二甲基联苯胺对斑马鱼早期胚胎发育的表观影响

研究了3,3'-二甲基联苯胺对斑马鱼早期胚胎发育的表观毒性影响.试验证明4.32 mg/L以上高浓度的二甲基联苯胺都能导致斑马鱼受精卵发生卵黄凝聚,使胚胎发育终止而死亡.低浓度下2.16 mg/L的二甲基联苯胺对斑马鱼受精卵的发育有一定的毒害作用,但有近一半的胚胎存活,这可能是影响其胚胎发育成活的一个临界浓度;还发现低...     

肌间刺缺失突变对斑马鱼胚胎发育过程中肌肉发育的影响

为阐明肌间刺缺失对斑马鱼(Danio rerio)胚胎发育时期肌肉发育的影响,采用qRT-PCR的方法,检测分析了斑马鱼不同肌间刺表型间5个肌肉特异性基因(mef2、myf5、myod、myog和sox6)在囊胚期(3hpf,hourspost fertilization)、原肠胚期(6 hpf)、体节期(12 hpf)、咽囊期(24 hpf)和孵化期(72 hpf)5个发育时期的表达情况,对比分析了肌间刺缺失对斑马鱼胚胎发育过程中肌肉发育特异性基因的时序表达差异和肌肉发育的影响,以及对受精率、孵化率和畸形率的影响。结果显示, 5个特异性基因在肌间刺缺失突变型与野生型斑马鱼各发育时期的表达量无显著性差异(P0.05)。其中,myf5、myod基因在原肠胚期表达量升高,在体节期和咽囊期开始逐步下降;mef2、myog和sox6基因在囊胚期和原肠胚期表达量很低,在体节期和咽囊期表达量迅速升高,并在孵化期开始下降。此外,受精率、孵化率和畸形率的统计结果显示,肌间刺突变型与野生型间亦无显著性差异(P0.05)。综上,推测肌间刺的缺失对斑马鱼胚胎发育及胚胎发育过程中肌肉的发育没有影响。     

斑马鱼早期胚胎发育囊胚sphere时期的蛋白组学研究

斑马鱼(Danio rerio)早期胚胎发育囊胚时期是胚胎发育过程的关键时期,利用i TRAQ蛋白质质谱分析技术,检测斑马鱼早期胚胎发育过程中囊胚sphere时期的蛋白质表达情况,并分析该时期表达的蛋白质的相应功能和参与调控的生物过程。以野生型斑马鱼发育至sphere时期即4 hpf的胚胎为样本,利用i TRAQ标记与LC-MS/MS串联质谱技术,结合数据库比对,对该时期表达的蛋白质进行定性和定量的鉴定分析。检测结果共鉴定到的总蛋白数为1 178个,利用生物信息学进行功能分析,发现这些蛋白广泛参与了细胞信号传递、细胞运动和细胞骨架构建、细胞增殖、细胞分化、物质合成与代谢等各项重要的生命活动过程。研究表明,利用i TRAQ标记的方法可以对4hpf时期的斑马鱼胚胎中的蛋白质进行有效的分离和鉴定,并初步建立了斑马鱼早期发育关键阶段sphere时期的蛋白质组表达图谱,以期为斑马鱼胚胎发育过程中蛋白质组学和调控机制的研究提供参考。     

UVB辐射导致斑马鱼早期胚胎发育异常和DNA损伤的初步研究

为研究UVB辐射对斑马鱼(Danio rerio)早期胚胎发育的影响,采用波长302nm,强度分别为120、240、310、420μW/cm2的UVB照射斑马鱼早期发育阶段的胚胎,照射时间分别为1、3、5、10min,用于研究UVB照射对形态学及DNA损伤的影响;将原肠期、体节期胚胎及孵化后2d仔鱼经UVB照射,设立未经UVB照射组为对照组,观察记录UVB照射后胚胎发育情况,统计死亡率、畸形率;应用单细胞凝胶电泳(SCGE)技术检测不同UVB照射条件对DNA损伤的影响。结果表明:(1)UVB照射对斑马鱼早期胚胎发育形态有明显的影响,能够造成尾部弯曲、围心腔扩大、脊柱扭曲等多种畸形甚至死亡;(2)在检测UVB照射与DNA损伤的关系时,发现UVB照射对胚胎细胞中的DNA能够产生比较明显的损伤,且DNA损伤随照射强度的增加和照射时间的延长而加剧。在所研究的3个发育阶段中,原肠胚受损最为严重,但未发现明显的剂量累积效应。结论认为,UVB照射对斑马鱼早期胚胎发育有明显影响,不同的照射强度和时间造成的影响差异显著;不同发育阶段的斑马鱼胚胎对UVB照射的耐受程度也不同。     

