银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。     

银鲫转录调控因子lbh-b cDNA克隆与表达分析

Lbh (Limb-bud and heart)基因是脊椎动物中高度保守的转录调控因子, 在早期胚胎发育及某些人类疾病的发病过程中发挥着重要作用。我们前期在银鲫(Carassius gibelio)垂体转录组中筛选到一个在垂体中大量表达的基因lbh-b。为了进一步研究lbh基因在银鲫的表达特征, 首先采用RACE方法克隆了银鲫lbh基因家族的成员lbh-b基因(Cglbh-b)。Cglbh-b的cDNA全长1526 bp, 开放阅读框549 bp, 共编码182个氨基酸。生物信息学分析表明CgLbh-b蛋白与其他脊椎动物的Lbh蛋白同源性在68%以上, 可能也是无序蛋白质家族的成员之一。成体组织RT-PCR分析表明Cglbh-b仅在银鲫的垂体、端脑、卵巢及眼睛中表达。不同胚胎发育时期的表达分析表明, 在受精卵至原肠胚中Cglbh-b转录产物是以母源形式存在的mRNA, 其合子转录起始于尾芽期。胚胎整体原位杂交结果显示从受精后2d到受精后3d, Cglbh-b大量表达于脑和眼睛。此外, 随着卵子成熟Cglbh-b在银鲫垂体中的表达上调。这些结果暗示, Cglbh-b可能在调控银鲫脑和眼睛的发育以及卵子成熟过程中发挥着重要作用。     

银鲫3个肌球蛋白轻链基因cDNA的克隆与特征分析

银鲫(Carassiusauratusgibelio),因具有雌核发育的生殖能力而其天然群体中又存在一定比例(约5%—20%)的雄性个体1, 被认为是进行进化遗传和个体发育调控研究的独特模型动物2。       

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A_1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料 ,提取总RNA ,分离mRNA ,进而反转录合成cDNA并定向插入λgtllSfi Not克隆载体 ,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到 3 1× 1 0 6(银鲫 )和 1 6× 1 0 6(彩鲫 )。进一步人工合成CyclinA1 保守引物 ,采用PCR扩增文库的方法 ,克隆了银鲫 (1 61 6bp)与彩鲫 (1 62 6bp)的CyclinA1 全长cDNA。序列分析结果表明 :两种鱼编码区长度均为 1 1 73bp ,起始于一个包含在脊椎动物起始密码子ANNATG基元内ATG的单一开放读码框 ,编码 391个氨基酸 ;5’ -端非编码区长度也同为 70bp ,3-’端非编码区长度略有不同 ,银鲫为 373bp ,而彩鲫则为 383bp ;二者 3’ -端均带有AATAAA的Poly(A)加尾信号以及 2 4bp(银鲫 )和 2 7bp(彩鲫 )的Poly(A)尾巴。比较银鲫、彩鲫和金鱼与人、爪蟾CyclinA1氨基酸序列同源性的结果表明 ,CyclinA1 在人、爪蟾与鱼类之间具有较高同源性 ;而在银鲫、彩鲫和金鱼之间 ,CyclinA1 仅在周期蛋白框外存在 5个氨基酸的差异 ,且这些差异均是由个别碱基的变异造成的。     

银鲫与彩鲫卵母细胞cDNA文库构建及周期蛋白A1的cDNA克隆

分别取行天然雌核发育繁殖的银鲫和两性生殖的彩鲫的卵母细胞为材料,提取总RNA,分离mRNA,进而反转录合成cDNA并定向插入λgtll Sfi-Not克隆载体,经体外包装构建了银鲫与彩鲫卵母细胞的表达型cDNA文库。测试结果表明库容量分别达到3.1×106(银鲫)和1.6×106(彩鲫)。进一步人工合成Cyclin A1保守引物,采用PCR扩增文库的方法,克隆了银鲫(1616bp)与彩鲫(1626bp)的Cyclin A1全长cDNA。序列分析结果表明:两种鱼编码区长度均为1173bp,起始于一个包含在脊椎动物起始密码子ANNATG基元内ATG的单一开放读码框,编码391个氨基酸;5'-端非编码区长度也同为70bp,3-'端非编码区长度略有不同,银鲫为373bp,而彩鲫则为383bp;二者3'-端均带有AATAAA的Poly(A)加尾信号以及24bp(银鲫)和27bp(彩鲫)的Poly(A)尾巴。比较银鲫、彩鲫和金鱼与人、爪蟾Cyclin A1氨基酸序列同源性的结果表明,Cyclin A1在人、爪蟾与鱼类之间具有较高同源性;而在银鲫、彩鲫和金鱼之间,Cyclin A1仅在周期蛋白框外存在5个氨基酸的差异,且这些差异均是由个别碱基的变异造成的。       

