灵活操控果蝇翅芽中wingless基因表达水平的策略

【目的】灵活操控靶基因的表达水平对于研究基因的功能十分重要。Gal4/UAS系统已被广泛应用于调控基因表达,可研究果蝇Drosophila等模式生物复杂的生物学问题。受采用载体的特性及插入位点的影响,Gal4或UAS转基因品系在构建好之后,其调控靶基因的能力基本是确定的。本研究旨在在现有Gal4/UAS系统的基础上,开发一种新的策略,实现在果蝇翅芽中灵活操控wingless(wg)基因的表达水平。【方法】用遗传学手段将黑腹果蝇Drosophila melanogaster品系的UAS-wg和UAS-wg-RNAi转基因重组到同一黑腹果蝇品系中。将该重组黑腹果蝇品系与dpp-Gal4黑腹果蝇品系杂交,同时驱动UAS-wg和UAS-wg-RNAi在果蝇幼虫翅芽中共表达。杂交子代幼虫分别放置在不同的温度(18, 25和30℃)下培养。将幼虫翅芽解剖并进行免疫组化染色,测量染色的荧光强度,分析翅芽中wg的表达水平。【结果】在低温(18℃)下,UAS-wg在基因表达调控中起主要作用,wg表现为超表达,但其超表达的效率可被UAS-wg-RNAi有效地削弱。相反,在高温(30℃)下,UAS-wg-RNAi起主导作用,wg的表达受到抑制。并且通过转换温度,可实现wg在翅芽发育的不同阶段在超表达和抑制之间相互转化,从而灵活地操控wg基因在翅芽中的表达水平。【结论】该方法可以灵活操控果蝇翅芽中wg基因的表达水平,对于调控转基因的表达有重要的意义。     

酵母转录因子Gal4研究进展

胁迫应答基因的转录激活是细胞应答胁迫作用的关键步骤。转录激活因子与启动子顺式作用元件结合是胁迫应答基因转录激活的关键环节。进化保守的Gal4是半乳糖代谢相关基因的转录激活因子。酵母Gal4通过其N端的DNA结合结构域识别并结合启动子UAS,通过其C端的激活结构域与转录因子作用,起始RNA聚合酶Ⅱ复合体的组装和转录。该过程不仅受转录调控因子Gal80和Gal3的调节,还与Gal4二聚体的形成有关。概述了酵母半乳糖代谢相关基因转录激活因子Gal4的研究进展。     

GAL4/UAS系统在果蝇Tis11基因RNA干扰中的应用

TTP在哺乳动物许多关键基因表达的转录后水平上起调控作用,Tis11是TTP蛋白在果蝇中的同源物.目前还没有现成的可用于研究Tis11功能的基因敲除或敲低的果蝇.为了获得肌动蛋白启动子或者热激蛋白启动子驱动表达Tis11 mRNA干扰序列的具有较高干扰效率的Tis11基因干扰果蝇,将肌动蛋白启动子或者热激启动子驱动表达的GAL4果蝇品系与融合有Tis11 mRNA干扰序列的UAS品系杂交,收集同时带有GAL4基因和UAS序列的子一代果蝇.提取所收集果蝇的总RNA,将其中的mRNA逆转录成cDNA,并设计检测Tis11基因的特异性引物,然后通过Real-time PCR检测Tis11 mRNA的表达情况.结果显示所收集的能表达Tis11基因干扰序列的子一代果蝇与不能表达Tis11基因干扰序列的对照果蝇相比,其体内Tis11 mRNA的表达水平下降明显.收集的果蝇其体内所表达的干扰序列对Tis11 mRNA干扰效果显著,我们成功获得了Tis11基因的RNA干扰果蝇.     

