绿色荧光蛋白及其在植物分子生物学研究中的应用

绿色荧光蛋白(GFP)是海洋生物水母(Aequoria victoria)体内的一种发光蛋白,近十年来成为在生物化学和细胞生物学研究和应用中用得最广泛的蛋白质之一。文章就绿色荧光蛋白的特性及其在植物分子生物学中应用的研究进展作了概述。     

A Photostable Green Fluorescent Protein Variant for Analysis of Protein Localization in Candida albicans

Fusions to the green fluorescent protein (GFP) are an effective way to monitor protein localization. However, altered codon usage in Candida species has delayed implementation of new variants. Examination of three new GFP variants in Candida albicans showed that one has higher signal intensity and increased resistance to photobleaching.The human fungal pathogen Candida albicans can cause severe infections, particularly in immunocompromised patients. Important insights into its pathogenesis have been obtained by analyzing fusions to green fluorescent protein (GFP) (8). Although GFP tagging has been very successful, many fusion proteins are not easily detected. New GFP variants with improved fluorescence and protein folding properties have been identified by genetic approaches in other organisms (2, 7, 8). However, these GFP variants have not been assessed in C. albicans and related species, presumably because of the added difficulties of attempting heterologous expression in C. albicans.To adapt GFP for effective use in C. albicans, Cormack et al. introduced three types of codon changes: the S65G S72A mutations to enhance fluorescence; the CTG codon 201 change to TTG, since CUG is translated as Ser instead of Leu in C. albicans; and the optimization of the other codons for translation in C. albicans (1). This variant, known as YeGFP3, was introduced into convenient vectors for creating gene fusions in C. albicans (4). Another version of eGFP known as mut2 (S65A V68L S72A Q80R) was adapted for C. albicans by changing the CTG codon but without further codon optimization (5). These obstacles to heterologous expression in C. albicans have presumably delayed implementation of newer versions of GFP. Therefore, in this study three different GFP variants were introduced into YeGFP3 and examined for function in C. albicans.The GFP variants were constructed using standard methods to introduce changes in the coding sequence of YeGFP3. In brief, mutagenic oligonucleotides were used to prime PCR synthesis of a plasmid carrying YeGFP3, the template DNA was then destroyed by digestion with DpnI, and then the resulting DNA was transformed into Escherichia coli. DNA sequencing (carried out by the Stony Brook University DNA Sequencing Facility) confirmed that the correct substitutions were present. The mutant GFP genes were then released as PstI-AscI fragments and then were subcloned to replace the corresponding GFP fragment of plasmid pFa-GFP-URA3 (6), which carries a PCR cassette module for creating GFP fusions in C. albicans. Because of the large number of changes, the mutants were given the more convenient names of CaGFPα (F64L S65T F99S M153T V163A), CaGFPβ (F64L S65T N149K M153T I167T; also known as emerald), and CaGFPγ (F64L S65C V163A I167T). The CaGFPγ was also introduced into vectors that contain selectable markers HIS1 and ARG4 (6). DNA sequences used to design primers for creating GFP fusions in C. albicans were as follows: forward primer, 5′ (region of homology)-GGTGCTGGCGCAGGTGCTTC-3′, and reverse primer, 5′ (region of homology)-TCTGATATCATCGATGAATTCGAG-3′.CDC11-GFP fusion genes were created in C. albicans by homologous recombination, as described previously (4, 6). In brief, long oligonucleotide primers with homology to the 3′ end of the CDC11 open reading frame were used to prime PCR synthesis of each of the corresponding GFP variant genes plus an adjacent selectable marker gene (URA3). These DNA elements were then introduced into C. albicans cells and allowed to recombine with the homologous region of the CDC11 gene in C. albicans to create the CDC11-GFP fusion genes. Sequences used for the design of PCR primers to amplify the pFa-GFP plasmids are shown above. Cells carrying the indicated CDC11-GFP fusion gene were grown overnight in log phase in synthetic medium (yeast nitrogen base plus amino acids and dextrose). Cdc11-GFP fluorescence intensity was analyzed with an Olympus BH2 microscope equipped with a Zeiss AxioCam camera run by Openlab software. The relative GFP signal was determined by measuring the intensity of GFP fluorescence of the septin ring and then subtracting the fluorescence of an area immediately adjacent to each ring. All samples were visualized under the same conditions.Samples were prepared for Western blot analysis by resuspending cells in TNE lysis buffer (10 mM Tris base, 1 mM EDTA, 100 mM NaCl) with 100× protease mix (40 mg/ml pepstatin A, 40 mg/ml aprotinin, 20 mg/ml leupeptin) and then agitating in the presence of glass beads. The supernatant was collected after low-speed centrifugation at 3,000 rpm for 1 min, protein concentrations were determined by the bicinchoninic acid (BCA) protein assay (Pierce), and then equal amounts of protein extract were separated by gel electrophoresis and transferred to a Protran nitrocellulose membrane (Whatman GmbH). The blots were incubated with mouse anti-GFP (Millipore), rabbit anti-glucose-6-phosphate dehydrogenase (anti-G6PD; Sigma), or rabbit anti-Cdc11 (Santa Cruz Biotechnology) primary antibodies; washed; and then incubated with either goat anti-mouse IRDye 800cw or goat anti-rabbit IRDye 680 (Li-Cor Biosciences, Lincoln, NE). The immunoreactive proteins were visualized with a Li-Cor fluorescence scanner run by Odyssey software.Three new GFP variants based on YeGFP3 were constructed by introducing mutations predicted to improve either