=-0.151 0x~2-0.301 6x+136.11。研究表明,在水温18~28℃条件下,可根据水温计算出鲢的催产时间,为在鳜人工繁育生产中进行开口饵料鱼的精准配套提供参考。     

碧流河水库斑鳜的食性及其渔业利用

对碧流河水库斑鳜胃含物分析结果表明：（１）斑鳜以小型野杂鱼类和虾类为主要食物，其中虎鱼、餐条和虾类的出现率分别为２４．６４％、１２．２３％和２２．０８％。鲢、鳙在斑鳜的食谱中也占有一定比例，出现率为５．６６％、２．７４％。（２）体长２１．５ｃｍ以上的斑鳜对鲢、鳙鱼种造成危害，可吞食１２．６ｃｍ以上的鱼种。（３）斑鳜的食物组成存在明显的季节变化，其中掠食鲢、鳙鱼种主要有两个时期，即鱼种放库后的１０－１１月和斑鳜生殖前的４月－６月上旬。对其在该水库渔业中的地位及渔业利用进行了讨论，并对放养鲢、鳙鱼种的规格、时间和方式作了探讨     

尼罗罗非鱼雌雄鱼肌肉组织差异表达基因的筛选

应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2-CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。     

乌鳢抽出物成分的研究

分析了乌鳢(Channaargus)即杀后背肉、腹肉、尾部肉、肝脏、生殖腺中ATP关联化合物、游离氨基酸、多胺、糖元及糖酵解中间代谢物、有机酸等的含量。ATP关联化合物在肌肉中的总量为7.5～8.0μmol·g-1。ATP在背肉含量为3.9μmol·g-1,腹肉为4.1μmol·g-1,尾部肉为4.7μmol·g-1。运动激烈的尾部肉ATP占63%,含量相当高。肝脏中有少量的腺苷与肌苷酸一起被检出,据此可认为ATP的分解存在两个途径。游离氨基酸总量在背肉中为436.0mg·(100g)-1,腹肉中为405.0mg·(100g)-1,尾部肉中为356.3mg·(100g)-1。牛磺酸和甘氨酸为主要氨基酸,占68%～73%。丙氨酸和谷氨酸也相当高的含量检出。肌肉中的多胺检出为精胺和亚精胺,肝脏和生殖腺中有较高浓度的腐胺及亚精胺和精胺检出。即杀后糖元的量约占肌肉的0.5%,还有相当多的葡萄糖和6磷酸葡萄糖的糖酵解中间代谢物以及其最终产物乳酸的大量检出。     

黄条魳(Seriola aureovittata)肌肉营养组成分析与评价

为进一步认识和评价黄条(Seriola aureovittata)的营养与食用价值,本研究测定了其肌肉中的水分、蛋白质、粗脂肪、灰分、氨基酸、脂肪酸和矿物质等成分,并对其养殖及野生个体的肌肉营养成分组成进行了比较分析.结果显示,黄条肌肉中蛋白质含量较高,必需氨基酸和鲜味氨基酸含量丰富,完全符合FAO/WHO推荐的理想蛋白质标准,是一种优质蛋白供给源.根据AAS和CS分值,黄条肌肉的第一限制氨基酸为蛋氨酸,第二限制氨基酸为缬氨酸.黄条肌肉的脂肪含量高于三文鱼(Salmo salar)、金枪鱼(Thunnus thynnus)、石斑鱼(Epinephelus sp.)等,且肌肉中含有丰富的不饱和脂肪酸,不饱和脂肪酸与饱和脂肪酸比值较高,特别是EPA+DHA的含量高,具有较优的口感鲜度和较高的营养价值.另外,黄条肌肉中含有多种机体新陈代谢所需的矿物质,经常食用可促进人体新陈代谢水平.比较发现,养殖黄条较野生黄条具有相对较高的脂肪含量和较低的水分含量,比能值(EP)分别为13.44 kJ/g和8.68 kJ/g,其他营养成分无显著差异,表明养殖与野生黄条肌肉营养价值相似.综上所述,黄条肌肉蛋白质和脂肪质量较高,口感鲜美,营养成分丰富,是一种值得大力开发养殖的海产经济鱼类.     

