斑马鱼(Danio rerio)的资源管理：综述

斑马鱼胚胎发育中神经元迁移的调控斑马鱼（Danio rerio）是一种被广泛用于生物学研究的水生动物，因其易于繁殖和培养，以及胚胎发育透明，方便研究和观察而备受关注。

在斑马鱼胚胎发育过程中，神经元的迁移是其中一个重要的过程，对于神经系统的形成与功能的发挥有着重要的影响。

因此，研究斑马鱼胚胎发育中神经元迁移的调控机制有着重要的科学意义。

神经元迁移的定义和重要性神经元迁移是指在神经系统发育中，神经元从胚胎前体向特定的位置前进而且改变形态的过程。

神经元迁移是神经系统正常发育过程中的一个关键环节。

在神经元迁移期间，神经元会通过定向移动和膜形态改变的方式进入其指定的位置，并与其他神经元组成神经电路。

这些神经电路是形成复杂神经网络的基础，控制着许多基本的生理行为和认知功能，如感觉、运动和行为学。

斑马鱼神经系统的发育斑马鱼胚胎发育的神经系统分为中央神经系统（CNS）和外周神经系统（PNS）两部分，CNS包括大脑和脊髓，而PNS包括不同形状和大小的神经节和神经丛。

斑马鱼胚胎发育的神经系统的结构和发育过程与哺乳动物相似，并且由于斑马鱼胚胎是透明的，这提供了研究神经系统发育和功能的难得机会。

神经元迁移的调控机制在斑马鱼神经系统的发育中，神经元迁移的调控是一个复杂、多因素的过程。

目前研究表明，神经元迁移涉及了多种信号通路和调节因子的参与，例如细胞间的信号分子和细胞自身的信号分子等。

在神经元迁移开始时，神经元需要定向向特定的位置前进，这个过程的调控是非常重要的。

许多分子参与了神经元定向移动和膜形态改变的过程。

其中一些分子是在神经元和周围细胞之间相互作用的信号分子，例如神经生长因子（NGF）和神经细胞粘附分子（NCAM）。

这些信号分子介导神经元与其周围环境的交流，确定神经元的运动方向和路径。

另一些重要的分子是细胞识别分子和结构蛋白，它们参与了神经元膜形态的改变和移动速率的控制。

但是，这些参与神经元迁移的分子的调控和相互作用模式仍然不完全了解。

斑马鱼卵黄原蛋白受体的功能分析斑马鱼卵黄原蛋白受体的功能分析摘要:斑马鱼（Danio rerio）作为一种重要的实验动物模型，近年来在生命科学研究中越来越受到关注。

卵黄原蛋白是斑马鱼早期发育过程中的关键调控因子，起着重要的生物学功能。

本文通过系统性的功能分析，探讨了斑马鱼卵黄原蛋白受体在胚胎发育、胚胎毒理学等方面的功能机制，为进一步深入研究斑马鱼卵黄原蛋白受体的作用提供了理论依据。

关键词：斑马鱼，卵黄原蛋白受体，胚胎发育，胚胎毒理学，功能分析引言在生物体早期发育过程中，卵黄原蛋白扮演着极为重要的角色，其中一类卵黄原蛋白是通过卵黄原蛋白受体介导结合和内化起作用的。

斑马鱼作为一种优秀的实验动物模型，卵黄原蛋白受体在斑马鱼的胚胎发育中起着非常重要的作用。

本文将重点从斑马鱼卵黄原蛋白受体的功能出发，探讨其在胚胎发育和毒理学过程中的生物学作用和机制。

一、胚胎发育中的功能1. 体轴形成斑马鱼胚胎的体轴形成过程包括胚中胚层的形成和胚胎体轴的形成。

通过实验观察发现，卵黄原蛋白受体的存在能够影响斑马鱼的胚中胚层形成，从而影响体轴形成的过程。

2. 胚胎发育的调控卵黄原蛋白受体对斑马鱼胚胎发育中多个关键调控因子的表达进行了调控。

例如，在胚胎脊椎骨发育过程中，卵黄原蛋白受体能够调控促细胞分化和生长因子的表达，从而影响脊椎骨的发育。

3. 神经发育卵黄原蛋白受体在斑马鱼神经发育中发挥着重要作用，它通过调控神经营养因子的表达和神经元的生成，影响神经元的迁移和突触形成。

二、胚胎毒理学的功能1. 胚胎毒理学研究需求胚胎毒理学是一门研究外界物质对胚胎发育影响的学科。

斑马鱼由于其繁殖周期短、胚胎发育透明等特点，被广泛应用于胚胎毒理学研究中。

卵黄原蛋白受体能够与一些有机或无机化合物结合，从而影响斑马鱼的胚胎发育过程。

2. 卵黄原蛋白受体对毒物的敏感性卵黄原蛋白受体作为特异性受体，对毒物的敏感性较高，能够很好地反应斑马鱼胚胎对毒物的毒性效应。

姓名：谭克强专业：生物技术学号：2009211803斑马鱼模式生物简介斑马鱼是在印度和巴基斯坦河里发现的一种鲤鱼。

成年3~4cm 长，有漂亮的花纹，群居生活。

在实验室里，斑马鱼广泛用于标准毒理学检验。

1996年，一系列论文报道斑马鱼胚胎发育的突变体的筛选及鉴定[1]，揭开了斑马鱼广泛应用于基因组功能分析、获得与脊椎动物发育和疾病相关的新基因研究的序幕。

斑马鱼(Danio rerio)作为水生脊椎动物的代表, 是现代遗传学、细胞生物学及发育生物学等研究的常用模式动物。

生物信息学预测认为, 斑马鱼基因组可能编码超过 400 种 miRNAs[2]。

通过构建不同发育阶段斑马鱼的小 RNA cDNA 文库发现, 斑马鱼miRNAs总数已经达到217个[2], 其中一些miRNAs在斑马鱼中的功能已经被解析。

miRNAs整体缺失对斑马鱼胚胎发育的影响研究结果表明, 缺失 miRNAs 的斑马鱼胚胎早期发育过程明显缓慢, 最初的 24 h 发育进程就被延迟了 3~4 h。

在原肠胚发育过程中, 突变胚胎不能进行正常的外包和内卷, 正常胚胎的索前板迁移发生在 80%外包时期, 而突变胚胎由于外包的推迟, 索前板迁移发生在 50%~60%外包时期。

此后, 由于体轴延伸减少导致胚胎缩短和脑部区域细胞的积累。

而在发育晚期, Dicer 突变胚胎后部卵黄延伸的范围也减少[3]。

Dicer 突变严重影响了神经胚形成。

由神经板发育成神经管的过程不能正常完成, 使神经管变成一个实心的棒状结构。

Dicer 突变胚胎脑内缺乏脑间隔而导致脑室数减少, 神经管腔的缺失和神经底板的减少表明脊髓发育也被干扰。

另外, 视网膜的发育也受到影响。

尽管神经系统发育畸形, 基因表达分析却发现, 神经管的前-后轴和背-腹轴图示均没有被完全破坏, 说明胚胎神经系统的图式形成和命运决定过程受到 miRNAs 的影响较少, 而脑的正常发育和神经细胞分化却需要 Dicer 酶的作用[3]。

