吡哆醛激酶基因在家蚕体内的时空表达特征

【目的】了解家蚕Bombyx mori维生素B₆关键代谢酶吡哆醛激酶（pyridoxal kinase, PLK）在家蚕不同组织间的表达差异。【方法】原核表达家蚕重组PLK, 获得目的蛋白制备多克隆抗体, 利用Western blot方法对PLK在家蚕不同发育时期、 5龄第3天幼虫不同组织及5龄不同日龄幼虫表皮、 头部和马氏管中的表达进行分析; 并采用实时荧光定量PCR的方法对PLK基因在家蚕不同组织中的mRNA转录水平进行比较。【结果】PLK在家蚕5龄幼虫的表达水平最高, 在5龄第3天幼虫各组织中的表达由高到低依次为： 精巢、 马氏管、 表皮、 中肠、 头部、 卵巢、 脂肪体、 丝腺; 5龄期不同日龄幼虫表皮组织的表达差异最大, 头部和马氏管中均为前期表达量稍高于后期。在转录水平方面, 精巢的表达量最高, 其次是马氏管和头部。【结论】PLK在家蚕不同发育时期及组织中的差异表达进一步证实PLK在家蚕VB6代谢中的生物学功能的重要性。     

nanos同源基因在黑脊倒刺鲃生殖细胞中的表达研究

目的:研究黑脊倒刺鲃scnanos的核苷酸序列及其在配子发生中的空间表达特征,以期为鱼类生殖发育研究提供一个有效的分子标记。方法:RT-PCR法首次克隆出scnanos基因,并检测其在不同组织中的表达水平,进而原位杂交法检测其在性腺中的表达水平。结果:scnanos开放阅读框长456bp,编码152个氨基酸。其具有较高的保守性,与鲤鱼、斑马鱼、家蚕、大熊猫、紫色球海胆的相似性分别为81%、63%、52%、50%、44%;scnanos仅在性腺表达,精子发生中,scnanos仅在精原细胞和精母细胞中表达。卵子发生的各时期都发现有scnanos的表达,且杂交信号的强度都随着配子发生的进行逐渐减弱。结论:该研究得到的scnanos是斑马鱼nanos的同源基因,而且此基因适合作为黑脊倒刺鲃及其它鱼类生殖发育研究的有效分子标记基因。     

家蚕磷酸吡哆醇氧化酶基因的表达谱分析

【目的】了解家蚕Bombyx mori维生素B6关键代谢酶磷酸吡哆醇氧化酶（pyridoxine- 5′-phosphate oxidase, PNPO）基因在家蚕不同发育阶段及5龄幼虫不同组织中的表达差异。【方法】将家蚕PNPO基因的重组表达质粒pET-22b(+)-PNPO转化入大肠杆菌Escherichia coli Rosetta中诱导表达, 纯化蛋白制备多克隆抗体。分别采用荧光定量PCR和Western blot方法对家蚕PNPO基因进行了转录水平和翻译水平的表达分析。【结果】在家蚕发育水平上, 5龄幼虫的PNPO翻译量为最高。PNPO基因在5龄幼虫各组织中的转录水平由高到低依次为精巢、 头、 中肠、 马氏管、 卵巢、 表皮、 脂肪体、 丝腺; 翻译量也以精巢为最高, 其次是头、 中肠和马氏管。【结论】明确了PNPO在家蚕各发育阶段及5龄幼虫各组织中的表达情况。     