Protein digestibility and amino acid availability of several protein sources for juvenile Chinese hairy crab Eriocheir sinensis H. Milne‐Edwards (Decapoda,Grapsidae)

Apparent and true values of protein digestibility (APD vs. TPD) and amino acid availability (AAAA vs. TAAA) of casein, gelatin, fish meal, shrimp meal, soybean meal and spirulina meal were determined for juvenile Chinese hairy crab Eriocheir sinensis H. Milne‐Edwards. Assay diets were prepared by incorporation of 20% of a protein source into a reference diet. A protein‐free diet (PFD) was used to estimate the metabolic faecal nitrogen (MFN) content and amino acid composition (MFAA). MFN content was 445.3 mg 100 g^?1 PFD. MFAA ranged from 19.6 mg 100 g^?1 PFD for tryptophan to 228.5 mg 100 g^?1 PFD for glutamic acid. Casein and gelatin trended towards higher APD, while fish meal had the lower APD and TPD. Although there was reasonable agreement between protein digestibility and average amino acid availability, protein sources had significant effects on AAAA and TAAA. For total amino acids and most individual amino acids, casein and gelatin were relatively higher both in AAAA and in TAAA, soybean meal and spirulina meal intermediate, while fish meal was lower. Data of AAAA and TAAA suggest that soybean meal and spirulina meal could be used as substitutes for animal proteins. Individual AAAAs and TAAAs were variable within and among protein sources. Those results suggest that determination of amino acid availabilities is necessary for the more accurate and economical feed formulation.     

Biochemical differences in lateral muscle of wild and farmed gilthead sea bream (series Sparus aurata L.)

Red and white muscle from specimens of wild and farmed gilthead sea bream (Sparus aurata) were analyzed for histochemical ATPase activity, total protein content, fatty acids, trace element concentrations and myosin isoforms. The fibre type composition of muscle samples was confirmed histochemically by the ATPase reaction, which did not show any differences between the two groups of animals. Myosin ATPase activities, myosin and protein yields were significantly higher in white muscle than in the red muscle and for the red muscle the latter two parameters were higher in wild fish. Fatty acid profiles revealed differences between the two groups of animals, probably because of the fatty acid composition of the diets. Zinc, copper and iron concentrations were higher in red muscle than in white muscle; muscles from wild fish were significantly richer in trace elements. No separation of fast and slow heavy chains of myosin could be obtained on SDS-gel electrophoresis, but two dimensional electrophoresis revealed the presence of three light chains in white muscle (LC1F, LC2F, LC3F), and two main types in red muscle (LC1S, LC2S). Small, variable percentages of LC3F were found in the red muscle samples, especially in the wild fish. It is concluded that the different environmental conditions, experienced by wild and farmed fish, have significantly influenced the biochemical composition of their lateral muscle.     

Fatty acid composition of the ingested food only slightly affects physicochemical properties of liver total phospholipids and plasma membranes in cold-adapted freshwater fish

The fatty acid content, fatty acid molecular species composition, and fluidity of phospholipid vesicles and plasma membranes of livers of fish feeding on phytoplankton (silver carp, Hypophthalmichtys molitrix), zooplankton, (big head carp, Hypophtalmitrix nobilis), higher plants, (grass carp, Ctenopharyngodon idella), and omnivorous fish, (common carp, Cyprinus carpio), adapted to reduced temperatures, was determined. Levels of long chain polyunsaturated fatty acids (PUFAs) were highest in silver and big head carp and lowest in common carp. Differences in long chain PUFAs were poorly reflected in fluidity, determined either by fluorescence anizotropy of 1,6-diphenyl 1,3,5-hexatriene or rotational correlation time of 16-doxyl- stearic acid, of phospholipid vesicles. Phospholipid vesicles of C. caripo and C. idella proved to be slightly more rigid than of H. molitrix and H. nobilis below ca. 15 °C. These differences were not seen with isolated plasma membranes assessed with 1,6-diphenyl 1,3,5-hexatriene. Using electron spin resonance spectroscopy plasma membranes from H. molitrix proved to be more fluid than those from C. carpio below ca. 15 °C. Phosphatidylethanolamines were abundant in 1-monounsaturated, 2-polyunsaturated (18:1/22:6, 18:1/20:4) species with H. molitrix and H. nobilis more rich in these species than C. carpio and C. idella. It is suggested that differences in fluidities of phospholipid vesicles below 15 °C is due to differences in levels of the above molecular species. In native membranes these differences may be masked by proteins and other membrane components. It is also suggested that it is not the fatty acid composition of the ingested food but the specific response of the fish to temperature that is the most important factor controlling the physicochemical properties of membranes during thermal adaptation.     

