从ATPase8-6基因研究杂交多倍体鱼线粒体母性遗传

异源四倍体鲫鲤是世界上首例人工培育的两性可育并形成群体的且能自然繁殖的四倍体鱼。本文采用质粒克隆测序法测定了红鲫、异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤的ATPase8和ATPase6基因全序列 ,结合鲤鱼、日本白鲫和斑马鱼的同源序列 ,对不同倍性水平鲤科鱼类的ATPase8和ATPase6基因进行了比较 ,分析了碱基组成、变异情况以及核苷酸和氨基酸序列差异。红鲫、鲤鱼、异源四倍体鲫鲤、日本白鲫、三倍体湘云鲫和三倍体湘云鲤之间的序列差异为 0 0 % - 1 3 4 % ,它们与外群斑马鱼之间的序列差异为 2 7 9% -31 0 %。用MEGA软件中的MP法、ME法、NJ法和UPGMA法构建分子系统树 ,得到了相似的拓扑结构。结果分析表明 ,人工杂交多倍体异源四倍体鲫鲤、三倍体湘云鲫和三倍体湘云鲤在线粒体ATPase8和ATPase6基因上具有严格的母性遗传特征。值得注意的是 ,异源四倍体鲫鲤经过 1 1代的繁育后 ,与其原始母本红鲫仍然保持了非常高的同源性 ,说明了新的异源四倍体基因库在线粒体ATPase8和ATPase6基因上拥有稳定的遗传特性。对不同倍性鲤科鱼类线粒体ATPase8和ATPase6基因的研究表明 ,ATPase8和ATPase6基因是杂交鱼后代遗传变异研究的一个很好的分子标记     

CaPKR-like在鲫鱼与草鱼组织中的表达特性分析

摘要：PKR是由干扰素诱导的最重要的抗病毒蛋白之一,能抑制细胞和病毒蛋白的合成。鲫鱼PKR基因(CaPKR-like)是鱼类第一个,也是非哺乳类脊椎动物第一个报道的PKR全长cDNA序列。本研究制备了CaPKR-like N-端包含Za结构域的多克隆抗体,采用Western blots检验PKR-like在鲫鱼和草鱼组织中的表达特性。Western blots显示在未诱导的鲫鱼和草鱼中该基因表达水平非常低,但是经Poly I:C免疫一周后,在鲫鱼和草鱼等8种组织中有较强的PKR-like蛋白产生。结果暗示：CaPKR-like与哺乳类PKR有相同的组织表达特性。。     

HIF-1α and iNOS levels in crucian carp gills during hypoxia-induced transformation

Hypoxia inducible factor 1 alpha (HIF-1α) initiates expression of a wide variety of genes, some of which are involved in apoptosis and cell cycle arrest. We have previously shown that crucian carp increases its respiratory surface area 7.5-fold in response to hypoxia. This change is due to apoptosis and cell cycle arrest in specific parts of its gills. Here we have characterized crucian carp HIF-1α, and measured mRNA, protein and DNA binding levels during hypoxia exposure in crucian carp gills. We have also measured an HIF-1α-induced gene, the inducible nitric oxide synthase (iNOS), which has the ability to initiate apoptosis and cell cycle arrest. Crucian carp HIF-1α was found to have all critical domains known to be important for function. Comparison of the peptide sequence with other species indicated high similarity with other cyprinid fish, but a pronounced variation compared to the salmonid, rainbow trout. Further, we found HIF-1α protein to be stabilized during hypoxia. Further, HIF-1α was often present in normoxia, and showed marked individual weight-dependent variation. We found no alteration of iNOS mRNA levels during hypoxia exposure. These findings suggest HIF-1α involvement in hypoxia-induced change of respiratory surface area in crucian carp gills. However, its activity does not seem to be mediated through iNOS.     