环境污染物对斑马鱼早期胚胎转座子转录活性的影响

转座子在生物基因组中占据重要的组成部分,是基因组中可移动和扩展的重要元件,其转座活性受外界环境因子调控。为了探讨环境应激对基因组转座子表达活性的影响,用环境污染物二噁英(TCDD)和重金属Cu~(2+)或Cd~(2+)处理斑马鱼早期胚胎,通过荧光定量PCR(q RT-PCR)比较处理前后9种结构完整的转座子转录活性变化,这9种转座子分别是DNA转座子Tc1家族的Tc-a、Tc-b、Tc-c、Tc-d、Tc-e,反转录转座子病毒家族ZB-ERV-1、ZB-ERV-2、反转录转座子LINE家族L1-323和L1-21。结果发现,TCDD使8个转座子转录活性明显下调,1个转座子显著上调;Cu~(2+)导致7个转座子上调,2个显著下调;Cd~(2+)导致6个转座子上调,3个显著下调。研究表明,斑马鱼转座子转录活性受环境因素影响显著,环境胁迫可能是转座子活性变化的原因之一。本研究对了解和评估环境因子对鱼类转座子活性的影响作用具有重要的意义,为进一步研究生物基因组的进化机制提供参考。     

jnk3基因在金鱼和斑马鱼不同发育时期胚胎与成体不同组织中的分化表达

JNKs是丝裂原活化蛋白激酶(MAPK)家族重要成员,参与应激反应和动物体轴的构建。为了进一步了解JNK家族在动物发育中的功能,首次分离和克隆了金鱼jnk3基因,研究了jnk3在金鱼和斑马鱼组织特异性和胚胎发育特异性表达状况。金鱼jnk3基因cDNA总长1 794 bp,其中阅读框1 293 bp,编码430个氨基酸。对jnk3基因序列及其推导的氨基酸序列进行同源性分析,结果显示,jnk3基因在脊椎动物较为保守,金鱼jnk3基因核苷酸序列与斑马鱼、人的同源性分别为94.1%和80.7%,金鱼jnk3基因氨基酸序列与斑马鱼、人的同源性分别为99.7%和93.4%。jnk3基因在鱼类成体中的表达存在着显著的组织差异,在金鱼和斑马鱼脑和精巢组织的表达具专一性,此外在斑马鱼心脏中也检测到jnk3的表达。RT-PCR检测结果显示,在鱼类胚胎发育中,神经胚期开始检测到jnk3基因mRNA表达,从神经胚到心跳期胚胎,jnk3基因表达水平逐渐增高,此后直至出膜一直保持较高的表达水平。研究表明,jnk3基因可能在鱼类后期的胚胎发育和成体脑、精巢与心脏发育中具有重要作用。     

鲥鱼的早期发育

本文报道在江西省峡江县赣江鲥鱼产卵场人工授精后鲥鱼卵的胚胎发育及仔稚鱼形态特征。     

重金属Cu~(2+)、Cd~(2+)、Hg~(2+)对斑马鱼胚胎发育的毒性效应

在半静态水的条件下,研究了铜离子(Cu~(2+))、镉离子(Cd~(2+))和汞离子(Hg~(2+))对斑马鱼(Danio rerio)胚胎发育的单一和联合毒性效应。采用48 h、72 h、96 h致死率和72 h、96 h孵化抑制率作为生理毒性终点,以其半数致死浓度(LC50)值作为毒性评价标准。结果表明:Cu~(2+)、Cd~(2+)和Hg~(2+)对斑马鱼胚胎的48 h-LC50分别为0.58 mg·L~(- 1)、0.72 mg·L~(- 1)和0.000 46 mg·L~(- 1),72 h-LC50分别为0.39 mg·L~(- 1)、1.15 mg·L~(- 1)和0.000 30 mg·L~(- 1),96 hLC50分别为0.08 mg·L~(- 1)、0.19 mg·L~(- 1)和0.000 18 mg·L~(- 1);Cu~(2+)、Cd~(2+)和Hg~(2+)的72 h孵化抑制率的LC50分别为0.05 mg·L~(- 1)、0.01 mg·L~(- 1)和0.000 04 mg·L~(- 1),96 h孵化抑制率的LC50分别为0.06 mg·L~(- 1)、0.05 mg·L~(- 1)和0.000 11 mg·L~(- 1);Cu~(2+)、Cd~(2+)和Hg~(2+)对斑马鱼胚胎毒性大小顺序为Hg~(2+)Cu~(2+)Cd~(2+)。Cu~(2+)、Cd~(2+)和Hg~(2+)对斑马鱼胚胎的致死率和孵化抑制率均具有明显的剂量-效应关系,72 h孵化抑制率可以作为斑马鱼胚胎最敏感的毒性终点指标。分别用单一毒性Cu~(2+)、Cd~(2+)和Hg~(2+)的96 h-LC50值,按毒性单位1∶1两两混合,对斑马鱼胚胎在第48、第72、第96小时的联合毒性均为协同作用;按毒性单位1∶1∶1混合共存时,对斑马鱼胚胎在第48、第72、第96小时的联合毒性均为拮抗作用。     

