斑马鱼胚胎发育过程中TGF-1基因的表达特征分析

为研究转化生长因子 (Transforming growth factor , TGF)1对斑马鱼胚胎发育的调控作用, 通过NCBI获得TGF-1基因序列, TGF-1 cDNA全长1571 bp, 编码377个氨基酸。系统进化树分析发现, TGF-蛋白按照不同的类型严格聚类, 斑马鱼TGF-1与其他鱼类的TGF-1聚集到一个分支, 在进化中非常保守。对斑马鱼胚胎进行RT-PCR和Real-Time PCR检测显示, TGF-1基因为母源表达基因, 在分节期之前的表达水平比较低, 而从咽囊期开始持续高水平的表达。胚胎整体原位杂交发现, TGF-1基因在斑马鱼24 hpf 胚胎中开始有特异信号出现, TGF-1基因的表达主要分布在腮弓、侧线原基、耳囊、嗅觉基板、心脏和前肾等处, 表明TGF-1基因可能参与斑马鱼胚胎免疫调节、循环系统发育和侧线形成。用低氧处理斑马鱼胚胎, 发现低氧处理24h后斑马鱼胚胎发育延迟。利用Real-Time PCR和胚胎整体原位杂交检测发现, 低氧处理后发育延迟的斑马鱼胚胎中TGF-1 mRNA表达量较常氧组显著降低。以上结果表明, TGF-1基因参与斑马鱼胚胎发育调控, 并且可能与低氧处理后斑马鱼胚胎发育延迟有关。研究结果将为深入研究斑马鱼TGF-1基因的功能奠定基础。       

斑马鱼DrSpin-1基因的克隆和特征分析

Spin蛋白家族是具有Spin/Ssty保守结构域并在配子发生过程中发挥关键作用的一类分子。研究利用简并引物PCR,从斑马鱼成熟卵母细胞SMART cDNA文库中筛选到260 bp的DrSpin-1和DrSpin-2部分序列,经序列同源性比对,斑马鱼DrSpin-1的部分氨基酸序列与银鲫CagSpin一致性高达81%。利用RACEPCR从该cDNA文库中获得斑马鱼DrSpin-1的全长cDNA序列。序列分析表明,DrSpin-1全长cDNA为1082 bp,开放阅读框771 bp,编码257个氨基酸,具有三个Spin/Ssty保守域,8个可能的磷酸化位点,初步确定斑马鱼DrSpin-1是Spin基因家族成员。斑马鱼DrSpin-1蛋白与已报道的鱼类Spin蛋白多重序列比对表明,DrSpin-1蛋白与银鲫CagSpin蛋白同源性最高。可以推测克隆得到的斑马鱼DrSpin-1与已知功能的银鲫CagSpin具有相近的表达谱和生物学功能,可能在配子发生和受精过程中发挥重要作用。     

α-L-岩藻糖苷酶基因在斑马鱼发育过程中的功能分析

α-L-岩藻糖苷酶(alpha-L-fucosidase,FUCA1/2)是糖基水解酶家族的成员,参与糖蛋白、糖脂等生物大分子的分解代谢反应.为了解α-L-岩藻糖苷酶在动物发育中的功能,检测了fuca1和fuca2在斑马鱼组织和胚胎发育中的特异表达模式.fuca1和fuca2基因在斑马鱼成体中的表达存在组织差异,在斑马鱼的精巢中表达量最高,此外肝脏中也检测到fuca1和fuca2的高表达.在斑马鱼胚胎发育中,在其整个胚胎发育时期均检测到了fuca1和fuca2的表达.构建了fuca1基因的反义表达载体,显微注射到斑马鱼受精卵.统计结果显示反义载体注射导致斑马鱼胚胎高致死率.研究表明,fuca1和fuca2基因在鱼类胚胎发育和成体的精巢、卵巢与肝脏发育中可能具有重要的作用.     

