Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

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 ) .     

Non-invasive quantitative detection and applications of non-toxic, S65T-type green fluorescent protein in living plants

Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.     

Measurement of green fluorescent protein concentration in single cells by image analysis

The gene encoding the green fluorescent protein (GFP) has been widely used in studies of gene expression. The GFP can be detected nondestructively in living cells or tissues by the green fluorescence of the protein under blue light. Solutions of enhanced GFP (EGFP) of known concentration were filled in glass capillaries and used to calibrate a method for quantitative determination of EGFP or GFP-S65T in plant cells. Images captured by a digital camera were analyzed to determine the linear range for measurement of EGFP expression. The value of the method was illustrated by analysis of the relative levels of GFP expression under control of different promoters in aleurone cells of barley.     

Red fluorescent protein DsRed from Discosoma sp. as a reporter protein in higher plants

GFP from Aequorea victoria is a standard genetic marker widely used to visualize cellular events in a noninvasive manner. For simultaneous imaging of different processes, in vivo mutants of GFP with shifted wavelength spectra (e.g., blue fluorescent protein) are conventionally used. The recently reported red fluorescent protein from Discosoma sp., DsRed, represents a new marker that can be used together with GFP variants for multicolor imaging. DsRed is an interesting marker protein for use in plants because of its red-shifted wavelength spectrum that will avoid damaging cells and tissues by excitation light. In this report, we show that DsRed is an excellent marker in higher plants in spite of the interfering red autofluorescence of chlorophyll, which can be eliminated by using the appropriate filter sets. Transient expression of DsRed1-C1 and a soluble-modified, red-shifted GFP variant has been carried out both individually and jointly in the epidermal cells of three different Nicotiana species and Chenopodium quinoa, which gives rise to dual labeling in plants. For this purpose, a human codon-optimized variant of DsRed has been adopted for expression in plants. Moreover, the DsRed reporter gene was expressed by using a labeled plant viral vector derived from an infectious full-length clone of potato virus X.     

基于GFP观察的花粉介导植物转基因方法的细胞学研究

花粉介导的植物转基因方法不需要组织培养过程,且操作简便易行。为明确该方法的细胞学基础,以离体下米（Zeamays）郑单958花粉为材料,在超声波作用下将含有绿色荧光蛋白（GFP）基因的质粒与花粉共处理,对处理过的仡粉进行体外培养及人工授粉,并利用荧光显微镜对GFP基因在玉米花粉粒、花粉管及胚中的表达进行示踪观察。结果表明：处珲绀和对照组均有部分花粉粒呈强烈的绿色荧光,因此通过观察花粉粒荧光来确定GFP基因是否表达不可靠;处理组化粉管较对照旱现强烈的绿色荧光：GFP基因在玉米胚中表达可以作为鉴定转化体的证据。该实验首次利用荧光观察为花粉介导植物转基因方法提供了可视的细胞学证据。     

Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants

Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.     

绿荧光蛋白及其在转基因动物研究中的应用

绿荧光蛋白（ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ,ＧＦＰ）是源于水母（Ｊｅｌｙｆｉｓｈ）、海笔（ＳｅａＰｅｎ,ＳｅａＰａｎｓｙ）等海洋无脊椎动物的一种蛋白质,这种蛋白质在体外经适当波长的光激发便可发出绿光,所发出的绿光用普通荧光显微镜或荧光激活细胞分拣器（ＦＡＣＳ）均可检测到。ＧＦＰ作为动、植物以及微生物基因工程研究上的一种选择标记具有检测灵敏度高,操作简便,对机体毒副作用小且不需要添加任何底物或辅助因子等优点,更重要的是检测ＧＦＰ无损于细胞或胚胎的完整性及活力。本文概括介绍ＧＦＰ的生化、发光光谱及遗传学特征及其在转基因动物研究上的应用。     

Limited utility of blue fluorescent protein (BFP) in monitoring plant virus movement