银鲫两个蛋白合成相关基因全长cDNA的克隆及其特征分析

通过构建雌核发育银鲫心跳期SMART cDNA质粒库并从库中随机挑选克隆测序,克隆得到银鲫翻译起始因子3亚单位2(GTIF3—S2)和翻译延伸因子1亚单位α(GEF-1α)基因全长cDNA。银鲫翻译起始因子3亚单位2基因cDNA全长1280bp,开放阅读框位于117—1091bp之间,编码325个氨基酸。其推断的氨基酸序列存在三个WD结构域。该基因在鱼类中为首次报道。银鲫翻译延伸因子1亚单位alpha基因cDNA全长1784bp,开放阅读框位于82—1467bp之间,编码462个氨基酸。RT-PCR表明,这两个基因在成熟卵母细胞和胚胎发育早期可以检测到少量的转录产物,在胚胎发育期间从原肠期开始转录,并随着发育进程逐渐增强。成鱼组织中除精巢表达较弱外,其他组织都表达较强。同源分析比较表明,TIF3-S2和EF-1α在物种进化过程中具有高度的进化保守性,在物种间的同源性很高。因此,作认为,这两个基因是研究物种间系统发育的优良对象。     

银鲫精巢特异的Ly-6/uPAR相关蛋白的分子克隆及其特征分析

Ly-6/uPAR基因超家族(Ly-6 SF)成员广泛地存在于后生动物中, 开展该家族相关功能基因研究具有重要的意义。研究从银鲫(Carassius auratus gibelio)中鉴定到一个该家族新成员, cDNA全长为570 bp, 其中开放阅读框长度为300 bp, 编码99个氨基酸, 生物软件预测该蛋白含有一个LU结构域, 不含GPI锚信号序列, N端含有信号肽, 表明其可能为Ly-6基因超家族中分泌型蛋白。组织表达分析显示, 该基因只在银鲫精巢中特异表达, 且又是Ly-6基因超家族中一员, 因此将其命名为银鲫精巢特异的Ly-6/uPAR相关蛋白(Carassius auratus gibelio testis-specific Ly-6/uPAR related protein, 简称CagTslurp)。原位杂交结果显示, 该基因在银鲫精巢的精原细胞, 初级精母细胞以及次级精母细胞中表达, 精子细胞中存在少量的表达, 而在体细胞中不表达。这种精巢特异的表达模式, 暗示CagTslurp在银鲫精子发生中可能发挥了作用。       

银鲫HIRA多克隆抗体的制备及组织特异性表达分析

Hir/Hira基因家族的成员广泛存在于多种生物体中,但有关其在生物体发育过程中的具体功能还不甚清楚.对果蝇的研究表明,dHira基因产物可能在受精时精核的解凝过程和雄性原核的正常形成过程中起重要作用.本研究组前期已经分别克隆出雌核发育银鲫和两性生殖彩鲫的Hira基因(cagHira 和caHira),本实验在银鲫cagHira基因的特异区域,设计一对引物,以银鲫成熟卵母细胞总RNA逆转录出的cDNA为模板,扩增出cagHira的特异片段.再将该片段克隆到原核表达载体pET-32a上,转化BL21(DE3)菌株,经诱导后表达出融合蛋白.分析表明,该融合蛋白主要以包涵体形式表达.以纯化的融合蛋白作为抗原去免疫小鼠,制备多克隆抗血清,经蛋白质印迹分析检测,该抗血清(稀释到1:2000)与包涵体蛋白识别反应良好,确定获得了具有高效价的特异性银鲫CAGHIRA多克隆抗体,为进一步研究HIRA在鱼类发育和雌核生殖过程中的作用奠定了基础.对银鲫HIRA蛋白的组织特异性表达分析发现,该蛋白仅在成熟卵巢组织中特异表达,故表明HIRA可能对鱼类卵子发生和/或早期胚胎发育具有重要作用.     

