光棘球海胆seali基因克隆及表达特性分析

seali基因隶属于PIWI超家族, 其编码的RNA结合蛋白在生殖细胞发育过程中发挥重要作用。研究经同源比对从光棘球海胆(Mesocentrotus nudus)性腺转录组数据库中筛选得到seali基因片段, 随后通过cDNA末端快速扩增技术(Rapid amplification of cDNA ends, RACE), 获得其全长cDNA序列。光棘球海胆seali基因cDNA全长3462 bp, 其中3′UTR长度为416 bp, 5′UTR长度为180 bp, 其中3′UTR的加尾信号并非经典的AAUAAA或AUUAAA, 而是较少见的AAUACA。开放阅读框(Open Reading Frame, ORF)2862 bp, 编码954个氨基酸, 具有保守的PIWI和PAZ结构域, 多重序列比对和系统进化分析结果表明其属于Argonaute家族的PIWI亚家族成员。荧光定量PCR技术检测结果表明, Mnseali基因在光棘球海胆性腺、肠、管足和体腔细胞中均有表达, 在性腺中表达量最高。此外, Mnseali基因为母源因子, 在整个胚胎发育时期均有表达。在卵巢中, 随着卵母细胞的成熟, Mnseali的表达量逐渐升高, 而仅在成熟期的精巢中表达量显著上调。RNA原位杂交结果表明, Mnseali在光棘球海胆性腺的生殖细胞中特异表达, 是光棘球海胆生殖细胞标记基因。该研究为海胆生殖细胞发育相关的研究提供了支撑。     

多倍体鲫鲤原始生殖细胞的标记及其迁移研究

原始生殖细胞(primordial germ cells, PGCs)的起源和迁移已在多种鱼类进行了研究,但多倍体鲫鲤原始生殖细胞的标记和迁移尚未见报道。本实验室前期通过远缘杂交方法成功获得了一个倍性明确、亲缘关系清晰的多倍体杂交鱼研究体系,该体系由异源四倍体鲫鲤及其二倍体父母本和三倍体杂交后代组成。本文利用RNA定点表达(localized RNA expression, LRE)技术,将来自于斑马鱼的nanos1-3'-UTR与GFP融合后生成的m RNA注射入多倍体鱼受精卵中,首次对多倍体鲫鲤原始生殖细胞进行标记,并观察了其迁移途径。结果显示,多倍体鲫鲤PGCs能被斑马鱼GFP-nanos1-3'-UTR标记,并且多倍体鲫鲤PGCs的迁移与斑马鱼类似。本实验结果为多倍体鲫鲤原始生殖细胞的产生和迁移的研究提供了一些基础数据。     

抑制Ligase4或Xrcc6活性增强斑马鱼原始生殖细胞中DNA同源重组的效率

利用斑马鱼作为体内模型, 研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells, PGCs)中同源重组(Homologous recombination, HR)的效率。首先, 将UAS:mRFP-nos1载体显微注射到Tg (kop:KalTA4) 转基因胚胎中标记转基因PGCs, 结果表明筛选PGCs特异表达mRFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系, 并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平, 而且在PGCs水平都能够显著提高HR的效率。研究表明Tg (kop:KalTA4) 转基因品系是开展HR介导的基因打靶的一个有效平台。       

Nanos3与生殖细胞发育分化

小鼠Nanos3基因是果蝇Nanos的同源基因,是Nanos基因家族的一员. Nanos3是一种RNA结合蛋白,靠近其C端有两个非常保守且连续的Cys-Cys-His-Cys特异锌指结构域.研究显 示,Nanos3在小鼠生殖细胞中特异表达,不仅在原始生殖细胞（primordial germ cells, PGCs）的维持方面发挥重要作用,而且是一种启动雄性生殖细胞分化程序的内源性因子, 其在精子发生中的重要作用已引起越来越多的关注.探讨Nanos3的生物学功能,有助于了解生殖细胞发育过程中的部分重要机制.本文就Nanos3维持生殖细胞更新和原始生殖细胞的 维持等作用作一综述.     

