草鱼Stefin克隆表达、纯化及活性特征鉴定

文章克隆了草鱼(Ctenopharyngodon idellus)Stefin c DNA全长序列,全长294 bp,编码97个氨基酸,无二硫键,N端存在高度保守的Gly(3、4)残基及QXVXG(45~49)序列,比对结果显示其氨基酸序列与Burton's mouthbrooder(Haplochromis burtoni)Stefin A1一致性最高,为47.5%。进化树分析表明草鱼Stefin A与Burton's mouthbrooder(Haplochromis burtoni)、southern platyfish(Xiphophorus maculatus)、Colisa chuna(Trichogaster chuna)、lamprologini(Neolamprologus brichard)、elephant shark(Callorhinchus milii)及bicolor damselfish(Stegastes partitus)Stefin A聚为一类。将构建的原核表达载体Stefin-Pet30a转入E.coli BL21,以1 mol·L-1IPTG诱导表达重组Stefin蛋白,而后经梯度尿素洗涤和镍亲和层析纯化,并分别利用SDS-PAGE和TSK-GEL G2000SWxl高效液相色谱检测诱导及纯化效果,SDS-PAGE结果显示重组Stefin蛋白得到高度纯化,最终呈现相对分子量11.4 k D的单一条带;其在高效液相上保留时间25.98 min处亦呈单一活性峰,纯度为96.28%。以荧光合成肽底物(Z-Phe-Arg-MCA)测活法鉴定重组草鱼Stefin对鲤鱼组织蛋白酶B、L的抑制活性,发现该重组蛋白对二者均体现了明显的抑制活性。     

草鱼肠道芽孢杆菌的鉴定及产酶能力分析

从100 g左右的健康草鱼(Ctenopharyngodon idellus)肠道中分离出3株疑似芽孢杆菌(Bacillus)菌株(G1、G2、G3),并通过对其进行生理生化特征、16S rDNA全序列和产酶能力的分析,筛选出1株高产酶能力的益生菌株。结果显示:G1、G2和G3菌株与枯草芽孢杆菌在16S rDNA序列相似性≥99%的水平上聚为同一分支,结合形态观察和生理生化特征,最终鉴定G1、G2和G3菌株均为枯草芽孢杆菌(Bacillus subtilis);以水解圈直径/菌落直径比值评定了G1、G2和G3菌株的产纤维素酶、淀粉酶和蛋白酶的能力,其中G1菌株产纤维素酶、淀粉酶和蛋白酶能力(比值分别为2.9、2.2和3.3)均高于G2、G3菌株,具有作为益生菌的潜力。     

草鱼ghrelin基因的分子克隆与组织分布及其摄食调控作用分析

ghrelin是一种在脊椎动物摄食调节过程中起重要作用的脑肠肽,具有明显的摄食促进作用。实验利用同源克隆技术获得了草鱼ghrelin基因的cDNA序列和DNA序列,其中cDNA序列全长506 bp,包括90 bp的5′端非编码区(5′-untranslated region,5′UTR),312 bp的开放阅读框(open reading frame,ORF),以及104 bp的3′端非编码区(3′-untranslated region,3′UTR)。开放阅读框编码的103个氨基酸的ghrelin前体肽,经剪切加工后形成含有19个氨基酸的成熟肽。氨基酸序列分析结果显示,草鱼ghrelin与硬骨鱼类ghrelin相似度最高,而与其他脊椎动物相似度较低,同时草鱼ghrelin成熟肽N端的"活性中心"(active core)为鲤科鱼类中常见的GTSF形式。与大多数硬骨鱼类的ghrelin基因结构相同,草鱼ghrelin基因也包括4个外显子和3个内含子。荧光定量PCR检测到ghrelin mRNA大量分布于草鱼的前肠和脾,脑、肾、肝、肌肉、皮和鳔等组织也有ghrelin mRNA分布。草鱼脑和肠中的ghrelin表达水平在摄食后下降,随着饥饿时间的延长表达水平逐步升高,最后维持在较高水平,表明ghrelin作为摄食启动信号对草鱼的摄食活动起到了促进作用。     