the fluorescence properties or protein folding (2, 7, 8). Because multiple changes were introduced into each variant, they were given the more convenient names of CaGFPα, CaGFPβ, and CaGFPγ (see above). The key mutations in CaGFPα and CaGFPβ have been described previously (2, 7, 8), but CaGFPγ represents a novel combination of mutations. The 3 new GFP variants plus the YeGFP3 and mut2 versions were compared by fusing them to the C terminus of the Cdc11 septin protein (3). The Cdc11 protein was selected because its restricted localization to the bud neck facilitated microscopic analysis and comparison of fluorescence properties. CDC11-GFP fusion genes were constructed in strain BWP17 (9) using PCR-generated modules with a URA3 selectable marker, as described previously (4, 6).Cells were grown in synthetic medium overnight to log phase at both 30°C and 37°C, temperatures that are commonly used to propagate C. albicans and that may affect the folding properties of GFP. GFP fluorescence was then analyzed by quantifying the intensity of the septin rings in digital images (Fig. ​(Fig.1A).1A). Septin rings were analyzed only if they were obviously in focus and at the same stage of the cell cycle (large budded). CaGFPγ gave a slightly stronger signal than the other variants, which was most obvious at 30°C (Fig. ​(Fig.1A).1A). At least two independent clones were analyzed for each CDC11-GFP variant, and the two gave similar results (data not shown).Open in a separate windowFIG. 1.Properties of Cdc11-GFP fusion proteins. Cells were grown to log phase overnight at the indicated temperature, and then Cdc11-GFP fluorescence was analyzed. (A) Signal intensity for the different versions of Cdc11-GFP was compared in three independent assays in which 50 septin rings per assay were quantified for each different Cdc11-GFP. The average fluorescence intensity was normalized to 100 for Cdc11-YeGPF3. The Cdc11-CaGFPγ variant gave a significantly stronger signal than the other variants (P < 0.001). (B) Western blot analysis comparing the levels of Cdc11-GFP produced in the indicated strains. The lane labeled “neg” refers to the negative-control strain (BWP17) that lacks GFP. Blots were probed with anti-GFP to detect Cdc11-GFP, anti-glucose-6-phosphate dehydrogenase (αG6PD) as a control, and anti-Cdc11 to detect the untagged version of Cdc11.The levels of the Cdc11-GFP proteins at both 30°C and 37°C were compared on two independent Western blots using anti-GFP antibody (Fig. ​(Fig.1B).1B). The relative levels of Cdc11-mut2GFP and Cdc11-CaGFPα were the lowest, consistent with their lower fluorescence intensity. The lower levels of Cdc11-mut2GFP are consistent with the fact that the codons in the mut2 version of GFP were not optimized for expression in C. albicans (5). The Cdc11-YeGFP3 and Cdc11-CaGFPγ were present at higher levels, and the Cdc11-CaGFPβ was produced at even slightly higher levels, consistent with reports that this latter version of GFP (also known as emerald) has improved folding properties (7). The Cdc11-GFP variants did not affect the production of the untagged Cdc11 protein (Fig. ​(Fig.1B1B).Photobleaching is also an important factor for GFP (7), especially in time-lapse studies or Z-stack analysis of different optical sections of cells. Photostability of the GFP variants was examined by taking pictures at 4-s intervals during 1 min of continuous exposure to the fluorescence excitation lamp (Fig. 2A and B). The fluorescence of YeGFP3, mut2GFP, and CaGFPα fused to Cdc11 decayed to 50% of original intensity within 15 to 30 s, and the rate of photobleaching was even higher for CaGFPβ. In contrast, Cdc11-CaGFPγ showed extended photostability at both 30°C and 37°C (half-life [t_1/2] of ∼2 min). Similar results were also obtained for CaGFPγ fused to the Golgi protein Vrg4 (data not shown), although the standard deviations were larger because the mobile Golgi compartments frequently moved out of the focal plane during the time course (data not shown). On a practical level, the Cdc11-GFPγ fluorescence was readily detectable after several minutes of continuous exposure (Fig. ​(Fig.2C),2C), demonstrating its clear advantage for allowing more time to observe protein localization before photobleaching becomes significant.Open in a separate windowFIG. 2.Photostability of GFP variants. (A and B) Relative fluorescence intensity of the GFP variants at 4-s intervals over a time course of 1 min of continuous exposure to the fluorescence excitation lamp after growth at 30°C (A) and at 37°C (B). CaGFPγ showed the best photostability (t_1/2 of ∼2 min). The relative fluorescence was normalized to 100 for each Cdc11-GFP variant at the start of the time course. The results represent the average of three independent assays in which three septin rings were analyzed for each mutant. Error bars indicate standard deviations. (C) Cells carrying Cdc11 fused to YeGFP3 or CaGFPγ were continuously exposed to the fluorescence excitation lamp, and then images of septin rings were captured at the indicated times.Altogether, Cdc11-CaGFPγ had the best overall properties based on protein levels, signal intensity, and photostability in C. albicans. The higher level of Cdc11-CaGFPβ production was apparently offset by increased photobleaching, resulting in no overall advantage for this variant. The Cdc11-CaGFPα was produced at relatively low levels, and it was less photostable compared to the other versions. Thus, CaGFPγ is a novel GFP variant that offers improved features for the study of protein localization in C. albicans and will likely also be useful for expression in other species.     