Estimating the ideal dietary indispensable amino acid pattern for growth of white sturgeon, Acipenser transmontanus (Richardson)

Juvenile white sturgeon (Acipenser transmontanus, Richardson) were fed or starved for 4 weeks, and their whole body amino acid composition determined at the beginning and the end of the study. The amino acid compositions of muscle, liver, gill and gastrointestinal tract, and the free amino acids in plasma of fed and starved fish were also determined. Very little variation was observed in the amino acid compositions of all the tissues examined after starvation. However, the plasma free amino acids were significantly (P < 0.05) decreased following starvation. On the basis of the amino acid composition and nitrogen retention values of the whole fish, daily indispensable amino acid requirements for protein accretion and obligatory nitrogen losses (mg amino acid 100 g body weighr^?1 day^?1) were estimated. The pattern of indispensable amino acids required for each of these is different, with the pattern for protein accretion containing much higher levels of lysine, but lower levels of tryptophan and sulfur amino acids. Individual amino acid retention rates varied from 33.3% of dietary tryptophan to 80.2% for arginine. The estimated dietary indispensable amino acid pattern for young growing white sturgeon (expressed as a percentage of total indispensable amino acids plus cystine and tyrosine) is: arginine, 14.0; histidine, 6.6; isoleucine, 8.8; leucine, 12.5; lysine, 15.8; methionine (plus cystine), 6.6; phenylalanine (plus tyrosine), 15.5; threonine, 9.7; tryptophan, 0.8; and valine, 9.7.     

罗非鱼无乳链球菌LrrG-Sip融合蛋白免疫原性研究

为探究无乳链球菌LrrG(Leucine-rich repeat protein from GBS)和表面免疫原性蛋白Sip(surface immunogenic protein)串联表达的LrrG-Sip重组融合蛋白的免疫原性,该研究将原核表达的LrrG-Sip重组融合蛋白分别以0.5μg·g~(-1)(R1组)、1.0μg·g~(-1)(R2组)和1.5μg·g-1(R3组)每尾200μL腹腔注射免疫尼罗罗非鱼(Oreochromis niloticus),同时以Sip蛋白(S组)、LrrG蛋白(L组)以及PBS(P组)作为对照。2周后对所有免疫鱼体进行无乳链球菌(Streptococcus agalactiae)人工攻毒,攻毒剂量为其半致死浓度(LD_(50):4.0×108CFU·mL~(-1))。结果显示LrrG-Sip重组融合蛋白R1组对尼罗罗非鱼的相对免疫保护率最高,达89.14%;且免疫后第14和第28天,该组鱼体血清抗体OD_(450nm)值分别达0.63和0.64,均显著高于单一蛋白对照组(S和L组)和PBS组(P0.05);R1组鱼体血清过氧化物酶(POD)和碱性磷酸酶(AKP)活性在上述2个时间点也显著高于其他组(P0.05);但溶菌酶(LZM)和总超氧化物歧化酶(T-SOD)活性与其他组之间差异不显著(P0.05)。初步表明LrrG-Sip重组融合蛋白具有良好的免疫原性,其免疫原性明显优于单个蛋白,且能有效减少免疫剂量。     

Estimating Amino Acid Requirement of Brazilian Freshwater Fish from Muscle Amino Acid Profile

