Identification of keratins and analysis of their expression in carp and goldfish: comparison with the zebrafish and trout keratin catalog

With more than 50 genes in human, keratins make up a large gene family, but the evolutionary pressure leading to their diversity remains largely unclear. Nevertheless, this diversity offers a means to examine the evolutionary relationships among organisms that express keratins. Here, we report the analysis of keratins expressed in two cyprinid fishes, goldfish and carp, by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot binding assay, and immunoblotting. We further explore the expression of keratins by immunofluorescence microscopy. Comparison is made with the keratin expression and catalogs of zebrafish and rainbow trout. The keratins among these fishes exhibit a similar range of molecular weights and isoelectric points, with a similar overall pattern on two-dimensional gels. In addition, immunofluorescence microscopy studies of goldfish and carp tissues have revealed the expression of keratins in both epithelial and mesenchymally derived tissues, as reported previously for zebrafish and trout. We conclude that keratin expression is qualitatively similar among these fishes, with goldfish and carp patterns being more similar to each other than to zebrafish, and the cyprinid fishes being more similar to each other than to the salmonid trout. Because of the detected similarity of keratin expression among the cyprinid fishes, we propose that, for certain experiments, they are interchangeable. Although the zebrafish distinguishes itself as being a developmental and genetic/genomic model organism, we have found that the goldfish, in particular, is a more suitable model for both biochemical and histological studies of the cytoskeleton, especially since goldfish cytoskeletal preparations seem to be more resistant to degradation than those from carp or zebrafish. This work was supported by grants to J.M. from the Stiftung Rheinland Pfalz für Innovation (836-386261/138) and the Deutsche Forschungsgemeinschaft (Ma 843/5-1) and a grant to D.G. from the National Science Foundation (INT-0078261).     

Positive selection on multiple antique allelic lineages of transferrin in the polyploid Carassius auratus

Transferrin polymorphism has been studied in the polyploid Carassius auratus by cloning and sequence analysis of cDNAs from its three subspecies C. auratus gibelio, C. auratus auratus, and C. auratus cuvieri. DNA polymorphism of extremely high extent was shown for the transferrin gene by the 248 segregation sites among coding region sequences of its alleles. The deduced amino acid sequences of the transferrin alleles showed variable theoretical physicochemical parameters, which might constitute molecular basis for their electrophoretic heterogeneity. Positive selection was inferred by the replacement/synonymous ratios larger than 1 in partial allelic lineages which was subsequently confirmed by likelihood simulation under neutral or selection models. Furthermore, the correspondent sites to these selected codons were collectively located at two planes in the crystallographic structure of rabbit transferrin, which suggested that the rapid evolution of C. auratus transferrin might correlate to its adaptation to variable environmental elements such as oxygen pressure. The minimal 26 recombination events were detected among coding sequences of C. auratus transferrin, with partial mosaic sequences and breakpoints identified by identity scanning and information site analyses. Phylogenetic analyses revealed multiple antique allelic lineages of transferrin, which was estimated to diverge fifteen to twenty MYA. All these features strongly suggested the role of balancing selection in long persistence of high transferrin polymorphism in C. auratus. Furthermore, owing to its particular evolutionary backgrounds, the silver crucian carp might possess a distinctive balancing selection mechanism.     

Sex steroid regulation of glutamate decarboxylase mRNA expression in goldfish brain is sexually dimorphic

Testosterone and oestradiol can modulate GABA synthesis in sexually regressed goldfish. Here we investigated their effects on the mRNA expression of two isoforms of the GABA synthesizing enzyme glutamate decarboxylase (GAD(65) and GAD(67), EC 4.1.1.15). Full-length GAD clones were isolated from a goldfish cDNA library and sequenced. Goldfish GAD(65) encodes a polypeptide of 583 amino acid residues, which is 77% identical to human GAD(65). Goldfish GAD(67) encodes a polypeptide of 587 amino acid residues and is 82% identical to human GAD(67). Goldfish GAD(65) and GAD(67) are 63% identical. Sexually regressed male and female goldfish were implanted with solid silastic pellets containing testosterone, oestradiol or no steroid. Semiquantitative PCR analysis showed that oestradiol significantly increased GAD(65) mRNA expression in female hypothalamus and telencephalon, while testosterone resulted in a significant increase only in telencephalon. GAD(67) mRNA levels were not affected by steroids in females. In contrast, both steroids induced significant decreases of GAD(65) and GAD(67) mRNA levels in male hypothalamus, but had no effect on GAD mRNA expression in male telencephalon. Our results indicate that modulation of GAD mRNA expression is a possible mechanism for steroid action on GABA synthesis, which may have opposite effects in males and females.     