果蝇翅芽特异表达Gal4的筛选及活力检测

筛选并鉴定在果蝇翅芽不同区域特异表达的Gal4,可为以果蝇翅为模型的相关研究提供更多的遗传工具。本研究对Bloomington Drosophila Stock Center (BDSC)未见详细报道的Gal4品系进行筛选，将其中12个候选Gal4品系的雄虫与UAS-GFP基因型的处女蝇分别杂交，检测Gal4在子代幼虫翅芽的表达位置。经筛选重点关注3个Gal4,并以翅芽特异隔间标记物为参照，精确表征Gal4在翅芽的表达部位。结果显示：c563a-Gal4在前隔间翅囊区边缘及部分铰链区表达；c804-Gal4在围绕翅囊的环形区域以及A/P边界的细胞条带处表达；GMR11F02-Gal4在整个翅囊区表达；利用G-TRACE技术对上述Gal4的细胞发育谱系进行追踪，进一步揭示了翅芽发育历程中开启Gal4表达细胞的子代细胞分布区域；同时，通过驱动特定靶基因myc的表达，检测Gal4激活靶基因表达的活力。结果表明，3种Gal4都可以有效激活靶基因myc。其中，GMR11F02-Gal4的活力最强，c563a-Gal4最弱。本研究为翅芽相关研究提供新的遗传工具选项。     

转飞蝗羧酸酯酶基因果蝇品系的构建及其在有机磷农药代谢解毒中的作用

【目的】在活体水平验证飞蝗Locusta migratoria羧酸酯酶基因LmCesA1和LmCesA2是否参与有机磷杀虫剂的代谢解毒。【方法】采用Gal4/UAS系统,借助转基因技术,构建两个转基因黑腹果蝇Drosophila melanogaster品系,选取3品系Gal4(act-Gal4, tub-Gal4和c601-Gal4)果蝇作为母本分别与两种转基因果蝇(UAS-LmCesA1和UAS-LmCesA2)以及一种亲本对照果蝇(RB0006{y v; attP40, y+})进行杂交。对子一代转基因果蝇从DNA和RNA水平进行验证,筛选出成功构建的品系。采用生物测定方法检测转基因果蝇与Gal4果蝇杂交后代对马拉硫磷的抗性。【结果】转基因果蝇DNA水平鉴定结果显示,转基因果蝇tub>LmCesA1和tub>LmCesA2中分别扩增到目的基因LmCesA1和LmCesA2,而对照组果蝇tub>attP40中未扩增到目的基因。转基因果蝇RNA水平的检测结果显示,这两个基因在相应的杂交后代中均有表达,表明转基因果蝇构建成功。目的基因在转基因果蝇成虫不同组织中的表达结果表明,两个目的基因LmCesA1和LmCesA2分别在转基因果蝇c601>LmCesA1和c601>LmCesA2的肠道中高表达; LmCesA1在c601>LmCesA1果蝇肠道中的表达量分别是脑和表皮中的7.6和16.7倍, LmCesA2在c601>LmCesA2果蝇肠道中的表达量分别是脑和表皮中的5.4和10.9倍。杀虫剂生物测定结果显示,与对照组果蝇(c601>attP40)相比,超表达LmCesA2的果蝇(c601>LmCesA2)对马拉硫磷的抗性显著提高,抗性倍数为1.67。【结论】本研究的结论与我们前期采用RNAi结合杀虫剂生测的研究结论一致,即羧酸酯酶基因LmCesA2可能参与飞蝗对马拉硫磷的代谢解毒过程。     