绿色荧光蛋白——现代细胞生物学与分子生物学研究领域的新标记物

从多管水母属A equoren v ictu ria 分离出的绿色荧光蛋白(GFP) , 因其特有的生物化学性质及该基因在异源细胞内的表达产物亦能产生强烈的绿色荧光, 使其在现代细胞生物学和分子生物学研究领域的应用具有广阔前景。本文就其研究进展及其应用进行简要综述。     

绿色荧光蛋白在蛋白质研究中的应用

绿色荧光蛋白（green fluorescent protein,GFP）自发现以来,由于具有自发荧光等特性,在分子生物学和细胞生物学领域得到广泛应用。GFP作为一种报道分子,在研究蛋白质相互作用和构象变化、检测蛋白质表达、蛋白质和细胞荧光示踪中,起到了重要的作用。该文通过对绿色荧光蛋白特性的分析．介绍其作为荧光标记在蛋白质研究中的应用,并展望进一步的研究前景。     

绿色荧光蛋白在生命科学研究中的应用

近年来,随着水母Ａｅｑｕｏｒｅａｖｉｃｔｏｒｉａ来源的绿色荧光蛋白（Ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ,ＧＦＰ）在各种异源细胞,如细菌、霉菌、线虫、酵母、果蝇、昆虫细胞、哺乳动物细胞及植物细胞中的表达,ＧＦＰ作为一种新型的报告物在生物学界...     

绿色荧光蛋白作为分子标记物在微生物学中的应用

荧光染料在微生物学中的应用受到广泛的关注。近年来 ,来源于发光性生物的荧光蛋白进一步丰富了微生物学的研究手段。其中绿色荧光蛋白 (Greenfluorescentprotein ,GFP ,来源于水母 )具有独特的应用价值。在活体研究中 ,GFP相对于其它报告蛋白 (如 β 半乳糖苷酶 )在原位、实时的微生物生理生化研究中有很多优越性。对GFP作为分子标记物在微生物学中的应用进行回顾 ,对GFP在微生物与宿主相互作用、生物膜(biofilm)、生物降解、细菌与原生动物相互作用、基因转导、基因表达、蛋白质定位以及生物传感器等领域的应用进行讨论 ,并扼要介绍了一些应用于荧光观察和定量分析的方法。     

Use of Green Fluorescent Protein in mouse embryos

Green Fluorescent Protein (GFP) has rapidly been established as a versatile and powerful cell marker in many organisms. Initial problems in using it in mammalian cells were solved by introducing mutations to increase its solubility at higher temperatures, such that GFP has now been used as a reporter in both gene expression and cell lineage studies, and to localize proteins within mammalian cells. GFP has two unique advantages: (i) the protein becomes fluorescent in an autocatalytic reaction, so that it can be introduced into any cell type simply as a cDNA or mRNA, or as protein; (ii) it is "bright" enough to be visualized in living cells under conditions that do not cause photodamage to the cells. In this article we outline the ways in which we have used GFP mRNA and cDNA in our studies of mouse cell lineages, and to characterize the behavior of proteins within the embryos.     

