绿色荧光蛋白及其在植物研究中的应用

绿色荧光蛋白(GFP)是海洋生物水母(Aequorea victoria)体内的一种发光蛋白,分子量27kD,由238个氨基酸组成。该蛋白65~67位Ser-Tyr-Gly三种氨基酸环化加氧形成特殊的生色团结构。野生型GFP发光较弱,而且gfp-cDNA含有隐蔽型剪切位点,而加工改造的GFP在植物中能够正常表达并且加强了荧光信号。GFP作为新的报告基因和遗传标记被广泛应用于植物研究之中。     

Green fluorescent protein (GFP) fusion constructs in gene therapy research

The history of green fluorescent protein (GFP) as a marker is less than 10 years old, but it has already made a major impact on many areas of natural sciences, especially on cell biology and histochemistry. GFP can be detected in living cells without selection or staining and it can be fused to other proteins to yield fluorescent chimeras. The potential of GFP has also been recognised by gene therapy researchers and various GFP-tagged therapeutic proteins have been constructed. These chimeric proteins have been used to determine the expression level, site and time course of the therapeutic gene, or the correlation between gene transfer rate and therapeutic outcome. This review summarises the status of the applications of GFP fusions in gene therapy research.     

Histological analysis of GFP expression in murine bone.

The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a beta-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein-protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

Understanding the folding of GFP using biophysical techniques

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein–protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.     

Green fluorescent protein as a molecular marker in microbiology

Molecular markers such as: lacZ (b-galactosidase), xylE (catechol 2,3-dioxygenase), lux (bacterial luciferase), luc (insect luciferase), phoA (alkaline phosphatase), gusA and gurA (beta-glucuronidase), gfp (green fluorescent protein), bla (beta-lactamase) and other antibiotic resistance markers, heavy metals resistance genes are commonly used in environmental microorganisms research (Errampaii et al., 1998; Kohler et al., 1999). Most of these markers require one or more substrates, complex media and/or expensive equipment for detection. The gfp gene is widely used as a marker because of its very useful properties such as high stability, minimal toxicity, non-invasive detection and the ability to generate the green light without addition of external cofactors and without application of expensive equipment. Various applications of that reporter gene were showed starting from monitoring of microorganism's survival in complex biological systems such as activated sludge to biodegradation of chemical compounds in soil. GFP allowed the detection, determination of spatial location and enumeration of bacterial cells from diverse environmental samples such as biofilm and water. The gfp as a biomarker was very useful in monitoring of gene expression and protein localisation in bacterial cells, too. The techniques with using gfp marker promise to supply a better understanding of environmental processes. It can make possible to use that knowledge in designing more effective and more efficient methods of biodegradation of toxic compounds from different environments.     

GFP imaging: methodology and application to investigate cellular compartmentation in plants

The cloning of the jellyfish gfp (green fluorescent protein) gene and its alteration for expression in subcellular locations in transformed plant cells have resulted in new views of intracellular organization and dynamics. Fusions of GFP with entire proteins of known or unknown function have shown where the proteins are located and whether the proteins move from one compartment to another. GFP and variants with different spectral properties have been deliberately targeted to separate compartments to determine their size, shape, mobility, and dynamic changes during development or environmental response. Fluorescence Resonance Energy Transfer (FRET) between GFP variants can discern protein/ protein interactions. GFP has been used as a sensor to detect changes or differences in calcium, pH, voltage, metal, and enzyme activity. Photobleaching and photoactivation of GFP as well as fluorescence correlation spectroscopy can measure rates of diffusion and movement of GFP within or between compartments. This review covers past applications of these methods as well as promising developments in GFP imaging for understanding the functional organization of plant cells.     

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual‐labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N‐terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin‐C promoter. mCherry is expressed in almost all tissues analyzed including pre‐ and post‐implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals. genesis 48:723–729, 2010. © 2010 Wiley‐Liss, Inc.     