异源四倍体银鲫外周血和精巢的组织学特征

通过比较D 系三倍体银鲫 (Carassius auratus gibelio Bloch) 与异源四倍体银鲫, 我们发现异源四倍体的外周血与精巢组织跟三倍体银鲫存在明显差异。HE 染色结果表明, 异源四倍体银鲫外周血红细胞有明显的分裂倾向。利用流式细胞术对D 系三倍体银鲫与异源四倍体银鲫外周血的DNA 直方图进行比较, 结果表明异源四倍体外周血的DNA 直方图有两个主峰。此外, 我们观察到异源四倍体银鲫精巢的三种类型, 其中Ⅰ型精巢可以产生正常精子, Ⅱ型可观察到精小囊结构, 但不能产生精子, Ⅲ型精巢未发育出精小囊结构。进一步用银鲫Vasa 抗体对精巢切片进行组织免疫荧光共聚焦显微分析, 结果表明, Ⅰ型精巢的生殖细胞完成了减数分裂, 能观察到精原细胞、初级精母细胞、次级精母细胞, 以及大量位于精小管中间的精子细胞和精子; 而Ⅱ型精巢的生殖细胞不能完成第二次减数分裂, 精小囊中存在大量的初级和次级精母细胞, 没有精子细胞产生。研究丰富了对异源四倍体银鲫生物学性状的认识。       

新疆额尔齐斯河水系银鲫克隆多样性研究

银鲫（Carassius auratus gibelio Bloch）由于其独特的遗传背景和繁殖方式而成为研究进化遗传学和选择育种的一个独特的模式生物。到目前为止,我们对新疆额尔齐斯河水系银鲫群体的多样性状况一直知之甚少。为了更好地了解额尔齐斯河水系银鲫群体的克隆多样性状况,本研究中,我们采集了来自新疆额尔齐斯河水系的4个鲫鱼群体。通过流式细胞术分析血细胞样品,结果证实这些鲫鱼均为三倍体银鲫。通过血清转铁蛋白电泳表型分析,我们从这些银鲫群体中鉴定出总共8个不同的克隆。在这些鉴定的克隆中,有4个克隆(克隆A、J、M和S)同于以前鉴定的克隆,而另外的4个克隆是新发现的。克隆A和M分布最广,出现在所调查的4个群体中;克隆J出现在2个不同的群体中;其余的5个克隆中每个克隆均为单个群体所拥有。不同克隆在群体中的这种分布谱式可能反映了银鲫的各个克隆可在不同水体之间迁移以及同一克隆在不同水体中生存能力存在有差异。在取样的银鲫群体中,发现有一个群体的克隆多样性水平明显低于其他3个群体,而这后3个群体的克隆多样性水平是与已报道过的银鲫群体相似的。这一结果可能暗示着修建水利工程和过度捕捞等人类活动的不利影响。本研究所揭示的克隆多样性将有助于进化遗传学和选择育种研究。同时,也反映了保护额尔齐斯河水系银鲫克隆多样性的重要性。     

饲料中豆粕替代鱼粉蛋白对异育银鲫生长、代谢及免疫功能的影响

本实验评价了饲料中豆粕替代鱼粉蛋白后对异育银鲫的生长、饲料利用、氮代谢和鱼体免疫力等的影响。实验设计4种等氮等能的饲料,每种3个重复,分别以豆粕替代饲料中鱼粉蛋白的0%（对照,D1）、20%（D2）、80%（D3）和100%（D4）。实验在半循环水养殖系统持续16周,鱼的初重约2.32g,实验期间水温23－30℃。结果表明,随着饲料中豆粕含量的升高,摄食率显著升高(P＜0.05),特定生长率、饲料转化效率、蛋白沉积率和能量沉积率显著降低(P＜0.05);蛋白表观消化率显著升高,干物质和能量表观消化率则显著降低(P＜0.05);总氮摄入量、表观氮摄入量、粪氮排出量、非粪氮排泄量、总氮沉积率均随着饲料中豆粕含量的升高呈显著降低到的趋势(P< 0.05),生产每千克鱼的氮排放量则随着饲料中豆粕含量的升高显著升高(P< 0.05);血清葡萄糖和甘油三酯的含量显著升高,而胆固醇的含量显著降低(P＜0.05);血清的溶菌酶显著降低,超氧化物岐化酶逐渐升高(P＜0.05)。     

