斑马鱼shh启动子指导绿色荧光蛋白基因在脊索中的表达

体外实验业已证明shh基因的启动子受HNF3β蛋白直接调控。为研究斑马鱼shh启动子在体内的作用模式,构建了由538bp斑马鱼shh启动子(包含两个HNF3β结合位点)与绿色荧光蛋白EGFP组成的表达载体,命名为pShh-EGFP。将pShh-EGFP注射到斑马鱼1-细胞期内的受精卵中,定期在倒置荧光显微镜下观察GFP的表达。GFP在原肠作用期就开始表达,主要发生在中轴下胚层中;在体节形成期,GFP在脊索细胞中表达,但未发现在神经底板细胞中表达。由此可见,含两个HNF3β结合区域的538bp的斑马鱼shh启动子具有脊索表达活性。     

Cyp19a1基因在中华鳖早期卵巢分化中的功能研究

芳香化酶是雌激素合成的关键酶,在非哺乳类脊椎动物的早期雌性性腺分化中起重要的调控作用.本研究首先分析了中华鳖(Pelodiscus sinensis)芳香化酶编码基因Cyp19a1在不同组织及不同发育时期雌雄胚胎性腺中的表达情况;其次,利用芳香化酶抑制剂(AI)和慢病毒RNA干扰及过表达技术,进行Cyp19a1基因的功能验证研究.研究结果表明,Cyp19a1基因在中华鳖成体卵巢组织中高度特异性表达,睾丸和心、肝、脾、肺、肾、肌、肠中未见表达.同时发现,Cyp19a1基因在第18期就呈现雌性性腺特异性表达,早于性腺分化启动时间(第19期).功能缺失实验表明,经过AI及Cyp19a1-shRNA处理后,ZW胚胎性腺皮质区退化,髓质区高度发育,形成原始性索结构,呈现雌性向雄性性逆转现象;同时雌性标记基因Foxl2的表达量下调,雄性标记基因Amh的表达量则显著上调.功能获得实验则表明,Cyp19a1过表达后的ZZ型性腺皮质区高度发育,髓质区退化,形成空洞结构,呈典型雌性特征,同时Foxl2基因的表达量上升,Amh的表达量显著降低.综上所述,本文证明了Cyp19a1是中华鳖早期卵巢分化的关键调控因子,为中华鳖性别分化机制研究奠定了基础.     

翘嘴鳜性腺型芳香化酶基因CYP19a的克隆及表达研究

采用同源克隆与SMART RACE技术首次分离和克隆了翘嘴鳜性腺型芳香化酶基因CYP19a的c DNA全长,并通过实时荧光定量PCR(real-time quantitative PCR,q PCR)技术分析了其在翘嘴鳜成体不同组织及性腺不同发育时期的表达特点。序列分析结果显示,CYP19a基因全长1 873 bp(Gen Bank登录号为KX911988),其中开放阅读框长1 581 bp,编码526个氨基酸。通过多序列比对,发现该基因编码的蛋白质具有P450arom A特定的功能保守序列,包括I-螺旋区、Ozol肽区、亚铁血红素结合区域以及芳香化酶特异性结合区域;同时,CYP19a基因序列的同源性分析结果显示,其在脊椎动物中较为保守。此外,q PCR检测结果显示,CYP19a基因在翘嘴鳜成体组织中的表达存在显著差异(P0.05),主要在性腺中表达,其次在脑和肝脏有少量表达;而且CYP19a基因在繁殖期性腺中的表达量显著低于非繁殖期性腺中的表达量(P0.05)。以上研究表明,CYP19a基因在鱼类性腺形成及发育过程中具有重要作用。     

