斑马鱼nrf基因时空表达分析及其在调控胰外分泌酶原基因表达中的作用研究

通过GenBank搜索发现,斑马鱼中有6个nrf同源基因,即nfe2、nrf1a、nrf1b、nrf2a、nrf2b和nrf3。为了研究6个nrf在物种之间的关系,实验对6个Nrf蛋白进行系统发育树分析,结果显示6个Nrf蛋白的分子进化趋势与物种进化趋势一致,尽管在硬骨鱼类中由于基因组倍增的原因,nrf1和nrf2基因各产生了两个拷贝,但Nrf蛋白仍然是比较保守的。此外,实验选取斑马鱼胚胎发育十个不同时期（1 cell、2 cell、3.5 hpf、6 hpf、12 hpf、24 hpf、36 hpf、48 hpf、72 hpf、96 hpf）,对其表达模式进行了详细的研究,结果显示:6个nrf在一细胞期都有转录信号,且胚胎发育早期（48 hpf之前）全身广泛表达。72 hpf后,nrf1b、nrf2a、nrf2b、nrf3和nfe2基因的表达部位主要集中在头部、部分躯干、肠处,nrf2b还在脊髓处有微弱的表达。总体来说,6个nrf基因的表达部位基本重叠。研究还利用双荧光素酶检测系统检测了6个Nrf蛋白对胰外分泌酶原基因（tryl）转录的调控,结果显示nrf1a、nrf2a、nrf2b与nfe2的过表达能使tryl的表达上调,而nrf1b与nrf3的过表达能抑制tryl的表达。研究结果将为深入研究斑马鱼6个nrf基因的功能叠加、互补及其功能歧化奠定基础。     

Sema4d基因在斑马鱼早期发育过程中的表达

目的:研究sema(semaphorin)4d基因在斑马鱼早期发育过程中的表达.方法:提取斑马鱼胚胎的总RNA,制备地高辛标记的sema4d RNA反义探针,WISH(整胚胎原位杂交)研究sema4d在斑马鱼早期发育过程中的表达.结果:成功合成sema4d基因探针,获得sema4d基因在斑马鱼早期发育过程中的表达情况:sema4d在0.75 hpf(hours post fertilization)、1.0 hpf、1.5 hpf、12 hpf前普遍性表达;17 hpf开始至24 hpf在头部表达较多,在脊髓、肌肉、中间细胞群ICM(intermediate cell mass)区处有特异性表达区处有特异性表达;48 hpf在头部和躯干肌肉持续表达.结论:Sema4d在早期参与了造血的发生,在脑部,脊髓,肌肉的发育中可能起到了重要作用.     

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。       

斑马鱼胚胎发育过程中FGF3基因的表达

为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PcR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T／FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示：FGF3P基因在2hp胚胎就有表达,并持续至胚胎孵化,12hpf胚胎FGF3表达量达到高峰（P〈0．01）;胚胎发育过程中心表达部位以头、尾、咽弓为主。由此得出结论,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。     

斑马鱼akt3 基因的克隆及其表达图谱与过表达分析

采用RT-PCR 和RACE 相结合的方法, 克隆得到了斑马鱼的akt3/pkb 基因, 其cDNA 全长为2874 bp,编码479 个氨基酸。斑马鱼akt3 具有akt 家族成员间保守的PH 结构域、催化活性结构域和调节结构域以及两个保守的磷酸化位点Thr305 和Ser472。与已发表的人、大鼠、小鼠的akt3 氨基酸序列比较, 相似性分别为95.8%、94.7%和95.4%。对斑马鱼早期胚胎进行RT-PCR 检测显示, akt3 在0-4hpf(hours post fertilization)含量水平较高, 6hpf 到12hpf 降低至较低水平, 16hpf 后表达量开始逐渐上升, 60hpf 至96hpf 则稳定在较高水平。原位杂交结果表明: akt3 在2hpf 至96hpf 的胚胎中整体都有表达, 没有组织特异性。在成鱼中, 除鳃部外, akt3 在所检测的其他各组织器官中均有表达, 在脑部和卵巢表达量较高; 利用显微注射持续表达myr-akt3 mRNA 研究其功能充分性时结果显示, 过量表达斑马鱼akt3 mRNA 能使斑马鱼胚胎发育滞后且伴随着尾部短粗、体节模糊、尾末端膨大甚至严重缩短等不同程度畸形。而在斑马鱼myr-akt3 注射组发育至24hpf 时观察(以排除akt3 造成的发育延迟的影响), 发现注射过akt3 的斑马鱼胚胎的脑部厚度较对照组显著增大, 表明akt3 对斑马鱼胚胎脑部尺寸发育有影响。       

