Cloning, expression and identification of GRD and LCCH3 subunits of GABA receptor in Drosophila melanogaster

昆虫GABA受体(γ-aminobutyric acid receptor,GABAR)是杀虫剂的重要靶标之一.本研究以黑腹果蝇Drosophila melanogaster整体组织的cDNA作为模板,采用RT-PCR技术扩增了黑腹果蝇GABA受体LCCH3亚基和GRD亚基的cDNA序列,并克隆至pET-32a表达载体上,测序结果表明获得的序列与基因库中已发表的序列一致性在99％以上,无移码突变.在IPTG的诱导下,LCCH3基因成功在大肠杆菌Escherichia coli中表达,而GRD基因未表达.通过包涵体洗涤、变性、Ni2+亲合层析纯化、稀释复性获得纯化的重组表达的LCCH3蛋白,并用圆二色谱测定了目标蛋白的二级结构,主要富含β结构.该研究结果为研究昆虫GABAR的结构和功能关系提供了重要的参考数据.     

本期重点推介

γ-氨基丁酸受体(γ-aminobutyric acid receptor,GABAR)是哺乳动物和昆虫神经系统中主要的抑制性神经递质受体,是氟虫腈和阿维菌素等多种杀虫剂的作用靶标。上海师范大学生命与环境科学学院张晓敏和任天瑞等采用RT-PCR方法对黑腹果蝇Drosophila melanogaster的GABA受体LCCH3亚基和GRD亚基的     

异育银鲫GABA A受体γ2亚基全长克隆及生物信息学分析

近年来GABA A受体亚基特异性在药物筛选、研发过程中的应用得到广泛地关注,其中有关α1、β2和γ2三种功能性亚基的研究最为深入。异育银鲫因其良好的生长、繁殖优势,在国内得到广泛养殖。采用RACE法克隆得到了异育银鲫GABA A受体γ2亚基基因全长cDNA,并进行了生物信息学分析。该基因长2 763 bp,其中CDS区长1437 bp,可编码477个氨基酸的前体蛋白。预测蛋白分子量55.3 k D,理论等电点9.13。异育银鲫体内GABA A受体γ2亚基氨基酸序列N端存在1个长度为35个氨基酸的信号肽,4个长度分别为23、20、23和23个氨基酸的跨膜区,3个N-糖基结合位点和2个O-糖基化位点,1个特异性结构域,其氨基酸序列具有明显的氯离子门控通道家族特征。氨基酸序列与其他物种氨基酸序列的同源性都在89%以上,表明该蛋白属于GABA A受体亚基家族。系统进化树表明异育银鲫与斑马鱼聚为一支,亲缘关系最近。     

二化螟乙酰胆碱受体a亚基的基因克隆与序列分析

烟碱型乙酰胆碱受体（nAChR）在昆虫的兴奋性突触传递中起着重要的作用,同时也是杀虫剂作用的重要靶标。近年来,二化螟对作用于昆虫nAChR的沙蚕毒素类杀虫剂杀虫单产生了高抗性。为了研究可能存在的靶标不敏感机制,我们采用RT-PCR技术,对二化螟nAChRa亚基全长cDNA进行了分子克隆。序列分析表明,这是1个新的a亚基基因,定名为Cs a 1。基因全长为1997个核苷酸,包含了1个开放阅读框,编码1个509氨基酸的成熟蛋白和1个24氨基酸的信号肽。Cs a 1与其他昆虫nAChR a亚基之间有52%～94%的同源性,高于与脊椎动物nAChR a亚基之间的同源性。     

桔小实蝇烟碱型乙酰胆碱受体β亚基基因的克隆及mRNA表达分析

烟碱型乙酰胆碱受体(nicotinic acetylcholine receptor,nAChR)在昆虫中枢神经系统的递质传递过程中起着重要作用。本研究采用RT-PCR和RACE技术,从桔小实蝇Bactrocera dorsalis(Hendel)体内克隆获得nAChRβ亚基的cDNA序列,命名为Bdβ3(GenBank登录号:JF974074)。测序结果表明,Bdβ3的cDNA序列全长1602bp,开放阅读框为1287bp,编码429个氨基酸残基,预测蛋白质分子量和等电点分别为48.8ku和5.81。通过对氨基酸同源性分析表明,Bdβ3具有nAChR亚基的典型特征,与其他昆虫nAChR亚基具有较高的氨基酸相似性,与黑腹果蝇nAChRβ3亚基具有49.78%的相似性。Bdβ3在桔小实蝇的不同发育时期和成虫的不同体段的实时定量PCR结果表明,Bdβ3在整个发育阶段均有表达,其中在成虫期的表达水平最高,这可能与Bdβ3主要在成虫期发挥作用有关;Bdβ3在桔小实蝇头部表达量最高,显著高于胸部和腹部。研究结果为深入分析桔小实蝇nAChR亚基的功能以及对多杀菌素的靶标抗性机制提供了基础数据。     