Ypel3基因在小鼠胚胎发育中的表达与调控

目的:探讨小鼠胚胎发育过程中Ypel3基因的时空特异性表达与调控,为后续功能研究奠定基础。方法:选取胎龄(E)10.5、12.5、14.5、16.5和18.5 d的小鼠胚胎,利用荧光定量RT-PCR技术研究Ypel3基因mRNA的时序性动态表达谱;采用原位杂交技术观察Ypel3基因mRNA在胚胎发育E11.5和E15.5的空间表达谱;应用定量RT-PCR技术检测表观遗传学修饰对Ypel3基因mRNA表达丰度的影响。结果:定量RT-PCR表明该基因从胚胎发育的早中期开始表达,到出生前表达量呈逐渐升高趋势;原位杂交显示E11.5信号出现在脑和心脏中,E15.5信号在脑、舌、心、肺、胸腺、肝、肾等主要脏器中均有表达;甲基化转移酶抑制剂5-氮胞苷(5-Aza)处理的Neuro-2a(N2a)细胞中,Ypel3的表达水平未产生显著变化,而去乙酰化酶抑制剂4-苯丁酸(4-PBA)处理后该基因表达显著升高,5-Aza和4-PBA联合处理后表达水平进一步升高。结论:Ypel3基因在小鼠胚胎发育各阶段有广泛的表达,提示其具有重要作用,且该基因的表达可能受到组蛋白乙酰化的调控。     

团头鲂nanos3基因的克隆鉴定

为了标记团头鲂(Megalobrama amblycephala)的原始生殖细胞(Primordial Germ Cells, PGCs), 首次克隆并鉴定了团头鲂nanos3基因(mananos3)。mananos3全长1027 bp, 包括48 bp 5′UTR (5′untranslated Region), 490 bp 3′UTR和489 bp开放阅读框(Open Reading Frame, ORF)。该基因编码162个氨基酸。通过序列比对发现Mananos3蛋白和其他物种Nanos蛋白一样, 存在一个保守的RNA结合功能域, 该功能域包含一个锌指基序(Motif)。系统发育树结果显示, Mananos3与鲤(Cyprinus carpio)的Nanos3最为相近。半定量和定量PCR结果表明, mananos3具有较高的母源表达, 并在胚胎发育早期高量表达, 而在1000细胞期之后表达量逐渐降低。在成体组织中, mananos3仅在卵巢中检测到表达。mananos3和斑马鱼(Danio rerio) nanos3 (zfnanos3)的3′UTR均可以介导绿色荧光蛋白特异标记团头鲂和斑马鱼胚胎发育早期的PGCs, 但是mananos3的3′UTR能够更特异地标记团头鲂的PGCs。通过比对mananos3和zfnanos3的3′UTR发现, mananos3 的3′UTR中有一个非经典的miR430识别位点(GCACTA)。通过对该位点的突变研究证实其有利于nanos3在非PGCs组织中的降解。综上所述, 团头鲂mananos3的3′UTR序列中的非经典miR430识别位点(GCACTA)可能与介导报告基因在PGCs中特异表达相关。     

小鼠生殖细胞的胚胎发育

本文综述了近年来小鼠胚胎发育过程中生殖细胞的起源、迁移与增殖、性别分化及其基因组修饰等方面的研究进展。小鼠生殖细胞在7~7.5dpc时由原始生殖细胞（PGC）演变而来,至12.5dpc时PGC全部迁移进入生殖嵴,到13.5dpc时停止分裂。Steel/c-kit信号途径在PGC迁移过程中起重要作用。生殖细胞的性别主要是由生殖腺中体细胞的微环境决定的。Y染色体上存在精子形成所必需的基因。生殖细胞的全基因组范围的重新甲基化晚于胚胎体细胞的重新甲基化,到18.5dpc时才完成。雌性生殖细胞的X染色体重新活化在14.5~15.5dpc时完成,并且与生殖嵴的性别分化无关。 Abstract:This paper reviewed the recent progress of the origin,migration and proliferation,sex determination,and genomic modification of murine germ cells during its embryonic development. Murine germ cells originate from primordial germ cells at about 7~7.5dpc. Then PGCs migrated into germinal ridge at about 12.5dpc during which Steel/c-kit signal pathway plays important roles and stopped division at 13.5dpc. The sex of germ cells was mainly determined by the soma microenvironment in the gonad. And there are essential genes for sperm formation on the Y chromosome. The de novo methylation of murine germ cells was much later than soma cells and was completed at about 18.5dpc. The X chromosome reactivation of female germ cells was finished at about 14.5~15.5dpc which was independent of sexual differentiation of germinal ridge.     