草鱼三、四核苷酸重复微卫星标记的分离与特征分析

采用磁珠富集法、通过质粒检测法结合同位素杂交检测获得草鱼(Ctenopharyngodon idellu)三、四核苷酸阳性克隆1 378个,测序705个,共得到846个微卫星座位,其中完美型632个（占74.7％）,非完美型114个(占13.48％),混合型100个(占11.82％).根据微卫星侧翼序列设计并合成100对微卫星引物,检测结果显示35对(35％)引物在邘江野生群体中表现出多态性.选择其中2对多态性稳定的引物进行遗传多样分析,结果显示,20个位点共检测到212个等位基因,平均观察杂合度(Ho)为0.681 1,平均期望杂合度（He）为0.797 5,平均多态信息含量为0.777 7.结果表明,所筛选的微卫星标记能够用于草鱼群体遗传学研究.本研究旨在为草鱼的种群遗传结构分析、遗传图谱构建等研究奠定基础.     

草鱼天然抗性相关巨噬蛋白基因全长cDNA的克隆与表达分析

天然抗性相关巨噬蛋白(Nramp)家族是一类抑制胞内寄生菌侵染的天然免疫相关蛋白.本研究克隆了草鱼(Ctenopharyngodon idellus)Nramp基因并进行了表达分析.该基因cDNA序列全长为3158 bp,编码1个含544个氨基酸的蛋白.该蛋白含有Nramp家族的特征序列:包含12个跨膜区(TM)、1个由20个氨基酸残基组成的胞质内转运结构域(CTM).草鱼Nramp同其他16个物种的Nramp氨基酸序列同源性在62.5%～90.2%之间.草鱼Nramp基因cDNA的独特结构是其3味端非翻译区(UTR)和5'UTR各有1个脊椎动物Nramp2中的铁反应控制蛋白结合位点(IRE).系统进化分析表明,草鱼Nramp和所有鱼类Nramp聚为一簇,与哺乳类Nramp2的亲缘关系较近.Nramp基因在单鱼头肾和脾脏中的表达量最高,在肌肉和皮肤中的表达量最低,在草鱼呼肠孤病毒(GCRV)感染的草鱼肾脏细胞系(CIK)中的表达量明显升高.     

草鱼(Ctenopharyngodon idellus)成纤维细胞生长因子8的cDNA克隆及特征分析

以草鱼(Ctenopharyngodon idellus)为实验对象,利用RACE技术克隆了草鱼成纤维细胞成长因子8(Fibro-blast Growth Factor8,FGF8)cDNA序列。实验结果显示,草鱼FGF8 cDNA序列长度为887 bp,其中5′-非翻译区长100 bp,3′-非翻译区长157 bp,开放阅读框(ORF)633 bp,编码210个氨基酸的前体蛋白,包含22个氨基酸的信号肽和188个氨基酸的成熟肽。利用已知的FGF8序列绘制系统发育树,发现草鱼与两种鲤科鱼(班马鱼、墨西哥脂鲤)聚为一簇,显示亲缘关系很近,而与其它脊椎动物如两栖类、鸟类、哺乳类的亲缘关系则相对较远,这与形态分类学的结果一致。此外,还通过生物信息学分析,揭示了草鱼FGF8的分子结构特征。     

Identification,characterization and feeding response of peptide YYb (PYYb) gene in grass carp (Ctenopharyngodon idellus)

The peptide YYb (PYYb) is a fish-specific peptide belonging to the neuropeptide Y (NPY) family. In the present study, the full-length cDNA sequence and genomic structure of PYYb (gcPYYb) from Ctenopharyngodon idellus have been isolated and characterized. The gcPYYb gene consists of three exons interspaced by two introns, opposing to the typical architecture of most NPY-family genes as well as its paralogs. Alignment of deduced amino acid sequence indicates that the fish PYYb is more variable compared to NPY and PYY as shown by more residue changes in teleosts lineage, suggesting mild selective pressure imposed on the peptide. Real-time quantitative PCR analysis shows the gcPYYb mRNA in developing larvae is increased during the mixed endo- and exogenous-feeding period and is widely distributed throughout the intestine of fully grown individuals. Following a single meal, the gcPYYb mRNA in foregut is increased at 3 h post-feeding and subsequently decreased before the foregut contents are cleared. These results suggest that the gcPYYb has an important role in the early life stages of grass carp and is involved in food intake by transmitting feeding-related signals.     