转飞蝗羧酸酯酶基因果蝇品系的构建及其在有机磷农药代谢解毒中的作用

【目的】在活体水平验证飞蝗Locusta migratoria羧酸酯酶基因LmCesA1和LmCesA2是否参与有机磷杀虫剂的代谢解毒。【方法】采用Gal4/UAS系统,借助转基因技术,构建两个转基因黑腹果蝇Drosophila melanogaster品系,选取3品系Gal4(act-Gal4, tub-Gal4和c601-Gal4)果蝇作为母本分别与两种转基因果蝇(UAS-LmCesA1和UAS-LmCesA2)以及一种亲本对照果蝇(RB0006{y v; attP40, y+})进行杂交。对子一代转基因果蝇从DNA和RNA水平进行验证,筛选出成功构建的品系。采用生物测定方法检测转基因果蝇与Gal4果蝇杂交后代对马拉硫磷的抗性。【结果】转基因果蝇DNA水平鉴定结果显示,转基因果蝇tubLmCesA1和tubLmCesA2中分别扩增到目的基因LmCesA1和LmCesA2,而对照组果蝇tubattP40中未扩增到目的基因。转基因果蝇RNA水平的检测结果显示,这两个基因在相应的杂交后代中均有表达,表明转基因果蝇构建成功。目的基因在转基因果蝇成虫不同组织中的表达结果表明,两个目的基因LmCesA1和LmCesA2分别在转基因果蝇c601LmCesA1和c601LmCesA2的肠道中高表达;LmCesA1在c601LmCesA1果蝇肠道中的表达量分别是脑和表皮中的7.6和16.7倍,LmCesA2在c601LmCesA2果蝇肠道中的表达量分别是脑和表皮中的5.4和10.9倍。杀虫剂生物测定结果显示,与对照组果蝇(c601attP40)相比,超表达LmCesA2的果蝇(c601LmCesA2)对马拉硫磷的抗性显著提高,抗性倍数为1.67。【结论】本研究的结论与我们前期采用RNAi结合杀虫剂生测的研究结论一致,即羧酸酯酶基因LmCesA2可能参与飞蝗对马拉硫磷的代谢解毒过程。     

结合GAL4/UAS系统与CyO-GFP平衡子获取表达RNA干扰序列果蝇幼虫并有效干扰Tis11表达

TIS11是转录后调控因子TTP在果蝇中的同源物,在果蝇幼虫免疫、发育和代谢等多种生理过程中都发挥重要作用.为研究TIS11的功能,需要利用GAL4/UAS系统获得整体高干扰效率的Tis11 RNAi果蝇幼虫．为平衡GAL4转基因果蝇高活性启动子的致死效应,需要使用带有成蝇卷翅标记CyO的第二染色体的平衡子,但CyO标记在幼虫中无可见表型,因此无法区分杂交幼虫的基因型．为解决这一问题,引入了带有CyO-GFP标记的平衡子．携带CyO-GFP平衡子的G-Actin果蝇与携带Tis11 RNAi序列的101765果蝇杂交,杂交幼虫可以通过GFP标记进行区分,剔除带有CyO-GFP平衡子的幼虫,从而挑选出表达Tis11 RNAi序列的幼虫,最后经real-time PCR检测所得幼虫具有整体高干扰效率．     

利用G-TRACE技术追踪果蝇后肠肠细胞谱系的发育模式

【目的】果蝇是完全变态昆虫,蛹期经历了幼虫组织解离和成虫组织重塑的过程。本研究旨在利用细胞谱系追踪方法 G-TRACE(Gal4 technique for real-time and clonal expression)这一新的遗传学技术,检测果蝇幼虫后肠肠细胞在蛹期发育过程中是否发生细胞迁移。【方法】采用黑腹果蝇Drosophila melanogaster engrailed-Gal4(en-Gal4)品系和G-TRACE品系杂交,并引入tub-gal80ts控制Gal4的开启时间,分别在果蝇幼虫期和蛹期进行细胞谱系追踪。幼虫期追踪:亲代产卵后将卵置于30℃培养,3龄中期转入18℃培养,成虫羽化1 d内进行检测。蛹期追踪:亲代产卵后将卵置于18℃培养,在蛹期不同发育阶段转入30℃培养,待虫体羽化后检测成虫肠道。【结果】当在果蝇幼虫期启动细胞谱系追踪,在蛹期停止追踪,发现中肠靠近中后肠边界处以及马氏管存在绿色肠细胞。而当在果蝇幼虫期关闭细胞谱系追踪,在蛹期开始追踪,则发现虫体中肠各部位及马氏管分布着绿色肠细胞。en基因在果蝇蛹期肠道中表达。【结论】结果表明,在果蝇蛹形成过程中,后肠的部分肠细胞迁移至中肠和马氏管,参与中肠和马氏管的重塑。本研究对于探索昆虫在变态发育过程中成虫器官的重塑机制具有重要的意义。     