异育银鲫幼鱼对饲料中赖氨酸的利用及需要量研究

以添加晶体氨基酸的半精制饲料饲喂异育银鲫幼鱼,通过69d的生长实验来确定其赖氨酸需要量。饲料以白鱼粉为主要蛋白源,饲料中的总赖氨酸含量分别为1.82%、2.32%、2.82%、3.32%3、.82%、4.32%和4.82%7个水平。实验在室内循环水养殖系统中进行,每种饲料随机3个重复。实验结果表明,异育银鲫能够利用饲料中的晶体赖氨酸、蛋氨酸。在投喂后3h,其血浆中的游离赖氨酸、蛋氨酸含量最高。当饲料中赖氨酸含量为3.32%时,异育银鲫的终末尾均重、特定生长率和鱼空壳占体重的百分比最高,肝体指数最低。当饲料中赖氨酸含量为3.82%时,异育银鲫的干物质表观消化率显著高于其他组(PPP>0.05)。血红蛋白含量以赖氨酸含量为2.82%的饲料组最高,4.82%组最低;随着饲料中赖氨酸含量的升高,异育银鲫红细胞数下降,血清脲氮含量升高,且血清脲氮含量具有组间显著性差异(P<0.05)。根据折线法,由异育银鲫的特定生长率同饲料中赖氨酸水平的相关性得出其赖氨酸需要量为3.27%,占饲料蛋白的8.52%。       

Morphological,histochemical and biochemical studies on germ cell mitochondria of normal rats

Summary Morphological changes in rat germ cell mitochondria are described. In diplotene and secondary spermatocytes and in the spermatids of the Golgi, cap and acrosomal phases, the mitochondria take on a rounded appearance with the inner space containing the matrix flattened against the outer membrane and the intracristal spaces considerably swollen (condensed mitochondria).Functional studies on condensed mitochondria isolated from the germ cells of normal rats have been performed. The following parameters have been evaluated: ADP/O ratio, respiratory control ratio (RCR) and ADP affinity. The ADP/O values found in the presence of various substrates are in agreement with the theoretical figures. The RCR is remarkably high. Moreover, the ADP affinity of these mitochondria is very high, as demonstrated by the low values of the apparent Km. These biochemical findings, which demonstrate a high oxidative capacity coupled with a marked phosphorylation, suggest that the condensed appearance of germ cell mitochondria is the expression of an active functional state.The work was partially supported by a grant from The Consiglio Nazionale delie Ricerche (C.N.R.), Rome, Italy     

饲料脂肪水平对长养殖周期异育银鲫“中科3号”生长和脂代谢的影响

实验以初重为(11.33±0.03) g的异育银鲫(Carassius auratus gibelio)为研究对象,分别投喂脂肪水平为4%(L4)、8%(L8)、12% (L12)、16% (L16)和20% (L20)的5种等氮饲料进行为期340d的长养殖周期实验,以探究饲料脂肪水平对长养殖周期异育银鲫生长性能、消化酶活性和脂代谢的影响。期间共取样5次,生长阶段分为63d(D63,幼鱼期)、110d(D110,养成前期)、223d(D223,越冬期)、275d(D275,越冬后)和340d(D340,养成中后期)。实验结果显示,以增重率为评价指标,幼鱼期D63的异育银鲫适宜脂肪水平为8%,养成前期D110的异育银鲫适宜脂肪水平为12%,而其他生长阶段饲料脂水平对增重率无显著影响。饲料脂肪水平对幼鱼期异育银鲫肠道消化酶活性有显著影响,脂肪酶活性随脂肪水平的升高呈现先降低后升高的趋势,幼鱼期(D63)异育银鲫肠道胰蛋白酶和淀粉酶活性高于越冬期和养成中后期的异育银鲫,表明幼鱼期的异育银鲫对脂肪的利用较低。幼鱼期(D63)异育银鲫脂肪合成相关基因pparγ和fas的表达量饲料脂肪水平升高呈先升高后下降的趋势,且在L8组表达量最高,而脂解基因lpl和cpt1a的表达量在低脂组L4显著低于其他各组。pparγ和cpt1a在越冬后期(D275)的表达量随饲料脂肪水平的升高呈现先上升后下降,而fas表达量在L4组显著高于其他组,表明不同生长阶段异育银鲫对饲料脂肪摄入的响应策略不一致,摄入过高或过低均会导致代谢紊乱。适宜的脂水平(8%—12%)可促进幼鱼期和养成前期异育银鲫的生长,增强脂肪利用率和脂代谢能力,而较大规格的异育银鲫对脂肪的变化不敏感。     

Minocycline up-regulates BCL-2 levels in mitochondria and attenuates male germ cell apoptosis

In this study, we determined the efficacy of minocycline, a second generation tetracycline, in preventing male germ cell apoptosis after withdrawal of gonadotropins and intratesticular testosterone (T). Groups of 5 male rats received one of the following treatments daily for 5 days: (i) daily sc injection of GnRH-A (1.6 mg/kg BW), (ii) oral administration of 30% gum acacia as a vehicle control, and (iii) GnRH-A + oral administration of 50 or 100 mg/kg BW of minocycline. Minocycline at both 50 and 100 mg dose levels significantly (P < 0.05) prevented GnRH-A -induced germ cell apoptosis by 59.4% and 62.2%, respectively, and fully prevented PARP cleavage. Minocycline-mediated protection occurred at the mitochondria, involving the restoration of the BCL-2 levels and, in turn, suppression of cytochrome c and DIABLO release. Minocycline was also effective in preventing human male germ cell apoptosis induced by hormone free culture condition.     

