细菌介导的发夹结构双链RNA对秀丽小杆线虫中par-1基因的RNA干扰

秀丽小杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前在C.elegans中发现的许多基因在序列和功能上与哺乳动物的基因有很高的同源性,对其基因功能的研究有助于阐明哺乳动物的基因功能。为检测细菌介导的通过发夹结构表达双链RNA(dsRNA)的RNA干扰(RNAi)在C.elegans中的作用效果,我们设计并构建了可以转录表达母源极性基因par-1的发夹dsRNA的RNAi载体,并转入E.coli HT115中,经过IPTG诱导后浓缩。浓缩菌液在25℃下喂食C.elegans48h后,观察C.elegans胚胎发育情况,同时以含有空载体的菌液作为对照,并与par-1突变体及野生型比较。结果显示:发夹结构表达的dsRNA对par-1基因进行RNAi后,虫体内par-1 mRNA几乎消失,C.elegans早期胚胎分裂不对称性丧失,par-1(RNAi)干扰率在95%以上。     

原位杂交检测pal-1 mRNA在秀丽小杆线虫野生型和突变体早期胚胎中的分布

pal-1是秀丽小杆线虫(Caenorhabditis elegans)早期胚胎发育中决定体细胞命运的重要基因,也是转录因子,调控后续基因的表达,凡含有该基因表达的细胞发育成体细胞。本文通过整体原位杂交技术检测pal-1mRNA在C·elegans野生型和par-1、par-2、par-3、par-4突变体、spn-4突变体、mex-5/mex-6突变体早期胚胎中的分布,探讨这些基因在胚胎发育早期对pal-1mRNA的影响。实验结果表明:par-1、par-3、par-4突变使4细胞胚胎pal-1mRNA完全丧失了野生型不对称分布模式,pal-1mRNA分布在所有卵裂球中;par-2对pal-1mRNA的分布影响较小,在par-2突变体4细胞胚胎中pal-1mRNA分布与野生型相同。spn-4、mex-5、mex-6也能影响pal-1mRNA的分布,使其分布丧失不对称性。在par-1、par-4突变的情况下,pal-1mRNA广泛存在,但PAL-1蛋白也不表达,显示对pal-1mRNA的翻译调控是PAL-1蛋白空间和时序不对称分布的主要原因[动物学报51(5):884-891,2005]。     

原位杂交检测mex-3 mRNA在C．elegans野生型胚胎发育中的分布

厦门大学生命科学学院杨玉荣博士在发育生物学中对有关胚胎发育的专题作了研究,指出Caenorhabditis elegans是发育生物学中的重要模式生物,也是全基因序列已知的惟一多细胞生物,如动物,许多基因参与C．elegans的细胞命运决定和介导细胞谱系发育。mex-3是C．elegans早期发育中细胞命运决定的重要基因。采用原位杂交技术检测mex-3 mRNA在C．elegans野生型胚胎发育中的分布,发现mex-3 mRNA在生殖腺和卵细胞的胞质都有广泛而丰富的分布,从受精开始mex-3 mRNA的分布开始发生了变化,     

C.elegans野生型和par突变体早期胚胎卵裂的观察

不对称分裂在动植物的发育中起到了非常重要的作用。Caenorhabditis elegans（C．elegans）胚胎最早的两次卵裂为研究控制不对称分裂的机制提供了很好的机会。用普通光学显微镜观察了野生型胚胎早期卵裂和par-1、par-2、par-3、par-4突变体胚胎的早期卵裂。野生型胚胎最早的分裂是不等的,产生了两个不同大小的子细胞。两个子细胞又以不同的方向进行第二次分裂。在C．elegans中任意一个par基因的缺失会使胚胎的第一次卵裂丧失不对称性。这会导致一些发育调控因子不能在特定的胚胎细胞中准确地定位,造成细胞分裂纺锤体方向的异常。par类基因参与不对称性的建立,这种不对称性决定了C．elegans身体的前后轴。     