Genetic Evidence for Gonochoristic Reproduction in Gynogenetic Silver Crucian Carp (Carassius auratus gibelio Bloch) as Revealed by RAPD Assays

Sex evolution has been a debating focus in evolutionary genetics. In lower vertebrates of reptiles, amphibians, and fish, a species or a bioform reproduces either sexually or asexually but never both. A few species were found to consist of all females in fish. These all-female species can propagate by asexual reproduction modes, such as gynogenesis and hybridogenesis. However, the coexistence of sexuality and asexuality in a single species was recently noted only in a cyprinid fish silver crucian carp, Carassius auratus gibelio. This fish had been demonstrated to be capable of gynogenesis stimulated by sperm from other related species. Surprisingly, natural populations of this fish consist of a minor but significant portion (approx. 20%) of males. As different clones with specific phenotypic and genetic characteristics have been found, and RAPD markers specific to each clone have recently been identified, this fish offers many advantages for analyzing whether or not genetic recombination occurs between different clones. In this study, artificial propagation was performed in clone F and clone D. Ovulated eggs from clone F were divided into two parts and respectively inseminated with sperm from a clone D male and from a red common carp (Cyprinus carpio) male. The control clone D individuals were selected from gynogenetic offspring of clone D activated by sperm of red common carp. The phenotype and sex ratio in the experimental groups were also observed. Using RAPD molecular markers, which allow for reliable discrimination and genetic analysis of different clones, we have revealed direct molecular evidence for gonochoristic reproduction in the gynogenetic silver crucian carp and confirmed a previous hypothesis that the silver crucian carp might reproduce both gynogenetically and gonochoristically. Therefore, we conclude that the silver crucian carp possesses two reproductive modes, i.e., gynogenetic and gonochoristic reproduction. The response mechanism of two reproductive development modes may be the first discovery in vertebrates. Additionally, we discuss the evolutionary implication between gynogenetic and gonochoristic reproduction modes and the contribution of the minor proportion of males to genetic flexibility in the gynogenetic silver crucian carp. Received: 5 January 2000 / Accepted: 3 August 2000     

Rapid genomic DNA changes in allotetraploid fish hybrids

Rapid genomic change has been demonstrated in several allopolyploid plant systems; however, few studies focused on animals. We addressed this issue using an allotetraploid lineage (4nAT) of freshwater fish originally derived from the interspecific hybridization of red crucian carp (Carassius auratus red var., ♀, 2n=100) × common carp (Cyprinus carpio L., ♂, 2n=100). We constructed a bacterial artificial chromosome (BAC) library from allotetraploid hybrids in the 20th generation (F₂₀) and sequenced 14 BAC clones representing a total of 592.126 kb, identified 11 functional genes and estimated the guanine–cytosine content (37.10%) and the proportion of repetitive elements (17.46%). The analysis of intron evolution using nine orthologous genes across a number of selected fish species detected a gain of 39 introns and a loss of 30 introns in the 4nAT lineage. A comparative study based on seven functional genes among 4nAT, diploid F₁ hybrids (2nF₁) (first generation of hybrids) and their original parents revealed that both hybrid types (2nF₁ and 4nAT) not only inherited genomic DNA from their parents, but also demonstrated rapid genomic DNA changes (homoeologous recombination, parental DNA fragments loss and formation of novel genes). However, 4nAT presented more genomic variations compared with their parents than 2nF₁. Interestingly, novel gene fragments were found for the iqca1 gene in both hybrid types. This study provided a preliminary genomic characterization of allotetraploid F₂₀ hybrids and revealed evolutionary and functional genomic significance of allopolyploid animals.     

