SDF-1α基因与绿色荧光蛋白融合载体的构建及亚细胞定位

目的构建SDF-1α基因与绿色荧光蛋白的融合蛋白表达载体,进而观察SDF-1α基因编码蛋白在细胞内的定位情况。方法用EcoRI内切酶从pMD-T18一SDF-1α重组载体中酶切分离SDF-1α基因的完整ORF,构建pEGFP-C1-SDF-1α的融合表达载体,脂质体转染COS-7细胞,并在荧光显微镜下观察表达的融合蛋白。结果SDF-1α基因在COS-7细胞中高效表达,激光共聚焦的结果显示,SDF-1α基因定位在细胞质内。结论成功构建了pEGFP-C1-SDF-1α的融合表达载体,SDF-1α基因主要在细胞质中表达。     

鸭梨多酚氧化酶基因反义表达载体的构建及农杆菌介导的遗传转化

根据鸭梨多酚氧化酶基因序列设计引物,PCR扩增该基因3′端450bp的片段,并将该片段反向插入真核表达载体pB I121的CaMV 35S启动子和NOS终止子之间,首次构建了鸭梨PPO基因的反义表达载体;其后,在农杆菌EHA105的介导下,成功实现了PPO反义基因对鸭梨组培苗的遗传转化。经Northern杂交和酶活检测证实,转基因鸭梨植株体内的多酚氧化酶基因转录和翻译水平均得到明显抑制,从而为耐褐化梨新品种的培育奠定了基础。     

拟南芥血红蛋白3的亚细胞定位

拟南芥的血红蛋白3（AtGLB 3）属于截短的血红蛋白。与拟南芥血红蛋白1相比,拟南芥血红蛋白3具有不同的起源、不同的生化特性和结构;但其功能还不清楚。蛋白质的定位与蛋白质的功能息息相关。为深入研究该基因功能,构建了拟南芥血红蛋白3基因与绿色荧光蛋白融合的植物表达载体pUCGFP/AtGLB3。利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白3在细胞中的定位。荧光显微镜检测结果表明,AtGLB3基因表达产物主要定位在细胞膜上。     

拟南芥血红蛋白1的亚细胞定位

为研究拟南芥的血红蛋白1(AtGLB1)基因的亚细胞定位,该实验构建了拟南芥血红蛋白1基因与绿色荧光蛋白基因融合的植物表达载体pUCGFP/ AtGLB1.利用基因枪转化法将重组载体转入洋葱表皮细胞瞬时表达,通过检测融合蛋白在洋葱表皮细胞中的分布来确定拟南芥血红蛋白1在细胞中的定位.荧光显微镜检测结果表明,AtGLB1基因表达产物主要定位在细胞核中,少量定位在细胞质中.     

GFP基因在棉花转化中的应用

以绿色荧光蛋白GFP基因为报道基因,用花粉管通道和农杆菌介导的转化方法将外源基因导入棉花（Gossypium hirsutum L.)分别获得转化幼胚,幼苗和转化愈伤组织,用手持紫外灯结合显微镜检术能够快速地对转化子进行活体筛选鉴定,比用GUS检测广阔圾明显的优越性,本研究不但为花粉管道道转化法的可行性提供了新的证据。同时也建立了GFP用于棉花基因工程研究的检测技术体系。     

拟南芥谷氨酸受体基因的亚细胞定位

为明确拟南芥谷氨酸受体1.3基因(AtGLR1.3)的亚细胞定位,该实验以拟南芥(Arabidopsis thalianaCo-lumbia ecotype)为材料,运用PCR方法从其基因组中扩增得到了AtGLR1.3的启动子和基因序列,将其连接到载体pBIsGFP上,构建成AtGLR1.3基因与绿色荧光蛋白基因融合的植物表达载体,通过农杆菌介导的花序浸润法将重组载体转化拟南芥野生型,转基因植株通过激光共聚焦扫描显微镜观察显示,GFP荧光信号存在于细胞质膜上,表明AtGLR1.3为细胞膜蛋白.该结果为进一步研究AtGLR1.3的作用机理奠定了基础.     

