E3泛素连接酶基因CG4911敲除和功能的初步研究

蛋白质的泛素化是一种重要的翻译后修饰过程,参与调控细胞周期、基因转录、信号转导、炎症反应和干细胞的维持等过程。泛素连接酶E3（ubiqutin ligase）是泛素化过程中关键酶。但许多E3基因在发育中的功能和作用机制还不明确。该研究以黑腹果蝇为模式动物,研究泛素连接酶家族一个重要基因CG4911的功能及分子机制。获得CG4911基因敲除果蝇,CG4911敲除果蝇纯合子可活。原位杂交结果显示,CG4911在胚胎发育早期表达。通过构建CG4911-pUAST-3HA重组子转染Hela细胞,确定CG4911定位于细胞质中,其表达并无修饰作用,并且过表达基因CG4911可导致背板发育缺陷。该研究首次获得了CG4911基因敲除果蝇和CG4911转基因果蝇,并初步探索了F-box基因CG4911的功能,为进一步阐明泛素连接酶的功能及分子机制提供了科学依据。     

睾丸优势表达的Boule蛋白影响果蝇发育

boule基因是屈指可数的高度保守、横跨后生动物的调控多个物种精子发生所不可或缺的基因.在果蝇中,boule基因突变体bol1可导致雄性不育,而boule基因的大片段缺失突变体boule40却表现了致死表型,提示boule可能在胚胎发育过程中也起到一定的作用.为了验证这一假设,首先,本团队利用RT-PCR检测了boule基因在不同组织中的表达情况,并构建了GFP敲入突变体检测Boule蛋白在不同组织中的分布,结果表明boule mRNA及Boule蛋白在发育过程及成蝇的头、胸、腹中均存在.其次,构建了boule基因完全缺失突变体,发现在不同发育阶段均有致死发生.为了探寻这种影响是来自boule基因产生的蛋白还是非编码RNA或是内含子产生的未发现的基因,本团队又构建了boule基因单碱基缺失的移码突变体,发现同样有致死现象.最后,全身性过表达Boule蛋白也可导致果蝇发育过程中的逐渐死亡.以上结果都说明,果蝇体细胞中的Boule蛋白在发育过程中起重要作用,并需要进行精确调控.这一睾丸以外的作用目前只在昆虫中发现.     

果蝇新基因CG7609的克隆与初步功能研究

通过生物信息学方法和分子克隆技术克隆了一个果蝇新基因CG7609．该基因属于WD40家族,具有7个典型的WD40重复结构域．与人类WDR24基因的同源度很高．胚胎原位杂交和RT-PCR显示其在胚胎早期的中胚层有弱表达,在胚胎晚期主要在肠中表达．通过心脏特异抗体检测CG7609突变体的表型,发现其心脏前体细胞的分化不受影响．但通过果蝇心力衰竭模型分析该基因在成体心脏中的功能,发现CG7609基因缺失后心力衰竭发生率明显高于野生型,而且在与pannier基因配对后心力衰竭率发生较大的变化,这个初步发现推测该基因可能在戍体心脏中具有功能．     

tap基因靶向表达导致果蝇神经系统异常发育

[目的]果蝇tap基因编码进化保守的碱性螺旋-环-螺旋(bHLH)蛋白,该研究初步探索其在果蝇神经系统发育中的功能。[方法]合成tap基因的编码序列并亚克隆至p UAST质粒,通过胚胎显微注射和mini-white标记基因筛选,获得UAS-tap转基因果蝇。将该转基因品系分别与ap-GAL4和GMR-GAL4果蝇品系杂交,采用定量RT-PCR方法检测杂交前后tap基因表达水平,并在显微镜下观察杂交后代的发育情况。[结果]成功构建了p UAS-tap重组质粒,通过显微注射与转基因果蝇筛选、平衡及定位,共获得5个独立转基因品系。tap基因靶向表达后引起成虫出现糙眼,翅膀出现异位刚毛和异位翅脉表型。[结论]tap基因可能通过原神经模式,也可能通过调节神经前体细胞分化而参与神经发育。对该基因功能与调控的深入研究,将与脊椎动物中其同源基因ngn的同类研究互相借鉴。     

pygo基因对心脏功能的调控及其研究进展

Wg/Wnt信号参与调控多种组织的发育,尤其在心脏发育和心脏衰老过程中发挥重要的作用。pygo作为Wnt信号途径的一个新成员,可能依赖于Wnt信号调控心脏发育,而最新发现pygo敲低品系引起的成体心脏功能缺陷与Wnt信号缺失在心脏中的表型具有显著性差异,表明pygo调控成体心脏功能不依赖于经典Wnt信号,可能存在新的调控机制。主要对pygo基因在调控心脏发育和心脏衰老中的功能以及在果蝇和哺乳动物中pygo基因调控心脏功能分子机制的研究进展进行了综述。虽然pygo不依赖经典Wnt信号在成体心脏中发挥作用,但显性负抑制TCF突变体引起严重的成体心脏生理功能缺陷,与pygo表型一致,暗示着pygo可能依赖与TCF类似因子相互作用发挥功能。其次,pygo能跨越TCF或Lgs直接与Wnt信号靶基因相互作用。此外,Pygo蛋白能与Lgs相互作用形成Pygo-BCL9/Lgs-H3K4me复合物调节Wnt信号靶基因,且甲基转移酶HMT核心组件WDR5与Pygo蛋白的相互作用能促进PHD结构域与H3K4的结合,表明pygo调节成体心脏功能与表观遗传学修饰也具有一定的相关性。     