斑马鱼雄核发育技术研究

为探索人工诱导斑马鱼雄核发育二倍体克隆鱼的途径和方法,运用紫外线照射卵子使其遗传物质失活,利用斑马鱼体表花纹形态作为遗传标记,使用热休克技术阻止受精卵的第一次卵裂加倍染色体。试验结果表明总辐射剂量为120mJ cm2紫外线照射斑马鱼卵可以使雌核遗传物质完全失活;体表花纹为豹斑花纹被证实是由纯合隐性基因控制的性状,可以作为雄核发育遗传标记;遗传失活卵受精后经2min,(41 5±0 5)℃的热休克后出现两个染色体加倍的高峰期,分别为受精后的11min和23min,且后一时期效果优于前一时期,最高二倍体诱导率达46 15%;经遗传分析,共获得正常存活的雄核发育二倍体克隆鱼19尾,说明经过优化的紫外线结合热休克等染色体操作技术,可以成为诱导雄核发育二倍体克隆鱼的有效手段。     

2-乙基己酯-4-甲氧基肉桂酸(EHMC)对斑马鱼早期生命阶段的毒性效应

在水温(28.0±1.0)℃下,将斑马鱼(Danio rerio)受精卵暴露于2-乙基己酯-4-甲氧基肉桂酸(2-ethylhexyl-4-methoxycinnamic acid,EHMC)中,浓度为0μg·L^-1(对照组,仅0.1%DMSO助溶剂)、2μg·L^-1、20μg·L^-1和200μg·L^-1,每个浓度三个平行。每24 h更换四分之一相应浓度的EHMC,48 h、72 h、96 h后观察死亡和畸形,测量鱼心率、体长等,120 h后测定幼鱼的生长激素(growth hormone,GH)和胰岛素样生长因子(insulin-like growth factor,IGF-Ⅰ)含量及GH/IGF-I轴相关基因的m RNA表达和转录水平。结果表明：EHMC暴露未显著影响斑马鱼的孵化率(P>0.05),但使心率显著下降(P<0.05);高浓度EHMC(200μg·L^-1)下斑马鱼畸形率升高,幼鱼体长下降(P<0.05)。EHMC诱导的生长抑制效应可能与鱼体内I...     

低温驯化下斑马鱼LINE1的表达检测

长散布核元件-1(Long spread nuclear element-1,LINE1)是跳跃基因。前期比较基因组研究发现,南极鱼经历漫长的低温适应进化后,与南极圈外的同亚目鱼类相比较,在基因水平上LINE1的扩增效率高达8~300倍,但LINE1的扩增与鱼类抵御寒冷之间的关系尚未明了。本实验对斑马鱼(Danio rerio)胚胎成纤维细胞ZF4进行了不同时间梯度的低温处理(18℃、5 d和18℃、30 d),同时对斑马鱼成鱼也进行了不同时间的低温处理(10℃,3 h、6 h、1 d、3 d、5 d)。采用RT-qPCR检测了LINE1的mRNA水平,并克隆了斑马鱼LINE1基因启动子区,利用Luciferase双荧光报告系统,在ZF4细胞中验证LINE15’UTR在低温压力下的生物活性。结果显示,短时间低温处理下,ZF4细胞中LINE1 mRNA水平有所降低,而在长时间低温处理中,LINE1的mRNA水平显著升高。在成鱼中,短时间低温处理下,LINE1 mRNA水平降低;长期低温处理下,LINE1 mRNA水平显著升高。在ZF4细胞中发现,LINE15’UTR具有生物活性。在低温处理(18℃,3 d)下,报告基因信号减弱,间接表明LINE1启动子活性减弱。研究结果表明,低温压力会影响LINE1在鱼类中的表达。本研究为进一步探究LINE1在鱼类适应低温环境中的作用机制奠定了基础。     