斑马鱼miR-196a-1基因敲除品系的构建

microRNA(miRNA)是一类内生的、长度约为19～23个核苷酸的非编码RNA,通过影响mRNA的稳定性和翻译,来参与基因表达的转录后调控。生物信息学分析表明该基因在各个物种中高度保守。为了阐明该基因在肠道发育中的作用,本文利用Cloning free CRISPR/Cas9基因编辑技术构建miR-196a-1基因敲除的斑马鱼品系。首先通过分析软件筛选出斑马鱼miR-196a-1基因的两个敲除位点,两个敲除位点相隔132 bp,利用PCR技术扩增miR-196a-1的向导DNA,再以向导DNA为模板转录得到miR-196a-1的sgRNA,将miR-196a-1基因的sgRNA和Cas9蛋白共同注射到斑马鱼胚胎1细胞期胚胎中。斑马鱼胚胎发育到36 hpf后进行基因编辑的有效性检测,研究结果显示,miR-196a-1基因出现102 bp碱基的缺失,表明CRISPR/Cas9系统对miR-196a-1基因的敲除有效。对其F0代、F1代、F2代进行筛选,成功获得斑马鱼miR-196a-1基因敲除品系,为研究miR-196a-1在肠道发育中的作用奠定了基础。     

斑马鱼心脏发育候选基因Pygo1多克隆抗体的制备及检测

Pygo1是心脏发育候选基因,可能在斑马鱼心脏发育过程中起着重要的调控作用.利用生物信息学选择斑马鱼Pygo1基因抗原亲水区,将扩增出的PCR片段克隆到原核表达pGEMT-4T-1载体中,转入E.coli中后通过IPTG(Isopropylβ-D-thiogalactoside)分别诱导表达GST融合蛋白.蛋白经纯化分离后用于免疫新西兰大白兔制备多克隆抗体.用Western Blot检测抗体的效价和特异性.     

斑马鱼和鲤miR-1-2/133a-1基因间序列活性和调控研究

分别以斑马鱼(Danio rerio)和鲤(Cyprinus carpio L.)为研究材料,通过克隆斑马鱼和鲤miR-1-2和133a-1的基因间增强子序列,利用活体和离体实验探讨其是否具有肌肉特异性;并通过荧光素酶报告系统等研究转录因子MyoD是否调控该序列。研究结果显示:在活体实验中,无论斑马鱼还是鲤, miR-1-2/133a-1基因间序列均有肌肉特异性,且保留有保守区域(cr,含有E-box)的序列,注射72h后GFP表达量明显高于其他突变体。体外细胞实验也显示,转染含有cr序列的实验组,分化后的荧光素酶活性明显高于分化前。进一步探讨MyoD对miR-1-2/133a-1基因间序列的调控作用结果显示:无论是斑马鱼还是鲤, miR-1-2/133a-1基因间序列活性均受MyoD调控。结果为完善鱼类肌肉发育机理,及未来的鱼类分子育种奠定基础。     

斑马鱼Zar1和Zar1-like基因的克隆及其表达分析

合子阻滞基因1(Zygote arrest 1,Zar1)及Zar1-like(Zar1L)作为母源基因,在小鼠卵子-合子转变过程中发挥着极其重要的作用。研究以斑马鱼为对象,采用cDNA末端快速克隆(RACE)技术获得了Zar1L cDNA全长,并采用RT-PCR技术检测Zar1和Zar1L在斑马鱼不同组织器官、卵母细胞和胚胎发育时期的表达情况。Zar1L cDNA的全长为1146 bp,编码311个氨基酸残基,5′非编码区20 bp,3′非编码区190 bp。多重序列比对结果显示,C-末端的序列非常保守,相似性高达74%,具有非典型的PHD基序(Plant homeo domain),并具有两个C4类型的锌指结构。在基因结构上,Zar1和Zar1L均由四个外显子构成,两个基因的每一个外显子在大小上也比较相近。分子进化表明斑马鱼Zar1和Zar1L属于两个不同的基因,可能是同一来源的祖先基因重复和进化的结果。RT-PCR结果表明斑马鱼Zar1和Zar1L的表达模式相似,都是卵巢特异表达基因,在卵母细胞及早期胚胎中可检测到大量转录本的存在,囊胚、原肠胚期后下降,但在整个胚胎发育期都可检测到转录本。斑马鱼Zar1和Zar1L mRNA的表达水平的一致性,似乎暗示着两个基因的功能基本一致。表达模式分析表明Zar1和Zar1L可能与斑马鱼卵子-合子转变有关,也可能与其他的胚胎发育过程有关。       

斑马鱼(Danio rerio)mapk1基因对tp53基因调控研究

旨在研究斑马鱼(Danio rerio)mapk1基因对tp53基因的调控作用.通过生信网站分析斑马鱼tp53启动子序列与mapk1基因CDS序列,构建斑马鱼tp53启动子荧光素酶报告基因质粒pGL3-tp53-Luc和mapk1表达质粒pCMV-Tag2B-mapk1.利用Luciferase实验验证tp53启动子报...     