绿色荧光蛋白在转基因研究中的应用

绿色荧光蛋白(green fluorescent prote in,GFP)是一种能够自身催化形成生色团并在蓝光或紫外光激发下发出绿色荧光的蛋白。有现代生物学北斗星之美誉的它,在生物学的很多领域都有广泛应用。GFP具有荧光稳定、易于检测、表达调控简单、生物安全性好等优点,在转基因研究中的各个方面均应用颇多。就GFP在转基因研究中的应用特点及应用进展做一综述。     

A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea

A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that ∼15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37°C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.     

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis¹. The primary target cells of MVV in vivo are considered to be of the monocyte lineage². Certain strains of MVV can replicate in other cell types, however^3,4. The green fluorescent protein is a commonly used marker for studying lentiviruses in living cells. We have inserted the egfp gene into the gene for dUTPase of MVV. The dUTPase gene is well conserved in most lentivirus strains of sheep and goats and has been shown to be important in replication of CAEV⁵. However, dUTPase has been shown to be dispensable for replication of the molecular clone of MVV used in this study both in vitro and in vivo⁶. MVV replication is strictly confined to cells of sheep or goat origin. We use a primary cell line from the choroid plexus of sheep (SCP cells) for transfection and propagation of the virus⁷. The fluorescent MVV is fully infectious and EGFP expression is stable over at least 6 passages⁸. There is good correlation between measurements of TCID₅₀ and EGFP. This virus should therefore be useful for rapid detection of infected cells in studies of cell tropism and pathogenicity in vitro and in vivo⁸.Download video file.^{(54M, mov)}     

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。     

Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix

Abstract. It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100× objective (0.8-μm spot diam) gave half-times for fluorescence recovery of 15–19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2–3 × 10⁻⁷ cm²/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid β-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 ± 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, ~0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse.     

Bimolecular Fluorescence Complementation Analysis of Inducible Protein Interactions: Effects of Factors Affecting Protein Folding on Fluorescent Protein Fragment Association

Bimolecular fluorescence complementation (BiFC) analysis enables visualization of the subcellular locations of protein interactions in living cells. Using fragments of different fluorescent proteins, we investigated the temporal resolution and the quantitative accuracy of BiFC analysis. We determined the kinetics of BiFC complex formation in response to the rapamycin-inducible interaction between the FK506 binding protein (FKBP) and the FKBP-rapamycin binding domain (FRB). Fragments of yellow fluorescent protein fused to FKBP and FRB produced detectable BiFC complex fluorescence 10 min after the addition of rapamycin and a 10-fold increase in the mean fluorescence intensity in 8 h. The N-terminal fragment of the Venus fluorescent protein fused to FKBP produced constitutive BiFC complexes with several C-terminal fragments fused to FRB. A chimeric N-terminal fragment containing residues from Venus and yellow fluorescent protein produced either constitutive or inducible BiFC complexes depending on the temperature at which the cells were cultured. The concentrations of inducers required for half-maximal induction of BiFC complex formation by all fluorescent protein fragments tested were consistent with the affinities of the inducers for unmodified FKBP and FRB. Treatment with the FK506 inhibitor of FKBP-FRB interaction prevented the formation of BiFC complexes by FKBP and FRB fusions, but did not disrupt existing BiFC complexes. Proteins synthesized before the addition of rapamycin formed BiFC complexes with the same efficiency as did newly synthesized proteins. Inhibitors of protein synthesis attenuated BiFC complex formation independent of their effects on fusion protein synthesis. The kinetics at which they inhibited BiFC complex formation suggests that they prevented association of the fluorescent protein fragments, but not the slow maturation of BiFC complex fluorescence. Agents that induce the unfolded protein response also reduced formation of BiFC complexes. The effects of these agents were suppressed by cellular adaptation to protein folding stress. In summary, BiFC analysis enables detection of protein interactions within minutes after complex formation in living cells, but does not allow detection of complex dissociation. Conditional BiFC complex formation depends on the folding efficiencies of fluorescent protein fragments and can be affected by the cellular protein folding environment.     