Bacterial communities in Chinese grass carp (Ctenopharyngodon idellus) farming ponds

Bacterial communities in the water column and sediment of 12 commercial grass carp (Ctenopharyngodon idellus) farming ponds were analysed. At the same time, physical and chemical environmental parameters were measured, including secchi depth (SD), total nitrogen (TN), total phosphorus (TP) and chemical oxygen demand (COD_Mn). From 12 water samples and 12 sediment samples, 39 different ribotypes were detected with PCR‐denaturing gradient gel electrophoresis (PCR‐DGGE). The ribotype richness showed that bacterial species in the water column differed among these ponds, and the result of sequencing further revealed the dominant and common bacterial species. In total, 32 bacterial species belonging to seven phyla (Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Acidobacteria, Fibrobacteres and Fusobacteria) were identified. Ordination via canonical correspondence analysis (CCA) on the DGGE data and environmental parameters indicated that the composition of bacterial community was significantly influenced by SD.     

氨对草鱼生长的危害

氨对草鱼生长的危害朱耘，吴圣杰，华丹（中国水产科学研究院淡水渔业研究中心，无锡２１４０８１）关键词氨，草鱼，生长ＴＨＥＨＡＲＭＦＵＬＥＦＦＥＣＴＯＦＴＨＥＡＭＭＯＮＩＡＯＮＴＨＥＧＲＯＷＴＨＯＦＧＲＡＳＳＣＡＲＰ（ＣＴＥＮＯＰＨＡＲＹＮＧＯＤＯＮＩＤ...     

草鱼鱼种对胆碱需要量的研究

用不含胆碱的饲料饲养草鱼鱼种，草鱼鱼种生长减慢，饲料效率降低，肝脏脂肪浸润，血浆总脂，甘油三脂，胆固醇和磷脂含量降低，随着饲料中胆碱含量的添加，草鱼生长和饲料效率改善，肝脏脂肪含量降低，血浆总脂、甘油三脂、胆固醇，磷脂含量回升。当饲料中胆碱添加量达到０．３％以上时，草鱼生长率最高，肝脏脂肪含量，血浆总脂、甘油三脂，胆固醇，磷脂与摄食天然饲料（浮萍）的草鱼基本相同。在本研究条件下，建议草鱼鱼种饲料中     

草鱼ATGL基因的表达及饲喂n-3 HUFAs对其的影响

实验获得了草鱼脂肪组织甘油三酯水解酶(ATGL) 部分cDNA序列(GenBank 登录号为HQ845211), 并进行了序列同源性分析; 采用实时定量反转录聚合酶链式反应(qRT-PCR)方法, 检测了ATGL基因在草鱼不同组织的表达状况; 研究了投喂n-3高不饱和脂肪酸(n-3 HUFAs)对草鱼肝胰脏ATGL基因时序表达的影响。结果显示, 所获得的草鱼ATGL基因部分cDNA序列长度为687 bp, 与人、牛、小鼠、长腭泥虎鱼、大黄鱼等物种的同源性为65%~75%; 该基因在草鱼心脏、肝胰脏、脾脏、鳃、肾脏、肌肉、腹腔脂肪组织、脑、小肠、精巢10个组织中均有表达, 其中在腹腔脂肪组织中表达丰度最高, 在肝胰脏和肌肉中表达丰度次之。处理组草鱼摄食n-3 HUFAs饲料后, ATGL基因的表达水平在第1周和第2周显著高于对照组, 第3周后, 该基因的表达水平在处理组与对照组间无显著差异。研究首次克隆得到草鱼ATGL基因部分cDNA序列, 并发现该基因在草鱼脂质蓄积及代谢较旺盛的组织中表达水平较高, 且其在肝胰脏中的表达受到n-3 HUFAs的影响, 其规律为先被诱导升高, 然后回复到正常水平。     

草鱼肾细胞中双氟沙星代谢酶的酶动学

细胞色素P450s(CYPs)主要参与动物体内药物代谢。水产养殖中不合理的联合用药常会导致治疗失败,这通常与CYP活性的诱导有关。然而,关于鱼类CYP的诱导却知之甚少。为获得有关CYP诱导的信息,实验采用反相高效液相色谱(RP-HPLC)的方法测定了草鱼肾细胞(CIK)中CYPs的特异性诱导剂对双氟沙星(DIF)的代谢作用及酶动学分析。对照组和β-萘黄酮(BNF)诱导组的酶动学方程分别为1/V=0.1375×1/[S]+0.003和1/V=0.0245×1/[S]+0.0013。DIF与经BNF处理的CIK共孵育后,其代谢量增加了1倍,酶动学参数Clint和Vmax值分别增加了7倍和2倍。BNF是CYP1A的特异性诱导剂,因此,CYP1A可能参与了DIF的代谢。     