GAL4/UAS系统在转基因技术中的应用研究进展

GAL4/UAS系统是一种转基因技术体系,其原理是利用特定的启动子或增强子,以组织特异性的方式激活酵母转录激活子GAL4的表达,GAL4又以同样的方式引起GAL4反应元件(UAS)-靶基因的转录。GAL4/UAS系统的关键点在于:GAL4基因和UAS-靶基因分别存在于两个转基因系中。GAL4转基因系中有转录激活子,但没有靶基因;在UAS-靶基因系中,转录激活子不存在,因而靶基因处于沉默状态,只有将GAL4转基因系与UAS-靶基因系进行杂交,才可能产生表达靶基因的后代。本文综述了GAL4/UAS系统的建立及其研究应用。     

Hsp22对SCA3/MJD转基因果蝇的神经保护作用研究

为了探讨Hsp22在SCA3/MJD发病机制中的作用．选用GMR-GAL4和elav-GAL4驱动子,利用经典的GAL4-UAS系统,将含有78个CAG重复扩增的ataxin-3蛋白片段(MJDtr-Q78)分别在果蝇眼睛和神经系统选择性表达,构建GMR-GAL4/UAS和elav-GAL4/UAS系统SCA3/MJD转基因果蝇模型, 然后利用遗传学方法和热休克反应使Hsp22在SCA3/ MJD转基因果蝇眼睛和神经系统以不同水平过表达．结果表明,Hsp22过表达显著抑制了MJDtr-Q78蛋白的神经毒性,果蝇眼睛视网膜光感受神经元变性明显缓解,果蝇存活能力也显著提高．Hsp22对SCA3/MJD具有保护作用,增强Hsp22表达对SCA3/MJD可能是一种潜在的治疗方法．     

Delaying Gal4-driven gene expression in the zebrafish with morpholinos and Gal80

The modular Gal4/UAS gene expression system has become an indispensable tool in modern biology. Several large-scale gene- and enhancer-trap screens in the zebrafish have generated hundreds of transgenic lines expressing Gal4 in unique patterns. However, the early embryonic expression of the Gal4 severely limits their use for studies on regeneration or behavior because UAS-driven effectors could disrupt normal organogenesis. To overcome this limitation, we explored the use of the Gal4 repressor Gal80 in transient assays and with stable transgenes to temporally control Gal4 activity. We also validated a strategy to delay Gal4-driven gene expression using a morpholino targeted to Gal4. The first approach is limited to transgenes expressing the native Gal4. The morphant approach can also be applied to transgenic lines expressing the Gal4-VP16 fusion protein. It promises to become a standard approach to delay Gal4-driven transgene expression and enhance the genetic toolkit for the zebrafish.     

Characterization of a dorsal‐eye Gal4 Line in Drosophila

In Drosophila, the Gal4‐UAS system is used to drive ectopic gene expression in a tissue‐specific manner. In this system, transgenic flies expressing tissue specific Gal4 are crossed to a line in which the gene to be expressed is under the control of a Gal4‐responsive UAS sequence. The resulting progeny express the gene of interest in the pattern of the particular Gal4 line. Since a given UAS‐transgene can be driven by any Gal4 line, this system is predominantly limited by available Gal4 lines. Here we report the characterization of a novel line, DE‐Gal4, which in the eye is expressed in the dorsal compartment for the majority of development. Furthermore, we use functional tests to show that the DE‐Gal4 line is a useful tool with which to manipulate gene expression in half of the developing eye. genesis 48:3–7, 2010. © 2009 Wiley‐Liss, Inc.     

