斑马鱼胚胎发育中细胞凋亡的研究进展

细胞凋亡是细胞在基因调控下发生的主动消亡过程,在脊椎动物胚胎发育过程中非常重要。斑马鱼作为一种十分理想的发育分子生物学研究模型,在有关细胞凋亡在诸如形态发生、性别分化等方面功能之活体在位研究中日益受到重视。目前,斑马鱼胚胎发育中主要凋亡通路研究已进行了不少工作,特别是caspase及其它凋亡调控基因在斑马鱼中已被成功克隆,通过转基因斑马鱼胚胎中胁迫诱导细胞凋亡并研究其信号通路以及斑马鱼胚胎形态发生的异常改变,为阐明这些凋亡调控基因与发育之间的关系提供了一个强有力的手段。     

A bioenergetic model for zebrafish Danio rerio (Hamilton)

A bioenergetics model was developed from observed consumption, respiration and growth rates for zebrafish Danio rerio across a range (18–32° C) of water temperatures, and evaluated with a 50 day laboratory trial at 28° C. No significant bias in variable estimates was found during the validation trial; namely, predicted zebrafish mass generally agreed with observed mass.     

斑马鱼对无氧运动训练的适应性变化

该研究以斑马鱼为(Danio rerio)对象,研究了四周无氧运动训练对斑马鱼行为、形态、生长、肌肉生化组分及代谢酶活性的影响。旨在探索斑马鱼对无氧运动训练的适应性变化,为进一步了解鱼类适应无氧运动训练的分子机制提供基础数据。结果发现:斑马鱼的日常活跃程度经四周无氧运动训练后显著降低,群聚程度增加;训练组个体体重和体长增长减缓,更利于运动;肌糖原含量显著增加,运动持久能力加强;肌肉乳酸脱氢酶(LDH)活性显著增高,柠檬酸合成酶(CS)活性显著降低,无氧代谢能力加强。即,斑马鱼无氧运动能力和无氧代谢能力在训练后得以明显提升。     

斑马鱼繁殖内分泌学研究进展

作为四大模式动物之一,斑马鱼(Danio rerio)广泛应用于胚胎学、发育生物学、毒理学、分子生物学等研究。但关于斑马鱼繁殖内分泌生理和环境毒理方面的研究少见报道,本文综述了近年来,在内分泌学方面以斑马鱼作为实验动物的研究概况。     

Biochemical and molecular characterization of galectins from zebrafish (Danio rerio): notochord-specific expression of a prototype galectin during early embryogenesis

Galectins are a family of beta-galactoside-binding lectins that on synthesis are either translocated into the nucleus or released to the extracellular space. Their developmentally regulated expression, extracellular location, and affinity for extracellular components (such as laminin and fibronectin) suggest a role in embryonic development, but so far this has not been unequivocally established. Zebrafish constitute an ideal model for developmental studies because of their external fertilization, transparent embryos, rapid growth, and availability of a large collection of mutants. As a first step in addressing the biological roles in zebrafish embryogenesis, we identified and characterized members of the three galectin types: three protogalectins (Drgal1-L1, Drgal1-L2, Drgal1-L3), one chimera galectin (Drgal3), and one tandem-repeat galectin (Drgal9-L1). Like mammalian prototype galectin-1, Drgal1-L2 preferentially binds to N-acetyllactosamine. Genomic structure of Drgal1-L2 revealed four exons, with the exon-intron boundaries conserved with the mammalian galectin-1. Interestingly, this gene also encodes an alternatively spliced form of Drgal1-L2 that lacks eight amino acids near the carbohydrate-binding domain. Zebrafish galectins exhibited distinct patterns of temporal expression during embryo development. Drgal1-L2 is expressed postbud stage, and its expression is strikingly specific to the notochord. In contrast, Drgal1-L1 is expressed maternally in the oocytes. Drgal1-L3, Drgal3, and Drgal9-L1 are expressed both maternally and zygotically, ubiquitously in the adult tissues. The distinct temporal and spatial patterns of expression of members of the zebrafish galectin repertoire suggest that each may play distinct biological roles during early embryogenesis.     

Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities

In May 2005, a disease outbreak was investigated at a zebrafish (Danio rerio) research facility experiencing severe losses. Mycobacterium haemophilum was isolated from these fish and the disease was subsequently recreated in experimentally infected zebrafish. Fish exhibited signs characteristic of mycobacteriosis, including granuloma formation and severe, diffuse, chronic inflammation. Bacteria were observed in multiple tissues, including the central nervous system. Biofilm samples from the outbreak facility were PCR positive for M. haemophilum, suggesting biofilms might act as a reservoir for infection. Zebrafish appear to be particularly vulnerable to M. haemophilum, and measures such as quarantine and treatment of incoming water should be implemented to minimize the likelihood of introduction of this bacterium to zebrafish research facilities. Zebrafish are already a well-established laboratory animal model for genetics, toxicology and disease, their susceptibility to M. haemophilum may make them useful for the study of this bacterium in the future.     

Gene-Gun-Mediated Transfer of Reporter Genes to Somatic Zebrafish (Danio rerio) Tissues

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-μm microcarriers and 1 μg of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 μg of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when using the biolistic DNA delivery parameters and target tissues described. Received August 20, 1999; accepted January 6, 2000.     

Validation of Zebrafish (Danio rerio) Reference Genes for Quantitative Real-time RT-PCR Normalization

The normalization of quantitative real time RT-PCR (qRT-PCR) is important to obtain accurate gene expression data. The most common method for qRT-PCR normalization is to use reference, or house- keeping genes. However, there is emerging evidence that even reference genes can be regulated under different conditions, qRT-PCR has only recently been used in terms of zebrafish gene expression studies and there is no validated set of reference genes. This study characterizes the expression of nine possible reference genes during zebrafish embryonic development and in a zebrafish tissue panel. All nine reference genes exhibited variable expression. The β-actin, EF1α and Rp113α genes comprise a validated reference gene panel for zebrafish developmental time course studies, and the EF1α, Rp113α and 18S rRNA genes are more suitable as a reference gene panel for zebrafish tissue analysis. Importantly, the zebrafish GAPDH gene appears unsuitable as reference gene for both types of studies.     

Comparative osteology of the Danio (Cyprinidae: Ostariophysi) axial skeleton with comments on Danio relationships based on molecules and morphology

Danio is a diverse group of small, colourful and easily bred freshwater fishes native to Southern Asia. Biological interest in danios has increased in recent years because the zebrafish, Danio rerio , has become an important model organism, particularly for studies of vertebrate developmental biology and genetics. Though several phylogenetic studies of Danio have been done on a subset of Danio species, the resulting phylogenies conflict in detail. To examine the utility of osteology for systematics of this group at the species level, we studied the axial skeleton for 11 species of Danio. We analyse our morphological data alone and combined with DNA sequence data for five gene sequences generated in earlier phylogenetic studies. The axial skeleton of Danio exhibits 14 characters that prove useful in phylogenetic analysis. Both molecular and morphological data support the monophyly of the danios included in our analysis and both data sets support the monophyly of two subclades: a deep-bodied group and a slender-bodied group. Separate analysis of molecular and morphological data sets show that the molecular data resolves relationships within the slender subclade whereas the topology of the deep-bodied subclade is determined by morphological data.  © 2002 The Linnean Society of London. Zoological Journal of the Linnean Society , 2002, 135 , 529–546.     

不同大菱鲆(Scophthalmus maximus)个体肠道菌群结构差异研究

目的:肠道微生物能够帮助宿主完成多种生理生化功能,对宿主的健康生长也起到非常重要的作用。大菱鲆(Scophthalmus maximus)是我国北方最重要的海水养殖品种之一。然而,细菌性疾病的频发造成大规模的鱼类死亡。此外,许多大菱鲆致病菌也被证实是人类潜在的致病菌。因此鉴定成年养殖大菱鲆肠道中所包含的核心菌群,并筛选可能与大菱鲆或人类健康密切相关的细菌尤为重要。方法:构建三个大菱鲆个体肠道16S rRNA文库,利用限制性片段长度多态性分析(RFLP)及生物信息学技术研究三个大菱鲆个体肠道菌群结构及多样性。结果:共得到758个阳性克隆,16个OTU类型。分类分析显示,变形菌门在大菱鲆肠道中占优势地位。进一步将细菌按属来分类显示,弧菌属所占的比例最高,约为95.3%。韦恩图显示大菱鲆三个个体共有的OTU类型数量为3,分别占三个大菱鲆个体肠道文库中阳性克隆总数的94.9%、96.7%以及93.5%。分类结果显示它们分别属于条件致病菌哈氏弧菌、副溶血弧菌和创伤弧菌。结论:大菱鲆的核心肠道菌群为弧菌属,它的主要成员哈氏弧菌、副溶血弧菌和创伤弧菌对大菱鲆及人类的健康起到关键的作用。在水产养殖过程中,通过监控这些弧菌种类的浓度变化能够及时对养殖环境进行评价,并及时调整温度、水质等因素,这为预防大菱鲆细菌性疾病,保证人类健康提供重要的理论依据。     