Developmental Expression of the Nfe2-Related Factor (Nrf) Transcription Factor Family in the Zebrafish, Danio rerioLarissa M. Williams1,2, Alicia R. Timme-Laragy1, Jared V. Goldstone1, Andrew G. McArthur3, John J. Stegeman1, Roxanna M. Smolowitz4, Mark E. Hahn1*1 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America,2 Biology Department, Bates College, Lewiston, Maine, United States of America,3 Andrew McArthur Consulting, Hamilton, Ontario, Canada,4 Department of Biology and Marine Biology, Roger Williams University, Bristol, Rhode Island, United States of AmericaIntroductionThe nuclear factor-erythroid-2 (NFE2)-related factor (NRF) genes encode transcription factors in the Cap’n’Collar basic leucine zipper (CNC-bZIP) gene family. Members of this family include NFE2 [1,2], NRF1 (NFE2L1) [3,4], NRF2 (NFE2L2) [5], and NRF3 (NFE2L3) [6]. (For nomenclature conventions, see footnote 3 of ref [7]..) These transcription factors occur in vertebrates from fish [8] to mammals [9,10], and are known to regulate metabolic [11,12], cell cycle [12-14] and cytoprotective processes [9,13,15-18]. In mice, Nrf proteins have been shown to play important and often essential roles in development. Nfe2-null mice lack circulating platelets [19], the Nrf1 knockout is embryonic lethal [20], Nrf2-null mice are highly sensitive to carcinogens and oxidative stress [21], and the Nrf3knockout does not show abnormalities under normal conditions but is susceptible to lymphoma induced by exposure to carcinogens [22].Although studies in mice have provided important information about the function of Nrf genes, viviparous development makes it difficult to directly observe the role and effects of genes throughout the embryonic period.Zebrafish are a powerful model for studying molecular mechanisms of vertebrate development. Genes and signaling pathways [23,24] and molecular mechanisms of developmental toxicity [25-29] are highly conserved between fish and mammals. Furthermore, due to the whole genome duplication that occurred during early teleost radiation [30-33], zebrafish have duplicated copies (paralogs) that are co-orthologs of many single copy human genes [30,34]. Paralogs that have undergone subfunctionalization provide an opportunity for newmechanistic insights and thus new understanding about the functions of their single human counterpart [32,35]. In zebrafish, there are several paralogs within the nrf gene family, including nrf1a and nrf1b as well as nrf2a and nrf2b [7]. In total, zebrafish have six nrf family genes.In mammals, NFE2 forms a heterodimeric transcription factor with small MAF proteins, regulates expression of globin genes [2,36], and may regulate the oxidative stress response in mature erythrocytes [17]. In zebrafish, nfe2is a single copy gene [8]; its duplicate appears to have been lost [7]. In embryos nfe2 is highly expressed in the intermediate cell mass during erythroid differentiation [8]. Apart from in situ hybridization during development and protein characterization, the expression and role of nfe2in development are largely unexplored.NRF1 in mammals is expressed in many tissues [4]; it plays a role in regulating redox balance in the liver [37] and in regulating genes involved in development [20], oxidative stress [38,39], cytoskeletal organization [40], and the proteasome [41]. In response to UVB damage, mammalian NRF1 regulates nucleotide excision repair and glutathione homeostasis, and may act as a tumor suppressor [13]. The two zebrafish nrf1 paralogs, nrf1a and nrf1b, are syntenic with the duplicated hoxb clusters, similar to the human NRF1 that is near the human HOXB cluster [7]. Prior to the current study, the only information about expression of zebrafish nrf1had been from EST data [38] and a limited assessment by qualitative RT-PCR [7].NRF2 has a broad tissue distribution in mammals and acts as a pleiotropic transcription factor involved in regulating the susceptibility to disease, in mediating the response to xenobiotic exposure and oxidant stress, and in proteome maintenance [42]. In zebrafish, two paralogs (nrf2a and nrf2b) are broadly expressed both in the embryo and adult and apparently have undergone subfunctionalization [7]. The primary role of nrf2a seems to be in the regulation of cytoprotective genes upon oxidative stress [7,9]. Conversely, nrf2b is a negative regulator of embryonic gene expression both basally and under oxidative stress conditions [7].NRF3 has been found to be expressed at high levels in mammalian placenta and the B cell lineage and at lower levels in the heart, lung, kidney, and pancreas [6]. NRF3 may play a protective role in regulating genes responding to oxidative stress, as Nrf3-deficient mice treated with an oxidant suffer acute lung damage [43]. Zebrafish have one nrf3 ortholog, and like nfe2, the duplicated gene appears to have been lost [7]. The expression and function of nrf3in zebrafish was completely unexplored prior to this study.In this paper, we address fundamental aspects of the expression and role of six nrf genes during vertebrate development using the zebrafish as a model system. We show that the expression of nrf genes varies temporally throughout development and that nrf genes are induced following a pro-oxidant exposure and are potentially cross-regulated by gene family members Nrf2a and Nrf2b. We also identify a novel role for nfe2 in the cellular organization of epithelia in the pneumatic duct.Experimental ProceduresFish husbandryZebrafish of the Tupfel/Longfin mutation (TL) wild-type strain were used in all experiments. Adults were maintained and embryos were collected as previously described [44]. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Woods Hole Oceanographic Institution Animal Care and Use Committee (Animal Welfare Assurance Number A3630-01).Cloning and confirmation sequencing of nrf genesIn order to determine nrf cDNA sequences in the TL wild-type strain, and identify strain-specific polymorphisms, nrf cDNAs were amplified with primer pairs (Table 1), using Advantage cDNA Polymerase. The cycling conditions were [94°C, 1 min], [94°C, 30 sec; 68°C, 3 min] for 35 cycles, followed by 68°C for 3 minutes and 10 minutes at 15°C. The PCR products were purified with the GENECLEAN kit (Qbiogene, Quebec, Canada), ligated into pGemT Easy Vector System (Promega, Madison, WI) and constructs were sequenced. Prior to confirmation of in vitro expression, gene products from pGemT were subcloned into pcDNA3.1/Zeo (+) (Invitrogen, Carlsbad, CA). Sequences of clones were deposited into Genbank and have accession numbers JX867113-JX867116.Sampling, Chemical exposure, RNA extraction, and cDNA synthesisEmbryos for Developmental Series.As described in Timme-Laragy et al. [7], 4 pools of 30 staged embryos from a single clutch were reared at low density at 28.5°C in 0.3x Danieau’s and flash frozen in liquid nitrogen at 6, 12, 24, 48, 60, 72, 96, and 120 hpf and stored at -80°C. Hatched and unhatched embryos were collected separately at the 48 and 60 hpf time points. Unfertilized eggs, representing the 0 hpf time point, were manually stripped from 3 females, combined, and flash frozen as 3 separate biological replicates and stored at -80°C. Although it limited our ability to determine individual variation amongst embryos, pooling of embryos was needed to provide sufficient amounts of RNA for measuring expression of multiple genes.Chemical exposure of embryos to tBOOH.Embryos (3 pools of 30 staged embryos) were exposed to tert-butylhydroperoxide (tBOOH). Embryos (96 hpf) were exposed to 0, 0.5 or 0.75 mM tBOOH for 6 hours in 150x20mM glass petri dishes. Following tBOOH exposure, embryos were immediately placed in RNAlater and stored at -80°C.RNA extraction and cDNA synthesis.Total RNA was isolated from pooled embryo samples using RNA STAT-60 (AMS Biotechnology, Abingdon, UK) following the manufacturer’s protocol. cDNA was synthesized from 1 µg of total RNA using random hexamers and the Omniscript cDNA Synthesis Kit (Qiagen, Valencia, CA).Quantitative real-time RT-PCR gene expression profilingUsing the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA), QPCR was conducted with the iQ SYBR Green Supermix (Bio-Rad) on nrf genes. For each sample, duplicate reactions in separate wells were run containing 5 ng of cDNA. The PCR conditions were 95°C for 3.5 minutes followed by 35 cycles of 95°C for 15 seconds and 25 seconds at a gene specific temperature. Gene-specific primers and temperatures are listed in Table 1. Following each run, a melt curve was generated to ensure the amplification of single product. All primers were tested for amplification efficiency using a calibration dilution curve and slope calculation approach [45]. β-actin 1 (actb1) was chosen as a housekeeping gene due to its limited variation in expression with embryonic development and chemical exposure [46]. We confirmed the suitability of this housekeeping gene with the set of samples used in this study. Expression of genes wasanalyzed using the comparative ∆∆CT method [47].Statistical analysis of QPCR dataDevelopmental Series. Data were analyzed with MicrosoftExcel using the ∆∆CTmethod to calculate fold change. Valuesare presented as mean ± SEM, and N is defined as the numberof pools of embryos. Relative expression was normalized to