印记基因Dlk1在小鼠胚胎发育中的表达分析

目的:研究印记基因Dlk1在小鼠胚胎发育过程中的动态表达模式,以揭示Dlk1与胚胎发育的关系。方法:通过半定量PCR和定量PCR分析Dlk1在小鼠胚胎发育E8.5～E19.5的基因表达模式,并选取Dlk1表达量最高的时期进行胚胎切片原位杂交和组织定量PCR分析。结果:在小鼠胚胎发育E8.5～E15.5时,Dlk1的表达逐渐升高,在E15.5时表达量达到最高;E15.5～E19.5时,Dlk1表达有所下降,但仍然维持较高水平。E15.5切片原位杂交显示,垂体、肺脏、软骨、舌和背侧肌肉组织中Dlk1表达较高,组织定量PCR实验进一步证实了原文杂交的结果。结论:Dlk1在小鼠胚胎发育中后期持续表达,并呈现一定的组织特异性,对胚胎发育可能起重要的调节作用。     

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。     

家蚕吡哆醛激酶基因干扰降低转氨酶基因的转录表达

【目的】维生素B₆在氨基酸代谢中是多种酶的辅酶,维持氨基酸代谢的正常运行。磷酸吡哆醛（pyridoxal-5′-phosphate, PLP）是维生素B₆的主要辅酶形式,吡哆醛激酶（pyridoxal kinase, PLK）是PLP的重要生成酶,本研究试图明确PLK基因与PLP依赖酶之间转录水平的调节关系。【方法】本研究采用RNA干扰（RNA interference, RNAi）方法对家蚕 Bombyx mori 的PLK基因进行干扰,通过体外合成PLK基因的3个干扰片段（siRNA1, siRNA2和siRNA3）,将siRNA从体腔注入5龄第3天的家蚕幼虫体内诱导RNAi。利用荧光定量PCR测定不同干扰片段、不同时间点及不同组织中PLK基因表达量的变化;并测定家蚕体内磷酸丝氨酸转氨酶（phosphoserine aminotransferase, SerB）和天门冬氨酸氨基转移酶（asparate aminotransferase, AST）基因的表达量。【结果】注射干扰片段后48 h干扰效果达到最佳。3个干扰片段干扰效果从高到低依次为siRNA1, siRNA2和siRNA3。RNAi效果最好的是中肠组织,其PLK基因的相对表达量下降了55%。RNA干扰PLK基因后,后部丝腺中SerB和AST基因相对表达量分别下降了90%和29%。【结论】本研究通过RNAi实现了家蚕PLK基因干扰,并进一步证明了家蚕PLK基因和SerB基因及AST基因存在联动调节关系。     

斑马鱼ca15b的克隆及在原始生殖细胞中的特异表达

高保真PCR克隆获得了ca15b基因的全长,利用胚胎整体原位杂交等技术研究了ca15b基因在斑马鱼早期发育过程中的动态表达。结果发现,ca15b在斑马鱼早期发育过程中存在显著的原始生殖细胞（Primordial germ cell,PGC）特异表达模式。ca15b是一个母源性表达的基因:在分裂期的胚胎中,其mRNA集中分布于位于分裂沟的生殖质（Germ plasm）;从囊胚期开始,可以观察到其在PGC中的特异表达;在原肠胚中,其mRNA在体细胞中急剧降解,仅特异表达于PGC,这一表达特征一直持续到受精后1d的胚胎。将体外合成的包含5'UTR和3'UTR的ca15b全长mRNA注射到斑马鱼胚胎后,仅能增强原肠期之前胚胎中ca15b的整体杂交信号;在原肠胚期之后,注射的mRNA在体细胞中快速降解。这提示在ca15b mRNA上可能存在某种转录后调控其在早期胚胎体细胞中降解而在PGC中稳定存在的机制。       

Cloning and characterization of nanos gene in silkworm Bombyx mori

Gene nanos is a maternal posterior group gene required for normal development of abdominal segments and the germ line in Drosophila. Expression of nanos-related genes is associated with the germ line in a broad variety of other taxa. In this study, the 5＇-RACE method and the in silico cloning method are used to isolate the new nanos-like gene of Bombyx mor/and the gene obtained is analyzed with bioinformatics tools. The putative protein is expressed in Escherichia coli and the antiserum has been produced in New Zealand white rabbits. The result shows that the nanos cDNA is 1,913 bp in full length and contains a 954 bp open reading frame. The deduced protein has 317 amino acid residues, with a predicted molecular weight of 35 kDa, isoelectric point of 5.38, and contains a conserved nanos RNA binding domain. The conserved region of the deduced protein shares 73% homology with the nanos protein conserved region of Honeybee （Apis mellifera）. This gene has been registered in the GenBank under the accession number EF647589. One encoding sequence of the nanos fragment has been successfully expressed in E. coli. Western blotting analysis indicates that homemade antiserum can specifically detect nanos protein expressed in prokaryotic cells.     