家蚕生殖细胞相关基因nanos的克隆表达及在胚胎发育中的表达模式

【目的】探讨nanos基因在家蚕Bombyx mori胚胎发育中的表达模式,为进一步研究该基因在家蚕胚胎发育中的功能奠定基础。【方法】根据本实验室提交到GenBank中家蚕nanos的cDNA序列(登录号EF647589)设计引物,扩增出了一条长684 bp的编码片段,对该片段进行了克隆和表达,亲和纯化表达的蛋白并免疫新西兰大白兔制备抗体。Western blot检测家蚕早期胚胎nanos的表达情况,荧光定量PCR检测nanos在整个家蚕胚胎发育中的表达情况。【结果】克隆并表达了一条长684 bp的编码片段,得到了分子量约33 kD的融合蛋白。用制备的抗血清对家蚕早期胚胎蛋白的Western blot检测表明,nanos在此阶段基本是恒定表达。荧光定量PCR结果显示刚产的卵中nanos的表达量最大,第2天开始急剧下降,此后到第10天表达量几乎没有变化。【结论】本实验克隆的nanos是家蚕中的一个同源物,该基因在家蚕胚胎发育中的表达模式与蜜蜂等有很大的不同,反映了昆虫生殖细胞形成机制的多样性。     

抑制Ligase4或Xrcc6活性增强斑马鱼原始生殖细胞中DNA同源重组的效率（英文）

利用斑马鱼作为体内模型,研究旨在提高斑马鱼原始生殖细胞(Primordial germ cells,PGCs)中同源重组(Homologous recombination,HR)的效率。首先,将UAS:m RFP-nos1载体显微注射到Tg(kop:Kal TA4)转基因胚胎中标记转基因PGCs,结果表明筛选PGCs特异表达m RFP的胚胎能够相对提高转基因的生殖系传递效率。随后建立了PGCs中HR效率的评估体系,并且证明抑制DNA ligase IV(Lig4)和Xrcc6(曾用名Ku70)的活性不但在全胚胎水平,而且在PGCs水平都能够显著提高HR的效率。研究表明Tg(kop:Kal TA4)转基因品系是开展HR介导的基因打靶的一个有效平台。     

转录因子GATA3 mRNA 5′非翻译区内部核糖体进入位点的活性

GATA3(GA-TA-binding protein-3)是锌指蛋白GATA家族成员之一,在细胞的增殖和分化中起着重要的作用,GATA3在细胞中的异常表达也是导致众多肿瘤形成的原因。通过对GATA3m RNA 5′非翻译区(untranslated region,UTR)进行分析,发现其UTR长达557 bp并且具有复杂的二级结构。将GATA3 m RNA 5′UTR克隆至双荧光素酶报告载体p RL-FL中,瞬时转染至细胞中然后对细胞进行无血清培养后,发现GATA3 m RNA 5′UTR介导的翻译明显升高。将GATA3 m RNA 5′UTR克隆至Δp RL-FL载体上,瞬时转染细胞后检测萤火虫荧光素酶的表达,发现GATA3 m RNA 5′UTR不具有隐含启动子,进而确定GATA3 m RNA 5′UTR具有内部核糖体进入位点(internal ribosome entry sites,IRES)元件;进一步对GATA3 m RNA 5′UTR进行序列截短分析,发现GATA3 m RNA 5′UTR中345~557 bp区间可能是抑制IRES活性的调控元件,而95~344 bp区间则是IRES元件的主要活性中心调控域,并且在不同的细胞系中GATA3 IRES元件的活性存在显著的差异。该研究结果表明,GATA3m RNA的5′UTR可参与GATA3的表达调控。     

池蝶蚌STAT基因的克隆及功能分析

为了探究池蝶蚌(Hyriopsis schlegelii)信号转导及转录激活因子STAT基因的初步功能, 通过RACE-PCR技术首次在池蝶蚌中获得STAT的cDNA全长序列(GenBank ID: KY123702), 命名为HsSTAT, HsSTAT全长为2752 bp, 5′-非编码区(5′UTR)为121bp, 3′-非编码区3′UTR为264 bp, 开放阅读框(ORF)为2367 bp, 编码788个氨基酸; 生物信息学分析发现该蛋白结构域主要由STAT_int、STAT_alpha、STAT_bind、SH2四个经典保守性功能域组成; 缬氨酸最多占12.9%。色氨酸使用频率为100%。二级结构中α螺旋最多占46.83%、β折叠最少占8.5%。HsSTAT蛋白亲水性指数为– 0.531, 是亲水性蛋白。预测到棕榈酰化修饰位点1个, 小泛素相关修饰序列3个, 磷酸化修饰位点22个。系统进化树分析显示, HsSTAT与光滑双脐螺STAT5B和盘鲍STAT5聚在一支; 亚细胞定位结果显示HsSTAT定位于血细胞的细胞质中; 荧光原位杂交结果显示HsSTAT主要在细胞核。qPCR结果显示HsSTAT在所有的组织中均有表达, 其在组织中的表达量在肝胰腺中最高, 在血细胞中最低。细胞增殖实验显实验组增殖率为119%, 高于对照组。实验已克隆到HsSTAT的全长序列, HsSTAT可能为STAT5亚型, 具有促进SMCC-7721细胞增殖的功能, 推测具有池蝶蚌细胞的信号转导功能参与池蝶蚌的先天免疫。     

