黑腹果蝇头部中RNA结合蛋白RBP9的互作蛋白筛选

【目的】筛选RNA结合蛋白RBP9在黑腹果蝇Drosophila melanogaster头部中的互作蛋白。【方法】利用CRISPR/Cas9基因组编辑技术，分别将3×Flag和V5标签编码序列插入到黑腹果蝇成虫头中RBP9和FNE基因起始密码子ATG的后面，构建黑腹果蝇转基因品系3×Flag-RBP 9/3×Flag-RBP9(3FRBP9)和V5-FNE/V5-FNE(VFNE)；利用免疫沉淀和质谱法鉴定黑腹果蝇3FRBP9和野生型品系成虫头部中RBP9的互作蛋白并进行生物信息学分析。利用免疫共沉淀方法检测RBP9与FNE蛋白之间的相互作用。【结果】质谱法从黑腹果蝇成虫头部共鉴定了190个与RBP9相互作用的蛋白，其中包括ELAV和FNE。KEGG富集分析显示这些蛋白的基因主要参与核糖体、碳代谢、三羧酸循环、氨基酸生物合成等通路。免疫共沉淀实验结果验证了RBP9与FNE蛋白之间存在相互作用。【结论】RNA结合蛋白ELAV家族成员在黑腹果蝇成虫头部中具有相互作用。本研究为RBP9在果蝇神经系统发育中的功能研究提供了重要实验依据。     

黑腹果蝇的性别控制

性别的形成包括两个过程,即性别决定和性别分化。果蝇的性别控制研究包括性别决定、性别分化、性别鉴定、性别诱导和性别控制5个方面。性别决定是在两种不同发育途径之间的选择,它提供了一个研究基因调控的模式系统。果蝇的性别决定问题已经研究得相当详细［1］。性别分化是使胚胎向着雌性或雄性发育的过程,决定了性别表型。果蝇的性别分化也取得了不少研究成果。近年来,许多重要的性别调控基因已被克隆和鉴定。随着果蝇基因组全序列测定的完成,果蝇的性别控制研究将会更为深入而完善。本文对与黑腹果蝇性别决定和性别分化相关的一些问题进行综述。     

沙丁胺醇对黑腹果蝇基因组稳定性、细胞凋亡和蛋白表达的影响

【目的】已有研究表明,食用了饲喂以沙丁胺醇为主要成分的瘦肉精的动物肉类后,瘦肉精成分会在人体内富集,摄入过量沙丁胺醇会对生物体造成不良影响,但是,其具体毒性作用机理目前尚不明确。作为一种模式生物,黑腹果蝇Drosophila melanogaster与哺乳动物的基因具有较高的同源性,且具有繁殖周期短、方便进行遗传操作等优势。因此,我们通过研究过量沙丁胺醇对黑腹果蝇基因组稳定性、细胞凋亡和蛋白表达的影响,来探究它对生物体毒性作用的机理。【方法】将野生型黑腹果蝇3龄幼虫用含沙丁胺醇（120 μg/mL）的饲料饲喂2 h后,对幼虫翅成虫盘进行H2Av抗体免疫染色。选取rpr-lacZ转基因黑腹果蝇1龄幼虫用含沙丁胺醇（40 和120 μg/mL）的饲料饲喂,对幼虫翅成虫盘进行lacZ活性测定。提取沙丁胺醇处理后的野生型3龄幼虫总蛋白,采用SDS-PAGE比较对照组和实验组蛋白表达的差异,并通过质谱分析差异蛋白的氨基酸序列。【结果】沙丁胺醇处理后,经免疫荧光染色发现野生型黑腹果蝇幼虫翅成虫盘部分细胞中组蛋白H2Av的量有显著增加。随着沙丁胺醇浓度的增加,转rpr-lacZ报告基因黑腹果蝇成虫盘细胞lacZ活性增强。采用SDS-PAGE和质谱分析表明,沙丁胺醇处理后黑腹果蝇肌动蛋白（Actin-87E）和异柠檬酸脱氢酶表达量上升。【结论】沙丁胺醇处理会引起黑腹果蝇细胞核DNA损伤,对基因稳定性有显著影响,并且会促进细胞凋亡和蛋白表达的改变。沙丁胺醇可能通过促进肌肉收缩和加速生物体能量代谢这两方面来减少脂肪积蓄。     

高效特异的CRISPR/dCas9转录激活系统——flySAM系统

正随着人类等物种的基因组计划的完成,关于基因组的研究已经从结构基因组学转向了功能基因组学。将基因组的序列信息转化为功能信息,解密生命的密码,完成基因组的功能注释对于全面理解生长发育、疾病衰老、学习记忆等过程具有重大意义。黑腹果蝇(Drosophila melanogaster)具有易于饲养、     