Generation of stella-GFP transgenic mice: a novel tool to study germ cell development

The relationship between germ cells and pluripotent embryonic stem (ES) cells is of particular interest, together with approaches to generate primordial germ cell (PGCs) from ES cells. A critical requirement in these experiments is the ability to unambiguously detect PGCs with the use of, for example, reporter genes. The currently available transgenic reporters do not show exclusive expression in PGCs at their earliest developmental stages. Here we describe the use of germline-restricted expression of stella, which is currently the best marker gene for PGCs. We generated two stella-GFP reporters and show that both transgenes surpass other reporters in terms of timing and specificity of expression in PGCs. Additionally, we demonstrate the usefulness of stella-GFP during the derivation of PGCs from ES cells.     

家蚕CyPA基因的克隆、表达谱及进化分析

CyP蛋白家族在蛋白质折叠过程中起着重要作用。本研究克隆了家蚕Bombyx mori CyPA基因(BmCyPA),该基因由2个外显子和1个内含子组成,推导开放阅读框编码165个氨基酸,分子量为19.4 kD,等电点为8.79。序列分析表明BmCyPA在不同物种间具有高度的保守性,含有肽酰脯氨酸顺反异构酶活性位点及与CsA侧链结合的氨基酸,提示BmCyPA可能具有肽酰脯氨酸顺反异构酶活性和与CsA结合的特性。组织表达谱及EST数据分析显示,BmCyPA在丝腺中高丰度表达。通过对不同物种来源的CyP基因的进化分析,进一步预测了BmCyP基因的功能,BmCyP可能与丝蛋白的正确折叠相关。     

家蚕羧酸酯酶基因Bmae35的克隆、 序列分析及表达

昆虫羧酸酯酶是一类能对外源化合物解毒和气味分子降解的重要酶系。本研究选取在家蚕Bombyx mori幼虫嗅觉感器中有表达, 并与蛀茎夜蛾Sesamia nonagrioides触角酯酶基因Snon-EST可能为直系同源基因的Bmae35进行克隆和外源表达研究。结果表明： 该基因编码区长1 581 bp, 共编码526个氨基酸。与其他昆虫触角酯酶的多序列比对分析发现, Bmae35编码的蛋白具有酯酶活性必须的催化残基Ser191, Glu313和His429, 也保持着α-酯酶家族特征基序Gly-x-Ser-x-Gly。RT-PCR分析显示, Bmae35在家蚕5龄第3天各组织中均有表达, 其中在头、 脂肪体、 马氏管、 体壁和丝腺中的表达量较高。Bmae35在雌蛾性信息腺中表达, 并与性信息素合成呈正相关, 暗示其在信息素合成中起重要作用。构建Bmae35与pET28(a) 重组载体, 经异丙基-β-D-硫代半乳糖苷诱导,电泳检测发现该基因以包涵体形式表达, 以镍亲和层析柱纯化, Western blotting鉴定证实Bmae35在大肠杆菌Escherichia coli中正确表达并得以纯化。本实验通过对家蚕Bmae35基因的克隆、 原核表达与纯化, 为进一步深入研究其表达定位和气味降解等功能奠定基础。     

Origin of primordial germ cells in the prestreak chick embryo

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII–XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vilelline membranes at stages VII–IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX–XIV with and without an STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII–IX. When individual stage IX–XIV embryos were dispersed and cultured without a feeder layer, 25–45 PGCs/embryo were detected only with stage X–XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX–XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI–XIV. © 1996 Wiley-Liss, Inc.     