Visualisation of plastids in endosperm,pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO,rice FtsZ and maize ferredoxin III proteins

The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.     

绿色荧光蛋白及其在GMOs 生态监测中的应用

绿色荧光蛋白 (green fluorescent protein, GFP)是在海洋无脊椎动物水母(Aequorea victoria)中获得的一种由238个氨基酸组成的多肽。该多肽通过翻译后加工形成生色基团, 产生稳定的荧光, 而且这种荧光很容易被检测。GFP作为动、植物以及微生物基因工程研究上的一种广泛的选择标记, 具有检测灵敏度高、操作简便、不需要添加任何底物或辅助因子等优点, 更重要的是利用GFP可对GMOs进行快速、原位、实时、活体监测。本文概括介绍了GFP的特性、改造及其检测, 并从生态学角度论述了GFP在GMOs生态监测研究中的应用及其发展前景。     

绿色荧光蛋白及其在GMOs生态监测中的应用

绿色荧光蛋白（green fluorescent protein,GFP）是在海洋无脊椎动物水母（Aequorea victoria）中获得的一种由238个氨基酸组成的多肽。该多肽通过翻译后加工形成生色基团,产生稳定的荧光,而且这种荧光很容易被检测。GFP作为动、植物以及微生物基因工程研究上的一种广泛的选择标记,具有检测灵敏度高、操作简便、不需要添加任何底物或辅助因子等优点,更重要的是利用GFP可对GMOs进行快速、原位、实时、活体监测。本文概括介绍了GFP的特性、改造及其检测,并从生态学角度论述了GFP在GMOs生态监测研究中的应用及其发展前景。     

Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography

GFP has often been used as a marker of gene expression, protein localization in living and fixed tissues as well as for protein targeting in intact cells and organisms. Monitoring foreign protein expression via GFP fusion is also very appealing for bioprocess applications. Many cells, including bacterial, fungal, plant, insect and mammalian cells, can express recombinant GFP (rGFP) efficiently. Several methods and procedures have been developed to purify the rGFP or recombinant proteins fused with GFP tag. However, most current GFP purification methods are limited by poor yields and low purity. In the current study, we developed an improved purification method, utilizing a FMU-GFP.5 monoclonal antibody (mAb) to GFP together with a mAb-coupled affinity chromatography column. The method resulted in a sample that was highly pure (more than 97% homogeneity) and had a sample yield of about 90%. Moreover, the GFP epitope permitted the isolation of almost all the active recombinant target proteins fused with GFP, directly and easily, from the crude cellular sources. Our data suggests this method is more efficient than any currently available method for purification of GFP protein.     

Engineered metal binding sites on green fluorescence protein

The ability to assay a variety of metals by noninvasive methods has applications in both biomedical and environmental research. Green fluorescent protein (GFP) is a protein isolated from coelenterates that exhibits spontaneous fluorescence. GFP does not require any exogenous cofactors for fluorescence, and can be easily appended to other proteins at the DNA level, producing a fluorescence-labeled target protein in vivo. Metals in close proximity to chromophores are known to quench fluorescence in a distance-dependent fashion. Potential metal binding sites on the surface of GFP have been identified and mutant proteins have been designed, created, and characterized. These metal-binding mutants of GFP exhibit fluorescence quenching at lower transition metal ion concentrations than those of the wild-type protein. These GFP mutants represent a new class of protein-based metal sensors.     

Generation and characterization of rgs5 mutant mice

Regulators of G-protein signaling (RGS) are involved in a wide variety of functions, including olfaction, vision, and cell migration. RGS5 has a perivascular expression pattern and was recently identified as a marker for brain pericytes. This suggests a role for RGS5 in vascular development and pericyte biology. We have created a mouse line which lacks the rgs5 gene and replaced it with a green fluorescent protein (GFP) reporter (rgs5(GFP/GFP)). The mice are viable and fertile and display no obvious developmental defects, and the vasculature appears to develop normally with proper pericyte coverage. Also, no differences were observed in the vasculature under pathological conditions, such as tumor growth and oxygen-induced retinopathy. The GFP expression in pericytes of rgs5(GFP) mice allows detection and sorting of these cells, thereby providing a valuable novel tool for pericyte research.     