the120 hpf value within a gene. As described in Timme-Laragy etal. [7], Statview for Windows (version 5.0.1; SAS Institute,Cary, NC) was used to determine differences in expression dueto hatching state. Data were log-normalized and six statisticaloutliers were removed from the nrf2b development series (onedata point from each of six time points: 0, 6, 24, 48(unhatched), 48 (hatched), and 96 hpf). If an ANOVA yieldedsignificance (p< 0.05), Fisher's protected least significantdifferences test was used as a post hoc test with Bonferronicorrection.Chemical Exposure and Nrf2a and Nrf2b crosstalk. Datawere analyzed with Statview for Windows (version 5.0.1; SASInstitute, Cary, NC) and presented as mean ± SEM where N isdefined as the number of pools of embryos. Significance inANOVA was determined with a p≤ 0.05. When significancewas yielded, Fisher’s protected least-significant differences testTable 1. Primers used for gene amplification, cloning, and qPCR of nrf genes.Gene Primer sequences (5’-3’)Amplicon size T (°C)RefR-GGTGAACTATCACTTTAATCAAACATnrf1a F-CAGTTCGCACGCCCTTATTTACTGAC237668-R-GCTGATGGACTTAACAGCAGACAGnrf1b F-CGTAACCTAATTTGGTTTGACG288568-R-GTCCTCCTTGACTTCCCATATCnrf3F-AAATTGAGCAGTTGCTCCCCTCC234668-R-CTCACCTCAAAGATAAAACTCACCnfe2-qPCR F-CAGAGTTTGAGGAACCCAATGAG12757-R-CACAAGTGGCTGGAATGGATTCnrf1a-qPCR F-CCAGAGTTGACAGGTCCTGG15864-R-CATAACCTGTGATTCCATGATAGACnrf1b-qPCR F-GCAGGACATGGAGGTGAACAATACG12066-R-GGATCGTGGGAGCCCAAAATTTCCnrf2a-qPCR F-GAGCGGGAGAAATCACACAGAATG8265[7] R-CAGGAGCTGCATGCACTCATCGnrf2b-qPCR F-GGCAGAGGGAGGAGGAGACCAT26968[7] R-AAACAGCAGGGCAGACAACAAGGnrf3-qPCR F-GCATGAGGATTTAGTGGTTAGTGG10864-R-GGAGTCAAAATCATCAAAGTCAGactb1-qPCR F-CAACAGAGAGAAGATGACACAGATCA14065[87] R-GTCACACCATCACCAGAGTCCATCACPrimer sequences are written 5’-3’ and amplicon size and melting temperature (T) are shown. For primers used previously, a reference is cited. Accession numbers for sequences newly reported here are: nrf1a (JX867114), nrf1b (JX867116), nrf3 (JX867113), nfe2 (JX867115).doi: 10.1371/journal.pone.0079574.t001was used as a post hoc test with Bonferroni correction. Relative expression was normalized to the water control for tBOOH. For Nrf2a and Nrf2b morpholino data, relative expression of each gene was normalized to the water control with control morpholino.Microarray gene expression profilingZebrafish embryo rearing, sampling, and microarray analysis were performed as previously reported [29]. Briefly, four replicate pools of 100 embryos were examined at 3, 6, 12, 24, 36, and 48 hpf. Agilent 4 × 44k DNA gene expression microarrays (part #GPL11077, WHOI Zebrafish 44k v1.0 custom-commercial Chemical Defensome array, Agilent Technologies, Santa Clara, CA), which included custom probes to ensure adequate coverage of the chemical defensome [29], were used to examine gene expression levels. Single color microarray data (Cy3) was normalized using the non-linear scaling method of Schadt et al. [48], where saturated probes and probes not above background in all replicated were removed. Because standard ANOVA is not appropriate for these data due to auto-correlation between time points, we used Bayesian Estimation of Temporal Regulation (BETR; [49]) to analyze the developmental time series. Normalized Cy3 values for each probe were log transformed, median-centered, and analyzed using BETR relative to 3 hpf. Normalized average Cy3 signals were compared for genes of interest among time points. All probes reported here had significant differential expression among sampling times. The microarray data have been deposited into the Gene Expression Omnibus (GEO) database with NCBI GEO accession number GSE24840.In situ hybridizationIn situ hybridization of nrf3 on whole embryos was performed using established procedures [50,51]. Sense and anti-sense nrf3 RNA probes were created by restriction digest of the nrf3 construct (in pGemT) with XhoI, yielding a 1080 bp sense probe and 1328 bp anti-sense probe. The nrf3 probe covers a 1240-bp region of the 3’ end of the cDNA and includes 87 base pairs of the 3’ UTR. In designing the probe, we carefully considered possible sequence identity with other nrf transcripts. Nucleotide sequence identities between nrf3and other nrf transcripts in the region covered by the probe were as follows: 45% with nfe2, 47% with nrf1a, 51% with nrf1b, 49% with nrf2a, and 45% with nrf2b. The overlap between the nrf3 probe and other nrf transcripts was typically interspersed throughout the sequence with no long runs of overlap (mainly, 4-5 nt at a time). We also conducted a Blast search using the nrf3 probe against the zebrafish genome; no significant overlap was found other than the nrf3 gene itself. Probes were labeled with digoxigenin-UTP by in vitro transcription (Roche San Francisco, CA) with either T7 (sense) or SP6 (anti-sense) polymerase. Pre-hybridization was carried out for 2 hours at 65°C. Hybridization was carried out overnight with 300 ng of probe per pool of seven embryos at a concentration of approximately 1.5 µg/mL. Embryos were blocked for 4 hours at room temperature and incubated with anti-digoxigenin alkaline phosphatase conjugated antibody (Roche, San Francisco, CA)at 4°C overnight. Embryos were imaged by light microscopy (Axio Observer.A1, Carl Zeiss, Oberkochen, Germany).Morpholino oligonucleotidesMorpholino antisense oligonucleotides (MO) were designed to block initiation of translation of zebrafish nfe2, nrf1a, nrf1b and nrf3 and obtained from Gene Tools, LLC (Philomath, OR). The first nfe2MO (AGTTCCTGCCAGGCCAAGTCCATCT) complements two residues of the 5’UTR, the start codon (underlined), and 20 residues of the coding region. A second, non-overlapping nfe2MO (AACGATGTGTCCGTAATCCAGTGAC) complements 25 residues of the 5’UTR and targets a region whose 3’ end is 15 residues upstream of the sequence targeted by the first nfe2 morpholino. The nrf1a MO (ATGGCCCAAACCATCACCGGCAGCA) complements 11 residues of the 5’UTR, the start codon (underlined), and 11 residues of the coding region. The nrf1b MO (AATCACGCAAACAAACGTCAAACCA) complements 25 residues of the 5’UTR and its 3’ end is located 34 residues upstream of the start codon. The nrf3MO (TTTTAACCTCAGGAGGCTTAAACGA) complements 25 of the 5’UTR and its 3’ end is located 14 residues upstream of the start codon. The standard control-MO from Gene Tools was also used (CCTCTTACCTCAGTTACAATTTATA). All MO were tagged at the 3’ end with fluorescein in order to detect successful injection and incorporation of MO into embryos. MOs targeting nrf2a and nrf2b are previously described [7]. Confirmation of MO translation inhibition by in vitro protein synthesisTo confirm the efficacy of morpholinos to inhibit translation of Nrf proteins, the TnT T7 Quick Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used to synthesize [35S]methionine-labeled zebrafish NF-E2, Nrf1a, Nrf1b and Nrf3 protein as per manufacturer’s protocols and as described previously [52]. Briefly, the TnT reaction included 1 μl of [35S]methionine (> 1000 Ci/mmol at 10 mCi/ml) and 2 μl of the respective nrf cDNA in pcDNA3.1/Zeo (+) (0.5 μg/μl) in a final volume of 25 μl with H2O. For reactions containing MOs, 0.5 μl of a 25 μM stock of the appropriate MO was added, resulting in a final concentration of 500 nM. Labeled protein products were resolved by SDS-PAGE, dried on Whatman filter paper, and visualized on film. To quantify the reduction in protein products, gel fragments containing a single protein band were excised and placed into 7 mL glass scintillation vials containing 5mL ScintiVerse II cocktail (Fisher Scientific, Pittsburg, PA). Counts were carried out for 5 minutes on a Beckman Coulter LS6500 Multi-purpose Scintillation counter (Brea, CA) and background was subtracted.Microinjection of zebrafish embryos with morpholinos Zebrafish embryos at the two- to four-cell stage were injected with 2.5-5 nL of a 0.1 mM solution of MO targeting nfe2, nrf1a, nrf1b, nrf2a, nrf2b, nrf3, or a control-MO, using a Narishige IM-200 microinjector (Tokyo, Japan). Injection volumes were calibrated as described previously [52]. The injections resulted in delivery of MO amounts ranging from 2-4 ng. At six to ninehpf, embryos were sorted and screened for successful fertilization and fluorescence. When no developmental phenotypes were observed, the concentration of the morpholino stock was increased to 0.15 mM, keeping the injection volume constant. MO-injected embryos were held in 0.3x Danieu’s solution. At 4 dpf, swim bladder inflation was scored as described earlier [53].Histological analysis of the swim bladderFour-day-old larvae from embryos injected with control morpholino (swim bladder inflated) or nfe2morpholino (swim bladder not inflated) were fixed in 4% formaldehyde in 1x phosphate buffer, dehydrated in ethanol and stored in 75% ethanol until embedding. Larvae were sent to Environmental Pathology Laboratories (Sterling, VA) where they were embedded laterally into either Technovit 7100 (Heraeus Kulzer, Hanau, Germany) or paraffin. Sagittal sections were made serially every 2 µm. Sections were mounted on superfrost glass slides and stained with hematoxylin and eosin. Histopathology was conducted by an Olympus BX 40 with an Olympus DP25 camera system.Analysis of blood flowTransgenic gata1dsRED embryos [54] (15 individuals per treatment) were injected with control or nfe2-MOs at one- to four-cell stages. As an index of blood flow, the number of red blood cells passing through the mesencephalic vein (MsV) per 15 s was determined by orienting 48 hpf zebrafish embryos laterally as described by Kubota et al. [55]. Blood flow was recorded with an AxioCam MRc5 camera on a Zeiss Axiovert 200 inverted microscope and analysis (counting of red blood cells) was performed using ImageJ (/ij/).Analysis of otic vesiclesAB embryos were injected at one- to four-cell stages with control-MO or nfe2-MO-1. At 30, 48 and 72 hpf, otic vesicles were imaged with an AxioCam MRc5 camera using a Zeiss Axiovert 200 inverted microscope.In silico Promoter Analysis and motif searchesTo determine whether Nrf2a or Nrf2b are directly involved in the transcriptional regulation of other nrf family members, in silico promoter analysis was carried out for AREs and XREs using a fuzzy search algorithm, fuzznuc [56]. 