An experimental study of body size and food size relations in crucian carp,Carassius carassius

Synopsis In dense, single-species assemblages of crucian carp, competition is intense and results in populations of stunted fish. To explore mechanisms underlying this competition, we measured handling times, return rates, and prey choice for five sizes of crucian carp feeding on six sizes of a standardized food. Handling times increased with prey size and decreased with fish size. Return rates (dry mass ingested per unit handling time) increased dramatically with fish size, and generally decreased with increasing prey size, especially for small fish. Patterns of return rates among size-classes suggested that one or more size-related shifts in feeding efficiency exist for crucian carp; combined with physiological stresses related to winter anoxia, the inability of fish to make these shifts may contribute to size structures observed in high-density populations. Comparisons of relations among fish size, prey size, and return rates for crucian carp and bluegill, Lepomis macrochirus, suggest that similar intraspecific competitive relations exist between generalist species with size-structured populations. Despite differences in return rates among prey sizes, the extent of food selectivity based on prey size exhibited by crucian carp in two types of choice trials was less than predicted. Crucian carp commonly take in several items before mechanically processing food with their pharyngeal apparatus; this multiple prey processing may contribute to disparities between observed choice patterns and those predicted based on return rates for single prey.     

斑马鱼一个IFIT 家族基因的鉴定及启动子分析

IFIT 家族由一类受干扰素诱导表达并具有TPR 结构域的蛋白组成, 但是在鱼类关于IFIT 基因的研究还很少。研究利用哺乳类IFIT 家族基因IFI56 的序列搜索斑马鱼基因组数据库鉴定出一个未知基因, 该基因具有哺乳类IFIT 家族保守的基因组结构, 编码蛋白具有保守的TPR 结构域, 暂命名为IFIT-A。RT-PCR 分析表明, Poly I:C 能够诱导IFIT-A 基因转录水平上调。与哺乳类IFIT 家族基因相似, 斑马鱼IFIT-A 启动子存在ISG 基因特有的典型ISRE 结构域。荧光素酶活性实验揭示Poly I:C 和重组IFN 蛋白能激活斑马鱼IFIT-A 启动子活性。此外, 过量表达IFN 调控因子IRF3 和IRF7 能诱导斑马鱼IFIT-A 启动子活性。实验结果证明IFIT-A基因是斑马鱼IFIT 家族成员, IRF3 和IRF7 在其诱导表中具有重要调控作用。       

Subcellular localization and functional characterization of a fish IRF9 from crucian carp Carassius auratus

Mammalian interferon (IFN) regulatory factor 9 (IRF-9) has long been recognized as the DNA sequence recognition subunit of IFN-stimulated gene factor 3 (ISGF3) complex, which is critical for type I IFN to induce the expression of IFN-stimulated genes (ISGs) against viral infection. Recent studies have shown that fish IFN exerts antiviral effects by induction of a number of ISGs and also of itself; however, little is known about the role of fish IRF9 in IFN signaling. Here we identify a fish IRF9 orthologue (CaIRF9) from IFN-producing cell line, crucian carp Carassius auratus blastulae embryonic (CAB) cells. Analysis of subcellular distribution of CaIRF9-green fluorescent protein indicates that CaIRF9 is constitutively present in the nucleus, which is driven by two nuclear localization signals (NLS), one locating within DNA-binding domain (DBD) of CaIRF9 and the other immediately behind DBD, although human IRF9 contains only one NLS analogous to the former of CaIRF9. Overexpression of CaIRF9 together with CaSTAT2 not only activates ISRE-containing promoter but also upregulates the expression of fish ISGs. Strikingly, CaIRF9 together with CaSTAT2 also exhibits an ability to activate crucian carp IFN promoter, and blockade of cellular CaIRF9 attenuates IFN itself-induced activation of crucian carp IFN promoter. Taken together, these data suggest that crucian carp IFN induces the expression of ISGs and also of itself possibly by the JAK-STAT signaling pathway that is conserved from fish to mammals.     