利用 CRISPR/Cas9 系统定向编辑黑腹果蝇Osiris24基因

Osiris基因在几丁质沉积过程中表达,可能参与昆虫表皮的发育。本研究利用CRISPR/Cas9 基因编辑系统对Osiris24基因进行编辑,进而观察Osiris24突变体果蝇的性状并且检测Osiris24的表达特征。在Osiris24第1外显子设计2个sgRNA靶位点,插入到pCFD4敲除载体骨架中,同时构建酵母Gal4蛋白序列的供体(donor)载体,将2个载体同时注射到nos-Cas9胚胎中获得G0代转基因果蝇。结果显示,G0代基因编辑阳性率为92.8%,Osiris24纯合突变体在胚胎或1龄幼虫期致死,杂合突变体未观察到可见表型。将阳性G0代雄虫与UAS-GFP雌虫杂交,检测不同龄期和不同组织GFP信号表达情况。结果发现,Osiris24在不同龄期幼虫中均有表达,幼虫期主要在体壁、气管、前肠和后肠高表达,蛹期主要在体壁和翅上表达,推测其在果蝇发育中发挥重要作用,本研究为深入探究Osiris基因功能提供了研究模型。     

果蝇spen蛋白的抗体制备、组织特异性表达及功能分析

Spen家族蛋白参与多种生物学过程,包括神经元细胞的命运、神经元突起延伸的调节、细胞周期调控等,并且是联系Notch信号途径和生长因子受体途径的关键分子。最近的研究表明spen基因在果蝇的眼睛、翅膀和腿组织中参与Wnt信号转导。但该基因在果蝇中的功能还有很多不明确之处。文章采用基因克隆、原核表达及亲和层析等方法制备并纯化了黑腹果蝇spen的C端6×His-spen融合蛋白,以纯化的融合蛋白免疫大鼠获得了抗spen的多克隆抗体。利用制备的抗体进行免疫染色结果显示spen蛋白定位于细胞核内,并且在大脑、脂肪体、血细胞、肠和唾液腺等组织中表达量较高。分析野生型和突变体果蝇血细胞的噬菌作用,发现spen蛋白低表达的突变体吞噬外来异物明显低于野生型,结果表明spen蛋白能够调节血细胞的吞噬功能。     

用“增强子陷阱”技术构造并筛选果蝇UAS/GAL4系统中GAL4新品系及脑基因表达图谱数据库的开发

"增强子陷阱"技术是建立果蝇脑全基因组表达图谱及其数据库的重要方法.筛选获得新特异表达的GAL4品系,可为进一步研究果蝇脑神经在学习记忆功能提供强有力的基因工具.通过"增强子陷阱"技术来获得果蝇突变体,并与报告转基因果蝇(UAS-EGFP)杂交,用荧光显微镜观察成年果蝇脑内荧光分布,从而获得该突变体的脑基因表达图谱,在此基础上利用JavaScript来建立果蝇脑全基因组表达数据库.目前获得基因突变体果蝇2 677种,大部分在果蝇脑中有表达,其中在果蝇嗅觉学习记忆相关脑区蘑菇体表达的基因有368个,且有部分基因特异地表达在某些传导通路上.这些果蝇基因突变体库及其表达图谱为进一步研究各基因的功能及作为遗传工具来研究各脑区结构和功能提供极大方便.     

细胞周期后期促进因子DIG9对植物侧根发生的调控研究

在植物体内,细胞周期对于植物的萌发、生长、开花、结实等各个生长发育阶段具有重要作用。细胞周期正常运转需要依赖一些细胞周期蛋白,但是目前关于细胞周期蛋白调控根发育的分子机制还不清楚。通过筛选模式植物拟南芥的根发育异常突变体,分离鉴定了1个突变体dig9（drought inhibition of lateral root growth）,该突变体表现为主根短、侧根少、发育迟缓、顶端分生组织变小、叶片扭曲、无主茎等表型。通过图位克隆,成功定位并克隆了DIG9基因,该基因编码一个细胞周期蛋白,是有丝分裂后期促进复合体的一个亚基APC8 （anaphase-promoting complex）。通过亚细胞定位发现DIG9定位于细胞核;qRT-PCR检测发现DIG9基因在根中有较高的表达量,进一步通过启动子-GUS报告系统发现DIG9在根尖、侧根和顶端分生组织等细胞分裂旺盛区域表达。外施IAA能恢复dig9突变体的侧根表型但不能恢复根短表型。dig9突变体对干旱及盐胁迫反应不敏感。研究结果表明DIG9基因可能通过影响IAA的产生来调控植物的侧根发育。     