10,000 base pairs upstream of the start site and the entire length of the gene were examined for putative zebrafish AREs (TGA(G/C)nnnTC [57]) and XREs (KNGCGTG [58]). Additional searches were carried out for Nfe2 binding sites [59] determined from ChIP-Seq studies. The zebrafish genome was searched for Nfe2 binding sites in the 10 kb upstream of the start codon of all known genes using a position-specific scoring matrix (PSSM) based on the empirical ChIP-Seq results of Wang et al [59] and the FIMO algorithm of the MEME/MAST package [60](p < 0.0001), with correction for background nucleotide frequencies of complete nuclear genome sequences.ResultsDevelopmental expression profilingTo begin to understand the role of the nrf genes during development, their expression was profiled from 0 to 120 hpf using qRT-PCR (Figure 1). Because the data were normalized within each gene, expression values can be compared at different times for each gene but not across genes. nfe2 transcripts were maternally deposited, but levels fell substantially at 6 hpf and did not increase again until 48 hpf. Expression remained relatively constant from 48 to 60 hpf and then decreased steadily until 120 hpf. Like nfe2, nrf3was maternally deposited and decreased by 6 hpf, but transcript levels increased again at 12 hpf. Expression decreased between 12 and 48 hpf, but then remained steady. nrf1a was expressed from 0-24 hpf but levels were reduced from 48-72 hpf, after which expression increased at 96 and 120 hpf. Conversely, nrf1b expression peaked between 12 and 24 hpf and then declined and remained relatively low from 48 to 120 hpf. Hatching did not have a significant effect on expression of nfe2, nrf3, nrf1a or nrf1b. For comparison, Figure 1 also includes data for expression of nfr2a and nrf2b, originally presented (in a slightly different format) by Timme-Laragy et al.[7]. These results showed that nrf2a expression increased steadily during development. The expression of nrf2b also increased through 48 hpf, but there was a significant effect of hatching on nrf2b expression, when expression decreased in hatched embryos compared to unhatched embryos of the same developmental age.Since molecule numbers were not available from the qRT-PCR data and data were normalized within each gene, a microarray was used to determine the expression of nrf genes relative to each other during the first 48 hours of development (Figure 2). Microarray and QPCR analyses were performed on samples obtained from independent experiments. The microarray data did not include a 0 hpf time point, so maternal transcript levels were not determined with this method. Gene expression profiles determined by QPCR and microarray for nfe2, nrf1a, nrf2a, and nrf2b displayed similar developmental patterns. The developmental patterns of nrf3expression measured by qPCR and microarray were similar, except that the pattern at 6 and 12 hpf was reversed. For nrf1b, the peak at 12 hpf measured by QPCR was not evident in the microarray data.Normalized Cy3 levels for nfe2, nrf1a, nrf1b, nrf2a, and nrf2b ranged from 100 to 1,800, whereas values for nrf3 ranged from 80,000 at 3 hpf to approximately 20,000 at 48 hpf (Figure 2). The nrf gene with lowest expression was nrf1a followed by nrf1b. Their average expression values were about half that of nfe2. The expression of nrf2a was up to10-fold greater than that of all other nrf genes except nrf3.Localization of nrf3 expression with ISHWe used in situ hybridization to examine the spatial expression of nrf3, the nrf gene most highly expressed during development; analysis was carried out in 12-hour intervals from 24 to 72 hpf. In situ gene hybridization results for nfe2, nrf1a, and nrf2a have been previously reported [8,61,62]. The mostprominent expression of nrf3 occurred at 24 and 36 hpf;expression was detected in the fore-, mid- and hindbrain as well as in the pectoral fin buds (Figures 3A, 3B, 3D, 3E). The high degree of staining seen is consistent with the relatively high level of nrf3 expression seen in the array and qRT-PCR data. At 48 hpf and 60 hpf, expression still was seen in the pectoral fin buds, and was less prominent in the forebrain andmore concentrated in both the mid- and hindbrain (Figures 3G,Figure 1. The expression of the nrf gene family during development as measured by quantitative real-time PCR. Black lines indicate expression pre-hatch and grey lines indicate expression post-hatch. Values were normalized to the 120 hpf time-point and β-actin 1 (actb1) was used as the housekeeping gene. Data are presented as the mean ± S.E.M. (error bars), and N = 4 pools of 30 embryos. Differences in expression between hatching state were assessed using ANOVA followed by Fisher's PLSD (*, p ≤0.05). Data for expression of nrf2a and nrf2b are from Timme-Laragy et al. [7] and are used with permission of the American Society for Biochemistry and Molecular Biology. Previously these data were presented as molecule number. Here we have normalized the data to β-actin 1 expression and to the 120 hpf time point and re-plotted for comparison to the new data on expression of other nrf family genes. Dashed lines indicate the value of 1.0 from the 120 hpf time point.doi: 10.1371/journal.pone.0079574.g0013H, 3J, 3K). Expression was also seen in the branchiogenic primordia. Expression at 72 hpf was almost exclusively in the mid/hindbrain junction (Figure 3M, 3N). There was no signal in sense controls at any time point assayed (Figures 3C, 3F, 3I,3L, 3O).Effect of nrf gene knockdown on developmentOne approach to identify the importance of nrf genes in developmental processes is to knock down their expression in the embryo. The in vitro translation-blocking efficiency of nrf morpholinos ranged from 65 to 68%, with the exception of morpholino 2 for nfe2 (22%; Figure 4). To determine whether any morphological abnormalities were associated with the reduction of Nrf proteins, embryos were injected with morpholinos and observed every 12 hours until 120 hpf. No gross morphological abnormalities were observed for control MO, nrf1a -MO, nrf1b -MO, a combination of nrf1a -MO and nrf1b -MO, or nrf3-MO. Additional experiments were conducted using higher concentration of morpholino (0.15 mM). However,at this concentration off-target effects were observed. Off-targeteffects observed in more than 50% of the MO-injected embryos included hatching gland deformities, pericardial edema, and bent tail post-hatching.In contrast to the lack of specific phenotypes in embryos injected with MOs targeting nrf1a, nrf1b , or nrf3, injection of an nfe2-MO (0.1 mM) resulted in the failure of the swim bladder to inflate at 96 hpf in 95% of the injected embryos (Figure 5B versus 5A) (Table 2). The specificity of this effect was confirmed using a second, non-overlapping nfe2 MO that also targeted the region surrounding the translation start site (Table 2).Histological analysis of larvae treated with control MO showed the normal pneumatic duct connection between the esophagus and the swim bladder (Figure 5C,D). Epithelial cells along the duct were orderly and cell morphology progressed from columnar at the entrance to the duct to cuboidal to squamous at the entrance to the swim bladder. In larvae in which Nfe2 had been knocked down, the pneumatic duct connection was intact but the number of epithelial cells and the regular progression from columnar to squamous was disrupted (Figure 5E). In the most severe example, the epithelialcellsFigure 2. The expression of the nrf gene family during development as measured by microarray analysis. Data are presented as normalized Cy3 levels, with average values for nfe2, nrf1a , nrf1b , nrf2a , and nrf2b displayed on the primary (left) y-axis. Values for nrf3 are displayed on the secondary (right) y-axis. Each time point represents the average normalized value ±SD of 4 replicates, each from 100 embryos. The Agilent probes from which these data were obtained are: nfe2 (A_15_P109440), nrf1a (A_15_P110831), nrf1b (A_15_P116909), nrf2a (CUST_121_PI358351581), nrf2b (CUST_122_PI358351581), nrf3(A_15_P116878). An additional probe for nrf2a (A_15_P109504) was present on the array and produced results very similar to those shown here for CUST_121_PI358351581. The microarray data have been deposited with NCBI GEO accession number GSE24840.doi: 10.1371/journal.pone.0079574.g002。