甜菜夜蛾烟碱型乙酰胆碱受体β1亚基基因的克隆与序列分析

烟碱型乙酰胆碱受体是昆虫体内重要的神经受体,同时也是杀虫剂作用靶标.从甜菜夜蛾Spodoptera exigua3龄幼虫体内提取总的RNA,经过反转录,利用RT-PCR获得了烟碱型乙酰胆碱受体6个α和1个β亚基基因的cD-NA序列片段,并利用cDNA末端快速扩增技术(RACE)获得了β亚基基因的cDNA序列全长.该基因命名为SenAChRβl,其长度为2231个碱基,含有一个1575个碱基的开放读码框,开放读码框编码524个氨基酸残基,预测的分子量为60 kDa.推导得到的氨基酸序列与其它昆虫特别是鳞翅目昆虫的烟碱型乙酰胆碱受体β亚基具有高度的同源性,并具有典型的烟碱型乙酰胆碱受体β亚基特征化位点.     

脊尾白虾血蓝蛋白大亚基基因的克隆及表达分析

应用RACE技术克隆脊尾白虾血蓝蛋白大亚基基因, 并通过攻毒实验揭示脊尾白虾血蓝蛋白基因的先天免疫防御作用, 为脊尾白虾(Exopalaemon carinicauda)的免疫防治研究提供依据和思路。研究成功克隆了脊尾白虾血蓝蛋白大亚基基因全长cDNA序列, 该大亚基cDNA全长 2192 bp, 开放式阅读框长 2034 bp, 5′非编码区长 21 bp, 3′非编码区长 137 bp, 将该基因命名为 EcHcL。EcHcL编码 667 个氨基酸, 前 21 个氨基酸组成信号肽, 推测成熟肽的分子量为 78.5 kD。Blast比对结果显示, 由脊尾白虾血蓝蛋白EcHcL序列推导的氨基酸序列与日本沼虾、凡纳滨对虾血蓝蛋白氨基酸序列的同源性分别达到 87%、73%, 其M结构域氨基酸序列与斑节对虾、日本对虾等物种同源性性高达 90% 左右, 由此推断该cDNA序列属于血蓝蛋白家族。组织表达分析结果显示, EcHcL基因在脊尾白虾鳃、卵巢、肝胰腺、心脏、肠、肌肉、胃、腹神经节、眼柄、血细胞中均有表达, 肝胰腺中相对表达量最高。Real-time PCR分析发现EcHcL基因在金黄色葡萄球菌、副溶血弧菌和对虾白斑综合征病毒(WSSV)感染后脊尾白虾肝胰腺和血细胞中的表达量显著增加, 并具有不同的时空表达模式, 推测脊尾白虾EcHcL基因在免疫防御中具有重要作用。     

意大利蝗血蓝蛋白亚基Ⅰ基因的克隆与原核表达

昆虫血蓝蛋白是在昆虫体内普遍存在的一种含Cu~(2+)的多功能蛋白,除参与呼吸外还具有能量贮存、抗菌和抗病毒等多种生物学功能。为了研究意大利蝗(Calliptanus italicus)血蓝蛋白亚基Cit Hc-1的结构和表达,通过RACE技术获得了Cit Hc-1的全长cDNA(2 335 bp)序列,其中开放阅读框2 079 bp,编码692个氨基酸,预测蛋白分子量79.88 k Da。序列比对结果显示Cit Hc-1基因cDNA序列与蝗总科其他物种血蓝蛋白亚基Ⅰ的cDNA序列的相似性为91%~94%。利用MEGA的NJ法构建昆虫纲血蓝蛋白亚基Ⅰ系统发育树,结果显示意大利蝗Cit Hc-1与东亚飞蝗Lmi Hc-1血蓝蛋白遗传距离最近形成姐妹群。为了研究血蓝蛋白的结构和功能,成功构建了意大利蝗Cit Hc-1基因活性区域的原核表达载体。融合蛋白p EASY-E1-Hc分子量约为32 k Da,与预期值一致,为进一步分析意大利蝗C.italicus Cit Hc-1的功能提供了理论基础。     