Ubiquitous expression of mRFP1 in transgenic mice

Fluorescent proteins provide a powerful means to track gene expression and cellular behaviors in the study of model organisms such as mice. Among the new generation of fluorescent protein markers, the monomeric red fluorescent protein mRFP1 is particularly attractive because of its rapid maturation and minimal interference with GFP and GFP-derived markers. Here we evaluate the utility of mRFP1 as a marker in transgenic mice. We show that high level and ubiquitous expression of mRFP1 does not affect mouse development, general physiology, or reproduction. mRFP1 expression can be readily detected with unaided eyes under daylight in transgenic mice on the albino background. The intensity of mRFP1 signals can be used to distinguish homozygous and heterozygous transgenic mice. Together, these features make mRFP1 an attractive marker for broad applications in transgenic research.     

Development of visible markers for transgenic plants and their availability for environmental risk assessment

Monitoring of transgenic plants in the field is important, but risk assessment has entailed laborious use of invisible marker genes. Here, we assessed three easily visible marker transgenes--green fluorescent protein (GFP), R, and Nicotiana tabacum homeobox (NTH) 15 genes--for their potential use as marker genes for monitoring genetically modified plants. Transgenic Arabidopsis thaliana plants for each of these genes were visibly distinguished from wild-type plants. We determined the germination rate, 3-week fresh weight, time to first flowering, and seed weight of the transgenic plants to evaluate whether the expression of these marker genes affected the growth of the host. Introduction of GFP gene had no effect on the evaluated parameters, and we then used the GFP gene as a marker to assess the outcrossing frequency between transgenic and two Arabidopsis species. Our results showed that the hybridization frequency between transgenic plants and Arabidopsis thaliana was 0.24%, and between transformants and Arabidopsis lyrata it was 2.6% under experimental condition. Out-crossing frequency was decreased by extending the distance between two kinds of plants. Thus, the GFP gene is a useful marker for assessing the whereabouts of transgenes/transformants in the field. We also demonstrated that the GFP gene is possibly applicable as a selection marker in the process of generation of transgenic plants.     

用绿色荧光蛋白监测转基因植物中选择标记基因的消除

绿色荧光蛋白(GFP)可直接进行活体观察,它的这个优点可被用于监测转基因植物中选择标记基因的消除。为此,构建了植物表达载体pGNG,将绿色荧光蛋白基因(gfp)和卡那霉素抗性基因表达盒(NosP-nptll-NosT)一起克隆在两个同向的lox位点间,在第一个lox位点上游置有CaMV 35S启动子以驱动GFP表达,第二个lox位点下游置有不含启动子的大肠杆菌β-葡萄糖醛酸酶(GUS)基因。首先在含卡那霉素(Kan)的培养基上筛选出转pGNG的烟草,借助绿色荧光可容易地检出表达GFP的转化体。然后用另一转化载体pCambia1300Cre二次转化表达GFP的转基因植物,利用另一选择标记基因潮霉素抗性基因(hpt)进行筛选,在获得的再生植株中,Cre重组酶的表达消除了转化体中两lox位点间的gfp和nptll。实验结果表明可借助GFP荧光的消失,快速选出nptII被消除的二次转化体,同时GUS(作为目的蛋白) 在CaMV 35S启动子驱动下获得表达。最后利用后代的分离将hpt和cre除去。     

绿荧光蛋白(GFP)研究进展

ＧＦＰ作为一种全新的标记基因,在生物学的各研究领域得到了广泛的应用,本文概述了的近年来相关方面的研究进展和重要应用,以及尚存在的不足。