斑马鱼模式生物简介斑马鱼（Danio rerio）是一种小型的鱼类，也是一种非常受欢迎的淡水观赏鱼，它是孔雀鱼科中的一员，是印度东北部和孟加拉国的淡水鱼类。

斑马鱼模式生物是国际上被广泛应用于基因工程和生物医学研究的模式生物之一，也可以作为重要的药物筛选平台和疾病模型使用。

那么，接下来就来逐一介绍斑马鱼的分类、形态特征、生活习性、模式生物优势及应用等方面的知识。

一、斑马鱼的分类斑马鱼属于脊索动物门、脊椎动物亚门、圆口纲、鲤形目、鲤科、孔雀鱼属的一种淡水鱼类，学名为Danio rerio。

斑马鱼还有各种不同的变种和突变体，例如白斑马鱼、红斑马鱼、紫斑马鱼、金斑马鱼等。

二、斑马鱼的形态特征1.外形特征斑马鱼身体呈纺锤形，通常长度为2.5-4厘米，雄性略大于雌性。

头部和尾部比身体稍大，呈楔形，头部较尖，口小，下颌略突出。

各种颜色和花纹的斑马鱼几乎可以说是身体通体条纹，由水平条纹串联俩个腹鳍之间的细密黑色点状线条逐渐变浓变宽而形成，其中背部、腹部及尾鳍的条纹分化明显。

因此，美国生物技术研究所把斑马鱼称为"斑马状鱼"（Zebra fish）。

2.内部结构斑马鱼的内部结构和人类相似，有头、躯干、尾等结构。

头部包括大脑、眼、鼻、口、耳等器官，身体包括骨架、肌肉、消化系统、心脏、循环系统、呼吸系统、泌尿系统等器官。

三、斑马鱼的生活习性1.水温适应性斑马鱼适应范围广，可在25摄氏度以下的水温生存，适应性强，而50摄氏度以上的水温则容易引起死亡。

通常在水温20度-28度之间生存。

2.繁殖生殖斑马鱼在2-3个月龄时性成熟，通常在水中产卵。

雌鱼体内产生卵子时，会有告诉性色素出现，这些色素可以帮助斑马鱼有效地控制自己和同伴的繁殖。

雄鱼会在巢穴内产生精子，和雌鱼配对后可以产生400-500颗卵。

在适当的条件下斑马鱼卵可以在69h后孵化。

3.食性习性斑马鱼性质温和，不挑食，食性杂，吃小型昆虫、蚯蚓、水虫以及水草、藻类等，在食物充足的情况下生长迅速，但在食物短缺时可以进入休眠状态。

斑马鱼动物模型的应用斑马鱼（Danio rerio）属于辐鳍亚纲（Actinopterygii）鲤科（Cyprinidae）短担尼鱼属（Danio）的一种硬骨鱼，原产于南亚，是一种常见的热带观赏鱼，因其体侧具有斑马一样暗蓝与银色相间的纹条而得名。

斑马鱼个体小，易于饲养，成体长4-5cm，雄鱼体修长，雌鱼体肥大。

可在有限空间里养殖相当大的群体，可满足样本需求量大的研究。

斑马鱼发育迅速，在28.5℃培养条件下受精后约40min完成第一次有丝分裂，之后大约每隔15min分裂一次，24h后主要器官原基形成，相当于28d的人类胚胎，幼鱼孵出后约3个月达到性成熟。

