Molecular characterization of heat shock protein 70 genes in the liver of three warm freshwater fishes with differential tolerance to microcystin-LR

Heat shock protein 70 (HSP70) protect cell from oxidative stress by preventing the irreversible loss of vital proteins and facilitating their subsequent regeneration. Silver carp (Hypophthalmichthys molitrix), grass carp (Ctenopharyngodon idellus), and Nile tilapia (Oreochromis nilotica) are three warm freshwater fishes with differential tolerance to microcystin-LR (MC-LR). Full-length cDNAs encoding the HSP70 were cloned from the livers of the three fishes. The HSP70 cDNAs of silver carp, grass carp, and Nile tilapia were 2356, 2348, and 2242 bp in length and contained an open-reading frame of 1950 bp (encoding a polypeptide of 649 amino acids), 1950 bp (649 amino acids), and 1917 bp (638 amino acids), respectively. Like mammalian HSP70, the HSP70 of the three fish was also composed of an ATPase domain from residues 1 to 383 (44 kDa), substrate peptide binding domain from residues 384 to 544 (18 kDa), and a C-terminus domain from residues 545 to 649 (10 kDa). The relatively high conservation of HSP70 sequences among different vertebrates is consistent with their important role in fundamental cellular processes. Using beta-actin as an external control, RT-PCR within the exponential phase was conducted to determine the constitutive and inducible expression level of HSP70 gene among the three fishes (6-12 g) intraperitoneally injected with MC-LR (50 μg kg(-1) body weight). Both constitutive and inducible liver mRNA levels of the fish HSP70 genes showed positive relationships with their tolerance to MC-LR: highest in Nile tilapia, followed by silver carp, and lowest in grass carp. The differential expression pattern of liver HSP70 genes in the three fish indicated a potential role of HSP70 in the detoxification process of MC-LR.     

Molecular characterization of glutathione peroxidase gene from the liver of silver carp, bighead carp and grass carp

The cDNAs encoding glutathione peroxidase (GPx) were cloned and sequenced from the liver of three Chinese carps with different tolerance to hepatotoxic microcystins, phytoplanktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), and herbivorous grass carp (Ctenopharyngodon idellus). Using genome walker method, a 750 bp 5'-flanking region of the silver carp GPx gene was obtained, and several potential regulatory elements were identified in the promoter region of the GPx gene. The silver carp GPx gene was widely expressed in all tissues examined. Despite phylogenetic analysis, assigning this newly described carp GPx to the group of mammalian GPx2, the carp GPx seems more similar to GPx1 from a physiological point of view. The constitutive expression pattern of the three carp liver GPx gene, shows a positive relationship with their tolerance to microcystins.     

草鱼α1-抗胰蛋白酶基因cDNA全长克隆与表达分析

采用RACE技术获得α1-抗胰蛋白酶基因cDNA全长序列为1 469 bp,开放阅读框为1 329 bp,可编码442个氨基酸。5′非编码区长19 bp,3′非编码区长121 bp。核苷酸序列分析表明,在N端可能存在一个由1～21位氨基酸残基组成的信号肽;与斑马鱼的同源性最好,其次是虹鳟;在系统进化上,与在斑马鱼、虹鳟共聚为一个大支。用半定量RT-PCR分析正常及细菌诱导下草鱼α1-抗胰蛋白酶基因在不同组织中的表达分布。结果显示:正常情况下,草鱼α1-抗胰蛋白酶在肝脏表达最丰富,在脾脏、前肾、前肠、中肠、后肠和也有少量表达;细菌诱导下,肝脏中表达最强,前肾、脾脏、肠道中表达均明显提高,心脏和后肾中也出现较高表达。提示α1-抗胰蛋白酶可能参与了机体对嗜水气单胞菌感染的免疫应答。     

Purification and characterization of alpha2-macroglobulin from grass carp Ctenopharyngodon idellus: cloning a segment of the corresponding gene

The plasma protein alpha2-macroglobulin (alpha2M) was purified by gel filtration and anion-exchange chromatography from grass carp plasma. The alpha2M consists of two different subunits of molecular weight 95 kDa and 80 kDa, respectively. The characteristics of grass carp alpha2M are similar to mammalian alpha2M, in that grass carp alpha2M exists in two forms: a fast-form and a slow-form. The former is complexed with protease. The sequence of grass carp alpha2M-conserved region and a region containing the bait region was determined by sequence analysis using polymerase chain reaction (PCR). The deduced amino acid sequence of the conserved region is similar to the alpha2M sequence of common carp, however, the bait region amino acid sequence is dramatically distinct from that of common carp. This may partially explain the differential ability of alpha2M of different species to inhibit different proteases. The alpha2M was able to inhibit Aeromonas hydrophila extracellular protease (AhECPase) and thus may play a role in resistance to infection by this pathogen.     