肌间刺缺失对斑马鱼骨骼发育的影响

利用斑马鱼(Danio rerio)野生型与肌间刺完全缺失突变型个体, 从骨骼染色和骨骼发育相关基因表达两方面, 初步评价了肌间刺缺失对斑马鱼骨骼发育的影响。通过骨骼染色对比观察了两种肌间刺表型个体受精后8dpf(days post fertilization, dpf)到56dpf的骨骼发育情况, 结果显示, 两种肌间刺表型除肌间刺外, 其他骨骼发育基本同步。此外, 通过qRT-PCR实验检测分析了6个骨骼发育相关基因(bmp2a、bmp4、smad1、smad4a、runx2a和sp7)在不同肌间刺表型5个胚胎发育时期(3hpf囊胚期、6hpf原肠胚期、12hpf体节期、24hpf咽囊期和72hpf孵化期)和5个胚后生长阶段(15、30、45、60和75dpf)的表达情况。结果显示:在胚胎发育时期, 野生型和突变型个体中bmp2a、bmp4、smad1、smad4a基因和突变型个体中sp7基因的表达均呈现先升后降的变化趋势, 且在体节期达到最高表达水平;野生型和突变型个体中runx2a基因和野生型个体中sp7基因则表现为逐渐上升的趋势。6个基因在囊胚期和原肠胚期表达量无显著差异, bmp2a的表达水平在体节期、咽囊期和孵化期无显著差异, 野生型个体bmp4、smad1、smad4a、runx2a基因在体节期、咽囊期和孵化期的表达水平明显高于突变型, 而sp7基因则表现为突变型明显高于野生型。胚后发育阶段 6个基因在5个生长阶段均呈现逐渐下降的趋势, 且在两种肌间刺表型间其表达仅在个别时期差异显著。综上所述, 肌间刺的缺失对斑马鱼骨骼发育表现型无显著影响, 只在胚胎发育时期影响骨骼相关基因表达水平的变化;结合骨骼染色结果, 推测肌间刺缺失对斑马鱼骨骼发育无显著影响。     

斑马鱼nr1d4a和nr1d4b基因的表达及其对不同环境刺激的响应

研究获得了斑马鱼nr1d4a和nr1d4b基因的cDNA,进行了序列比对和系统进化分析,并采用实时定量RT-PCR（qPCR）方法研究了其表达模式及对不同环境刺激的转录反应。研究发现,斑马鱼nr1d4a和nr1d4b是由基因复制产生的旁系同源基因,具有高度保守的DNA结合结构域和配体结合结构域。斑马鱼nr1d4a和nr1d4b的表达模式具有明显的差别。nr1d4a在胚胎发育早期的表达量很低,72 hpf时开始显著升高;而nr1d4b具有较高水平的母源性表达,6 hpf时的表达量明显降低,但也在72 hpf显著回升。nr1d4a在脑和肾脏中表达量最高,其次是鳃、卵巢、精巢和眼,在肝脏中的表达量最低;nr1d4b在卵巢中表达量最高,其次是精巢和脑,在肠道和心脏中表达量最低。斑马鱼nr1d4a和nr1d4b都能被多种环境刺激瞬时诱导表达。16℃低温处理0.5h就能显著诱导斑马鱼nr1d4a和nr1d4b基因的表达,但处理6h后其诱导效应开始下降并逐渐消失。除低温外,重金属（2 mol/L镉）、缺氧（5%氧气）和盐度（5）处理均能瞬时诱导nr1d4a和nr1d4b的表达,说明nr1d4a和nr1d4b基因可能参与斑马鱼对多种环境刺激的适应性反应。研究为深入揭示鱼类nr1d4a和nr1d4b基因的生物学功能及其表达调控机制奠定了基础。       