雌雄鱼通过调控光周期控制14:10（光照:黑暗）产卵时间，成熟鱼每周可产卵一次，一尾雌鱼每次可产卵100-300枚。

胚胎体外受精，体外发育，胚体透明，易于观察。

受精卵直径约1mm，易于进行显微注射和细胞移植等操作。

一、斑马鱼的品系经过30多年的研究应用和系统发展，已有约20个斑马鱼品系，斑马鱼基因数据库-ZFIN （http://zfin/org）里有相关的资料可供查询和下载。

目前研究中常用的斑马鱼野生型品系主要为AB 品系、Tuebingen（Tu）品系、WIK 品系，斑马鱼基因组计划所用品系是Tu。

AB 品系是实验室常用的斑马鱼品系，由单倍体细胞经早期加压法获得。

Tu品系斑马鱼具有胚胎致死突变基因，用于基因组测序前敲除该致死突变基因。

WIK品系较Tu品系具有更多的形态多样性。

此外，还保存有3000多个突变品系和100多个转基因品系。

这些品系资源对于利用斑马鱼开展各种科学研究起着很大的推动作用。

二、斑马鱼突变品系的筛选斑马鱼突变的方法主要有三种：已基亚硝脲（ENU）化学诱导、γ或χ射线照射和插入诱变。

ENU是一种DNA烃基化试剂，在生殖细胞减数分裂前诱导碱基对的替换，诱导产生的突变率为0.1%-0.2%，涉及单个基因的突变。

射线照射导致染色体大片段的缺失或染色体重排，产生突变率达1%。

斑马鱼(Danio rerio)养殖综述Christian Lawrence布莱根妇女医院卡帕研究实验室马萨诸塞州美国收稿日期：2007年7月16日；改修日期：2007年8月24日；接受日期：2007年8月25日。

摘要：斑马鱼(Danio rerio)是脊椎动物研究的模式生物。

斑马鱼的优良性状有利于研究人类疾病的发生与传播。

较强的繁殖力、短小的体型、繁殖周期短、胚胎早期光学透明性强等特性使得斑马鱼成为许多研究者研究其他项目青睐的对象，包括研究动物行为、鱼类生理机能和水产养殖毒性试验。

尽管如此，科学严谨的斑马鱼养殖技术还是很不发达。

尽管斑马鱼有很大的需求量，包括直接或者间接养殖。

斑马鱼的养殖缺乏应用养殖标准。

本文试图将已有的关于斑马鱼生物学和养殖的科学信息进行综述，可以用来提高这一重要的模型动物使用检索的效率。

本研究还强调了该领域还需要进一步研究的内容。

关键词：斑马鱼；Danio rerio；饲养；水产养殖；管理目录：1、引言 (2)2、斑马鱼自然生活史 (3)2.1、分布和栖息地 (3)2.2、生殖和行为 (3)2.3、寿命 (5)2.4、食物 (5)3、斑马鱼养殖 (5)3.1、水化学参数 (5)3.1.1、温度 (6)3.1.1、pH (6)3.1.3、硬度 (7)3.1.4、盐度 (7)3.1.5、溶解氧 (8)3.1.6、含氮废弃物 (8)4、营养、食物、投喂方式 (8)4.1、营养需求 (8)4.2、食物 (9)4.3、投喂 (11)5、繁殖和育种技术 (11)5.1、繁殖 (11)5.2、育种技术 (12)5.3、产卵效率 (13)6、幼鱼饲养 (13)6.1、幼鱼生物学 (13)6.2、食物和营养 (13)6.3、水质 (14)6.4、生长率和存活率 (15)7、成鱼培育 (15)7.1、放养密度 (15)7.2、遗传育种 (16)8、结语 (16)鸣谢 (16)参考文献 (16)1、引言在过去的二十年间，斑马鱼(Danio rerio)广泛作为遗传学和发育学研究的优良模式生物(Fishman,2001)，最近斑马鱼还应用于的人类疾病和治疗药物筛选的研究当中(Penberthy et al.,2002;Sumanasa and Lin, 2004)。

HEREDITAS (Beijing)2013年4月,35(4):547―548ISSN 资料库网络出版时间:2013-1-309:54:36URL:/kcms/detail/11.1913.R.20130130.0954.001.htmlDOI:10.3724/SP.J.1005.2013.00547斑马鱼研究计划与相关资源简介肖安,张雨田,张博北京大学生命科学学院,细胞增殖与分化教育部重点实验室,北京100871斑马鱼(Danio rerio )是目前遗传发育研究领域最为活跃的模式脊椎动物之一,有多个大型研究计划正在进行中。

本文选择其中的一部分进行简要介绍。

1斑马鱼基因组测序计划斑马鱼基因组测序计划始于2001年,最初由英国Sanger 研究所主持,现在转由参考基因组协会(Genome Reference Consortium,GRC)主导,跟人类、小鼠两个基因组计划一起成为GRC 的三个主要目标。

测序数据来自基于克隆图谱测序法和全基因组鸟枪法的整合结果。

随着测序进度的发展,主持单位会定期更新斑马鱼基因组的拼装,最新版本为2010年7月发布的Zv9。

目前斑马鱼基因组计划即将完成,已进入缺口(gap)修补和未定位片段定位的阶段,GRC 计划于2013年晚期发布新的拼装版本GRCz10。

在GRC 的计划页面上可以看到不断更新的缺口修补进度和其它信息。

斑马鱼基因组注释计划主要分为自动注释和人工注释两部分,分别发布于Ensembl 和Vega 数据库,此外,还有相当多方面的注释内容由UCSC Genome Browser 首先提供,但各个数据库间均相互共享数据。

2斑马鱼突变计划及相关的数据库(1)ZMP(Zebrafish Mutation Project)计划：原称Zebrafish mutation resource 计划,是由Sanger 研究所主持的一项基于TILLING(Targeting Induced Local Lesions IN Genomes)技术的斑马鱼突变筛选计划。

模式动物斑马鱼斑马鱼（Danio rerio）属于辐鳍亚纲（Actinopterygii）鲤科（Cyprinidae）短担尼鱼属（Danio）的一种硬骨鱼，原产于南亚，是一种常见的热带观赏鱼，因其体侧具有斑马一样暗蓝与银色相间的纹条而得名。

斑马鱼个体小，易于饲养，成体长4-5cm，雄鱼体修长，雌鱼体肥大。

可在有限空间里养殖相当大的群体，可满足样本需求量大的研究。

斑马鱼发育迅速，在28.5℃培养条件下受精后约40min完成第一次有丝分裂，之后大约每隔15min分裂一次，24h后主要器官原基形成，相当于28d的人类胚胎，幼鱼孵出后约3个月达到性成熟。

雌雄鱼通过调控光周期控制14:10（光照:黑暗）产卵时间，成熟鱼每周可产卵一次，一尾雌鱼每次可产卵100-300枚。

胚胎体外受精，体外发育，胚体透明，易于观察。

受精卵直径约1mm，易于进行显微注射和细胞移植等操作。

一、斑马鱼的品系经过30多年的研究应用和系统发展，已有约20个斑马鱼品系，斑马鱼基因数据库-ZFIN （http://zfin/org）里有相关的资料可供查询和下载。