黄体酮对成年斑马鱼下丘脑-垂体-性腺轴相关基因转录表达的干扰效应

黄体酮(P4)是一种类固醇激素。为了探究P4的内分泌干扰效应, 选择成年斑马鱼(Danio rerio)作为受试生物, 研究了P4对斑马鱼下丘脑-垂体-性腺轴(HPG轴)相关基因转录表达影响。成年斑马鱼在不同浓度P4(2、11和16 ng•L–1)下处理21 d。结果显示: 暴露于高浓度组的P4能够抑制雌鱼大脑中促性腺激素释放激素2(gnrh2)、促性腺激素释放激素3(gnrh3), 卵泡刺激素(fshb)、雌激素受体1(esr1)基因的转录表达; 然而诱导了雄鱼大脑中fshb、黄体生成素(lhb)、雄激素受体(ar)基因的转录表达, 这些转录变化暗示了P4对成年斑马鱼有潜在的弱雄激素效应。此外, P4暴露对雌鱼卵巢和雄鱼精巢类固醇合成途径中固醇激素合成急性调节蛋白(star)、细胞色素p450介导侧链裂解酶(cyp11a1)、17α羟化酶(cyp17)、卵巢细胞色素P450芳香化酶(cyp19a1a)、11β羟化酶(cyp11b)、羟基类固醇3β脱氢酶(hsd3b)、羟基类固醇20β脱氢酶(hsd20b)、羟基类固醇17β脱氢酶3(hsd17b3)、羟基类固醇11β脱氢酶2(hsd11b2)以及受体信号途径中孕激素受体(pgr)、esr1、ar基因的转录表达没有显著影响。可见, 在P4暴露下, 斑马鱼大脑比性腺更加敏感。总而言之, P4能够改变斑马鱼大脑中HPG轴相关基因的转录表达水平, 进而对斑马鱼的内分泌系统具有潜在的危险。     

利用GFP/RFP双荧光指示载体鉴定特异性启动子功能

在基因表达定位或启动子调控模式的研究中, 多以gusA作为报告基因。但由于部分组织中高内源GUS背景活性或转化手段的限制, 使判断基因表达定位或调控时存在很大误差。为了解决上述问题, 本实验将报道基因绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)融合构建双荧光标记瞬时表达载体pBI221-RFP/GFP。该载体以CaMV35S启动子驱动GFP确定转化效率, 通过鉴定阳性个体的红色荧光活性分析目的基因或启动子的表达模式。并通过番茄E8和西瓜AGPL1果实特异启动子验证了该载体在启动子调控模式研究中的应用可行性。结果表明pBI221-RFP/GFP是一个可以在基因和启动子功能验证中应用的高效瞬时表达载体。     

应用2A肽策略构建双基因共表达转基因斑马鱼的研究

目的以Tol2为骨架载体,以绿色荧光蛋白(GFP)、Cherry为报告基因,探讨采用2A肽双基因载体构建策略构建单启动子双基因共表达质粒的方法;将B细胞刺激因子(BAFF)分别置于2A序列前后位置,分析位置效应对跨膜融合蛋白的表达与剪切的影响,探讨多基因共表达转基因斑马鱼构建技术。方法以In Fusion法将GFP-2A-Cherry序列构建到Tol2质粒上,所得p Tol-GFP-2A-Cherry质粒转染He La细胞、显微注射1-细胞期斑马鱼受精卵;倒置荧光显微镜观察He La细胞、斑马鱼幼鱼体内GFP与Cherry蛋白的表达,Western blot法验证GFP和Cherry蛋白的表达量与剪切情况;分别构建p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry质粒,Western blot法检查BAFF的表达与剪切情况。结果 p Tol2-GFP-2A-Cherry质粒转染的He La细胞,GFP与Cherry均可单独表达且表达呈现时空一致性;GFP-2A-Cherry融合蛋白可被剪切为GFP与Cherry,且成等比例表达趋势。p Tol2-GFP-2A-Cherry质粒显微注射1-细胞期斑马鱼受精卵可获得可单独表达GFP与Cherry蛋白的转基因斑马鱼;p Tol2-GFP-2A-BAFF与p Tol2-BAFF-2A-Cherry于斑马鱼体内均有融合蛋白的表达,且BAFF序列位于2A序列后更易于融合蛋白的剪切。结论通过2A肽策略构建可实现在斑马鱼体内单一载体、单一启动子调控双基因表达目的。发现编码跨膜分泌蛋白的功能基因位于2A序列的不同位置会直接影响蛋白的剪切,功能基因位于2A序列后易于跨膜蛋白的剪切。     