饲喂不同浓度黄曲霉毒素B1饲料对异育银鲫成鱼的生长和毒素积累的影响

以含不同浓度黄曲霉毒素B1(AFB1)的配合饲料饲喂异育银鲫(Carassius auratus gibelio)成鱼56d,研究异育银鲫成鱼[(122.3±0.7)g]生长、生理反应、肝脏组织学变化、卵巢发育以及鱼体各组织中的AFB1的毒素积累状况。实验分为5个实验组,不同实验组饲料中AFB1含量分别为0、5、20、50、500μg/kg饲料(实测值分别为2.59、4.12、12.39、46.23、454.07μg/kg饲料),每个处理3个平行。在整个实验过程中各实验组均未表现出外部形态和行为异常,各组存活率均达到100%。各实验组异育银鲫成鱼终末体重、摄食率(FR)、特定生长率(SGR)和饲料效率(FE)均无显著差异。饲料AFB1水平对异育银鲫血清总胆固醇(TC)含量、血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)和碱性磷酸酶(AKP)活性均无显著影响。各毒素组血清超氧化物岐化酶(SOD)活性与对照无显著差异。各毒素组肝脏和卵巢均未见明显的组织学病理变化。肌肉和性腺中的AFB1积累量低于FDA食品安全限定标准(5μg/kg)。肝胰脏中的AFB1积累和饲料中的AFB1水平呈对数关系。饲喂AFB1≥50μg/kg饲料使异育银鲫成鱼肝脏AFB1积累超过安全限量标准。结果表明,异育银鲫成鱼至少可耐受AFB1含量达500μg/kg饲料(实测值:454.07μg/kg饲料)56d。     

双氟沙星在人工感染嗜水气单胞菌的异育银鲫体内的药代动力学

为了阐明双氟沙星在健康与处于患病状态下的异育银鲫(Carassius aurutus gibelio)体内的药代动力学特征差异,为双氟沙星的正确合理用药提供参考,本研究通过人工创伤感染的方式采用最佳浓度的嗜水气单胞菌(Aeromonas hydrophila)感染异育银鲫,在此基础上进一步以双氟沙星在健康异育银鲫体内的药代动力学特征为对照,采用反相高效液相色谱法测定双氟沙星在感染嗜水气单胞菌的异育银鲫体内的药代动力学特征。实验结果表明,以鱼体重的20 mg/kg口灌给药后,双氟沙星在人工感染嗜水气单胞菌的异育银鲫与健康异育银鲫体内的总药时曲线均符合一级吸收开放性二室模型,其药动学方程分别为C=6.227e^-0.109t-8.074e^-2.752t+1.847e^-0.006t和C=110.295e^-0.331t+1.533e^-0.01t-111.828e^-0.412t,但与双氟沙星在健康异育银鲫体内的药代动力学参数相比,双氟沙星在人工感染嗜水气单胞菌的异育银鲫体内的吸收、分布、消除速度减慢,其在人工感染嗜水气单胞菌的异育银鲫体内的分 布半衰期、消除半衰期、吸收速率常数、曲线下面积分别增加了4.25 h、36.17 h、2.34/h和74.52 mg·h/L,达峰时间延长了5.75 h,峰浓度降低了61.16%,且未出现重吸收现象。本研究证实嗜水气单胞菌感染能够导致异育银鲫肝肾功能损伤,因而双氟沙星在人工感染嗜水气单胞菌的异育银鲫体内的吸收、分布、代谢和消除均会减慢。     

双氟沙星对异育银鲫血脑屏障渗透性及消除规律简

以异育银鲫(Carassais auratus gibebio)为研究对象,采用组织匀浆法和高效液相色谱法,研究了双氟沙星(Difloxacin,DIF)通过异育银鲫血脑屏障情况,并比较分析了大脑和外周组织中DIF消除差异。结果显示,根据DIF 96h半数致死剂量(2840 mg/kg b.W)给药后,第96h时异育银鲫大脑组织匀浆中DIF的含量为(10.49±0.35)μg/g;同时在临床推荐用药剂量(20 mg/kg)给药后的15个时间点(0?960h)上均能从大脑组织匀浆中检测出DIF。上述结果表明DIF能渗透通过血脑屏障而进入异育银鲫大脑组织。另外,在大脑和外周组织消除过程上,以大脑组织中的DIF消除过程最为平缓(按照20 mg/kg给药)。到试验第960h,大脑组织中DIF含量最高,为(0.392±0.007)μg/g,且大脑中的消除半衰期最长,为1157.713h。因此,异育银鲫大脑组织可作为DIF药物残留分析的靶组织。另根据欧盟关于食品中DIF最大残留限量(MRL)之规定,实验条件下DIF休药期至少为25d。结果为研究鱼类血脑屏障作用,DIF神经毒性及其在水产养殖上的临床应用提供了参考。     