斑马鱼Zar1和Zar1-like基因的克隆及其表达分析

合子阻滞基因1(Zygote arrest 1,Zar1)及Zar1-like(Zar1L)作为母源基因,在小鼠卵子-合子转变过程中发挥着极其重要的作用。研究以斑马鱼为对象,采用cDNA末端快速克隆(RACE)技术获得了Zar1L cDNA全长,并采用RT-PCR技术检测Zar1和Zar1L在斑马鱼不同组织器官、卵母细胞和胚胎发育时期的表达情况。Zar1L cDNA的全长为1146 bp,编码311个氨基酸残基,5′非编码区20 bp,3′非编码区190 bp。多重序列比对结果显示,C-末端的序列非常保守,相似性高达74%,具有非典型的PHD基序(Plant homeo domain),并具有两个C4类型的锌指结构。在基因结构上,Zar1和Zar1L均由四个外显子构成,两个基因的每一个外显子在大小上也比较相近。分子进化表明斑马鱼Zar1和Zar1L属于两个不同的基因,可能是同一来源的祖先基因重复和进化的结果。RT-PCR结果表明斑马鱼Zar1和Zar1L的表达模式相似,都是卵巢特异表达基因,在卵母细胞及早期胚胎中可检测到大量转录本的存在,囊胚、原肠胚期后下降,但在整个胚胎发育期都可检测到转录本。斑马鱼Zar1和Zar1L mRNA的表达水平的一致性,似乎暗示着两个基因的功能基本一致。表达模式分析表明Zar1和Zar1L可能与斑马鱼卵子-合子转变有关,也可能与其他的胚胎发育过程有关。       

活细胞内人AGTR1-3′UTR的荧光标记及应激定位分析

利用噬菌体衣壳蛋白MS2和带有序列特异性茎环结构(含有MS2蛋白结合位点)的RNA之间的高度亲和力,对外源性人血管紧张素1型受体(angiotensin II receptor type 1,AGTR1)mRNA 3′端非翻译区(3′untranslated region,3′UTR)片段进行红色荧光标记,进而在活细胞(HeLa)内研究该mRNA片段的应激生物学行为。通过在pSG5空载体质粒上先后插入两个双链DNA目的片段AGTR1-3′UTR和24×MS2,构建重组质粒pSG5/AGTR1-3′UTR/24×MS2,并将该质粒与重组质粒pERFP/MS2和pEGFP/C1-G3BP共转染入Hela细胞。荧光显微成像结果显示,AGTR1-3′UTR-24×MS2 mRNA片段能够携带具有入核信号的MS2-RFP融合蛋白离开胞核进入胞浆,而且在亚砷酸盐刺激下,红色荧光标记的AGTR1-3′UTR-24×MS2 mRNA片段可在胞浆中形成与应激蛋白G3BP-GFP共定位的颗粒。该结果表明,针对AGTR1-3′UTR片段的MS2-RFP荧光标记系统构建成功,该荧光标记系统能有效避免假阳性的荧光信号。在细胞受到氧化应激时,AGTR1-3′UTR会被招募至胞浆中的应激颗粒结构中,启示了AGTR1-3′UTR区域对于调控AGTR1 mRNA在细胞内的应激定位具有重要作用。     

Conserved Mechanisms for Germ Cell-Specific Localization of nanos3 Transcripts in Teleost Species with Aquaculture Significance