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments.The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms.Driven by a given tissue-specific Gal4,expressing UAS-transgene or UAS-RNAi(RNA interference)could be used to up-or down-regulate target gene expression,respectively.However,the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock.Here,we describe a simple way to modulate optomotor blind(omb)expression levels in its endogenous expression region of the wing disc.We co-expressed UAS-omb and UAS-omb-RNAi together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch.The repression effect is more sensitive to temperature than that of overexpression.At low temperature,overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi.In contrast,at high temperature RNAi predominates in gene expression regulation.By this strategy,we could manipulate omb expression levels at a moderate level.It allows us to manipulate omb expression levels in the same tissue between overexpression and repression at different stages by temperature control.     

Development of the bi-partite Gal4-UAS system in the African malaria mosquito, Anopheles gambiae

Functional genetic analysis in Anopheles gambiae would be greatly improved by the development of a binary expression system, which would allow the more rapid and flexible characterisation of genes influencing disease transmission, including those involved in insecticide resistance, parasite interaction, host and mate seeking behaviour. The Gal4-UAS system, widely used in Drosophila melanogaster functional genetics, has been significantly modified to achieve robust application in several different species. Towards this end, previous work generated a series of modified Gal4 constructs that were up to 20 fold more active than the native gene in An. gambiae cells. To examine the Gal4-UAS system in vivo, transgenic An. gambiae driver lines carrying a modified Gal4 gene under the control of the carboxypeptidase promoter, and responder lines carrying UAS regulated luciferase and eYFP reporter genes have been created. Crossing of the Gal4 and UAS lines resulted in progeny that expressed both reporters in the expected midgut specific pattern. Although there was minor variation in reporter gene activity between the different crosses examined, the tissue specific expression pattern was consistent regardless of the genomic location of the transgene cassettes. The results show that the modified Gal4-UAS system can be used to successfully activate expression of transgenes in a robust and tissue specific manner in Anopheles gambiae. The midgut driver and dual reporter responder constructs are the first to be developed and tested successfully in transgenic An. gambiae and provide the basis for further advancement of the system in this and other insect species.     

Design and isolation of temperature-sensitive mutants of Gal4 in yeast and Drosophila

Little is known about mechanisms responsible for the temperature-sensitive (ts) phenotype, or of the transferability of ts mutants of a specific gene between organisms. Using a structure-based approach, nine ts mutants of Gal4 were generated in yeast by mutating four DNA binding residues. Two of these nine yeast ts mutants were cloned into P element vectors under control of the Elav and GMR promoters and transgenic Drosophila lines were generated. These were crossed to UAS reporter lines and progeny were characterized for reporter gene expression as a function of temperature. Both of these yeast ts mutants show a ts phenotype in Drosophila and result in rapid induction of reporter gene expression upon shifting to the permissive temperature. Exposed, functional residues involved in protein-ligand or protein-protein interactions appear to be attractive candidate sites for generating ts mutants that are transferable between organisms.     

Development and evaluation of a Gal4-mediated LUC/GFP/GUS enhancer trap system in Arabidopsis

Background  

Gal4 enhancer trap systems driving expression of LacZ and GFP reporters have been characterized and widely used in Drosophila. However, a Gal4 enhancer trap system in Arabidopsis has not been described in the primary literature. In Drosophila, the reporters possess a Gal4 upstream activation sequence (UAS) as five repeats (5XUAS) and lines that express Gal4 from tissue specific enhancers have also been used for the ectopic expression of any transgene (driven by a 5XUAS). While Gal4 transactivation has been demonstrated in Arabidopsis, wide use of a trap has not emerged in part because of the lack of detailed analysis, which is the purpose of the present study.