锦鲫幼鱼的社群等级地位及其与标准代谢率、血糖和临界游泳能力的关系

在(25.0±0.5)℃实验室条件下,考查标记后体重相近的30尾锦鲫幼鱼(3.5±0.1g)的标准代谢(standard metabolic rate,SMR),然后将实验鱼分为10个组(每组3尾);分别以好斗行为和摄食成功率两个指标将各组实验鱼评估为3个不同的社群等级;再分别测定各等级地位实验鱼的SMR、血糖浓度和临界游泳速度(critical swimming speed,Ucrit)。结果显示:锦鲫幼鱼3个等级地位由高到低的摄食成功率分别为(51.2±1.61)%、(29.3±1.87)%、(19.5±0.73)%;好斗行为指标得分分别为(8.1±0.30)、(1.9±0.30)、0.0,两个评估指标均存在显著性差异(P0.05);Ucrit分别为(35.5±0.22)、(33.7±0.66)、(29.8±1.02)cm/s,血糖浓度分别为(3.4±0.22)、(4.2±0.30)、(4.5±0.33)mmol/L(P0.05);而评估前、后各等级之间实验鱼的SMR均没有显著差异(P0.05)。结果表明:锦鲫幼鱼存在着明显社群等级分化的现象;好斗行为和摄食成功率两个指标分别对社群等级地位的评估结果具有显著的一致性;实验鱼等级地位越高其游泳能力越强,血糖浓度越低;这一现象提示,高等级地位的鱼受到较低的社群胁迫且具有较高的生理适合度。     

天然三倍体鲫鱼突变体萍乡肉红鲫线粒体细胞色素b基因序列分析

用细胞色素b(Cyt b)基因特异性引物,对萍乡肉红鲫(Carassius auratus var.pingxiangnensis)的线粒体Cyl b基因进行PCR扩增和双向测序.在12个个体中均得到序列一致的Cyt b基因全序列,长度为1 140 bp.其A、T、G、C含量分别为28.2%(321)、28.8%(328)、14.8%(170)和28.2%(321),A+T含量(57%)明显高于G+C含量(43%).与其他水生动物相同基因片段碱基含量相似.该基因中密码子第1位核苷酸中4种碱基组成较为均衡;第2位核苷酸中T的使用率较高,为41.8%,G的使用率较低,为13.2%;密码子第3位A的使用率较高,为39.7%,而G的使用率较低,仅为6.3%.BLAST结果显示,萍乡肉红鲫与其他鲫鱼类的Cyt b基因具有较高的同源性,其中与银鲫(C.a.gibelio)的相似性为93%,与普通鲫鱼(C.carassius)的相似性为98%.邻接法构建的分子系统进化树表明,萍乡肉红鲫与普通鲫鱼亲缘关系最近.     

银鲫原肠胚差异表达基因的筛选

胚胎发育是基因组中各个基因在时间和空间上选择性表达的结果。为了鉴定参与鱼类早期胚胎发育和胚层分化的调控因了,分别构建了银鲫(Carassiusauratusgibelio)原肠胚和成熟卵子的SMARTcDNA文库,并采用差异筛选的方法,从银鲫原肠胚SMARTcDNA文库中筛选不同于成熟卵子的差异表达基因。通过菌斑和PCR产物的两轮斑点杂交,从大约1500个克隆中筛选出131个阳性克隆,从中选择58个克隆测序并将测得的序列进行了数据库比对分析,结果显示这些差异表达基因大部分为参与转录和翻译的调控因子和核糖体蛋白以及一些在胚胎早期大量表达的参与脂类代谢的脂蛋白和一些未知的新基因。接着采用RTPCR技术,对其中6个基因在胚胎发育不同阶段的表达特征进行了分析,进一步证实这些基因在胚胎发育过程中存在表达差异;研究还从分析的基因中揭示出4类不同的表达模式。通过本研究,已筛选出一批在银鲫胚胎发育早期开始表达的调控基因,为开展鱼类胚胎发育早期表达基因的功能研究奠定了前期工作基础。       

异育银鲫c型溶菌酶全长cDNA序列的生物信息学分析及其组织表达分析

利用RACE及克隆等方法获得了异育银鲫（Carassius auratus gibelio）c型溶菌酶基因全长cDNA序列．序列分析表明,所克隆的异育银鲫溶菌酶的cDNA全长751 bp,包括溶菌酶基因开放阅读框（ORF）438 bp,5′ 非编码区（UTR）为109 bp和3′ UTR为204 bp．438 bp ORF共编码146个氨基酸,其成熟肽的分子量预测值为14　543.6,理论等电点为8.86．通过ClustalW软件,将异育银鲫和其它多个物种c型溶菌酶的氨基酸序列进行多序列比对发现,所克隆的异育银鲫溶菌酶编码的氨基酸序列中存在c型溶菌酶的活性中心（Glu53和Asp69）,且与活性位点相邻的氨基酸序列高度保守．同时,8个保守的半胱氨酸残基也与其它物种的c型溶菌酶相一致．结合BLASTN分析的结果,可以确认所获得的异育银鲫溶菌酶cDNA序列属于c型溶菌酶．异育银鲫c型溶菌酶和人c型溶菌酶（pdb 1at6_）在蛋白质序列上有50%相似性,其三维（3-D）结构非常类似．通过氨基酸空间位置比较发现,两者具有类似的酶活中心,异育银鲫c型溶菌酶只能形成3个二硫键,比人少1个．荧光定量RT-PCR检测和溶菌酶活性测定显示,异育银鲫头肾和脾脏c型溶菌酶mRNA的表达量约为肝胰脏的2.9 倍和1.7 倍,异育银鲫头肾和脾脏的溶菌酶活性约为肝胰脏的6.2 倍和4倍．