草鱼免疫相关基因Prkrip1的克隆和表达分析

随着草鱼养殖规模的扩大, 草鱼的病毒性疾病极大地影响着草鱼的产量。开展鱼类病毒免疫反应相关功能基因的研究意义重大。研究首先通过同源克隆的方法从草鱼中克隆到了一段Prkrip1基因的EST序列, 进一步通过RACE、长片段PCR和Genome walking的方法获得了该基因的全长cDNA序列、基因组DNA序列和启动子区序列。氨基酸序列分析显示, Prkrip1含有3个核定位信号和一个双链RNA结合区, 并具有与PKR结合的保守N端区; 荧光报告基因的表达证实我们所克隆到的启动子区是有活性的, 可用于后续该基因的转录调控分析; Real-time PCR分析发现, Prkrip1 基因在草鱼的肝和血中表达量最高, GCRV感染后在大部分免疫组织中均上调表达, 说明该基因确实与病毒感染相关。研究结果为Prkrip1基因在硬骨鱼类的功能研究提供了线索, 也为鱼类天然免疫反应中调控PKR信号通路的系统研究提供了理论依据。       

Molecular cloning and characterization of alpha-class glutathione S-transferase gene from the liver of silver carp, bighead carp, and other major Chinese freshwater fishes

Two full-length cDNAs encoding glutathione S-transferase (GST) were cloned and sequenced from the hepatopancreas of planktivorous silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis). The silver carp and bighead carp GST cDNA were 920 and 978 bp in length, respectively, and both contained an open reading frame that encoding 223 amino acids. Partial GST cDNA sequences were also obtained from the liver of grass carp (Ctenopharyngodon idellus), crucian carp (Carassius auratu), mud carp (Cirrhinus molitorella), and tilapia (Oreochromis nilotica). All these GSTs could be classified as alpha-class GSTs on the basis of their amino acid sequence identity with other species. The three-dimensional structure of the silver carp GST was predicted using a computer program, and was found to fit the classical two-domain GST structure. Using the genome walker method, a 875-bp 5'-flanking region of the silver carp GST gene was obtained, and several lipopolysaccharide (LPS) response elements were identified in the promoter region of the phytoplanktivorous fish GST gene, indicating that the GST gene expression of this fish might be regulated by LPS, released from the toxic blue-green algae producing microcystins. To compare the constitutive expression level of the liver GST gene among the six freshwater fishes with completely different tolerance to microcystins, beta-actin was used as control and the ratio GST/beta-actin mRNA (%) was determined as 130.7 +/- 6.6 (grass carp), 103.1 +/- 8.9 (bighead carp), 92.6 +/- 15.0 (crucian carp), 72.3 +/- 7.8 (mud carp), 58.8 +/- 11.5 (silver carp), and 33.6 +/- 13.7 (tilapia). The constitutive expression level of the liver GST gene clearly shows that all the six freshwater fishes had a negative relationship with their tolerance to microcystins: high-resistant fishes (phytoplanktivorous silver carp and tilapia) had the lowest tolerance to microcystins and the high-sensitive fish (herbivorous grass carp) had the highest tolerance to microcystins. Taken together with the reciprocal relationship of constitutive and inducible liver GST expression level in some of the tested fish species to microcystin exposure, a molecular mechanism for different microcystin detoxification abilities of the warm freshwater fishes was discussed.     

Molecular cloning and characterization of carp (Cyprinus carpio L.) CD8beta and CD4-like genes

Partial cDNA sequences of both CD8beta and CD4-like (CD4L) genes of common carp (Cyprinus carpio L.) were isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently the full length cDNAs of carp CD8beta and CD4L were obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp CD8beta is 1164 bp and encodes 207 amino acids including a signal peptide region of 24 amino acids, a transmembrane region of 23 amino acids from aa 167 to aa189 and an immunoglobulin V-set from aa 19 to aa 141. Similar to other species CD8betas, carp CD8beta also lacks p56(lck) domain in the cytoplasmic region. The full length cDNA of carp CD4L is 2001 bp and encodes 458 amino acids including four immunoglobulin (Ig)-like domains in the extracellular region, a transmembrane region of 23 amino acids at the C-terminal region from aa 402 to aa 424 and a cytoplasmic tail. Similar to mammalian, avian CD4s and fugu CD4L, carp CD4L also has the conserved p56(lck) tyrosine kinase motif (C-X-C) in the cytoplasmic region. RT-PCR analysis demonstrated that carp CD8beta and CD4L genes were both expressed predominantly in thymus. The results from this study can be used to understand the evolution of both the CD8beta and CD4 molecules which can be used as markers for cytotoxic and helper T cells in carp.     