PTC下游翻译重启导致NMD途径的逃逸

无义突变介导的mRNA降解(nonsense-mediated mRNA decay,NMD)途径是真核生物体内一种重要的mRNA监督质控机制,它降解含有由无义突变、错误剪接、移码突变等产生的提前终止翻译密码子(premature translation termination codon,PTC)的mRNA,从而防止这种mRNA翻译产生的截短型蛋白质对机体造成的伤害.研究发现,一些含有PTC的mRNA发生了NMD途径逃逸,但具体机制仍不清楚.本研究将成视网膜细胞瘤基因1(retinoblastoma gene 1,RB1)作为NMD途径的靶基因,构建mini-RB1基因,包括外显子1~14(c DNA)、内含子14-外显子15-内含子15和外显子16~27(c DNA)的三部分序列,将其构建到真核表达载体pc DNA 3.1(-)中.根据人类基因组突变数据库选择3个突变位点W99X、G310X和R467X,构建相应无义突变体.分别将mini-RB1基因野生型和无义突变体转入He La细胞进行表达.用qRT-PCR检测发现,W99X无义突变体与野生型相比mRNA的水平无显著差异.为了进一步证实mini-RB1(W99X)发生了NMD逃逸,利用NMD途径抑制剂放线菌酮和转录抑制剂放线菌素D,分别处理转入野生型的mini-RB1基因及其无义突变体mini-RB1(W99X)的哺乳动物细胞,发现mini-RB1基因无义突变体的mRNA水平与野生型无明显差异,说明含有W99X无义突变的mini-RB1基因的mRNA发生了NMD逃逸.Western印迹检测mini-RB1基因的蛋白质表达发现,在无义突变位点W99X下游重新起始了蛋白质的翻译,因此,PTC下游蛋白质翻译的重新起始可能是导致无义mRNA逃逸NMD途径监控的主要原因.     

PTC下游翻译的重新起始导致NMD途径的逃逸

无义突变介导的mRNA降解（nonsense mediated mRNA decay, NMD）途径是真核生物体内一种重要的mRNA监督质控机制, 它降解含有由无义突变、错误剪接、移码突变等产生的提前终止翻译密码子（premature translation termination codon, PTC）的mRNA, 从而防止这种mRNA翻译产生的截短型蛋白质对机体造成的伤害. 研究发现, 一些含有PTC的mRNA发生了NMD途径逃逸, 但具体机制仍不清楚.本研究将成视网膜细胞瘤基因1 (retinoblastoma gene 1, RB1)作为NMD途径的靶基因, 构建mini-RB1基因,包括外显子1～14(cDNA)、内含子14 外显子15 内含子15和外显子16～27(cDNA) 的三部分序列, 将其构建到真核表达载体pcDNA 3.1(-)中.根据人类基因组突变数据库选择3个突变位点W99X、G310X和R467X, 构建相应无义突变体.分别将mini RB1基因野生型和无义突变体转入HeLa细胞进行表达.用qRT-PCR检测发现, W99X无义突变体与野生型相比mRNA的水平无显著差异.为了进一步证实mini- RB1（W99X）发生了NMD逃逸, 利用NMD途径抑制剂放线菌酮和转录抑制剂放线菌素D, 分别处理转入野生型的mini RB1基因及其无义突变体mini-RB1（W99X）的哺乳动物细胞, 发现mini-RB1基因无义突变体的mRNA水平与野生型无明显差异, 说明含有W99X无义突变的mini-RB1基因的mRNA发生了NMD逃逸.Western印迹检测mini-RB1基因的蛋白质表达发现, 在无义突变位点W99X下游重新起始了蛋白质的翻译, 因此,PTC下游蛋白质翻译的重新起始可能是导致无义mRNA逃逸NMD途径监控的主要原因.     

黄瓜花叶病毒2b蛋白C末端无抑制局部RNA沉默的活性

目的:黄瓜花叶病毒 (Cucumber mosaic virus,CMV) 编码的2b蛋白具有RNA沉默抑制子的功能,其C末端氨基酸序列非常保守。为了明确2b蛋白C末端保守序列在RNA沉默抑制中的作用,构建了CMV Q株系野生型2b及其C末端缺失突变体2b^delC的植物瞬时表达载体。通过农杆菌共渗滤法对野生型2b及其C末端突变体的沉默抑制子活性进行了分析。结果与结论:烟草接种叶片中野生型2b及其C末端突变体的Western blot检测表明,野生型2b蛋白与其C末端突变体在植物中积累水平变化不大,说明2b蛋白C末端氨基酸残基在维持2b蛋白在植物细胞中的稳定性方面无作用。在整株、细胞和分子水平上分别比较了野生型2b及其突变体2b^delC对共表达GFP的表达量影响,结果表明在所有的测定结果中二者均无明显地差异,说明2b蛋白C末端94-111位氨基酸在抑制局部RNA沉默上无生物学活性,讨论推测C末端应不存在与小RNA结合的结构域。     

拟南芥bso-1突变体的基因定位及表型分析

通过EMS化学诱变在拟南芥Columbia(Col-0)野生型突变体库中筛选获得1株器官显著增大的突变体,命名为big size organ1(bso-1)。遗传分析表明,bso-1受单个隐性核基因控制。表型观察发现,突变体植株的幼苗、花、果荚及种子与野生型相比都表现出明显的增大。组织切片结果显示,突变体种子的增大主要由胚细胞个体增大导致胚体积增大而实现,因此突变体种子的重量也较野生型有明显增加。利用图位克隆方法将相关基因初步定位在4号染色体上SSLP标记T5L19与F28M11之间58kb区间内,生物信息学分析显示此区间内未见调控植物器官大小发育相关的已知基因的报道。该研究结果为进一步克隆bso-1突变体相关基因及探讨其在控制植物器官发育尤其是种子发育过程中的作用奠定了基础。     