植物乳杆菌促进黑腹果蝇生长发育

【目的】检测乳酸菌对果蝇发育历期的影响,进一步探讨其对果蝇促生长的分子机制。【方法】利用选择性培养基MRS从黑腹果蝇Drosophila melanogaster体内分离乳酸菌,利用革兰氏染色、生化方法及16S rRNA基因进行鉴定;通过体内定植和世代传递实验验证该菌是黑腹果蝇的共生菌;采用悉生模型检测乳酸菌对黑腹果蝇发育的促生长作用;利用实时定量PCR技术检测黑腹果蝇体内促前胸腺激素基因PTTH和胰岛素通路相关基因InR的表达水平;利用葡萄糖试剂盒检测血淋巴液葡萄糖浓度。【结果】从黑腹果蝇中分离到的菌株鉴定为植物乳杆菌Lactobacillus plantarum FY1菌株(Gen Bank登录号:KY038178),可在黑腹果蝇肠道内定植,每个肠道定植量约为104CFU,并能在世代间稳定传递。FY1菌株体外发酵可降低p H值至5.2,可诱导无菌果蝇卵至蛹发育时间由20.0 d缩短至6.9 d,卵至成虫发育时间由30.0 d缩短至10.7 d,其生长速率是无菌果蝇的约2倍。实时定量PCR结果表明,FY1菌株显著地提前了PTTH表达高峰期,同时降低果蝇中InR表达水平,血淋巴液葡萄糖浓度从5.1 mg/mL降低至2.7 mg/mL。【结论】植物乳杆菌是黑腹果蝇的一种益生菌,推测能通过胰岛素信号通路促进宿主黑腹果蝇的生长和发育。     

黑腹果蝇头部响应高盐摄入的基因可变剪接分析

高盐摄入不仅会带来高血压、糖尿病、自身免疫病等风险,而且会对大脑产生负面影响。可变剪接(Alternative Splicing,AS)作为基因表达调控的重要方式,其导致单个基因编码多种蛋白质,大大增加基因组编码的蛋白质的多样性,参与几乎所有的生物学过程。但是,关于高盐摄入是否改变神经系统中基因的可变剪接事件目前还未报道。本文以黑腹果蝇作为材料,利用RNA测序分析其头部响应高盐摄入的基因可变剪接变化。结果发现高盐摄入改变黑腹果蝇头部信号转导、离子稳态、离子通道活性、离子转运和钙调蛋白结合等重要信号相关基因的可变剪接。进一步分析具体发生变化的基因的功能,发现它们在多个层面对神经系统功能的调控起着重要的作用。该研究证明高盐摄入改变神经系统重要基因的可变剪接,为理解高盐摄入对神经系统的影响提供更多的依据和线索。     

黑腹果蝇CG18853基因编码蛋白的生物信息学分析

目的:利用生物信息学方法分析黑腹果蝇CG18853基因编码蛋白的结构和功能。方法:基于NCBI数据库中黑腹果蝇CG18853基因编码蛋白的氨基酸序列,从蛋白质的理化性质、跨膜区、信号肽、亚细胞定位、结构域、三维结构及不同物种间同源蛋白进化关系等方面进行分析。结果:果蝇CG18853蛋白的理论分子量约38.5 kDa,理论等电点为8.80。CG18853蛋白为不稳定亲水性蛋白,无跨膜区和信号肽,具有DNA光修复酶FAD结合结构域。果蝇CG18853蛋白与模板3umv.1.A有60.87%的氨基酸序列一致;CG18853蛋白与长鼻袋鼠、金鱼、拟南芥、粳稻的编码产物高度同源。结论:黑腹果蝇CG18853蛋白具有DNA光修复酶家族的典型结构,可能在细胞核中参与DNA损伤修复过程。     

敲低piwi基因对黑腹果蝇血细胞增殖及分化的影响

【目的】探究敲低piwi基因对黑腹果蝇Drosophila melanogaster血细胞增殖及分化的影响。【方法】利用黑腹果蝇e33C-Gal4和Hml-Gal4-UAS-2×EGFP品系分别与野生型w¹¹¹⁸和UAS-piwi RNAi品系杂交,实现在黑腹果蝇游离血细胞或淋巴腺中降低piwi基因的表达;采用免疫荧光染色方法检测Piwi蛋白在血细胞中的定位及其对黑腹果蝇血细胞增殖与分化的影响。【结果】Piwi蛋白在黑腹果蝇游离血细胞及整个淋巴腺中都表达,且主要定位在细胞质;敲低piwi基因导致游离血细胞数量明显增加,处于有丝分裂M期的细胞数量增加,但未影响游离血细胞中浆细胞及薄层细胞的分化;敲低piwi基因对淋巴腺血细胞增殖无影响,但导致浆细胞过度分化及薄层细胞的产生。【结论】piwi基因在果蝇游离血细胞中的缺失可引起血细胞过度增殖,而在淋巴腺中敲低可引起血细胞的异常分化。     