The importance of the aquaculture production is increasing with the declining global fish stocks, but early sexual maturation in several farmed species reduces muscle growth and quality, and escapees could have a negative impact on wild populations. A possible solution to these problems is the production of sterile fish by ablation of the embryonic primordial germ cells (PGCs), a technique developed in zebrafish. Cell-specific regulation of mRNA stability is crucial for proper specification of the germ cell lineage and commonly involves microRNA (miRNA)-mediated degradation of targeted mRNAs in somatic cells. This study reports on the functional roles of conserved motifs in the 3′ untranslated region (UTR) of the miRNA target gene nanos3 identified in Atlantic cod, Atlantic salmon, and zebrafish. The 3′UTR of cod nanos3 was sufficient for targeting the expression of green fluorescent protein (GFP) to the presumptive PGCs in injected embryos of the three phylogenetically distant species. 3′UTR elements of importance for PGC-specific expression were further examined by fusing truncated 3′UTR variants of cod nanos3 to GFP followed by injections in zebrafish embryos. The expression patterns of the GFP constructs in PGCs and somatic cells suggested that the proximal U-rich region is responsible for the PGC-specific stabilization of the endogenous nanos3 mRNA. Morpholino-mediated downregulation of the RNA-binding protein Dead end (DnD), a PGC-specific inhibitor of miRNA action, abolished the fluorescence of the PGCs in cod and zebrafish embryos, suggesting a conserved DnD-dependent mechanism for germ cell survival and migration.     

DAZL Relieves miRNA-Mediated Repression of Germline mRNAs by Controlling Poly(A) Tail Length in Zebrafish

Background

During zebrafish embryogenesis, microRNA (miRNA) miR-430 contributes to restrict Nanos1 and TDRD7 to primordial germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational repression of nanos1 and tdrd7 mRNAs in somatic cells. The nanos1 and tdrd7 3′UTRs include cis-acting elements that allow activity in PGCs even in the presence of miRNA-mediated repression.

Methodology/Principal Findings

Using a GFP reporter mRNA that was fused with tdrd7 3′UTR, we show that a germline-specific RNA-binding protein DAZ-like (DAZL) can relieve the miR-430-mediated repression of tdrd7 mRNA by inducing poly(A) tail elongation (polyadenylation) in zebrafish. We also show that DAZL enhances protein synthesis via the 3′UTR of dazl mRNA, another germline mRNA targeted by miR-430.

Conclusions/Significance

Our present study indicated that DAZL acts as an “anti-miRNA factor” during vertebrate germ cell development. Our data also suggested that miRNA-mediated regulation can be modulated on specific target mRNAs through the poly(A) tail control.     

Translational repression restricts expression of the C. elegans Nanos homolog NOS-2 to the embryonic germline

Members of the nanos gene family are evolutionarily conserved regulators of germ cell development. In several organisms, Nanos protein expression is restricted to the primordial germ cells (PGCs) during early embryogenesis. Here, we investigate the regulation of the Caenorhabditis elegans nanos homolog nos-2. We find that the nos-2 RNA is translationally repressed. In the adult germline, translation of the nos-2 RNA is inhibited in growing oocytes, and this inhibition depends on a short stem loop in the nos-2 3'UTR. In embryos, nos-2 translation is repressed in early blastomeres, and this inhibition depends on a second region in the nos-2 3'UTR. nos-2 RNA is also degraded in somatic blastomeres by a process that is independent of translational repression and requires the CCCH finger proteins MEX-5 and MEX-6. Finally, the germ plasm component POS-1 activates nos-2 translation in the PGCs. A combination of translational repression, RNA degradation, and activation by germ plasm has also been implicated in the regulation of nanos homologs in Drosophila and zebrafish, suggesting the existence of conserved mechanisms to restrict Nanos expression to the germline.     

Maternal Nanos‐Dependent RNA Stabilization in the Primordial Germ Cells of Drosophila Embryos

Nanos (Nos) is an evolutionary conserved protein expressed in the germline of various animal species. In Drosophila, maternal Nos protein is essential for germline development. In the germline progenitors, or the primordial germ cells (PGCs), Nos binds to the 3′ UTR of target mRNAs to repress their translation. In contrast to this prevailing role of Nos, here we report that the 3′ UTR of CG32425 mRNA mediates Nos‐dependent RNA stabilization in PGCs. We found that the level of mRNA expressed from a reporter gene fused to the CG32425 3′ UTR was significantly reduced in PGCs lacking maternal Nos (nos PGCs) as compared with normal PGCs. By deleting the CG32425 3′ UTR, we identified the region required for mRNA stabilization, which includes Nos‐binding sites. In normal embryos, CG32425 mRNA was maternally supplied into PGCs and remained in this cell type during embryogenesis. However, as expected from our reporter assay, the levels of CG32425 mRNA and its protein product expressed in nos PGCs were lower than in normal PGCs. Thus, we propose that Nos protein has dual functions in translational repression and stabilization of specific RNAs to ensure proper germline development.     