不同捕食者暴露对鲫幼鱼行为、运动和形态特征的影响

自然界中鱼类的捕食者种类繁多,为验证猎物鱼针对不同捕食者是否会做出相异的反捕食策略选择,选取鲫（Carassius auratus）幼鱼为猎物鱼,乌鳢（Channa argus）和胡子鲶（Clarias fuscus）为捕食者,将鲫幼鱼分别暴露于空白（对照）、乌鳢、胡子鲶和双捕食者（同时存在乌鳢和胡子鲶）环境中持续2个月,随后考查不同组鲫幼鱼的生长、行为特征、形态和运动能力等指标之间的差异。结果表明：经过2个月的捕食胁迫处理,鲫幼鱼的自发运动、隐蔽场所利用率和勇敢性等行为指标在4个组之间均未表现出显著性差异。相比对照组,乌鳢、胡子鲶和双捕食者暴露组鲫幼鱼的快速启动反应时滞显著缩短（P<0.05）;但所有处理组中仅乌鳢暴露组鲫幼鱼的快速启动120ms移动距离（S_120ms）和体高（BD）显著高于对照组（P<0.05）。另外鲫幼鱼的临界游泳速度（U_crit）和活跃代谢率（M_O2active）在各组之间也未表现出显著差异。乌鳢暴露组鲫幼鱼快速启动能力的提升可能与形态上体高的提高有关;另外相比临界游泳能力,快速启动能力可能在鱼类逃避捕食者过程中更为关键;鲫幼鱼表现出对乌鳢的反应最为明显,可能与乌鳢口裂较大,给鲫幼鱼带来的威胁更大有关。整体而言,应对捕食胁迫时,鲫幼鱼仅表现出形态和快速启动能力的反捕食响应,其行为特征的保守性可能是对生长作出的妥协,这对于鱼类适应生境中长期存在的捕食胁迫十分关键。     

Effects of thermal acclimation on the relaxation system of crucian carp white myotomal muscle.

Many cyprinid fish are able to compensate for a decrease in ambient temperature by process of physiological adaptation in the function of muscles. In the winter habitat of crucian carp (Carassius carassius L.), low temperature is associated with simultaneous oxygen shortage. Because of the oxygen deprivation, there is probably little space for compensatory adaptation because positive thermal compensation would increase energy demand and accelerate depletion of glycogen reserves. Thus, we assumed that the crucian carp, unlike many other cyprinid fish, would not show positive thermal compensation but either no compensation or inverse compensation in muscle function. To test this hypothesis in the relaxation system of skeletal muscles, we determined the parvalbumin content and the activity of sarcoplasmic reticular (SR) Ca-ATPase in white myotomal muscle of winter- and summer-acclimated crucian carp. In the laboratory, the winter fish were kept at 2 degrees C and the summer fish at 22 degrees C for a minimum of 3 weeks before the experiments. The specific activity of SR Ca-ATPase at low experimental temperature (2 degrees C) was similar in summer- and winter-acclimated fish (0.26 +/- 0.04 vs. 0.25 +/- 0.04 mM/mg/min; P > 0.05). Because of the bigger Q(10) of cold-acclimated carp, the enzyme activity at 30 degrees C was higher in cold-acclimated winter fish than in warm-acclimated summer fish (7.42 +/- 0.90 vs. 5.18 +/- 0.53 mM/mg/min; P < 0.05). In contrast, the yield of SR protein was 70% higher in summer than winter fish (0.315 +/- 0.045 vs. 0.187 +/- 0.017 mg/g; P < 0.001). Because of these opposing changes, total Ca-ATPase activity of SR (per gram muscle weight) remained relatively constant. Similarly, the parvalbumin content of the myotomal muscle was not different between summer (4.09 +/- 0.95 mg/g) and winter (3.70 +/- 0.60 mg/g) fish. Although there were no seasonal changes in the total relaxing system of the crucian carp white myotomal muscle, the same activity of SR Ca-ATPase in winter fish was obtained with less amount of SR pump protein, owing to the increased catalytic activity of the enzyme. The higher catalytic activity of winter fish SR Ca-ATPase might be caused by differences in fatty acid composition noted in membrane lipids; i.e., fewer saturated fatty acids and more n-6 polyunsaturated fatty acids (PUFAs), at the expense of n-3 PUFAs, were present in the SR of cold-acclimated winter fish. Temperature-induced changes in enzyme protein, however, cannot be excluded. Thus, the present results indicate the absence of positive thermal compensation in the relaxing system of crucian carp white muscle. It seems, however, that lipid composition of SR membranes and temperature dependence of SR Ca-ATPase are altered by seasonal acclimation.     