大豆茸毛突变体的鉴定及相关基因表达分析

茸毛是大豆的重要形态性状,对其自身的生长发育有着重要作用,也与百粒重等诸多农艺性状相关,因此对大豆茸毛的研究显得尤为重要。本研究从大豆EMS突变体库中筛选到4个茸毛发育异常的突变体,分别是短茸毛突变体ddsp1,无茸毛突变体ddsp2,茸毛部分空瘪突变体ddsp3和茸毛完全空瘪突变体ddsp4。扫描电镜观察结果表明这些突变体中茸毛的长度或形态等都发生了明显的变化,同时突变体的籽粒大小、百粒重等也发生了明显的改变。选取了10个可能与大豆茸毛发育相关的同源基因,分别是参与正调控茸毛发育起始的基因GL1、GL2、GL3和TCL1,参与核内复制负调控的基因RBR1、SIM、KAK和SPY以及参与核内复制正调控的基因CPR5和RHL,通过qRT-PCR分析了这10个基因在4个茸毛突变体和野生型中的表达情况。结果表明,控制茸毛发育起始的基因除了GL1和GL2分别在突变体ddsp2和ddsp4中显著下调表达之外,其余控制茸毛发育起始的基因均显著上调或无显著变化。参与核内复制负调控的基因中,除了RBR1基因只在突变体ddsp1中显著上调表达之外,SIM、KAK和SPY在这4个突变体中的表达水平均显著升高。而参与核内复制正调控的基因CPR5和RHL在4个突变体中的相对表达量均显著增加。本研究通过对茸毛相关基因的表达分析,为进一步挖掘控制大豆茸毛发育的基因以及研究基因的作用机理和调控模式奠定了基础。     

MicroRNA在哺乳动物精子发生中的作用

MicroRNAs(miRNAs)是一类长20~24 nt的单链非编码调控RNA序列。miRNA作为基因转录后表达调控分子,通过碱基互补配对的方式与靶mRNA结合,从而导致靶mRNA的降解或抑制其翻译过程。从最早发现存在于秀丽隐杆线虫Caenorhabditis elegans中的miRNA lin-4和let-7至今20多年里,研究人员已陆续从不同的种属中发现了大量的miRNA。近年来随着基因克隆、表达和功能研究技术的应用和发展,通过分析不同动物物种睾丸组织中miRNA的变化表明miRNA与精子发生过程密切相关。此外,miRNA相关的Dicer、Drosha等蛋白在初级精母细胞减数分裂粗线期所发挥的调控功能通过大量啮齿动物基因敲除模型得到证实。本文从miRNA的合成、作用机制和精子发生过程中的调控作用进行综述。     

Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation

The heterochronic gene lin-28 of the nematode Caenorhabditis elegans controls the relative timing of diverse developmental events during the animal's larval stages. lin-28 is stage-specifically regulated by two genetic circuits: negatively by the 22-nt RNA lin-4 and positively by the heterochronic gene lin-14. Here, we show that lin-28 is repressed during normal development by a mechanism that acts on its mRNA after translation initiation. We provide evidence that lin-14 inhibits a negative regulation that is independent of the lin-4 RNA and involves the gene daf-12, which encodes a nuclear hormone receptor. The lin-4-independent repression does not affect the initiation of translation on the lin-28 mRNA, and like the lin-4-mediated repression, acts through the gene's 3'-untranslated region. In addition, we find that lin-4 is not sufficient to cause repression of lin-28 if the lin-4-independent circuit is inhibited. Therefore, the lin-4-independent circuit likely contributes substantially to the down-regulation of lin-28 that occurs during normal development. The role of lin-4 may be to initiate or potentiate the lin-4-independent circuit. We speculate that a parallel lin-4-independent regulatory mechanism regulates the expression of lin-14.     