玻璃化冻存对斑马鱼卵母细胞的影响

采用比色法测定了在不同玻璃化冻存液中冻存1、2、4、8d时斑马鱼卵母细胞中谷胱甘肽含量和三磷酸腺苷酶的活性,以研究玻璃化冻存对卵母细胞谷胱甘肽含量和线粒体三磷酸腺苷酶活性的影响。试验结果显示,V4组斑马鱼(玻璃化液组分是:1.5mol/L甲醇、6.0mol/L乙二醇、0.25mol/L蔗糖)卵母细胞冻存1、2、4、8d后存活率最高[分别为(48.89±2.58)%、(39.35±1.34)%、(24.18±2.32)%和(14.56±1.64)%],显著高于其他组。经玻璃化冻存后,斑马鱼卵母细胞谷胱甘肽含量和线粒体三磷酸腺苷酶活性均随冻存天数的增加而显著下降(P0.05)。V4组的谷胱甘肽含量[(13.37±0.59)mg/g]和线粒体三磷酸腺苷酶活性较高,线粒体Ca~(2+)-三磷酸腺苷酶[(3.823±0.144)U/mg]、Mg~(2+)-三磷酸腺苷酶[(4.503±0.144)U/mg]和Na~+/K~+-三磷酸腺苷酶[(7.520±0.198)U/mg]活性最高。各组细胞的活性随着冻存天数的增加而逐渐下降。试验结果表明,玻璃化冻存不能完全避免低温和冷冻保护剂的毒性和对卵母细胞的损伤。     

Zebrafish (Danio rerio) as an animal model for bath infection by Flavobacterium psychrophilum

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease and rainbow trout syndrome in freshwater salmonid fish worldwide, generating injuries and high mortality rates. Despite several studies on this bacterium, the infection mechanism remains unknown due to limitations in the employed animal models. In this work, we propose using zebrafish (Danio rerio) as a model for studying bacterial pathogenicity. To substantiate this proposal, zebrafish infection by F. psychrophilum strain JIP 02/86 was characterized. Zebrafish larvae were infected using the bath method, and morphological changes and innate immune system activation were monitored using transgenic fish. Salmonid-like infection phenotypes were observed in 4.74% of treated larvae, as manifested by fin, muscle and caudal peduncle damage. Symptomatic and dead larvae accounted for 1.35% of all challenged larvae. Interestingly, infected larvae with no infection phenotypes showed stronger innate immune system activation than specimens with phenotypes. A failure of function assay for myeloid factor pu.1 resulted in more infected larvae (up to 43.5%), suggesting that low infection rates by F. psychrophilum would be due to the protective actions of the innate immune system against this bacterium in zebrafish larvae. Our results support the use of zebrafish as an infection model for studying F. psychrophilum. Furthermore, the percentage of infected fish can be modulated by disturbing, to varying extents, the differentiation of myeloid cells. Using this evidence as a starting point, different aspects of the infection mechanism of F. psychrophilum could be studied in vivo.     

Effects of Pseudoloma neurophilia infection on the brain transcriptome in zebrafish (Danio rerio)

Laboratory zebrafish are commonly infected with the intracellular, brain-infecting microsporidian parasite Pseudoloma neurophilia. Chronic P. neurophilia infections induce inflammation in meninges, brain and spinal cord, and have been suggested to affect neural functions since parasite clusters reside inside neurons. However, underlying neural and immunological mechanisms associated with infection have not been explored. Utilizing RNA-sequencing analysis, we found that P. neurophilia infection upregulated 175 and downregulated 45 genes in the zebrafish brain, compared to uninfected controls. Four biological pathways were enriched by the parasite, all of which were associated with immune function. In addition, 14 gene ontology (GO) terms were enriched, eight of which were associated with immune responses and five with circadian rhythm. Surprisingly, no differentially expressed genes or enriched pathways were specific for nervous system function. Upregulated immune-related genes indicate that the host generally show a pro-inflammatory immune response to infection. On the other hand, we found a general downregulation of immune response genes associated with anti-pathogen functions, suggesting an immune evasion strategy by the parasite. The results reported here provide important information on host–parasite interaction and highlight possible pathways for complex effects of parasite infections on zebrafish phenotypes.     