A review of intestinal microbes in grass carp Ctenopharyngodon idellus (Valenciennes)

Grass carp, Ctenopharyngodon idellus, harbours complex intestinal bacterial communities, which are important in several physiological processes of their host. Intestinal microbiota of grass carp have been previously described in numerous studies. However, an overview on the bacterial community diversity, including their establishment, their functions in host's nutritional processes and immune‐related responses, and use as probiotics, is absent. This study aimed to summarize the current understanding of the grass carp intestinal microbiota. In this review, we provide general information on the establishment and composition of intestinal microbial communities and factors influencing the diversity of gut microbiota. Also, this review covers the dietary effects of probiotics, prebiotics and/or synbiotics on the grass carp intestinal microbial communities and physiological characteristics. Although our knowledge of the grass carp intestinal microbiota is expanding rapidly, further studies on the factors affecting the diversity of intestinal microbes, interactions between intestinal microbiota and their hosts and application of probiotics/prebiotics/synbiotics in aquaculture industry, are needed.     

Quantifying the dietary potassium requirement of subadult grass carp (Ctenopharyngodon idellus)

A growth trial lasting for 12 weeks was conducted in 21 net cages to determine the dietary potassium (K) requirement of subadult grass carp (Ctenopharyngodon idellus) (Average weight: 331.3 g). Seven isonitrogenous and isoenergetic semi‐purified diets were compounded with different dietary K level. The specific growth rate (SGR) of fish was significantly (P < 0.05) improved by dietary K supplementation, SGR and the gill Na⁺‐K⁺ ATPase activity increased first and then decreased (P < 0.05) as dietary K level increased. The highest SGR and gill Na⁺‐K⁺ ATPase activity values were both observed at 6.38 g kg^?1 group. Dietary K level showed significant (P < 0.05) effect on serum superoxide dismutase (SOD) and glucose (GLU), the maximum values of SOD and GLU were in 8.42 and 6.38 g kg^?1 group, respectively. The body lipid content of the 6.38 g kg^?1 group was significantly (P < 0.05) lower than that of the control. However, the ash content in the 8.42 g kg^?1 group was significantly higher than those in the 1.21, 2.21, 4.41 and 6.38 g kg^?1 group. When dietary protein was 320 g kg^?1 and the waterborne potassium ranged from 6.86 to 9.10 mg L^?1, the dietary K requirement for subadult grass carp judged from SGR and gill Na⁺‐K⁺ ATPase activity is 5.38 and 7.41 g kg^?1 diet, respectively.     

草鱼脆化过程中肌肉品质变化

为了研究草鱼(Ctenopharyngodon idellus)脆化过程中肌肉品质变化对脆肉鲩(C.idellusC.et V)脆性形成规律的影响,在草鱼脆化过程中定期采样,比较研究了肌肉基本化学成分、质构特性、鱼肉蛋白质组成、肌肉氨基酸组成及营养价值评价等的变化规律。结果显示：脆肉鲩的水分、粗脂肪质量分数均显著低于普通草鱼(P0.05),粗蛋白、粗灰分质量分数比普通草鱼高;与普通草鱼相比,脆化后肌肉的肌原纤维蛋白、肌浆蛋白和基质蛋白质量分数分别增加了10.88%、15.41%和80.53%;脆肉鲩的硬度、咀嚼性、弹性和粘着性显著高于普通草鱼(P0.05);脆肉鲩肌肉的TAA、EAA、UAA最高分别为203.5gL-1、85.6 gL-1、79.1 gL-1,均显著高于普通草鱼(P0.05)。研究表明在草鱼脆化过程中,肌肉品质指标均有不同程度的变化,且这些变化是导致脆肉鲩脆性改变的重要因素。     