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.     

基于密码子优化策略的斑马鱼FOXP3A分子的原核表达及多克隆抗血清制备

目的:构建斑马鱼FOXP3A融合蛋白的原核表达系统,诱导表达并制备多克隆抗血清。方法:选取斑马鱼foxp3a基因cDNA的894 bp特异区段(s-foxp3a),对该片段进行密码子优化后构建原核表达载体pET-32a-s-foxp3a,转化大肠杆菌BL21(DE3),诱导表达斑马鱼S-FOXP3A-His融合蛋白并纯化,纯化后的蛋白免疫家兔,制备斑马鱼FOXP3A蛋白的多克隆抗血清,4次免疫后取血清,采用间接ELISA法检测抗血清效价。结果:在16℃经0.5 mmol/LIPTG诱导10 h的条件下,SDS-PAGE分析表明斑马鱼S-FOXP3A-His融合蛋白以包涵体形式产生;间接ELISA检测结果显示,FOXP3A蛋白多克隆抗血清的效价在1.6×10~5以上。结论:制备了高效价的斑马鱼FOXP3A多克隆抗血清,为进一步解析斑马鱼FOXP3A蛋白的功能提供了基础工具。     

九香虫(椿象)体内可培养细菌的多样性分析

【目的】认识药用昆虫九香虫(Aspongopus chinesis Dallas)成虫体内可培养细菌资源多样性。【方法】运用纯培养法、反转录重复因子扩增(BOXA1R-PCR)分析技术、16S r RNA基因测序和系统发育分析对样品中可培养细菌进行多样性研究,测定了分离菌株的抗菌特性、吲哚乙酸(IAA)含量和产淀粉酶活性等指标。【结果】通过6种不同培养基共分离得到52株菌落特征不同的细菌菌株。基于菌落特征和BOXA1R-PCR图谱选取12株代表菌株用于16S r RNA基因序列测定。16S r RNA基因序列系统发育分析显示,52株菌株分属于芽孢杆菌属(Bacillus)、假单胞菌属(Pseudomonas)、寡养单胞菌属(Stenotrophomonas)和伯克霍尔德氏菌属(Burkholderia)4个属,其中芽孢杆菌属(Bacillus)为优势菌属。分离到的52株细菌有44株(占总分离菌株的84.6%)表现出对供试病原菌具有较好的抑制作用,高达94.2%的分离菌株能产IAA,有43株(占总分离菌株的82.7%)表现出淀粉酶活性。【结论】九香虫内细菌种群较为多样,具有潜在应用价值。     

Genome Analysis of Pseudoloma neurophilia: A Microsporidian Parasite of Zebrafish (Danio rerio)

Microsporidia are highly successful parasites that infect virtually all known animal lineages, including the model Danio rerio (zebrafish). The widespread use of this aquatic model for biomedical research has resulted in an unexpected increase in infections from the microsporidium Pseudoloma neurophilia, which can lead to significant physical, behavioral, and immunological modifications, resulting in nonprotocol variation during experimental procedures. Here, we seek to obtain insights into the biology of P. neurophilia by investigating its genome content, which was obtained from only 29 nanograms of DNA using the MiSeq technology and paired‐end Illumina sequencing. We found that the genome of P. neurophilia is phylogenetically and genetically related to other fish‐microsporidians, but features unique to this intracellular parasite are also found. The small 5.25‐Mb genome assembly includes 1,139 unique open‐reading frames and an unusually high number of transposable elements for such a small genome. Investigations of intragenomic diversity also provided strong indications that the mononucleate nucleus of this species is diploid. Overall, our study provides insights into the dynamics of microsporidian genomes and a solid sequence reference to be used in future studies of host–parasite interactions using the zebrafish D. rerio and P. neurophilia as a model.     