果蝇研究技术与资源的开发

黑腹果蝇(Drosophila melanogaster)是进行生物医学研究的理想模型,但研究过程往往受到遗传工具和品系资源的限制,无法有效地调控目的基因表达,阻碍实验的深入开展。为了方便果蝇领域的实验室开展研究,我们首先开发并优化了基于CRISPR/Cas9的基因组编辑技术,随后研发了转录激活系统和新一代转基因干扰技术,最终可以简单高效、准确特异地调控目的基因表达。同时,利用这些遗传学新技术,我们构建了相应的基因敲除、转基因转录激活、转基因干扰的果蝇品系资源库,使清华大学果蝇中心成为世界上最重要的果蝇技术和资源中心之一。这些新技术以及相应的果蝇资源,正在被国内外果蝇研究领域的实验室所应用,对发育和疾病等相关研究发挥着广泛的促进作用。     

果蝇蛋白质组学研究进展

蛋白质组学旨在阐明基因组所表达的真正执行生命活动的全部蛋白质的表达规律和生物功能。随着人类基因组学计划的逐渐成熟,分子水平的实验技术不断发展,蛋白质组学的研究被提高到了前所未有的高度。果蝇是生命科学领域最为常用的一种模式生物,长期的系统研究也使果蝇的基因组成为至今注释最好的基因组之一,为功能基因组研究奠定了基础。但由于技术的限制,迄今有关果蝇蛋白质组学研究的报道尚不多见。近年来果蝇蛋白质组学的研究主要包括表达谱、修饰谱、比较蛋白质组学和疾病模型蛋白质组等四个方向,为进一步开展人类疾病临床蛋白质组学研究奠定了基础。     

Characterization of Dir: a putative potassium inward rectifying channel in Drosophila

Potassium channels vary in their function and regulation, yet they maintain a number of important features - they are involved in the control of potassium flow, cell volume, cell membrane resting potential, cell excitability and hormone release. The potassium (K(+)) inward rectifier (Kir) superfamily of channels are potassium selective channels, that are sensitive to the concentration of K(+) ions. They are termed inward rectifiers since they allow a much greater K(+) influx than efflux. There are at least seven subfamilies of Kir channels, grouped according to sequence and functional similarities (Curr. Opin. Neurobiol. 5 (1995) 268; Annu. Rev. Physiol. 59 (1997) 171). While numerous Kir channels have been discovered in a variety of organisms, Drosophila inward rectifier (Dir) is the first putative inward rectifier to be studied in Drosophila. In fact, there are only three genes (including Dir) encoding putative inward rectifiers in the Drosophila genome. Though there are other known potassium channels in Drosophila such as ether-a-go-go and shaker, most are voltage-gated channels. As an important first step in characterizing Kir channels in Drosophila, we initiated studies on Dir.     

Constitutive heterochromatin and transposable elements in Drosophila melanogaster

Several families of transposable elements (TEs), most of them belonging to the retrotransposon catagory, are particularly enriched in Drosophila melanogaster constitutive heterochromatin. The enrichment of TE-homologous sequences into heterochromatin is not a peculiar feature of the Drosophila genome, but appears to be widespread among higher eukaryotes. The constitutive heterochromatin of D. melanogaster contains several genetically active domains; this raises the possibility that TE-homologous sequences inserted into functional heterochromatin compartments may be expressed. In this review, I present available data on the genetic and molecular organization of D. melanogaster constitutive heterochromatin and its relationship with transposable elements. The implications of these findings on the possible impact of heterochromatic TEs on the function and evolution of the host genome are also discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purifying selection maintains highly conserved noncoding sequences in Drosophila