Caspase-9-dependent pathway to murine germ cell apoptosis: mediation by oxidative stress,BAX, and caspase 2

Ischemia-reperfusion (IR) of the testis results in germ-cell-specific apoptosis (GCA) and a reduction in daily sperm production. This has been correlated with and is dependent upon neutrophil recruitment to the testis. In a rat model of testicular IR, this has also been correlated with an increase in reactive oxygen species (ROS). We have investigated ROS in the mouse testis after IR and determined whether the observed GCA is mediated via a mitochondrial caspase-9-dependent pathway involving the upstream mediators caspase 2 and BAX. Mice were subjected to a 2-h period of testicular ischemia followed by reperfusion. An accumulation of 8-isoprostane, a marker of oxidative stress, occurred 4 h after reperfusion. Activation of a mitochondrial dependent pathway to GCA after testicular IR was determined based on the observations that both BAX and caspase 2 translocated to the mitochondria, and that an increase occurred in cytoplasmic cytochrome c. Moreover, microinfusion of a specific caspase 9 inhibitor significantly reduced active caspase 3 after testicular IR and the number of apoptotic germ cells. These results suggest that oxidative stress products accumulate in the testis following IR and demonstrate that the observed GCA is stimulated through a mitochondrial caspase-9-dependent pathway. The identification of the germ-cell apoptotic pathway induced after testicular IR, including the key players in the pathway subsequent to ROS (BAX, caspase 9, and caspase 2), aids our understanding of IR injury in the testis and provides a wider background for the development of therapeutic interventions to rescue testis function. The work was supported by grants P50 DK45179 and DK53072.     

Tudor and its domains： germ cell formation from a Tudor perspective

In many metazoan species, germ cell formation requires the germ plasm, a specialized cytoplasm which often contains electron dense structures. Genes required for germ cell formation in Drosophila have been isolated predominantly in screens for maternal-effect mutations. One such gene is tudor (tud); without proper tud function germ cell formation does not occur. Unlike other genes involved in Drosophila germ cell specification tud is dispensable for other somatic functions such as abdominal patterning. It is not known how TUD contributes at a molecular level to germ cell formation but in tud mutants, polar granule formation is severely compromised, and mitochondrially encoded ribosomal RNAs do not localize to the polar granule. TUD is composed of 11 repeats of the protein motif called the Tudor domain. There are similar proteins to TUD in the germ line of other metazoan species including mice. Probable vertebrate orthologues of Drosophila genes involved in germ cell specification will be discussed.     

The argininosuccinate lyase gene of Chlamydomonas reinhardtii: cloning of the cDNA and its characterization as a selectable shuttle marker

We describe the cDNA sequence for ARG7, the gene that encodes argininosuccinate lyase – a selectable nuclear marker – in Chlamydomonas reinhardtii. The 5′ end of the cDNA contains one more exon and the organisation of the mRNA is different from that predicted from the genomic sequence. When expressed under the control of the endogenous RbcS2 promoter, the 2.22-kb cDNA complements the arg7 mutation as well as the genomic DNA. A linear cDNA fragment lacking promoter sequences is also able to complement, suggesting that it could be used in promoter-trapping experiments. Despite the presence of a sequence encoding a potential chloroplast transit peptide in the cDNA the protein is not targeted to the chloroplast, nor can it complement the arg7 mutation when expressed there. By inserting a T7 bacteriophage promoter into the plasmid, a version of the cDNA which is able to complement both the C. reinhardtii arg7 mutant and the Escherichia coli argH mutant has been created. This modified Arg7 cDNA provides two advantages over the genomic DNA currently in use for gene tagging: it is shorter (6.2 kb versus 11.9 kb for pARG7.8φ3), and the selectable marker used in C. reinhardtii is the same as that used in E. coli, making plasmid rescue of the tag much more likely to succeed. Received: 2 June 1998 / Accepted: 25 September 1998