Visualization of the Xenopus primordial germ cells using a green fluorescent protein controlled by cis elements of the 3' untranslated region of the DEADSouth gene

We succeeded in visualization of the primordial germ cells (PGCs) in a living Xenopus embryo. The mRNA of the reporter Venus protein, fused to the 3' untranslated region (UTR) of DEADSouth, which is a component of the germ plasm in Xenopus eggs, was microinjected into the vegetal pole of fertilized eggs and then the cells with Venus fluorescence were monitored during development. The behavior of the cells was identical to that previously described for PGCs. Almost all Venus-expressing cells were Xdazl-positive in the stage 48 tadpoles, indicating that they were PGCs. In addition, we found three sub-regions (A, B and C) in the 3' UTR, which were involved in the PGC-specific expression of the reporter protein. Sub-region A, which was identified previously as a localization signal for the germ plasm during oogenesis, participated in anchoring of the mRNA at the germ plasm and the degradation of the mRNA in the somatic cells. Sub-regions B and C were also involved in anchoring of the mRNA at the germ plasm. Sub-region B participated in the enhancement of translation.     

Effects of specific and prolonged expression of zebrafish growth factors,Fgf2 and Lif in primordial germ cells in vivo

Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3′UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3′UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks. The results show that expression of Fgf2 significantly increased PGC number at 14- and 21-dpf while Bmp4 resulted in severe ventralization and death of the embryos by 3 days. Expression of Lif resulted in a significant disruption of PGC migration. Mopholino knockdown experiments indicated that Lif illicited its effect on PGC migration through Lif receptor a (Lifra) but not Lifrb. The general approach described in this study could be used to achieve prolonged PGC-specific expression of other proteins to investigate their roles in germ cell and gonad development. The results also indicate that zebrafish PGCs have a mechanism to stabilize and prolong the expression of mRNA that carries nanos3 3′UTR. Understanding this mechanism may make it possible to achieve prolonged RNA expression in other cell types.     

vasa and nanos expression patterns in a sea anemone and the evolution of bilaterian germ cell specification mechanisms

Most bilaterians specify primordial germ cells (PGCs) during early embryogenesis using either inherited cytoplasmic germ line determinants (preformation) or induction of germ cell fate through signaling pathways (epigenesis). However, data from nonbilaterian animals suggest that ancestral metazoans may have specified germ cells very differently from most extant bilaterians. Cnidarians and sponges have been reported to generate germ cells continuously throughout reproductive life, but previous studies on members of these basal phyla have not examined embryonic germ cell origin. To try to define the embryonic origin of PGCs in the sea anemone Nematostella vectensis, we examined the expression of members of the vasa and nanos gene families, which are critical genes in bilaterian germ cell specification and development. We found that vasa and nanos family genes are expressed not only in presumptive PGCs late in embryonic development, but also in multiple somatic cell types during early embryogenesis. These results suggest one way in which preformation in germ cell development might have evolved from the ancestral epigenetic mechanism that was probably used by a metazoan ancestor.     

Isolation of teleost primordial germ cells using flow cytometry

Primordial germ cells (PGCs) generate gametes, the only cells that can transmit genetic information to the next generation. A previous report demonstrated that a fusion construct of green fluorescent protein (gfp) and zebrafish nos 1 3UTR mRNA could be used to label PGCs in a number of fish species. Here, we sought to exploit this labeling strategy to isolate teleost PGCs by flow cytometry (FCM), and to use these isolated PGCs to examine germ cell migration to the gonadal region. In zebrafish, medaka and goldfish, the PGCs were labeled by injecting the gfp-nos1 3UTR mRNA into 1- 4 cell embryos. When the embryos had developed to the somitogenesis or later stages, they were enzymatically disaggregated and GFP positive cells isolated using FCM. PGCs in the different species clustered in the same segments of the FCM scatter diagrams for total embryonic cells produced by plotting the forward scatter intensity against GFP intensity. In situ hybridization showed that the sorted zebrafish cells expressed vasa RNA in their cytoplasm, suggesting that they were PGCs. When the migration ability of the sorted cells from zebrafish was examined in an in vivo transplantation experiment, approximately 30% moved to the gonadal region of host embryos. These observations demonstrate that PGCs can be isolated without use of transgenic fishes and that the isolated PGCs retain the ability to migrate. Our data indicate that this technique will be of value for isolating PGCs from a range of fish species.