斑马鱼nr1d4a和nr1d4b基因的表达及其对不同环境刺激的响应简

研究获得了斑马鱼nr1d4a和nr1d4b基因的cDNA,进行了序列比对和系统进化分析,并采用实时定量RT-PCR(qPCR)方法研究了其表达模式及对不同环境刺激的转录反应。研究发现,斑马鱼nr1d4a和nr1d4b是由基因复制产生的旁系同源基因,具有高度保守的DNA结合结构域和配体结合结构域。斑马鱼nr1d4a和nr1d4b的表达模式具有明显的差别。nr1d4a在胚胎发育早期的表达量很低,72 hpf时开始显著升高;而nr1d4b具有较高水平的母源性表达,6 hpf时的表达量明显降低,但也在72 hpf显著回升。nr1d4a在脑和肾脏中表达量最高,其次是鳃、卵巢、精巢和眼,在肝脏中的表达量最低;nr1d4b在卵巢中表达量最高,其次是精巢和脑,在肠道和心脏中表达量最低。斑马鱼nr1d4a和nr1d4b都能被多种环境刺激瞬时诱导表达。16℃低温处理0.5h就能显著诱导斑马鱼nr1d4a和nr1d4b基因的表达,但处理6h后其诱导效应开始下降并逐渐消失。除低温外,重金属(2μmol/L镉)、缺氧(5%氧气)和盐度(5‰)处理均能瞬时诱导nr1d4a和nr1d4b的表达,说明nr1d4a和nr1d4b基因可能参与斑马鱼对多种环境刺激的适应性反应。研究为深入揭示鱼类nr1d4a和nr1d4b基因的生物学功能及其表达调控机制奠定了基础。     

Tol2转座子介导斑马鱼rps26基因附近增强子捕获及注解分析

随着人类等生物大规模基因组测序工作的完成,认识和理解基因组上表达调控元件成为后基因组时代的重要研究任务,增强子捕获技术是一种鉴定基因组上增强子元件及其对基因表达调控机制的有效方法。本研究选择Tol2转座子系统介导制备的稳定增强子捕获品系TK4系(头部和躯干特异性GFP表达),利用Splinkerette PCR(sp-PCR)、原位杂交和比较基因组学等技术手段进行所捕获增强子的解析研究。将TK4系的F1代与野生型斑马鱼杂交,收集受精卵,于6 hpf(Hour post fertilization)、24 hpf、48 hpf、3 dpf(Day post fertilization)、4 dpf、5 dpf六个发育阶段通过荧光显微镜检测绿色荧光蛋白报告基因的表达模式;然后通过sp-PCR方法克隆到Tol2转座子插入位点斑马鱼基因组侧翼序列,经比对分析表明插入位点位于基因组23号染色体27749253位置,在rps26基因的intron1中,且报告基因插入方向与基因方向相反。在插入位点100 kb的基因组范围内有7个基因,分别为arf3a、wnt10b、wnt1、rps26、IKZF4、dnajc22和lmbr1l。通过VISTA程序对不同脊椎动物基因组同源序列比对结果显示,在rps26基因下游有2个潜在保守的非编码序列区CNS1(Conserved non-coding sequence)和CNS2,为可能的增强子元件。胚胎原位杂交表明:rps26基因的两个转录本有母源性表达,rps26-201在合子中的表达早于rps26-001,而TK4系斑马鱼的GFP在早期(6 hpf)不表达,后期rps26与GFP的表达模式既存在相似性,也存在差异性,提示两者可能既接受共同的增强子调控,但也存在不同增强子调控,所获得的2个潜在的增强子(CNS1和CNS2)可能对附近的基因(包括rps26)发挥差异的时空表达调控作用。本研究首次成功获得rps26基因附近2个潜在增强子,为深入研究这两个增强子对基因组附近基因的表达调控机制奠定基础,本研究所采用的结合技术手段也为增强子解析提供参考。     

团头鲂MSTN基因cDNA结构、表达及过表达对胚胎发育的影响

研究通过cDNA末端快速扩增法(RACE)克隆得到团头鲂生长抑制素(MSTN)基因的cDNA全长并分析了MSTN基因在团头鲂胚胎、成鱼组织中表达以及MSTN基因在胚胎中过表达情况。结果表明团头鲂MSTN基因的cDNA全长为2187 bp, ORF(开放阅读框)大小为1128 bp, 编码376个氨基酸。组织逆转录PCR (RT-PCR)结果显示, MSTN基因在肌肉、脑和精巢组织中大量表达, 肝脏、脾脏和卵巢组织中的少量表达, 肠、腮、心、眼和肾组织中的微量表达。胚胎逆转录PCR (RT-PCR)结果显示, 在0—44 hpf胚胎发育阶段, MSTN基因表达量较低; 而在48—52 hpf胚胎发育阶段, MSTN基因表达量逐渐升高。整胚原位杂交(WISH)结果显示, 胚胎发育的16 hpf时期MSTN基因主要在脊索中表达, 胚胎发育的28 hpf和55 hpf时期MSTN基因在脑中表达。MSTN基因过表达结果显示, 胚胎在体节发生期出现前-后轴拉长, 背-腹轴变短; 脊索发生扭曲, 强烈抑制体节发育而导致不分化等现象。研究为后续团头鲂MSTN基因的功能研究及团头鲂分子育种提供相关参考依据。     