鲤鱼和斑马鱼同义密码子使用偏性分析

本研究利用鲤鱼454测序所获得的基因的表达序列和GeneBank中斑马鱼全部基因的编码序列,采用多重变量分析软件CodonW对鲤鱼和斑马鱼影响密码子用法的因素进行了分析.结果表明,两个在59种同义密码子中有27种为最优密码子.伺时还指出,最优密码子的使用频率(Fop)与基因的G+C含量(GC),尤其是第三位密码子的G+C百分含量(GC3S)、密码子适应指数(CAI)和密码子偏爱指数(CBI)之间均呈现极显著的正相关(鲤鱼和斑马鱼的相关系数分别为r=0.4108和0.4087、0.5354和0.5628、0.7547和0.7771、0.9803和0.9741),而与有效等位基因数ENc存在显著负相关(相关系数r=-0.2832和-0.2390).表明基因GC含量直接影响密码子使用的偏性,同时基因的表达水平越高,对密码子的使用偏向性越强.     

邻苯二甲酸丁基苄酯（BBP）对斑马鱼的毒性效应研究

经急性毒性试验,求得邻苯二甲酸丁基苄酯（BBP）对斑马鱼96小时半致死浓度（LC50）为3.98 mg•L-1。在此基础上,设置4个浓度梯度：0、0.332、0.665、1.33 mg•L-1,研究了在不同的时间（7、14、21、28d）暴露下,BBP对斑马鱼超氧化物歧化酶（SOD）、乙酰胆碱酯酶（AChE）、过氧化氢酶（CAT）活性的影响。结果表明,随着时间的延长,SOD活性呈现先上升后下降的趋势,在14d时,各处理组SOD活性达到最高水平;实验期间（0-28d）,各处理组AChE活性被显著抑制,整体呈现下降趋势;CAT活性,在7d时,除0.665 mg•L-1浓度组有极显著抑制作用（p＜0.01）外,其余各浓度组CAT活性均与对照接近,无显著差异,14d开始,除了0.332 mg•L-1浓度组高于对照组,其他浓度组与对照组相比均呈现先下降后上升的趋势。BBP对斑马鱼SOD、AChE、CAT活性产生的影响,其变化可作为水体污染的分子生物标记物,为制订BBP水质标准及对其进行生态风险评价提供科学依据。     

Effects of nutritional status and diet composition on fatty acid transporters expression in zebrafish (Danio rerio)

This study investigated the expression of zebrafish (Danio rerio) fatty acid transporters, such as scavenger receptor CD36, plasma membrane fatty acid‐binding protein (FABPpm), and fatty acid transport protein family (FATPs), in responses to nutritional status and diet composition. For diet composition study, three isonitrogenous (43% crude protein) purified diets were formulated to contain 6%, 12% and 18% crude lipid levels. Meanwhile five isonitrogenous and isoenergetic purified diets were formulated to contain different lipid sources including fish oil (FO; rich in n‐3 HUFA), palmitic acid (PA; rich in C16:0), olive oil (OO; rich in C18:1n‐9), sunflower oil (SO; rich in C18:2n‐6) or perilla oil (PO; rich in C18:3n‐3) as the sole lipid source. Results showed that liver CD36, FABPpm‐a, FABPpm‐b, FATP1‐a and FATP6, intestine CD36, FABPpm‐a and FABPpm‐b, and muscle CD36 and FATP1‐b were significantly increased during postprandial period. During starvation, liver CD36, FABPpm‐a, FABPpm‐b and FATP1‐a, intestine FABPpm‐a, FABPpm‐b, FATP4 and FATP6, and muscle FATP1‐b, FATP4 and FATP6 were significantly increased. The expression of some fatty acid transporters (including liver CD36 and FABPpm‐b, intestine FABPpm‐a, and muscle CD36, FABPpm‐a, FATP1‐a, FATP2 and FATP4) was up‐regulated by refeeding, while the expression of other fatty acid transporters (liver FABPpm‐a and intestine FATP1‐a, FATP4 and FATP6) was down‐regulated by refeeding. The expression of several fatty acid transporters (liver FATP2 and FATP4, and intestine CD36, FATP2 and FATP4) was significantly increased by high‐lipid diet, whereas CD36, FATP1‐b, FATP2 and FATP4 expression in the muscle were reduced by increasing dietary lipid level. Compared with FO group, the muscle of fish fed the diet with OO had a higher CD36 mRNA abundance, while the muscle of fish fed the diet with OO and PO had a higher FATP1‐b expression. Compared with the FO group, the hepatic FATP2 mRNA was significantly increased in the PA group, while the intestine FATP2 mRNA was significantly increased in the SO and PO groups. In addition, intestine FATP4 expression in the PA and OO groups was lower compared with the FO group. The results indicate that zebrafish fatty acid transporters play a central role in coordinating the mobilization of fuel substrates in responses to nutritional status and diet composition.