Information on nutritional requirement of some Brazilian farmed fish species, especially essential amino acids (EAA) requirements, is scarce. The estimation of amino acids requirements based on amino acid composition of fish is a fast and reliable alternative. Matrinxa, Brycon amazonicus, and curimbata, Prochilodus lineatus, are two important Brazilian fish with potential for aquaculture. The objective of the present study was to estimate amino acid requirements of these species and analyze similarities among amino acid composition of different fish species by cluster analysis. To estimate amino acid requirement, the following formula was used: amino acid requirement = [(amount of an individual amino acid in fish muscle tissue) × (average totalEAA requirement among channel catfish, Ictalurus punctatus, Nile tilapia, Oreochromis niloticus, and common carp, Cyprinus carpio)]/(average fish muscle totalEAA). Most values found lie within the range of requirements determined for other omnivorous fish species, in exception of leucine requirement estimated for both species, and arginine requirement estimated for matrinxa alone. Rather than writing off the need for regular dose–response assays under the ideal protein concept to determine EAA requirements of curimbata and matrinxa, results set solid base for the study of tropical species dietary amino acids requirements.     

Amino acids composition difference and nutritive evaluation of the muscle of five species of marine fish, Pseudosciaena crocea (large yellow croaker), Lateolabrax japonicus (common sea perch), Pagrosomus major (red seabream), Seriola dumerili (Dumeril's amberjack) and Hapalogenys nitens (black grunt) from Xiamen Bay of China

A study was conducted to investigate the muscle amino acid profiles of five species of marine fish, Pseudosciaena crocea (large yellow), Lateolabrax japonicus (common sea perch), Pagrosomus major (red seabream), Seriola dumerili (Dumeril's amberjack) and Hapalogenys nitens (black grunt). These carnivorous fishes, all of which belong to the Perciforms, were sampled from Xiamen Bay. Chemical analysis shows significant difference (P < 0.01) among the five species in the muscle total amino acids (TAA). Lateolabrax japonicus has the highest level of TAA in muscle tissues. Significant difference in muscle total essential amino acid (TEAA) and total non‐essential amino acid (TNEAA) were also found among the five species. Lateolabrax japonicus has the highest TEAA level, and H. nitens has the highest level of TNEAA level. Significant difference (P < 0.01) existed in specific EAA except for lysine (P > 0.05). The significant difference (P < 0.05) of the A/E ratios [(each essential amino acid content/total essential amino acid content including cystine and tyrosine) ×1000] based on the essential amino acid composition of muscle tissue from the five species were found in lysine, histidine, methionine, cystine, valine, leucine, isoleucine, and arginine, except for threonine, phenylalanine and tyrosine. These differences indicate that the amino acids profile is species specific for the five species and their essential amino acid requirements are greatly different although they were fed similar feed. In contrast to the reference amino acid profile recommended by FAO/WHO, muscle proteins of each fish were all rich in lysine, the S‐containing amino acids and threonine, but histidine, valine, leucine, isoleucine, phenylalanine and tyrosine were deficient for children's diets. The first limiting amino acid was histidine for P. crocea and L. japonicus, valine for P. major and H. nitens, leucine for S. dumerili. The amino acid score was 66.8, 76.7, 78.4, 84.0 and 95.7 for P. crocea, L. japonicus, P. major, H. nitens and S. dumerili, respectively. In the adult human diet the muscle protein of the five species of marine fish can almost fulfil the requirements of all essential amino acids except for the histidine of P. crocea and L. japonicus and thus can serve as supplemental source of protein in cereal‐based adults diets if we reinforce the histidine correspondingly. Therefore, increasing the proportion of marine fish in the diet of the people in the area where paddy rice and wheat are the main protein sources is an effective way to enhance the nutrition value of food and improve the nutrition status of the developing countries. Moreover, all of five marine fish species contained a comparatively high content of glutamic acid and arginine, which are beneficial to the patients under such conditions as trauma, burn injury, massive small‐bowel resection and renal failure.