A Window to the Subsurface: Microbial Diversity in Hot Springs of a Sulfidic Cave (Kaklik,Turkey)

Caves are windows to the extreme habitats of deep subsurface, and provide answers of unknowns about the underground life. Furthermore, sulfidic caves are important analogues for the early Earth environments, since some environmental conditions are common, such as high sulfur concentration, high temperature and oxygen-poor conditions. Kaklik Cave (Denizli, Turkey) with its travertine formation, carbonate- and sulfur-rich thermal springs, exhibits a unique ecosystem as a sulfidic cave. This study represents the first molecular survey of the microbial community in the Kaklik Cave, Turkey using high-throughput 16S rRNA gene amplicon sequencing analysis. An average of 859–2,416 operational taxonomic units per sample were observed including 25 bacterial phyla and 3 archaeal phyla. The bacterial diversity profiles were generally dominated by Epsilonproteobacteria and Gammaproteobacteria. At the carbonate-rich hot spring, that formed travertine structure, 9.7% of sequence reads affiliated with Thiofaba spp. In contrast, 38.74% of the total sequence reads at the sulfidic hot spring samples associated with the genus Sulfurimonas and Sulfurovum. In the archaeal community composition, Thermoplasmata was the most abundant group in all sampling areas. The 454-pyrotag results provide leads about ammonia-, nitrite- and sulfur-oxidation as well as sulfur-reduction, carbon dioxide fixation, and nitrogen fixation.     

基于线粒体12S和16S rRNA基因部分序列探讨蚱科12种的系统发育关系

用ABI377自动测序仪测定了蚱科5属11个种的12s和16S rRNA基因部分序列,并从GenBank获得1属1种的同源序列;用Clustal X1．81比较其同源性,用Mega2．1计算序列变异性和遗传距离。在获得的736bp序列中,A T含量为71．2％～77．5％,平均为73．9％;G C含量为22．5％～28．8％,平均为26．1％。经Clustal X1．81软件比对,共得到755个位点,其中简约信息位点185个。以Cylindraustralia kochii为外群,构建NJ、MP和ML分子系统树,结果表明：(1)蚱属并非一个单系群,而是一个并系群;(2)环江柯蚱Coptltettix huanjiangensis和贡山柯蚱C.gongshanensis为同一个种,即贡山柯蚱,而环江柯蚱是贡山柯蚱的同物异名。     

Effects of diet,habitat, and phylogeny on the fecal microbiome of wild African savanna (Loxodonta africana) and forest elephants (L. cyclotis)

The gut microbiome, or the community of microorganisms inhabiting the digestive tract, is often unique to its symbiont and, in many animal taxa, is highly influenced by host phylogeny and diet. In this study, we characterized the gut microbiome of the African savanna elephant (Loxodonta africana) and the African forest elephant (Loxodonta cyclotis), sister taxa separated by 2.6–5.6 million years of independent evolution. We examined the effect of host phylogeny on microbiome composition. Additionally, we examined the influence of habitat types (forest versus savanna) and diet types (crop‐raiding versus noncrop‐raiding) on the microbiome within L. africana. We found 58 bacterial orders, representing 16 phyla, across all African elephant samples. The most common phyla were Firmicutes, Proteobacteria, and Bacteroidetes. The microbiome of L. africana was dominated by Firmicutes, similar to other hindgut fermenters, while the microbiome of L. cyclotis was dominated by Proteobacteria, similar to more frugivorous species. Alpha diversity did not differ across species, habitat type, or diet, but beta diversity indicated that microbial communities differed significantly among species, diet types, and habitat types. Based on predicted KEGG metabolic pathways, we also found significant differences between species, but not habitat or diet, in amino acid metabolism, energy metabolism, and metabolism of terpenoids and polyketides. Understanding the digestive capabilities of these elephant species could aid in their captive management and ultimately their conservation.