目前研究中常用的斑马鱼野生型品系主要为AB 品系、Tuebingen（Tu）品系、WIK 品系，斑马鱼基因组计划所用品系是Tu。

AB 品系是实验室常用的斑马鱼品系，由单倍体细胞经早期加压法获得。

Tu品系斑马鱼具有胚胎致死突变基因，用于基因组测序前敲除该致死突变基因。

WIK品系较Tu品系具有更多的形态多样性。

此外，还保存有3000多个突变品系和100多个转基因品系。

这些品系资源对于利用斑马鱼开展各种科学研究起着很大的推动作用。

二、斑马鱼突变品系的筛选斑马鱼突变的方法主要有三种：已基亚硝脲（ENU）化学诱导、γ或χ射线照射和插入诱变。

ENU是一种DNA烃基化试剂，在生殖细胞减数分裂前诱导碱基对的替换，诱导产生的突变率为0.1%-0.2%，涉及单个基因的突变。

射线照射导致染色体大片段的缺失或染色体重排，产生突变率达1%。

斑马鱼研究报告（Zebrafish,Danio rerio）目录一﹑斑马鱼二﹑斑马鱼基因与人类极为相似三﹑利用斑马鱼作为模式动物四﹑斑马鱼的心生五﹑突变斑马鱼及转基因斑马鱼表現类似人类疾病六﹑斑马鱼胚胎研究七﹑研究者在活的斑马鱼身上直接观看器官的发育与病变八﹑解开人与体內活菌共生秘密九﹑斑马鱼胚胎基因有助研究癌症、阿滋海默和帕金森症十﹑视网膜有自我修复潜能十一﹑培育变种斑马鱼可望用于人脸整型十二﹑基因決定人类肤色十三﹑斑马鱼的运动情形肌肉的发育及肌肉与运动神经的调控机制十四﹑细胞如何储存脂肪有助減肥新疗法十五﹑类固醇荷尔蒙的功能与调控十六﹑中神经细胞形成之分子调控机制十七﹑斑马鱼基因调控网路十八﹑干扰斑马鱼基因的技术十九﹑培育环保转基因斑马鱼二十﹑改造斑马鱼基因來试水质二十一﹑利用斑马鱼作为人类疾病模型及药物节选二十二﹑药物节选二十三﹑抗癌药物节选斑马鱼原产于东印度恒河流域，亦分布于巴基斯坦、尼泊尔、缅甸。

斑马鱼成鱼体长约4～5公分，体呈纺锤形，稍侧扁。

体侧从头至尾布满多条蓝色条纹，酷似斑马，故得名斑马鱼。

1.斑马鱼是研究发育生物学的新兴模式动物。

2.斑马鱼由于具有饲育容易、胚胎透明、体外受精、突变种多、遗传学工具成熟等诸多优点，近年来已成为研究脊椎动物发育与人类遗传疾病的新兴模式动物。

3.与其他脊椎动物相较下，斑马鱼最大的优点就是具有多达6000多种的遗传突变种，这些突变种的建立大致上是利用X射线、ENU或反转录病毒的感染造成基因组的突变，之后再经由多次的子代筛选所得。

4.突变种的表征包含如胚层分化，器官发育，生理调适与行为表现等多方面，所以可提供研究人员极佳的正向遗传学材料来进行发育机制上的研究。

5.在斑马鱼系统中也开发出阻断基因功能的工具-Morpholino，可快速以逆向遗传学手法来验证基因的功能。

所以正向遗传学与逆向遗传学的巧妙利用，可以正确推导出斑马鱼遗传发育途径，也是目前斑马鱼成为研究人类疾病新兴模式动物的主要原因。

斑马鱼及其研究应用作者：杜颖指导老师：张源淑摘要：斑马鱼作为一种新兴的重要模式动物之一，体外受精、胚胎透明，因此可在显微镜下直接观察发育过程及检测药物引起的内脏组织变化，在生命科学领域中应用前景十分广阔。

斑马鱼体型小,适合高通量研究,还具有生长繁殖周期短及其与人类高度相似的基因组等优点，已经广泛用于人类疾病模型的建立、新药研发和药物的筛选，此外，斑马鱼还被应用于毒理学、发育生物学和遗传学等的研究。

因为斑马鱼对污染物反应灵敏，现已用于监测环境污染物及污水检测。

本文主要从几个方面对斑马鱼的研究进展进行了整理和归纳。

关键字：斑马鱼模式动物科学研究发育感染药物Zebrafish and Its ApplicationAbstract:Zebrafish as an important model animal emerging, its in vitro fertilization, transparent embryo, internal organs can be directly observed during the development and testing organ change caused by drugs under the microscope, has very broad application prospects in the field of life sciences. zebrafish also has a live, high-throughput, growth and short reproduction cycles and highly similar to the human genome, etc., it is widely used in modeling human diseases, drug screening, and secondly, zebrafish also is applied in toxicology research, developmental biology and genetics, etc.. Because of its sensitivity, it has been used to monitor environmental pollutants and water testing.This paper mainly from several aspects of zebrafish research progress has been collated and summarizedKey words:zebrafish Animal models Scientific research Development Infection Drug斑马鱼(Danio rerio)又名蓝条鱼、花条鱼、蓝斑马鱼、印度鱼、印度斑马鱼,产于孟加拉、印度东部、巴基斯坦、缅甸、尼泊尔等地,是一种常见的热带淡水硬骨鱼。

江西省永丰中学2013届高三一轮复习英语单元验收试题（9）【新人教】命题范围：综合第Ⅰ卷第一部分听力（共两节，满分30分）第一节（共5小题；每小题l．5分，满分7．5分）听下面五段对话。

每段对话后有一小题，从题中所给的A、B、C三个选项中选出最佳选项，并在答题卡上将该项涂黑。

听完每段对话后，你都有10秒钟的时间来回答有关小题和阅读下一小题。

每段对话仅读一遍。

1．Where is the barber’s shop?A． On this side of the street．B． On the opposite side of the street．C． At the end of the street．2． Whom do they want to ask?A． The announcer．B．The captain．C． Stewardesses．3． What dress does the woman get?A． An order-made dress．B． A hand-made dress．C． A self-made dress．4． Where probably are the two speakers?A． At a restaurant．B．At a studio．C． At a concert．5． What is the reward for the woman?A． Five days off．B．Four days off．C． Three days off．第二节（共15 小题；每小题1．5分，满分22．5分）听下面5段对话或独白。

每段对话或独白后有几个小题，从题中所给的A、B、C三个选项中选出最佳选项，并标在试卷的相应位置。

听每段对话或独白前，你将有时间阅读各个小题，每小题5秒钟；听完后，各小题将给出5秒钟的作答时间。

每段对话或独白读两遍。

听第6段材料，回答第6、7题。

斑马鱼模型的基因组及其在发育研究中的应用斑马鱼（Danio rerio）在科学界已经成为了广泛使用的模式生物之一，其具有繁殖快、易于维护、生长快等特点，可以在研究发育生物学、遗传学、神经学等领域发挥重要的作用。

斑马鱼的基因组研究与分析，更是为生物学研究提供了丰富的资源。

本文将介绍斑马鱼基因组的构成及其在发育研究中的应用。

一、斑马鱼基因组的构成斑马鱼的基因组约为1.5亿个碱基对，分为25对染色体。

这种基因组主要由DNA组成，DNA又由核苷酸组成，核苷酸包括葡萄糖、磷酸和四种碱基（腺嘌呤、鸟嘌呤、胸腺嘧啶和鳕嘧啶）。

与其它生物一样，斑马鱼的基因组也可以被分为编码区和非编码区，编码区指的是直接编码功能蛋白的DNA序列，而非编码区则指由DNA转录产生但不被翻译成蛋白质的RNA序列。