绿色荧光蛋白基因作为报告基因在水稻基因转化中的应用研究

本研究中 ,构建了含有编码绿色荧光蛋白的改进型基因质粒pJPM5。用基因枪法分别把pJPM5和另一带有绿色荧光蛋白基因的质粒pSBG70 0转入水稻TNG6 7愈伤组织。用South ern杂交法证实了转基因的存在 ,而且表明多数转基因植株含有 1到 8个拷贝的转基因。取 2个月的转基因植株上的叶片用于分析绿色荧光蛋白基因表达。用SLM - 80 0 0荧光分析仪定量测定绿色荧光蛋白。多数转基因植株具有很高的绿色荧光蛋白信号。虽然水稻植株有少量自发荧光 ,但是绿色荧光蛋白基因表达出的绿色荧光蛋白信号比植株的自发荧光强得多 ,其测定不会受自发荧光的太大影响。在荧光显微镜下观察到了绿色荧光蛋白基因的表达。借助观察分析绿色荧光蛋白基因的瞬时表达 ,本研究还发现基因枪法转化中 ,如果两枪的气压为90 0psi& 135 0psi,比两枪的气压都为 90 0psi或者 135 0psi更好 ,因其能使质粒进入更多的细胞。研究结果表明 ,绿色荧光蛋白基因可以作为水稻 (甚至小麦、玉米 )转基因研究中的报告基因。研究还显示 ,MAR序列能明显增强绿色荧光蛋白基因的表达能力 (这一结果在另文讨论 ) .     

转SCaM—GFP融合基因烟草中钙调素分泌特性的研究

钙调素是一种重要的Ｃａ2 结合蛋白 ,在细胞内发挥着重要的生物学功能。一系列实验证实 ,钙调素也普遍存在于动植物细胞外[1- 3 ] ,并且具有重要的生物学功能[4 - 6] 。我们实验室的研究表明 :胞内外钙调素在Ｃａ2 依赖性以及在靶酶激活特性等方面基本相同 ,但在Ｃａ2 亲合力等方面存在差异[2 ,7] 。近年来 ,在植物细胞中发现存在多种钙调素亚型 ,特别是在大豆中对钙调素亚型研究得比较清楚[8] 。通过对大豆中钙调素亚型亚细胞定位的研究 ,一方面可以阐明钙调素是否具有向胞外主动分泌的特性 ,另一方面可以确定向细胞外分泌的钙调素是…     

基于农杆菌介导瞬时转化法的AtGLR1.4亚细胞定位分析

将拟南芥基因AtGLR1.4启动子驱动的AtGLR1.4基因与绿色荧光蛋白(GFP)基因融合后,利用根瘤农杆菌介导瞬时转化法(Fast Agro-mediated Seedling Transfomation,FAST)浸染拟南芥幼苗,对其进行亚细胞定位的研究。转基因植株通过激光共聚焦扫描显微镜的观察,发现GFP绿色荧光在叶片表皮细胞的细胞膜上特异表达,表明At-GLR 1.4蛋白定位于细胞质膜上,为其后续的功能研究提供了线索。     

人SDCT2蛋白亚细胞定位信号分析

为了确定人高亲和力钠离子依赖性二羧酸共转运蛋白（high-affinity sodium-dependent dicarboxylate co-transporter, SDCT2,NaDC3）在细胞内的定位,构建了SDCT2与增强型绿色荧光蛋白（EGFP）的融合蛋白表达载体,并转染肾小管上皮细胞LLC-PK1,激光共聚焦显微镜观察显示,SDCT2蛋白主要定位于细胞的基底侧膜上.同时将SDCT2-EGFP融合基因mRNA显微注射到爪蟾卵母细胞中表达,可见融合蛋白的绿色荧光仅分布在细胞膜上.为了进一步确定该蛋白质的亚细胞定位信号序列,将SDCT2基因的N端及C端分别缺失,并构建缺失突变体与EGFP的融合蛋白表达载体,将它们转染到LLC-PK1中,观察SDCT2 缺失体在细胞内的分布情况.结果显示,N端缺失的SDCT2蛋白主要位于细胞质中,顶膜和基底侧膜上也有表达;C端缺失的SDCT2蛋白主要位于基底侧膜上,顶膜几乎没有表达,细胞质中表达很少.免疫组化结果也显示,SDCT2只表达于人近端肾小管上皮细胞的基底侧膜.这表明SDCT2蛋白的N端序列对其亚细胞定位是必需的,人SDCT2蛋白的基底膜定位信号位于N端序列中.     