家蚕雌激素相关受体基因的克隆与表达分析

【目的】黑腹果蝇 Drosophila melanogaster 雌激素相关受体（estrogen-related receptor, ERR）通过调节糖酵解过程进而控制果蝇的能量代谢。本研究在克隆家蚕Bombyx mori ERR 基因 (BmERR) 的基础上,对其分子特性和系统演化进行生物信息学分析, 并检测该基因在家蚕生殖腺中的表达,为进一步研究ERR功能奠定基础。【方法】采用PCR技术克隆 BmERR 基因的全长cDNA序列,进行生物信息学分析;利用半定量RT-PCR检测该基因在停食后家蚕幼虫生殖腺中的表达情况。【结果】BmERR 基因全长cDNA序列为1 296 bp,编码431个氨基酸残基;具有ERR蛋白家族典型的结构特征;系统进化分析显示BmERR与其他昆虫ERR氨基酸序列一致性较高;半定量 RT-PCR 检测表明,BmERR 在家蚕上簇到化蛾期间的精巢和卵巢中均有表达,表达具有时期特异性,化蛹第1天达到表达高峰。【结论】本研究首次从鳞翅目昆虫中克隆获得ERR cDNA序列。ERR基因在家蚕生殖腺中表达量无明显性别差异,但具有发育时期特异性。     

珊瑚藻R-藻红蛋白γ亚基基因的克隆(英文)

根据珊瑚藻(Corallina afficinalis L.)R-藻红蛋白γ亚基N末端部分氨基酸序列(P83592)设计简并引物,结合RACE方法,扩增获得g亚基的全长cDNA序列。结果表明,序列全长为2308 bp(AY209894),5′非编码区长1203bp,3′非编码区长145 bp,编码区长960 bp,编码320个氨基酸组成的前体,包含71个氨基酸构成的信号肽和249个氨基酸组成的成熟蛋白。成熟蛋白序列内部存在重复序列与前人的报道一致。珊瑚藻亚基cDNA序列不同克隆子的测序结果表明,g亚基cDNA序列存在不同的3′末端,说明该基因可能存在多个拷贝或存在转录后加工。此外,扩增获得g亚基DNA序列(AY308999),比较表明编码区内部没有内含子存在。本文是对珊瑚藻R-藻红蛋白g亚基基因序列的首次报道。     

类大麦属高分子量谷蛋白基因Kx的序列测定及表达

利用SDS-PAGE检测了2份类大麦属(Crithopsis delileana)材料的高分子量谷蛋白亚基组成,并对其中1份材料的x型亚基进行了克隆和测序。结果表明,2份材料具有完全相同的蛋白电泳图谱。在小麦的高分子量区域仅检测到一条蛋白质带,与小麦y型亚基的迁移率接近,但克隆测序表明其为x型高分子量谷蛋白亚基,其编码基因命名为Kx。Kx基因编码区序列长度为2052bp．编码长度为661个氨基酸残基的蛋白质,其序列具有典型的x型高分子量谷蛋白亚基的特征。Kx基因能在原核表达系统内正确表达,其表达蛋白与来源于种子中的Kx亚基的迁移率完全一致。Kx亚基与小麦属A、B和D,山羊草属C和U以及黑麦属R染色体组编码的高分子量谷蛋白亚基氨基酸序列非常相似,但在N和C保守区的氨基酸组成以及重复区长度上与它们存在明显差异。聚类分析可将Kx与Ax1聚类为平行的分支。由此可见,来源于C．delileana的Kx基因为一新的x型高分子量谷蛋白亚基基因。     

Distribution of two GABA receptor-like subunits in theDrosophila CNS

Previously we have described the distribution of theRdl GABA receptor subunit in theDrosophila CNS. Knowing thatRdl can coassemble with LCCH3 (aDrosophila GABA receptor-like subunit showing sequence similarity to vertebrate subunit GABA_A receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast,Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列.表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似.在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区.但是,本研究所克隆的沉默型1Av基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白.讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及lAy基因沉默的机制.     