Drosophila acid DNase is a homolog of mammalian DNase II

Mammalian DNase II enzymes and the Caenorhabditis elegans homolog NUC-1 have recently been shown to be critically important during engulfment-mediated clearance of DNA. In this report, we describe the cloning and characterization of the gene encoding Drosophila DNase II. Database queries using the C. elegans NUC-1 protein sequence identified a highly homologous open reading frame in Drosophila (CG7780) that could encode a similar enzyme. Analysis of crude protein extracts revealed that wild-type Drosophila contain a potent acid endonuclease activity with cleavage preferences similar to DNase II/NUC1, while the same activity was markedly reduced in an acid DNase hypomorphic mutant line. Furthermore, the pattern of cleavage products generated from an end-labeled substrate by hypomorphic-line extracts was significantly altered in comparison to the pattern generated by wild-type extracts. Sequence analysis of CG7780 DNA and mRNA revealed that the hypomorphic line contains a missense mutation within the coding region of this gene. Additionally, Northern analysis demonstrated that CG7780 expression is normal in the mutant line, which in combination with the lowered/altered enzymatic activity and sequencing data suggested a defect in the CG7780 protein. To conclusively determine if CG7780 encoded the Drosophila equivalent of DNase II/NUC-1, transgenic lines expressing wild-type CG7780 in the mutant background were generated and subsequently shown to complement the mutant phenotype. Our results, therefore, provide compelling evidence that the predicted gene CG7780 encodes Drosophila DNase II (dDNase II), an enzyme related in sequence and activity to mammalian DNase II. Interestingly, overexpression of CG7780 both ubiquitously and in specific tissues failed to elicit any discernable phenotype.     

Drosophila spoonbill encodes a dual-specificity A-kinase anchor protein essential for oogenesis

spoonbill is a Drosophila female-sterile mutation, which interferes with normal egg patterning during oogenesis. Previous analyzes linked the mutation to a number of seemingly unrelated pathways, including GRK/EGFR and DPP, two major pathways essential for Drosophila and vertebrate development. Further work suggested that spoonbill may also function in actin polymerization and border-cell migration. Here we describe the molecular cloning of the spoonbill gene and characterize new mutant alleles, further demonstrating that spoonbill's function is essential during oogenesis. We found spoonbill to be allelic to CG3249 (also known as yu), which encodes the only known dual-specificity A-kinase anchor protein in Drosophila. Our data indicate that similar to mammalian AKAPs, Spoonbill protein contains a number of potential kinase and phosphatase binding motifs, and is targeted, in the ovary, to mitochondria and Golgi. Finally, we address some of spoonbill's mutant phenotypes from the perspective of the published data on the AKAP protein family.     

利用果蝇模型研究人类心脏早期发育的分子机理(英文)

近年来 ,果蝇心脏特化的遗传机制已初步研究清楚 ,但控制人类心脏早期发育的基因尚待鉴定。因为调控果蝇和脊椎动物早期心脏细胞命运定型的途径具有保守性 ,果蝇是一种探讨人类心脏早期发育的分子机理的理想动物模式。为此目的 ,我们采用P转座子和EMS诱变技术建立了约 3 0 0 0个隐性致死基因平衡系。通过心脏前体细胞特异性抗体免疫组化筛选 ,我们检出 2 0 0余个表现心脏突变表型的平衡致死系。我们进一步利用RNAi技术对一些基因的功能进行了初步的研究 ,证明这些基因表现RNAi的突变表型 ,该类突变表型与基因突变时表现的表型相似 ,即心管呈缺陷型或无心脏前体细胞形成。利用果蝇和人类基因组计划获得的成果 ,我们从果蝇心脏侯选基因中初步克隆和鉴定了 5 0个人类同源基因 ,其中 2 0个是新基因。Northen印迹分析表明 ,一部分人类基因在心脏组织中有表达 ,从而为研究这些基因在人类心脏早期发育中的作用提供了信息。目前 ,我们正在建立转基因果蝇 ,以此为模型研究这些基因是否对心肌细胞发生或心肌功能起调控作用。产生心肌细胞突变类型的基因如果类似于人类心脏病综合症 ,则可以作为人类心脏疾病侯选基因作进一步的分析。     

A large-scale screen for mutagen-sensitive loci in Drosophila

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.     

Identification of the Drosophila core 1 beta1,3-galactosyltransferase gene that synthesizes T antigen in the embryonic central nervous system and hemocytes

T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.