半滑舌鳎脑芳香化酶基因cDNA克隆及表达分析

为研究脑型芳香化酶(P450aromB)在半滑舌鳎性别分化中的作用,采用同源克隆策略,从半滑舌鳎脑分离了2184bp长的脑型芳香化酶的全长cDNA,该基因编码498个氨基酸。氨基酸序列和系统发育分析表明,P450aromB属于脑型P450arom,P450aromB的氨基酸序列与其他鱼类脑型P450arom的同源性较高(48.3%-66.1%),与性腺型P450arom的同源性较低(34.2%-49.9%),与自身的性腺型芳香化酶同源性为45.1%。RT-PCR分析表明:P450aromB mRNA的表达具有明显组织特异性,P450aromB只在性腺、脑、鳃和皮肤中表达,且脑中表达量远高于性腺,而在雌雄鱼的其他组织中都不表达。经过甲基睾酮浸浴处理和高温诱导半滑舌鳎由雌性性反转为雄性后,脑中P450aromB的表达量降低,这些结果表明P450aromB参与了半滑舌鳎的性腺分化和性别决定过程。     

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.     

用于化学预防剂筛选的转基因细胞模型的建立

建立TK启动子以及抗氧化反应元件(ARE)增强子调控报告基因GFP在HepG2细胞中稳定表达的细胞模型。人工合成ARE增强子序列,经退火和磷酸化后插入pTK-GFP载体的TK启动子上游,构建pARE-TK-GFP重组质粒。PCR法扩增TK和ARE-TK目的片段克隆到pEGFP-N1上,构建TK启动子启动以及上游由ARE增强子调控的报告基因表达载体pTK-GFP/Neo和pARE-TK-GFP/Neo。脂质体转染法转染人HepG2肝癌细胞后加G418筛选出阳性克隆。经扩大培养的克隆细胞中加入化学预防剂PDTC和香菇多糖作用48h后检测细胞中GFP荧光强度,结果显示pARE-TK-GFP/Neo表达载体中的GFP基因受ARE增强子的调控,其表达水平高于对照载体且在一定范围内与化学预防剂的浓度呈剂量效应关系,从而表明所构建的细胞模型可用于各种天然或人工合成的化学预防剂的初步筛选。     

鸡卵清蛋白基因启动子调控GFP基因在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞的表达

特异性扩增家鸡卵清蛋白基因上游调控序列 1340bp～ +16 5 5bp片段和第一内含子 +49bp～ +16 5 5bp片段 ,去除pG FP N2载体自身的CMV启动子 ,分别构建了P_2.9koval GFP和P_1.5koval GFP两种表达载体 ,经测序和酶切鉴定表达载体构建正确。采用脂质体转染法分别将这两种载体、pGFP N2 (阳性对照 )质粒及阴性对照转染鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞。用荧光倒置显微镜观测绿色荧光蛋白的表达。结果表明 :两种表达质粒在鸡原代输卵管上皮细胞和中国仓鼠卵巢细胞中都可以表达荧光蛋白。结果既显示卵清蛋白第一内含子对基因的表达起到一定的调控作用 ,也显示卵清蛋白启动子对输卵管上皮细胞和卵巢细胞不存在特异性 ,并且不存在种属差异性。     

Overexpression of Aromatase Alone is Sufficient for Ovarian Development in Genetically Male Chicken Embryos

Estrogens play a key role in sexual differentiation of both the gonads and external traits in birds. The production of estrogen occurs via a well-characterised steroidogenic pathway, which is a multi-step process involving several enzymes, including cytochrome P450 aromatase. In chicken embryos, the aromatase gene (CYP19A1) is expressed female-specifically from the time of gonadal sex differentiation. To further explore the role of aromatase in sex determination, we ectopically delivered this enzyme using the retroviral vector RCASBP in ovo. Aromatase overexpression in male chicken embryos induced gonadal sex-reversal characterised by an enlargement of the left gonad and development of ovarian structures such as a thickened outer cortex and medulla with lacunae. In addition, the expression of key male gonad developmental genes (DMRT1, SOX9 and Anti-Müllerian hormone (AMH)) was suppressed, and the distribution of germ cells in sex-reversed males followed the female pattern. The detection of SCP3 protein in late stage sex-reversed male embryonic gonads indicated that these genetically male germ cells had entered meiosis, a process that normally only occurs in female embryonic germ cells. This work shows for the first time that the addition of aromatase into a developing male embryo is sufficient to direct ovarian development, suggesting that male gonads have the complete capacity to develop as ovaries if provided with aromatase.     