乌拉尔图小麦(Triticum urartu)1Ay高分子量麦谷蛋白亚基基因编码区的克隆与鉴定(英文)

应用简并性引物和基因组PCR反应从乌拉尔图小麦(Triticum urartu)不同种质材料中获得并测定了表达型和沉默型1Ay高分子量麦谷蛋白亚基基因全长编码区的基因组DNA序列。表达型1Ay基因编码区的序列与前人已发表的y型高分子量麦谷蛋白亚基基因编码区的序列高度同源,由其推导的1Ay亚基的一级结构与已知的高分子量麦谷蛋白亚基相似。在细菌细胞中,表达型1Ay基因编码区的克隆序列可经诱导而产生1Ay蛋白,该蛋白与种子中1Ay亚基在电泳迁移率和抗原性上类似,表明所克隆的序列真实地代表了表达型1Ay基因的全长编码区。但是,本研究所克隆的沉默型1Ay基因的编码区序列因含有3个提前终止子而不能翻译成完整的1Ay蛋白。讨论了表达型1Ay基因在小麦籽粒加工品质改良中的潜在利用价值以及1Ay基因沉默的机制。     

Expression analysis of Arabidopsis thaliana NAD-dependent isocitrate dehydrogenase genes shows the presence of a functional subunit that is mainly expressed in the pollen and absent from vegetative organs

NAD-dependent isocitrate dehydrogenase (IDH) is a Krebs cycle enzyme situated in mitochondria. In Arabidopsis thaliana, five genes encode functional IDH subunits that can be classed into two groups based on gene structure and subunit amino acid sequence. Arabidopsis contains two 'catalytic' and three 'regulatory' subunits according to their homology with yeast IDH. To date, an active IDH is believed to be heteromeric, containing at least one of each subunit type. This was verified in Arabidopsis by the complementation of yeast IDH mutants with the different Arabidopsis IDH-encoding cDNAs. Indeed, a single 'catalytic' and 'regulatory' subunit was sufficient to restore acetate growth of the yeast IDH double mutant. To gain information on possible IDH subunit interactions in planta, Arabidopsis IDH gene expression was analysed by Northern blot, PCR on cDNA libraries, in silico and in 'promoter'-reporter gene transgenic plants. Four of the IDH genes were expressed in all plant organs tested, while one gene (At4g35650) was not expressed in vegetative organs but was mainly expressed in the pollen. In leaves, the IDH genes were highly expressed in the veins, and to a lesser extent in mesophyll cells. The data are discussed with respect to IDH in other plant species.     

Molecular Cloning and Differential Expression Patterns of Avian Glutamate Receptor mRNAs

Abstract: We have identified and studied potential ionotropic glutamate receptor genes in pigeon brain. Three cDNA clones exhibit significant amino acid sequence identity to members of a rodent ligand-gated ion channel family. One of them, GluP-II, encodes a full-length AMPA-sensitive glutamate receptor GluR2 (GluR-B) homologue, whereas the other two partial clones, designated as GluP-III and -IV, are nearly identical to rodent GluR3 (GluR-C) and GluR4 (GluR-D) receptor subunits. Northern analysis demonstrated that the avian genes are widely expressed in the brain. Within the brain regions analyzed by in situ hybridization histochemistry, the three avian GluR subunits showed distinct and regionally specific mRNA expression patterns in the adult. Most of the differences in their expression were observed in cell types of the telencephalon, certain thalamic nuclei, the optic tectum, and the cerebellar cortex. A particularly striking finding was the expression of GluP-II in Golgi epithelial/Bergmann glial cells. In contrast, Bergmann glial cells in rat cerebellum do not express GluR2 (GluR-B) subunit genes. Immunoreactivity for a monoclonal sequence-specific antipeptide antibody was widespread and most prominent in Purkinje cell perikarya and their dendrites, neuronal cell bodies of the ectostriatum, and the deep optic tectum. These results demonstrate the existence of multiple subunits of the ionotropic glutamate receptor channel family in avians. Excitatory amino acid receptor genes appear to be highly conserved during evolution.     

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Isolation and characterization of the beta and epsilon subunit genes of mouse muscle acetylcholine receptor

The genes coding for the beta and epsilon subunits of the mouse muscle nicotinic acetylcholine receptor (nAChR) were mapped by Southern blot analysis, and the entire loci for both genes cloned. The results indicate that they are single-copy genes. Both were sequenced to determine their size and structural organization. The beta subunit gene spans approximately 8 kilobases and is organized into 11 exons. A region containing cysteines, which are thought to form a disulfide bond and which are highly conserved, is encoded by one exon in all muscle acetylcholine receptor genes with the exception of the beta subunit gene, where it is split into two exons. The epsilon subunit gene spans 4.3 kilobases and contains 12 exons; it has the same structure as the gamma and delta nAChR genes. The intron-exon boundaries and exonic organization of the five known nAChR genes were compared. The analysis showed that the first 4 exons and the last exon of all muscle and brain nAChR subunit genes have the same boundaries, with the exception of a nAChR-related gene in Drosophila.