红鲫实验动物含肉率及营养成分分析

目的探讨红鲫实验动物的营养需要、科学配制红鲫饲料。方法用常规方法测定红鲫幼鱼、红鲫成鱼的含肉率及肌肉营养成分。结果红鲫幼鱼、红鲫成鱼的含肉率为46．34％、42．35％;红鲫幼鱼、红鲫成鱼肌肉（鲜样）中分别含水分79．11％、78．49％,粗蛋白质12．47％、14．72％,粗脂肪6．95％、4．37％,粗灰分1．01％、1．41％,无氮浸出物0．47％、1．07％,能量5．65kJ／g、5．24kJ／g,其E／P值（能量蛋白比）为45．32kJ／g、35．69kJ／g。结论红鲫幼鱼、红鲫成鱼含肉率、肌肉营养成分有一定差异。     

Comparative antioxidant enzyme study in freshwater fish with different types of feeding behaviour

Comparative studies were made of the activities of Cu,Zn-superoxide dismutase, Mn-superoxide dismutase, catalase and glutathione peroxidase in organ homogenates from two herbivorous (grass carp and silver carp) and three omnivorous fish (barbel, crucian carp and common carp). The protein contents and lipid peroxidation of the organ homogenates were also compared. These comparative measurements provided control values for subsequent toxicological examinations. For the herbivorous fish, the highest total superoxide dismutase activity was observed in the kidney, followed in turn by the liver or spleen. In contrast, the highest total superoxide dismutase activity in the barbel was found in the roe, in the crucian carp in the liver, and in the common carp in the liver too. The quantitative distributions of the two peroxide metabolism enzymes glutathione peroxidase and catalase in the organ homogenates present very varied pictures. The results of the lipid peroxidation are similarly difficult to fit into one framework, even for fish following the same diet. It appears that the enzyme activities mentioned in points 4 and 5, together with the lipid peroxidation, depend not on the species, but on the variety.     

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps.

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis.     

氨氮胁迫下不同食性鱼类仔鱼抗氧化和非特异性免疫的差异响应

为探究氨氮胁迫对不同食性鱼类的影响,研究选取不同食性的4种鱼类(鲢Hypophthalmichthys molitrix、草鱼Ctenopharyngodon idella、团头鲂Megalobrama amblycephala和黄颡鱼Pelteobagrus fulvidraco)仔鱼为研究对象,探讨不同浓度氨氮(0、1、2和3 mg/L)短期胁迫(96h)对其生长、抗氧化和非特异性免疫响应的影响及其分子机制。结果显示:(1)氨氮胁迫导致4种仔鱼的体长生长速度呈现剂量依赖性减缓;(2)不同浓度氨氮导致4种仔鱼体内T-AOC、CAT和GPx含量显著降低(P<0.05), 2和3 mg/L氨氮暴露显著降低了草鱼、团头鲂和黄颡鱼仔鱼体内SOD活力(P<0.05),仅检测到黄颡鱼gpx及鲢sod转录水平出现显著性下调(P<0.05);(3)在不同浓度氨氮胁迫下, 4种仔鱼相关免疫基因转录水平均呈现一定的上调,仅鲢il1β转录水平显著下降(P<0.05),相对地草鱼仔鱼LYZ含量显著性下降,黄颡鱼仔鱼LYZ含量在2 mg/L氨氮组显著性上升(P<0.05)。双因素...     

Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio)

Background

The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system.

Methodology/Principal Findings

In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified.

Conclusions/Significance

This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of teleost fish.