Development of Retroviral Vectors for Tissue-Restricted Expression in Chicken Embryonic Gonads

The chicken embryo has long been a useful model organism for studying development, including sex determination and gonadal differentiation. However, manipulating gene expression specifically in the embryonic avian gonad has been difficult. The viral vector RCASBP can be readily used for embryo-wide transgene expression; however global mis-expression using this method can cause deleterious off-target effects and embryo-lethality. In an attempt to develop vectors for the over-expression of sequences in chicken embryonic urogenital tissues, the viral vector RCANBP was engineered to contain predicted promoter sequences of gonadal-expressed genes. Several promoters were analysed and it was found that although the SF1 promoter produced a tissue-restricted expression pattern that was highest in the mesonephros and liver, it was also higher in the gonads compared to the rest of the body. The location of EGFP expression from the SF1 promoter overlapped with several key gonad-expressed sex development genes; however expression was generally low-level and was not seen in all gonadal cells. To further validate this sequence the key testis determinant DMRT1 was over-expressed in female embryos, which due to insufficient levels had no effect on gonad development. The female gene aromatase was then over-expressed in male embryos, which disrupted the testis pathway as demonstrated by a reduction in AMH protein. Taken together, although these data showed that the SF1 promoter can be used for functional studies in ovo, a stronger promoter sequence would likely be required for the functional analysis of gonad genes that require high-level expression.     

Real-time PCR analysis of ovary- and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development

Two forms of cytochrome P450 aromatase, acting in both the brain and the ovary, have been implicated in controlling ovarian development in fish. To better understand the expression of these two enzymes during sexual differentiation in Atlantic halibut (Hippoglossus hippoglossus), real-time PCR was used to quantify the mRNA levels of ovary- (cyp19a) and brain-type cytochrome P450 aromatase (cyp19b) genes in the gonad and brain during gonadal development. Both enzymes showed high levels of expression in both tissues in developmental stages prior to histologically detectable ovarian differentiation (38 mm fork length), with increased expression occurring slightly earlier in the brain than the gonad. Cyp19a showed a second peak of expression in later stages (> 48 mm) in the gonad, but not the brain. Cyp19b expression was generally higher in the brain than the gonad. These results suggest that sexual differentiation may begin in the brain prior to gonadal differentiation, supporting the idea that steroid hormone expression in the brain is a key determinant of phenotypic sex in fish. In an examination of sexually immature adults, cyp19a was highly expressed in female gonad while cyp19b was very highly expressed in the pituitary of both sexes. The ratio of cyp19a to cyp19b expression was much higher in ovaries than in testes in the adult fish, so this ratio was analyzed in the developing gonads of juvenile halibut in an attempt to infer their sex. This was only partially successful, with about half the fish in later developmental stages showing apparently sex-specific differences in aromatase expression.     

两种泥鳅芳香化酶基因的克隆与时空表达

鱼类的性别分化易受发育环境的影响。向性成熟的泥鳅和大鳞副泥鳅个体注射绒毛膜促性腺激素,获得卵子和精子进行人工授精。把胚胎分别置于20℃、25℃和30℃条件下,使其发育。经性腺检查发现随着温度的升高两种泥鳅中雄性个体所占的比例明显升高,获得明显的偏雄比率群体。根据已知细胞色素P450芳香化酶CYP19 b基因序列设计嵌套简并引物用巢式PCR扩增并克隆出了两种泥鳅的CYP19 b的DNA片段。MaCYP19 b片段和Pd-CYP19 b片段分别长1337bp和1473bp。在此基础上用各自的特异引物克隆出两种泥鳅CYP19 b的相应cDNA片段。通过基因组DNA和cDNA序列的比较证明两种泥鳅的CYP19 b基因均包含三个内含子和四个外显子,编码的蛋白质序列长145氨基酸残基。以GAPDH基因为对照,分别对两种泥鳅成体组织和不同发育阶段的胚胎的CYP19 b进行了半定量RT-PCR表达分析,结果表明泥鳅CYP19 b基因只在成体泥鳅卵巢、肾以及原肠胚和神经胚中表达。大鳞副泥鳅CYP19 b基因在成体的脑、卵巢和肾以及神经胚和卵黄吸收期表达。这些结果为揭示细胞色素P450芳香化酶基因与环境性别决定机制的关系奠定了基础。