地衣芽孢杆菌De在优质草鱼养殖中的应用研究

运用综合对比分析法探讨了地衣芽孢杆菌Bacillus licheniformis De在优质草鱼（Ctenopharyngodon idellus）养殖中的应用效果,其评价指标分别为成活率、水体pH、透明度、溶解氧及水中氨氮、硝酸盐浓度等。结果表明,施用地衣芽孢杆菌De可在一定程度上使水体环境和养殖生产性能得到优化,提高养殖草鱼的成活率,显著降低水体透明度及水中氨氮、硝酸盐含量（P〈0．05）,使水体pH、溶解氧有利于草鱼的生长。其中施菌组较对照组的成活率、水体pH、溶解氧分别提高了3．2％、3．9％、25．5％,而水体透明度、氨氮及亚硝氮浓度则分别降低了38．5％、74．6％、69．3％。     

Analysis of bacterial diversity in the intestine of grass carp (Ctenopharyngodon idellus) based on 16S rDNA gene sequences

In the current study, we assessed bacterial diversity in the gut content of pond-reared grass carp (Ctenopharyngodon idellus), in the associated habitat environments (pond water and sediment) and in the ingested food (commercial feed and the reed Phragmites australis) by analysing 16S rDNA sequences from clone libraries. The highest bacterial diversity was observed in the gut content and was determined by the total number of operational taxonomic units, Shannon diversity index (H), Shannon equitability index (E_H), Coverage (C_good) and rarefaction curves calculated from the 16S rDNA gene libraries. Our data indicated that allochthonous gut microbes of grass carp were distinctively different from the corresponding environmental microbes. The pairwise similarity coefficient (C_s) for microbe communities between gut content and ingested food was higher than for those between the gut content and habitats, indicating that the allochthonous microbiota identified in the intestines of grass carp were phylogenetically closer to those in the ingested food than to those in the habitat. Based on our study and previous research, we suggest that the digesta of grass carp harbours a microbiota phylogenetic core of Proteobacteria and Firmicutes and this observation deserves further investigations with respect to a potential pool of probiotics to grass carp.     

Effects of Bacillus preparations on immunity and antioxidant activities in grass carp (Ctenopharyngodon idellus)

This experiment was conducted to study the effects of Bacillus preparations on immunity and antioxidant activities in grass carp. A total of 315 grass carp, with similar initial weight (average weight of fish 45?g), were randomly divided into three groups with three replicates. The control group was fed the basal diet without Bacillus; treatment group 1 was added Bacillus preparation no. 1 with 1?×?10⁸?cfu/m³ per 7?days in culture water and also fed the basal diet; treatment group 2 was fed the basal diet mixed with 0.5?% Bacillus preparation no. 2, and the culture water was added 1?×?10⁸?cfu/m³ Bacillus preparation no. 1 per 7?days. After 4?weeks of culture, 12 grass carp from each replicate were taken randomly for the determination of immune response and oxidization resistance indices. The results showed that compared with control, the level of globulin and IgM of treatment group 2 was significantly increased (P?<?0.05), which was also significantly higher (P?<?0.05) than that of group 1. For the non-specific immunity index, compared with control, the lysozyme activity and complement (C3) content of treatment group 1 significantly increased (P?<?0.05), and the level of myeloperoxidase and C3 of treatment group 2 was significantly higher (P?<?0.05) than that of control, which was also significantly higher (P?<?0.05) than that of group 1. In the serum, compared with the control, the level of total antioxidant activity (T-AOC), antisuperoxide anion free radical (ASAFR) and glutathione (GSH) of two treatment groups was significantly increased (P?<?0.05); GSH content of treatment group 2 was significantly (P?<?0.05) higher than that of treatment group 1 and control. There was no significant difference between T-AOC and ASAFR among the two treatment groups; no significant effect was found on glutathione peroxidase (GSH-Px) and maleic dialdehyde (MDA) among the three groups. In the liver, T-AOC, SOD, ASAFR, GSH-Px and GSH from two treatment groups were significantly higher (P?<?0.05) than those of control; T-AOC, SOD and catalase from treatment group 2 were significantly higher (P?<?0.05) than those of treatment group 1; and MDA of two treatment groups was significantly decreased (P?<?0.05) as compared with the control. The results indicate that Bacillus preparations added into water can increase serum immunoglobulin levels and most of non-specific immune parameters content and enhance the antioxidant ability of grass carp, while adding Bacillus preparation into the water and feed is much better.