The majority of metazoan genomes consist of nonprotein-coding regions, although the functional significance of most noncoding DNA sequences remains unknown. Highly conserved noncoding sequences (CNSs) have proven to be reliable indicators of functionally constrained sequences such as cis-regulatory elements and noncoding RNA genes. However, CNSs may arise from nonselective evolutionary processes such as genomic regions with extremely low mutation rates known as mutation "cold spots." Here we combine comparative genomic data from recently completed insect genome projects with population genetic data in Drosophila melanogaster to test predictions of the mutational cold spot model of CNS evolution in the genus Drosophila. We find that point mutations in intronic and intergenic CNSs exhibit a significant reduction in levels of divergence relative to levels of polymorphism, as well as a significant excess of rare derived alleles, compared with either the nonconserved spacer regions between CNSs or with 4-fold silent sites in coding regions. Controlling for the effects of purifying selection, we find no evidence of positive selection acting on Drosophila CNSs, although we do find evidence for the action of recurrent positive selection in the spacer regions between CNSs. We estimate that approximately 85% of sites in Drosophila CNSs are under constraint with selection coefficients (N(e)s) on the order of 10-100, and thus, the estimated strength and number of sites under purifying selection is greater for Drosophila CNSs relative to those in the human genome. These patterns of nonneutral molecular evolution are incompatible with the mutational cold spot hypothesis to explain the existence of CNSs in Drosophila and, coupled with similar findings in mammals, argue against the general likelihood that CNSs are generated by mutational cold spots in any metazoan genome.     

Approach for functional analysis of glycan using RNA interference

The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C.elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose beta1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.     

Systematic analysis of the physiological importance of deubiquitinating enzymes

Deubiquitinating enzymes (DUBs) are proteases that control the post-translational modification of proteins by ubiquitin and in turn regulate diverse cellular pathways. Despite a growing understanding of DUB biology at the structural and molecular level, little is known about the physiological importance of most DUBs. Here, we systematically identify DUBs encoded by the genome of Drosophila melanogaster and examine their physiological importance in vivo. Through domain analyses we uncovered 41 Drosophila DUBs, most of which have human orthologues. Systematic knockdown of the vast majority of DUBs throughout the fly or in specific cell types had dramatic consequences for Drosophila development, adult motility or longevity. Specific DUB subclasses proved to be particularly necessary during development, while others were important in adults. Several DUBs were indispensable in neurons or glial cells during developmental stages; knockdown of others perturbed the homeostasis of ubiquitinated proteins in adult flies, or had adverse effects on wing positioning as a result of neuronal requirements. We demonstrate the physiological significance of the DUB family of enzymes in intact animals, find that there is little functional redundancy among members of this family of proteases, and provide insight for future investigations to understand DUB biology at the molecular, cellular and organismal levels.     

The sex chromosome that refused to die

Chromosomes that harbor dominant sex determination loci are predicted to erode over time--losing genes, accumulating transposable elements, degenerating into a functional wasteland and ultimately becoming extinct. The Drosophila melanogaster Y chromosome is fairly far along this path to oblivion. The few genes on largely heterochromatic Y chromosome are required for spermatocyte-specific functions, but have no role in other tissues. Surprisingly, a recent paper shows that divergent Y chromosomes can substantially influence gene expression throughout the D. melanogaster genome.1 These results show that variation on Y has an important influence on the deployment of the genome.     

Functional proteomics of the active cysteine protease content in Drosophila S2 cells

The fruit fly genome is characterized by an evolutionary expansion of proteases and immunity-related genes. In order to characterize the proteases that are active in a phagocytic Drosophila model cell line (S2 cells), we have applied a functional proteomics approach that allows simultaneous detection and identification of multiple protease species. DCG-04, a biotinylated, mechanism-based probe that covalently targets mammalian cysteine proteases of the papain family was found to detect Drosophila polypeptides in an activity-dependent manner. Chemical tagging combined with tandem mass spectrometry permitted retrieval and identification of these polypeptides. Among them was thiol-ester motif-containing protein (TEP) 4 which is involved in insect innate immunity and shares structural and functional similarities with the mammalian complement system factor C3 and the pan-protease inhibitor alpha2-macroglobulin. We also found four cysteine proteases with homologies to lysosomal cathepsin (CTS) L, K, B, and F, which have been implicated in mammalian adaptive immunity. The Drosophila CTS equivalents were most active at a pH of 4.5. This suggests that Drosophila CTS are, similar to their mammalian counterparts, predominantly active in lysosomal compartments. In support of this concept, we found CTS activity in phagosomes of Drosophila S2 cells. These results underscore the utility of activity profiling to address the functional role of insect proteases in immunity.     

Of flies and men--studying human disease in Drosophila

During the past year, the Drosophila genome has been sequenced. More than 60% of genes implicated in human disease have Drosophila orthologues. Developments in RNA-mediated interference and homologous recombination have made 'reverse genetics' feasible in Drosophila. Conventional Drosophila genetics is being used increasingly to place human disease genes of unknown function in the context of functional pathways.