TransientAgrobacterium-mediated gene expression in theArabidopsis hydroponics root system for subcellular localization studies

To a great extent, the cellular compartmentalization and molecular interactions are indicative of the function of a protein. The development of simple and efficient tools for testing the subcellular location of proteins is indispensable to elucidate the function of genes in plants. In this report, we assessed the feasibility ofAgrobacterium-mediated transformation of hydroponically grown roots to follow intracellular targeting of proteins fused to green fluorescent protein (GFP). We developed a simple in planta assay for subcellular localization of proteins inArabidopsis roots via transient transformation and tested this method by expressing a GFP fusion of a known nuclear protein, IQD1. Visualization of transiently expressed GFP fusion proteins in roots by means of confocal microscopy is superior to the analysis of green tissues because the roots are virtually transparent and free of chlorophyll autofluorescence.     

普通烟草CESA基因家族成员的鉴定、亚细胞定位及表达分析

纤维素合成酶蛋白(cellulose-synthase proteins, CESA)是一类质膜定位蛋白,以蛋白复合体的形式存在于质膜上合成纤维素,在细胞壁建成和植物生长发育过程中起着非常重要的作用。本研究利用CESA蛋白保守域序列PF03552检索普通烟草(Nicotiana tabacum L.)蛋白序列,并通过拟南芥(Arabidopsis thaliana)10个CESA蛋白序列在普通烟草基因组数据库中利用TBLASTN程序进行比对,共获得21条NtCESA基因候选序列,对这些序列进行蛋白序列理化性质分析、系统进化树构建、基因结构分析、保守结构域及跨膜区分析和组织表达模式分析,并对NtCESA9和NtCESA14两个蛋白进行了亚细胞定位实验。结果表明：获得的21条NtCESA蛋白序列的理化性质相似;系统进化分析将21个NtCESA基因和10个AtCESA基因分成5个分支,每一个分支各成员之间的进化相对保守,基因结构类似,不同分支之间的基因结构差异也较小;NtCESA蛋白结构域相对保守,都含有CESA蛋白典型的N端锌指结构、C端跨膜区和DDD-QXXRW保守功能域;组织表达分析结果表明,大部分NtCESA基因在幼苗和成熟期烟草的根、叶、胚芽和愈伤组织中都有表达,同一个分支中的基因表达模式基本一致,并且NtCESA基因参与初/次生细胞壁纤维素的合成与该基因编码蛋白的跨膜区数目存在关联,表明NtCESA基因家族成员功能上的复杂性;亚细胞定位结果证实NtCESA9和NtCESA14为质膜定位蛋白。本研究为烟草CESA基因家族功能的深入研究奠定了基础。     

Cellular localization and biochemical characterization of a novel calcium-dependent protein kinase from tobacco

By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.     

Prediction of protein subcellular localization by weighted gene ontology terms

We develop a new weighting approach of gene ontology (GO) terms for predicting protein subcellular localization. The weights of individual GO terms, corresponding to their contribution to the prediction algorithm, are determined by the term-weighting methods used in text categorization. We evaluate several term-weighting methods, which are based on inverse document frequency, information gain, gain ratio, odds ratio, and chi-square and its variants. Additionally, we propose a new term-weighting method based on the logarithmic transformation of chi-square. The proposed term-weighting method performs better than other term-weighting methods, and also outperforms state-of-the-art subcellular prediction methods. Our proposed method achieves 98.1%, 99.3%, 98.1%, 98.1%, and 95.9% overall accuracies for the animal BaCelLo independent dataset (IDS), fungal BaCelLo IDS, animal Höglund IDS, fungal Höglund IDS, and PLOC dataset, respectively. Furthermore, the close correlation between high-weighted GO terms and subcellular localizations suggests that our proposed method appropriately weights GO terms according to their relevance to the localizations.     

小麦铝胁迫基因TaAIP的克隆表达分析及亚细胞定位

以小麦抗逆相关蛋白TaMAPK2作为诱饵,利用酵母双杂交系统进行筛库,获得互作蛋白TaAIP。氨基酸序列分析发现TaAIP具有wali保守区,并且与一些物种的铝诱导蛋白相似。实时荧光定量PCR分析显示,TaAIP基因受到铝、干旱以及高盐胁迫上调表达。半定量RT-PCR结果表明,TaAIP在小麦茎中表达,在根部、叶片以及花中没有表达。亚细胞定位实验发现,TaAIP定位在细胞膜上。这些结果为深入分析TaAIP的抗逆性作用机理打下基础。     

Functional expression of human DNA topoisomerase I and its subcellular localization in HeLa cells