青鱼β-actin基因克隆及其启动子功能的初步检测

高保真PCR克隆青鱼β-actin基因开放阅读框和5’端侧翼序列,DNA测序结果表明：青鱼β-actin基因开放阅读框编码一段含375个氨基酸的蛋白,与其他物种actin家族相比较具有高度保守性。青鱼β-actin与鲤鱼、草鱼及斑马鱼的同源性均为100％,而与人和Norway鼠β-actin的同源性均为99.2％,与鸡和Kenyan爪蟾β-actin的同源性分别为98．9％和98．1％。将青鱼β-actin基因5’端启动调控区插入不含启动子的pEGFP1载体构建青鱼β-actin启动子／EGFP表达载体,与第一次卵裂之前显微注射该重组质粒入泥鳅受精卵,同时也用该重组质粒转染HeLa细胞系。观察结果表明：GFP在50％的泥鳅胚胎和2／3的HeLa细胞有所表达。GFP在泥鳅胚胎的各个部分均有表达,且在某些胚胎中GFP的表达遍布全身。因此,以EGFP为报告基因证实了青鱼β-actin基因启动子为一种非特异性表达的启动子。     

Cloning of Black Carp β-actin Gene and Primarily Detecting the Function of Its Promoter Region

A 3 338 bp DNA fragment including the open reading frame and 5′-flanking region of β-actin gene for black carp genome was obtained through PCR amplification. Analysis of the sequencing results indicated the ORF of black carp β-actin gene encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The black carp β-actin sequence showed 100% identity to common carp, grass carp, and zebrafish, 99.2% identity to human and Norway rat β-actin gene, 98.9% and 98.1% identity to chicken and Kenyan clawed frog β-actin gene, respectively. The promoter region of black carp β-actin gene was inserted into the promoterless pEGFP₁ vector. The recombinant plasmid was microinjected into the fertilized eggs of mud loach before two-cell stage as well as transfected into HeLa cell line. GFP expression was found in 50% of mud loach embryos and 2/3 HeLa cells. The GFP expression could be observed in every part of the mud loach embryos, and in some embryos, the GFP was expressed in the whole body. Thus, the usefulness of black carp β-actin promoter as a ubiquitous expression promoter was confirmed using the EGFP as a reporter gene.     

草鱼与鲢鱼肥胖基因克隆与序列分析

肥胖基因产物leptin是调节哺乳动物摄食、能量代谢等生命活动的重要细胞因子。 应用RT-PCR和RACE法获得了草鱼(Ctenopharyngodon idellus)和鲢鱼(Hypophthalmichthys molitrix) leptin基因的全长cDNA序列分别为1 096 bp和1 176 bp,编码173和172个氨基酸。氨基酸序列同源性分析表明,草鱼和鲢鱼的leptin序列与其它鲤科鱼类leptin的同源性较高,而与其他鱼类的leptin同源性很低,但所有鱼类的leptin均含有用于形成二硫键的高度保守的半胱氨酸。系统进化树分析显示,草鱼和鲢鱼leptin与其他鱼类leptin聚于一进化分支。应用PCR和Genome Walker方法,进一步获得了草鱼和鲢鱼leptin基因的内含子和5′侧翼区序列。结果表明,获得的草鱼和鲢鱼leptin基因长度分别为2 129 bp和2 192 bp,含有与其他脊椎动物leptin相似的基因结构（含三个外显子和两个内含子）。本研究为深入研究鱼类肥胖基因结构功能关系与鱼类抗肥胖品系定向遗传选育奠定了良好的基础。     

Structural conservation and food habit-related liver expression of uncoupling protein 2 gene in five major Chinese carps

The full-length cDNA of grass carp (Ctenopharyngodon idellus) and silver carp (Hypophthalmichthys molitrix) uncoupling protein 2 (UCP2) was obtained from liver. The grass carp UCP2 cDNA was determined to be 1152 bp in length with an open reading frame that encodes 310 amino acids. Five introns (Intron 3, 4, 5, 6 and 7) in the translated region, and partial sequence of Intron 2 in the untranslated region of grass carp UCP2 gene were also obtained. Gene structure comparison between grass carp and mammalian (human and mouse) UCP2 gene shows that, the UCP2 gene structure of grass carp is much similar to that of human and mouse. Partial UCP2 cDNA sequences of bighead carp (Aristichthys nobilis) and mud carp (Cirrhinus molitorella), were further determined. Together with the common carp (Cyprinus carpio) UCP2 sequence from GenBank (AJ243486), multiple alignment result shows that the nucleotide and amino acid sequences of the UCP2 gene, were highly conserved among the five major Chinese carps that belong to four subfamilies. Using beta-actin as control, the ratio UCP2/beta-actin mRNA (%) was determined to be 149.4 +/- 15.6 (common carp), 127.4 +/- 22.1(mud carp), 96.7 +/- 12.7 (silver carp), 94.1 +/- 26.8 (bighead carp) and 63.7 +/- 16.2 (grass carp). The relative liver UCP2 expression of the five major Chinese carps, shows a close relationship with their food habit: benthos and detritus-eating fish (common carp and mud carp) > planktivorious fish (silver carp and bighead carp) > herbivorous fish (grass carp). We suggest that liver UCP2 might be important for Chinese carps to detoxify cyanotoxins and bacteria in debris and plankton food.