斑马鱼HO1基因的表达特征及功能研究

实验对血红素加氧酶1（HO1）在斑马鱼发育中的功能进行了研究。多重序列比对结果显示,斑马鱼HO1与哺乳类、鸟类及其他鱼类的HO1氨基酸序列的总体相似性为44.1%86.8%,血红素结合标签相似性为87.5%95.8%。对斑马鱼早期胚胎和成鱼各组织进行RT-PCR检测,结果显示HO1转录本母源存在,HO1 mRNA的表达水平在尾芽期前较低,到咽囊期迅速上升并稳定在较高水平。HO1基因在斑马鱼成鱼多个组织中均有表达,在肝脏、脾、鳃、肾中的表达量较高。WISH结果显示,HO1基因在斑马鱼胚胎的卵黄合胞层、眼和血液中的表达量较高。利用超表达和基因敲降技术发现,注射HO1 mRNA使HO1基因过表达对斑马鱼早期胚胎发育无明显影响。注射HO1 MO使HO1基因表达抑制可导致斑马鱼胚胎出现发育迟缓、围心腔水肿、尾部消失等不同程度的畸形。HO1 MO导致的斑马鱼胚胎发育异常可被HO1 mRNA回复。利用Real-Time PCR研究发现,HO1基因表达抑制可导致IGF1表达量显著下降,IGFBP1表达量显著升高。这些结果表明斑马鱼HO1基因可通过调节IGF信号途径调控胚胎的正常发育。       

人p17.3基因及其推导蛋白的结构和功能分析

用生物信息学技术研究人类神经元蛋白p17.3的基因结构、染色体定位、组织表达特征及其推导蛋白的理化特性、空间结构和功能等。以高通量基因组序列(HTGS)数据库及SAGE文库为基础对p17.3基因进行染色体定位及组织表达谱分析。通过DAS和TMpred等程序预测其蛋白质结构及功能。利用PDB程序模拟其三维空间结构。研究表明p17.3基因定位于人类染色体Xp11.2-3,在多种组织中广谱表达,但在前列腺表达量最高。其编码蛋白含有一个PEST区域、一个线粒体靶向序列和一个潜在“跨膜螺旋”结构。p17.3基因可能是一个参与神经元发育调控的基因。     

Large-Scale Forward Genetic Screening Analysis of Development of Hematopoiesis in Zebrafish

Zebrafish is a powerful model for the investigation of hematopoiesis.In order to isolate novel mutants with hematopoietic defects, large-scale mutagenesis screening of zebrafish was performed.By scoring specific hematopoietic markers,52 mutants were identified and then classified into four types based on specific phenotypic traits.Each mutant represented a putative mutation of a gene regulating the relevant aspect of hematopoiesis,including early macrophage development,early granulopoiesis,embryonic myelopoiesis,and definitive erythropoiesis/lymphopoiesis.Our method should be applicable for other types of genetic screening in zebrafish.In addition,further study of the mutants we identified may help to unveil the molecular basis of hematopoiesis.     

封闭群斑马鱼的遗传生化标记研究

目的通过对AB、LF、ST（short tail,本地短尾群）三个封闭群斑马鱼的研究比较,建立封闭群斑马鱼的遗传生化标记。方法依照国标GB／T14927.1-2008的遗传操作规程优化实验条件,对三种群斑马鱼的7个遗传生化位点进行研究,以得到其遗传生化图谱。结果6-磷酸葡萄糖脱氢酶（Gpd）、过氧化氢酶（Ce）、苹果酸脱氢酶（Mod）、葡萄糖磷酸异构酶（Gpi）、酯酶（ES）位点表现出多态性。对三种群多态性位点进行卡方分析,Gpd、Gpi位点群间差异显著（P〈0.01）,Ce、Mod有群间差异（P〈0.05）。碱性磷酸酶（AKP）、乳酸脱氢酶（LDH）位点,各品系谱带较一致,未见同工酶多态性。结论Gpd、Ce、Mod、Gpi、ES可作为3种封闭群斑马鱼群间评价的生化标记,通过进一步研究,可建立封闭群斑马鱼的遗传标准。     