Embryonic lethality and fetal liver apoptosis in mice lacking all three small Maf proteins

Embryogenesis is a period during which cells are exposed to dynamic changes of various intracellular and extracellular stresses. Oxidative stress response genes are regulated by heterodimers composed of Cap'n'Collar (CNC) and small Maf proteins (small Mafs) that bind to antioxidant response elements (ARE). Whereas CNC factors have been shown to contribute to the expression of ARE-dependent cytoprotective genes during embryogenesis, the specific contribution of small Maf proteins to such gene regulation remains to be fully examined. To delineate the small Maf function in vivo, in this study we examined mice lacking all three small Mafs (MafF, MafG, and MafK). The small Maf triple-knockout mice developed normally until embryonic day 9.5 (E9.5). Thereafter, however, the triple-knockout embryos showed severe growth retardation and liver hypoplasia, and the embryos died around E13.5. ARE-dependent cytoprotective genes were expressed normally in E10.5 triple-knockout embryos, but the expression was significantly reduced in the livers of E13.5 mutant embryos. Importantly, the embryonic lethality could be completely rescued by transgenic expression of exogenous MafG under MafG gene regulatory control. These results thus demonstrate that small Maf proteins are indispensable for embryonic development after E9.5, especially for liver development, but early embryonic development does not require small Mafs.     

Isolation, characterization, and expression analysis of zebrafish large Mafs

Large Maf proteins, which are members of the basic leucine zipper (b-Zip) superfamily, are involved in the determination and control of cellular differentiation. The expression patterns of various vertebrate large Maf mRNAs were described previously. Here, we report the cloning of a novel zebrafish large Maf cDNA, SMaf1 (Somite Maf1), and other zebrafish large Mafs, the N-terminus domains of which possess transactivational activity. We also analyzed the expression patterns of SMaf1 and SMaf2 (Somite Maf2)/Krml2 as well as MafB/Val and c-Maf during zebrafish embryogenesis. In particular, the robust expression of the novel SMaf1 mRNA, which overlapped that of MyoD, in somitic cells during somitogenesis was noteworthy. In addition, the expression patterns of SMaf2 and MafB in the blood-forming regions, and those of c-Maf and MafB in the lens cells showed spatial redundancy, although the temporal appearance of these genes at these sites differed. These data indicate that SMafs may play important roles in somitogenesis, and that Maf proteins may have overlapping and yet specific functions as to the determination and differentiation of cell lineages.     

MafT, a new member of the small Maf protein family in zebrafish

Small Maf proteins play critical roles on morphogenesis and homeostasis through associating with CNC proteins. To date, three small Maf proteins, MafF, MafG, and MafK, have been reported in vertebrates, which share redundant functions. In this study, we tried to identify and characterize small Maf proteins in zebrafish to elucidate their conservation and diversity in the fish kingdom. We identified homolog genes of MafG and MafK but not MafF in zebrafish, indicating the former two are conserved among vertebrates. In addition, a novel type of small Maf protein MafT was identified. MafT protein bound MARE sequence as a homodimer or heterodimers with zebrafish Nrf2 or p45 Nfe2. Co-overexpression of MafT and Nrf2 synergistically activated MARE-mediated gene expression in zebrafish embryos. These results indicated that MafT is a new member of small Maf proteins and involved in the Nrf2-dependent gene regulation in cellular defense system.     

Genetic evidence that small maf proteins are essential for the activation of antioxidant response element-dependent genes

While small Maf proteins have been suggested to be essential for the Nrf2-mediated activation of antioxidant response element (ARE)-dependent genes, the extent of their requirement remains to be fully documented. To address this issue, we generated mafG::mafF double-mutant mice possessing MafK as the single available small Maf. Induction of the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene was significantly impaired in double-mutant mice treated with butylated hydroxyanisole, while other ARE-dependent genes were less affected. Similarly, in a keap1-null background, where many of the ARE-dependent genes are constitutively activated in an Nrf2-dependent manner, only a subset of ARE-dependent genes, including NQO1, were sensitive to a simultaneous deficiency in MafG and MafF. Examination of single and double small maf mutant cells revealed that MafK also contributes to the induction of ARE-dependent genes. To obtain decisive evidence, we established mafG::mafK::mafF triple-mutant fibroblasts that completely lack small Mafs and turned out to be highly susceptible to oxidative stress. We found that induction in response to diethyl maleate was abolished in a wider range of ARE-dependent genes in the triple-mutant cells. These data explicitly demonstrate that small Mafs play critical roles in the inducible expression of a significant portion of ARE-dependent genes.