DNA topoisomerase (topo) I plays an important role in DNA metabolism by relieving the torsional restraints of DNA topology through ATP-independent single-strand DNA breakage. In the present study, we expressed human topo I in HeLa cells by fusing it to enhanced green fluorescent protein (EGFP). The EGFP-topo I fusion protein is functionally active in that it relaxes supercoiled plasmid DNA; forms complexes with DNA, as revealed by band depletion assays; and increases the sensitivity of cells to topo I inhibitors such as topotecan, as determined by growth inhibition assays. In contrast, a mutant form of the EGFP-topo I fusion protein, in which the active Tyr has been replaced by Phe (Y723F), has no such activities. Furthermore, the fusion protein localizes to the nucleus at interphase and completely associates with chromatids at every stage of mitosis. Of importance, the mutant fusion protein (Y723F) displays a pattern of subcellular localization identical to that of the wild-type fusion protein, although the mutant fusion protein is catalytically inactive. These results suggest that in addition to its role in DNA metabolism, topo I might also play a structural role in chromosomal organization; moreover, the association of topo I with chromosomal DNA is independent of its catalytic activity. Finally, the fusion constructs may provide a useful tool to study drug action in tumor cells, as demonstrated by nucleolar delocalization of the fusion proteins in response to treatment with the topo I inhibitor topotecan.     

Prediction of protein subcellular localization

Because the protein's function is usually related to its subcellular localization, the ability to predict subcellular localization directly from protein sequences will be useful for inferring protein functions. Recent years have seen a surging interest in the development of novel computational tools to predict subcellular localization. At present, these approaches, based on a wide range of algorithms, have achieved varying degrees of success for specific organisms and for certain localization categories. A number of authors have noticed that sequence similarity is useful in predicting subcellular localization. For example, Nair and Rost (Protein Sci 2002;11:2836-2847) have carried out extensive analysis of the relation between sequence similarity and identity in subcellular localization, and have found a close relationship between them above a certain similarity threshold. However, many existing benchmark data sets used for the prediction accuracy assessment contain highly homologous sequences-some data sets comprising sequences up to 80-90% sequence identity. Using these benchmark test data will surely lead to overestimation of the performance of the methods considered. Here, we develop an approach based on a two-level support vector machine (SVM) system: the first level comprises a number of SVM classifiers, each based on a specific type of feature vectors derived from sequences; the second level SVM classifier functions as the jury machine to generate the probability distribution of decisions for possible localizations. We compare our approach with a global sequence alignment approach and other existing approaches for two benchmark data sets-one comprising prokaryotic sequences and the other eukaryotic sequences. Furthermore, we carried out all-against-all sequence alignment for several data sets to investigate the relationship between sequence homology and subcellular localization. Our results, which are consistent with previous studies, indicate that the homology search approach performs well down to 30% sequence identity, although its performance deteriorates considerably for sequences sharing lower sequence identity. A data set of high homology levels will undoubtedly lead to biased assessment of the performances of the predictive approaches-especially those relying on homology search or sequence annotations. Our two-level classification system based on SVM does not rely on homology search; therefore, its performance remains relatively unaffected by sequence homology. When compared with other approaches, our approach performed significantly better. Furthermore, we also develop a practical hybrid method, which combines the two-level SVM classifier and the homology search method, as a general tool for the sequence annotation of subcellular localization.     

人类GABARAPL2基因的亚细胞定位

为了对GABARAPL2（GABAA受体相关蛋白相似蛋白2）基因的功能进行初步分析,首先通过同源比较的方法将序列与其同源物进行比较,发现GABARAPL2的氨基酸序列与GABARAP（GABAA受体相关蛋白）高度同源,而GABARAP已证实通过结合细胞骨架的微蛋白,使GABAA受体聚集,定位在细胞膜上,本文采用PCR法从人脑组织的cDNA文库中扩增出GABARAPL2的cDNA,克隆至T质粒载体中进行测序验证,然后以此为模板引物中引入酶切位点再次PCR,扩增出GABARAPL2的开放阅读框,并将其插入到加强型绿色荧光蛋白融合表达载体EGFP中,将绿色荧光蛋白标记的GABARAPL2和GABARAP分别转染HLF细胞株,结果两种蛋白的分布情况基本一致,在细胞质内和核内均有分布,而且核内的分布较胞质为多,结构功能域分析表明,GABARAPL2含有蛋白激酶C磷酸化位点和酪氨酸激酶磷酸化位点,可能通过磷酸化参与细胞骨架的变化,结论 GABARAPL2和GABARAP不仅在胞质中作为受体相关蛋白协助受体的聚集、定位,还参与体内许多其它重要的生理过程。