Analysis of Nuclear Localization Signals Using a Green Fluorescent Protein-Fusion Protein Library

We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical NLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture.     

绿色荧光蛋白gfp基因在钝顶螺旋藻(Spirulina platensis)A9藻株中的表达

将钝顶螺旋藻(Spirulina platensis)A9藻株在24℃培养,经2 mmol/L的EDTA预处理24 h;采用功率300 W的超声波处理70 s获得单细胞样品,以本实验室构建的携带gfp基因的质粒p215t转化A9藻株单细胞藻液,利用Amp作为选择标记,使单细胞在平板上再生长出单藻落,获得17株具有Amp抗性的转化藻株,转化率3.73‰。在390 nm紫光激发下,生长30天的转化藻丝体发出稳定绿色荧光;培养45天后具有绿色荧光的藻丝出现断裂、具有荧光藻丝长度缩短的现象。实验结果初步表明:报告基因gfp在螺旋藻中得到稳定有效的表达,可以采用单细胞再生形成单藻落技术进行螺旋藻的基因克隆。     

绿色荧光蛋白在草鱼肾细胞中的表达

应用阳离子脂质体介导法,将含绿色荧光蛋白（GFP）基因的质粒pEGFP-N1转染到培养成单层的草鱼肾细胞（CIK）中,通过荧光倒置显微镜和特异性RT-PCR方法检测GFP的表达.在荧光倒置显微镜下可见CIK细胞的胞质和胞核均呈现绿色荧光,且细胞核的绿色荧光强度强于细胞质.转染细胞中的转录产物经RT-PCR扩增后,凝胶电泳鉴定出与GFP基因片段分子量大小一致的条带,经测序证明其为GFP基因序列.结果表明,GFP基因可以在草鱼CIK细胞内高效率成功表达,为构建以GFP为报告基因的真核重组质粒及研究草鱼出血病DNA疫苗奠定了重要的基础.     

以绿色荧光蛋白为报告基因的原核启动子检测体系构建

以质粒pMUTIN-GFP 扩增获得的目的gfp 基因为报告基因,将其克隆到大肠杆菌-枯草芽孢杆菌穿梭载体pBE2,构建成一个具有启动子活性检测功能的重组质粒pBE2-GFP .将组成型启动子P43和诱导型启动子Pspac克隆入pBE2-GFP ,得到重组表达载体pBE-GFP-P43和pBE-GFP-Pspac,转化至大肠杆菌和枯草芽孢杆菌.荧光显微镜检测GFP 蛋白的表达情况.结果 表明,2种不同类型的启动子均能在大肠杆菌BL21和枯草芽孢杆菌1A751中启动gfp 基因的表达.     

绿色荧光蛋白在植物细胞生物学中的应用

克隆于海洋动物水母 (Ａｅｑｕｏｒｅａｖｉｃｔｏｒｉ ａ)的绿色荧光蛋白 (ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ ,ＧＦＰ)作为一种新型的非酶性报告基因具有检测简便 ,结果真实可靠 ,不需要任何外源底物或辅助因子的特点 ,自出现以来它已引起人们的广泛兴趣 ,目前已经应用于烟草、柑橘、拟南芥、玉米、水稻、大豆、苜蓿等多种植物材料的研究中。ＧＦＰ含有特殊的六肽生色团结构 ,用蓝紫光激发即能发出肉眼清晰可见的绿色荧光 ,而无需任何底物或辅助因子。ＧＦＰ能与多种不同蛋白质的Ｎ端或Ｃ端融合而保持与天然蛋白质相似的荧…     

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 hirsutumL.),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检测方法有明显的优越性。本研究不但为花粉管通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。 Abstract：With the Green Fluorescent Protein gene (GFP) as a reporter gene, the transgenic embryos, seedlings and calli of cotton(Gossypium hirsutum L.) were obtained by the method of pollen tube pathway and Agrobacterium-mediated techniques separately. The GFP gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright?green fluorescence easily detectable and screenable in cotton tissue by fluorescence microscopy and a hand-held ultraviolet lamp. The screenable marker aided and facilated the rapid segregation of individual transformation events, drastically reduced the quantity of tissue to be handled. The GFP can be screened in vivo without destroying the materials, so it is more practical and useful than GUS. The use of GFP could advance the development of cotton gene engineering.