Crystal Structure of Zebrafish Hatching Enzyme 1 from the Zebrafish Danio rerio

Fish hatching enzymes are zinc metalloproteases that digest the egg envelope (chorion) at the time of hatching. The crystal structure of zebrafish hatching enzyme 1 (ZHE1) has been solved at 1.10 Å resolution. ZHE1 is monomeric, is mitten shaped, and has a cleft at the center of the molecule. ZHE1 consists of three 3₁₀-helices, three α-helices, and two β-sheets. The central cleft represents the active site of the enzyme that is crucial for substrate recognition and catalysis. Alanine-scanning mutagenesis of the two substrate peptides has shown that AspP1′ contributes the most and that the residues at P4-P2′ also contribute to the recognition of the major substrate peptide by ZHE1, whereas GluP3′ and the hydrophobic residues at P4-P2, P2′, and P5′ contribute significantly to the recognition of the minor substrate peptide by ZHE1. Molecular models of these two substrate peptides bound to ZHE1 have been built based on the crystal structure of a transition-state analog inhibitor bound to astacin. In substrate-recognition models, the AspP1′ in the major substrate peptide forms a salt bridge with Arg182 of ZHE1, while the GluP3′ in the minor substrate peptide instead forms a salt bridge with Arg182. Thus, these two substrate peptides would be differently recognized by ZHE1. The shapes and electrostatic potentials of the substrate-binding clefts of ZHE1 and the structurally similar proteins astacin and bone morphogenetic protein 1 are significantly dissimilar due to different side chains, which would confer their distinctive substrate preferences.     

T淋巴瘤侵袭转移诱导因子1的结构与功能

T淋巴瘤侵袭转移诱导因子1(T-lymphoma invasion and metastasis inducing factor 1，Tiam1)是Ras相关的C3肉毒素底物1(Ras-Related C3 botulinum toxin substrate 1，Rac1)的特异性鸟苷酸交换因子(guanine nucleotide ex-change factor，GEF)。在细胞外信号刺激下Tiam1可以促进Rac1从无活性的GDP结合状态向有活性的GTP结合状态转换，有活性的Rac1-GTP与不同的下游效应分子相互作用，从而影响多种细胞事件。     

Phylogenetic Analysis of Zebrafish Basic Helix-Loop-Helix Transcription Factors

The basic helix-loop-helix (bHLH) proteins play important regulatory roles in eukaryotic developmental processes including neurogenesis, myogenesis, hematopoiesis, sex determination, and gut development. Zebrafish is a good model organism for developmental biology. In this study, we identified 139 bHLH genes encoded in the zebrafish genome. Phylogenetic analyses revealed that zebrafish has 58, 29, 21, 5, 19, and 5 bHLH members in groups A, B, C, D, E, and F, respectively, while 2 members were classified as “orphan.” A comparison between zebrafish and human bHLH repertoires suggested that both organisms have a certain number of specific bHLH members. Eight zebrafish bHLH genes were found to have multiple coding regions in the genome. Two of these, Bmal1 and MITF, are good anchor genes for identification of fish-specific whole-genome duplication events in comparison with mouse and chicken genomes. The present study provides useful information for future studies on gene family evolution and vertebrate development. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Zebrafish Cytosolic Carboxypeptidases 1 and 5 Are Essential for Embryonic Development

The cytosolic carboxypeptidases (CCPs) are a subfamily of metalloenzymes within the larger M14 family of carboxypeptidases that have been implicated in the post-translational modification of tubulin. It has been suggested that at least four of the six mammalian CCPs function as tubulin deglutamylases. However, it is not yet clear whether these enzymes play redundant or unique roles within the cell. To address this question, genes encoding CCPs were identified in the zebrafish genome. Analysis by quantitative polymerase chain reaction indicated that CCP1, CCP2, CCP5, and CCP6 mRNAs were detectable between 2 h and 8 days postfertilization with highest levels 5–8 days postfertilization. CCP1, CCP2, and CCP5 mRNAs were predominantly expressed in tissues such as the brain, olfactory placodes, and pronephric ducts. Morpholino oligonucleotide-mediated knockdown of CCP1 and CCP5 mRNA resulted in a common phenotype including ventral body curvature and hydrocephalus. Confocal microscopy of morphant zebrafish revealed olfactory placodes with defective morphology as well as pronephric ducts with increased polyglutamylation. These data suggest that CCP1 and CCP5 play important roles in developmental processes, particularly the development and functioning of cilia. The robust and similar defects upon knockdown suggest that each CCP may have a function in microtubule modification and ciliary function and that other CCPs are not able to compensate for the loss of one.