通过对斑马鱼基因组的研究，科学家们已经发现了许多与人类病理有关的基因（如自闭症等）以及与发育有关的基因。

二、斑马鱼在发育研究中的应用斑马鱼在发育研究中的应用非常广泛。

典型的应用案例包括模拟人类疾病、研究肢体再生及衰老机制等。

其中，模拟人类疾病是利用斑马鱼具有与人类基因组高度相似的特性，来研究某些疾病。

例如，斑马鱼可以被制成自闭症模型，通过这一模型可以研究自闭症的神经学特性以及潜在的药物治疗。

将这些与人类疾病相关的斑马鱼研究结果应用到临床上，有望为人们提供新的预防和治疗方法。

肢体再生也是斑马鱼发育研究的一方面。

斑马鱼的可再生能力非常高，能够在受损的组织区域新生组织甚至再生失去的组织。

科学家们通过对斑马鱼肢体再生过程中参与的基因进行研究，有望为人类组织工程学领域提供新的思路和策略。

此外，斑马鱼还能被用于衰老研究。

斑马鱼短时间的寿命与其生长发育周期短、繁殖快、适应能力强等特点有关，这使得斑马鱼成为了衰老研究领域的理想候选。

科学家们通过对斑马鱼衰老过程中参与的基因进行研究，可以为人类寿命延长和老年病的治疗提供新的思路和策略。

三、结语综上所述，斑马鱼基因组的研究及其在发育研究中的应用，为人类研究提供了极大的帮助。

发育生物学研究中的模式动物——综述摘要：模式生物在生命科学研究中有重要的作用，不仅能回答最差不多的生物学问题，对人类的疾病治疗也有借鉴意义。

近年来随着分子生物学、发育生物学的进展及功能基因组打算的开展，模式生物的作用便显得越来越重要。

本文着重介绍了斑马鱼、秀丽线虫及果蝇三种经典模式生物的研究历史、研究优势及进展等，进而简要阐述了模式生物在今天生命科学进展中的重要地位和推动生命科学及医学进步的不可替代的庞大潜力．关键词：模式生物；发育生物学；引言：在生物学进展之初，人们发觉假如把关注的焦点集中在相对简单的生物上则发育的现象难题能够得到部分解答。

因为这些生物的细胞数量更少，分布相对单一，变化也较好观看。

同时由于进化的缘故，细胞生命在发育的差不多模式方面具有相当大的同一性，因此利用较低等级的物种来研究发育的共通规律是具有一定的可行性。

专门是当我们在有不同发育特点的生物中发觉共同的形状形成和变化特点时，就能够以此来建立发育的普遍原理，因此这种生物就显得尤为重要，我们称之为“模式生物”。

模式生物具有许多共同的特点，如形体相对较小，在实验室内易于培养和繁育，世代周期短，形状结构相对比较简单，繁育系数高（后代数量众多）等，而且通常情形下它的基因组会比较小。

前两点是出于实验室空间考虑，而世代周期短是出于研究时刻的考虑；形状结构的简单性能够减少特有生命现象的干扰，以便使人们更用心于生物遗传发育的差不多规律。

目前一些物种被大伙儿公认为是优良的模式生物，如线虫、果蝇、非洲爪蟾、蝾螈、小鼠、斑马鱼、噬菌体、大肠杆菌、酿酒酵母、海胆等。

它们在人口与健康领域应用范畴比较广。

而在植物学研究中比较常用的有，拟南芥、水稻、烟草等。

1.几种经典模式生物概况：1.1海胆第一个被用作模式生物的是海胆，它的胚胎对早期发育生物学的进展有举足轻重的作用。

早在一八七五年，奥斯卡‧赫特维格（Oscer Hertiwig, 1849-1922）就开始以海胆为材料研究受精过程中细胞核的作用，一八九○年后，海胆更在受精和早期胚胎发育的研究中担任重要角色。

斑马鱼的饲养与营养调控斑马鱼，又称斑马魟鱼，是一种常见的淡水观赏鱼类。

它以其独特的斑纹和鲜艳的颜色受到了许多人的喜爱。

要让斑马鱼在饲养环境中保持健康，并展现出最佳的色彩和行为，正确的饲养和营养调控是至关重要的。

本文将介绍斑马鱼的饲养技巧和营养需求，帮助读者更好地照料这些可爱的宠物。

一、饲养环境斑马鱼是淡水鱼类，对水质的要求相对较高。

为了提供一个适宜的饲养环境，有以下几个关键要点需要注意。

1. 水质：斑马鱼的饲养水质应保持清洁和稳定。

过滤器的使用是必不可少的，可以有效去除水中的异物和有害物质。

此外，定期检测水质的PH值、氨氮和硝酸盐等指标，并及时进行调整和处理。

2. 水温：斑马鱼适宜的生活温度范围为24-28摄氏度。

使用恒温器可以帮助稳定水温，保持恒定的温度对斑马鱼的生长和健康至关重要。

3. 水量：斑马鱼的饲养水箱大小要适合群养，一般建议每只斑马鱼提供2-3升的水空间，保证斑马鱼的活动和游泳。

二、饲料选择正确的饲料选择对于斑马鱼的健康和生长起着重要的作用。

以下是几种常见的斑马鱼饲料和饲养技巧。

1. 干饲料：斑马鱼适合食用干饲料，如颗粒状或片状的人工制作饲料。

在选择干饲料时，应注意其成分和口感，确保提供充足的蛋白质和维生素。

2. 活体饲料：斑马鱼也可以食用活体饲料，如蚕蛹、水蚤、糯米虫等。

这些活体饲料可以提供丰富的营养，但需要注意及时清理未被食用的残渣，以免引起水质问题。

3. 饲料频次：斑马鱼每日喂食2-3次，每次食量不宜过大。

过量喂食容易导致水质变差，并增加鱼类疾病的风险。

适量喂食有助于维持斑马鱼的健康并保持其活力和色彩。

三、营养调控为了保持斑马鱼的健康和营养平衡，有几个关键的营养需求需要注意。

1. 蛋白质：蛋白质是斑马鱼生长和维持身体功能的重要营养元素。

合理的饲料中应包含高质量的蛋白质源，如鱼粉、虾粉等，以满足鱼类的需求。

2. 维生素和矿物质：斑马鱼需要各种维生素和矿物质来维持正常的生理功能。

饲料中应包含适量的维生素和矿物质，也可以通过添加维生素片剂或液体来补充。

斑马鱼
别名：蓝条鱼、花条鱼、斑马担尼鱼
英文名：Zebra Danio
学名：Danio rerio (Hamilton, 1822)
原产地/分布：印度、孟加拉国
成鱼体长：5.0 - 7.0 cm
性格：温和
适宜温度：18.0 - 26.0 ℃
酸碱度：PH 6.5 - 7.2
硬度(DGH)： 4.0 - 12.0°N
活动水层：中层
繁殖方式：卵生
斑马鱼简介
体呈纺锤形。

背部橄榄色，体侧从鳃盖后直伸到尾未有数条银蓝色纵纹，臀鳍部也有与体色相似的纵纹，尾鳍长而呈叉形。

雄鱼柠檬色纵纹；雌鱼的蓝色纵纹加银灰色纵纹。

性情温和，小巧玲珑，几乎终日在水族箱中不停地游动。

易饲养，可与其他品种鱼混养。

在水温11~15℃时仍能生存，对水质的要求不高。

日常饲养时，在水族箱底部放些鹅卵石，使水质清澈。

雄鱼体狭长，活泼好动，体色较深；雌鱼腹部膨大，活动摇摇摆摆，体色较淡。

注：Danio rerio为其有效种名，Brachydanio rerio 为同种异名。