家蚕Piwi亚家族基因的克隆、表达及进化分析

为克隆家蚕Bombyx mori Piwi亚家族蛋白基因cDNA全长序列,分析其分子特征和表达模式,探究Piwi亚家族蛋白在家蚕中的生理功能,本研究利用已知物种的Piwi亚家族蛋白搜索家蚕基因组,预测获得家蚕Piwi亚家族蛋白基因siwi1和siwi2,采用RACE技术克隆siwi1和siwi2的全长cDNA序列,利用ORFfinder、Gene-Explorer、InterPro等分析其分子特征;其次,利用已知的所有物种Piwi蛋白及其类似物Piwil构建系统发育树;最后,通过荧光定量PCR技术检测了siwi1和siwi2在丝腺、马氏管、中肠、头部、卵巢和精巢以及不同发育时期(卵、1～5龄幼虫、蛹、成虫)的表达水平,结果显示,克隆获得了siwi1 cDNA全长3 277 bp,包含部分5′UTR、完整的开放阅读框ORF和3′UTR,获得了siwi2的部分序列,其中siwi1对应BmPiwi,siwi2对应BmAgo3。系统发育结果显示,家蚕Piwi亚家族蛋白与乳草长蝽Oncopeltus fasciatus、黑腹果蝇Drosophila melanogaster、橘小实蝇Bactro...     

家蚕细胞周期蛋白基因BmCcnl1的克隆及表达分析

家蚕Bombyx mori由受精卵到完成胚胎发育孵化的过程中, 细胞进行大量的分裂和分化, 然而滞育性卵的胚胎细胞分化至G2期便停滞在此阶段。为了探索这一发育阶段细胞内的分子调控, 本研究以人Homo sapiens的细胞周期蛋白基因cyclin L1为模板, 成功克隆了家蚕同源基因BmCcnl1（GenBank登录号: FJ889988）。BmCcnl1基因开放阅读框（open reading frame, ORF）全长1 254 bp, 编码417个氨基酸。利用Protean软件分析得出BmCcnl1蛋白预测分子量为49 kDa, 等电点为9.84。利用DNA重组技术构建了BmCcnl1基因的重组表达载体pET-21d-BmCcnl1, 对其进行原核表达, 其表达的蛋白以包涵体形式存在。利用RT-PCR技术分析了BmCcnl1基因在胚胎发育过程中的转录水平, BmCcnl1基因在非滞育性卵的胚胎发育阶段基本保持相对稳定的转录表达, 而滞育性卵从蛾体产下经过72 h后已经检测不到BmCcnl1基因的转录。结果提示, BmCcnl1基因与胚胎期滞育及非滞育性卵的发育调控相关。对该基因的克隆和表达分析为今后研究家蚕胚胎发育及细胞周期调控奠定了基础。     

Expression of vasa and nanos3 during primordial germ cell formation and migration in Atlantic cod (Gadus morhua L.)

Primordial germ cells (PGCs), progenitors of gametes, are specified very early in embryonic development and undergo an active migration to the site where the future gonads will form. While the developmental pattern of PGCs during embryogenesis has been documented in few model teleost fishes, there is currently no information available for any representative of Superorder Paracanthopterygii. This includes Atlantic cod (Gadus morhua), which is a historically important food fish in both fisheries and aquaculture industries. In the present study, we cloned and characterized vasa and nanos3 and used them as germ cell markers in Atlantic cod. Sequencing results showed prospective vasa and nanos3 mRNA contained the domains used to describe their respective protein family. Furthermore, phylogenetic analysis using the amino acid sequence placed Atlantic cod Vasa distinct from representatives of three other taxonomic Superorders. Atlantic cod Nanos3 was placed with other homologues from the Nanos3 subfamily. Expression of both genes was detected from the first cleavage division; both were specifically expressed in Atlantic cod PGCs from the 32-cell stage. While nanos3 expression ceased during early somitogenesis, vasa was strongly expressed throughout embryonic development. Using vasa as a marker, we described the Atlantic cod PGC migration pattern. We demonstrated that Atlantic cod PGCs migrate ventral to the trunk mesoderm. With the exception of Pacific herring (Clupea pallasii), PGCs in other described teleost fishes migrate lateral to the trunk. The results from this study are the first step toward understanding germ line formation in Atlantic cod.