A hierarchy of regulatory genes controls a larva-to-adult developmental switch in C. elegans

The heterochronic genes lin-4, lin-14, lin-28, and lin-29 control the timing of specific postembryonic developmental events in C. elegans. The experiments described here examine how these four genes interact to control a particular stage-specific event of the lateral hypodermal cell lineages. This event, termed the "larva-to-adult switch" (L/A switch), involves several coordinate changes in the behavior of hypodermal cells at the fourth molt: cessation of cell division, formation of adult (instead of larval) cuticle, cell fusion, and cessation of the molting cycle. The phenotypes of multiply mutant strains suggest a model wherein the L/A switch is controlled by the stage-specific activity of a regulatory hierarchy: At early stages of wild-type development, lin-14 and lin-28 inhibit lin-29 and thus prevent switching. Later, lin-4 inhibits lin-14 and lin-28, allowing activation of lin-29, which in turn triggers the switch in the L4 stage. lin-29 may activate the L/A switch by regulating genes that control cell division, differentiation, and stage-specific gene expression in hypodermal cells.     

lin-28 controls the succession of cell fate choices via two distinct activities

lin-28 is a conserved regulator of cell fate succession in animals. In Caenorhabditis elegans, it is a component of the heterochronic gene pathway that governs larval developmental timing, while its vertebrate homologs promote pluripotency and control differentiation in diverse tissues. The RNA binding protein encoded by lin-28 can directly inhibit let-7 microRNA processing by a novel mechanism that is conserved from worms to humans. We found that C. elegans LIN-28 protein can interact with four distinct let-7 family pre-microRNAs, but in vivo inhibits the premature accumulation of only let-7. Surprisingly, however, lin-28 does not require let-7 or its relatives for its characteristic promotion of second larval stage cell fates. In other words, we find that the premature accumulation of mature let-7 does not account for lin-28's precocious phenotype. To explain let-7's role in lin-28 activity, we provide evidence that lin-28 acts in two steps: first, the let-7-independent positive regulation of hbl-1 through its 3'UTR to control L2 stage-specific cell fates; and second, a let-7-dependent step that controls subsequent fates via repression of lin-41. Our evidence also indicates that let-7 functions one stage earlier in C. elegans development than previously thought. Importantly, lin-28's two-step mechanism resembles that of the heterochronic gene lin-14, and the overlap of their activities suggests a clockwork mechanism for developmental timing. Furthermore, this model explains the previous observation that mammalian Lin28 has two genetically separable activities. Thus, lin-28's two-step mechanism may be an essential feature of its evolutionarily conserved role in cell fate succession.     

Multiple mechanisms are involved in regulating the expression of the developmental timing regulator lin-28 in Caenorhabditis elegans

The timing of postembryonic developmental programs in Caenorhabditis elegans is regulated by a set of so-called heterochronic genes, including lin-28 that specifies second larval programs. lin-66 mutations described herein cause delays in vulval and seam cell differentiation, indicating a role for lin-66 in timing regulation. A mutation in daf-12/nuclear receptor or alg-1/argonaute dramatically enhances the retarded phenotypes of the lin-66 mutants, and these phenotypes are suppressed by a lin-28 null allele. We further show that the LIN-28 protein level is upregulated in the lin-66 mutants and that this regulation is mediated by the 3'UTR of lin-28. We have also identified a potential daf-12-response element within lin-28 3'UTR and show that two microRNA (miRNA) (lin-4 and let-7)-binding sites mediate redundant inhibitory activities that are likely lin-66-independent. Quantitative PCR data suggest that the lin-28 mRNA level is affected by lin-14 and miRNA regulation, but not by daf-12 and lin-66 regulation. These results suggest that lin-28 expression is regulated by multiple independent mechanisms including LIN-14-mediated upregulation of mRNA level, miRNAs-mediated RNA degradation, LIN-66-mediated translational inhibition and DAF-12-involved translation promotion.     

The bromodomain protein LIN-49 and trithorax-related protein LIN-59 affect development and gene expression in Caenorhabditis elegans

We have molecularly characterized the lin-49 and lin-59 genes in C. elegans, and found their products are related to Drosophila trithorax group (trx-G) proteins and other proteins implicated in chromatin remodelling. LIN-49 is structurally most similar to the human bromodomain protein BR140, and LIN-59 is most similar to the Drosophila trx-G protein ASH1. In C. elegans, lin-49 and lin-59 are required for the normal development of the mating structures of the adult male tail, for the normal morphology and function of hindgut (rectum) cells in both males and hermaphrodites and for the maintenance of structural integrity in the hindgut and egg-laying system in adults. Expression of the Hox genes egl-5 and mab-5 is reduced in lin-49 and lin-59 mutants, suggesting lin-49 and lin-59 regulate HOM-C gene expression in C. elegans as the trx-G genes do in Drosophila. lin-49 and lin-59 transgenes are expressed widely throughout C. elegans animals. Thus, in contrast to the C. elegans Polycomb group (Pc-G)-related genes mes-2 and mes-6 that function primarily in the germline, we propose lin-49 and lin-59 function in somatic development similar to the Drosophila trx-G genes.