While the green fluorescent protein (GFP) is a routinely used marker gene in higher plants, there are only a few data concerning the use of blue fluorescent protein (BFP). These proteins together are used for dual colour tagging experiments in various biological systems; however, the benefits of this technique in plant virology have not been exploited yet. In this work, our aim was to determine whether the BFP is a suitable second marker in conjunction with GFP for following the progress of virus infection. Nicotiana clevelandii, N. benthamiana and N. tabacum cv. Xanthi-nc plants were infected with potato virus X vector carrying the GFP or the Y66H type BFP gene. While GFP was brightly fluorescent in all species, the fluorescence intensity of BFP varied widely, from the bright fluorescence observed in N. clevelandii to the absence of fluorescence in N. tabacum cv. Xanthi-nc. Since at even mild acidic pH BFP rapidly fades, the more acidic cytosol of N. tabacum could be responsible for impaired in vivo fluorescence. After infiltration of the infected leaves of N. clevelandii with pH 5 phosphate buffer, the fluorescence faded thus confirming this situation.     

In Planta visual monitoring of green fluorescent protein in transgenic rice plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used reporter that can be directly visualized in the living cells in both animals and plants. We inserted a synthetic gene (sgfp) encoding a modified form of the GFP into expression vector, Act1-sgfp for the direct expression of GFP which is easily detectable in rice plants. Green fluorescence emitted from GFP could be visualized in calli, dry seeds, roots and seedlings with green shoots of transgenic rice plants. In our visualization system with a charge-coupled device camera, band-pass filters and a light source, the presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of GFP. These results demonstrate that GFP could be used as a non-destructive visual selection marker for examining gene expression in transformed calli, dry seeds and young plants.     

Generation of stable cell line by using chitosan as gene delivery system

Establishing stable cell lines are useful tools to study the function of various genes and silence or induce the expression of a gene of interest. Nonviral gene transfer is generally preferred to generate stable cell lines in the manufacturing of recombinant proteins. In this study, we aimed to establish stable recombinant HEK-293 cell lines by transfection of chitosan complexes preparing with pDNA which contain LacZ and GFP genes. Chitosan which is a cationic polymer was used as gene delivery system. Stable HEK-293 cell lines were established by transfection of cells with complexes which were prepared with chitosan and pVitro-2 plasmid vector that contains neomycin drug resistance gene, beta gal and GFP genes. The transfection efficiency was shown with GFP expression in the cells using fluorescence microscopy. Beta gal protein expression in stable cells was examined by beta-galactosidase assay as enzymatically and X-gal staining method as histochemically. Full complexation was shown in the above of 1/1 ratio in the chitosan/pDNA complexes. The highest beta-galactosidase activity was obtained with transfection of chitosan complexes. Beta gal gene expression was 15.17 ng/ml in the stable cells generated by chitosan complexes. In addition, intensive blue color was observed depending on beta gal protein expression in the stable cell line with X-gal staining. We established a stable HEK-293 cell line that can be used for recombinant protein production or gene expression studies by transfecting the gene of interest.     

Use of green fluorescent protein as A non-destructive marker for peanut genetic transformation

Summary The ability to non-destructively visualize transient and stable gene expression has made green fluorescent protein (GFP) a most efficient reporter gene for routine plant transformation studies. We have assessed two fluorescent protein mutants, enhanced GFP (EGFP) and enhanced yellow fluorescent protein (EYFP), under the control of the CaMV35S promoter, for their transient expression efficiencies after particle bombardment of embryogenic cultures of the peanut cultivar, Georgia Green. A third construct (p524EGFP.1) that expressed EGFP from a double 35S promoter with an AMV enhancer sequence also was compared. The brightest and most dense fluorescent signals observed during transient expression were from p524EGFP. 1 and EYFP. Optimized bombardment conditions consisted of 0.6 μm diameter gold particles, 12410 kPa bombardment pressure, 95 kPa vacuum pressure, and pretreatment with 0.4 M mannitol. Bombardments with p524EGFP.1 produced tissue sectors expressing GFP that could be visually selected under the fluorescence microscope over multiple subcultures. Embryogenic lines selected for GFP expression initially may have been chimeric since quantitative analysis of expression sometimes showed an increase when GFP-expressing lines, that also contained a hygromycin-resistance gene, subsequently were cultured on hygromycin. Transformed peanut plants expressing GFP were obtained from lines selected either visually or on hygromycin. Integration of the gfp gene in the genomic DNA of regenerated plants was confirmed by Southern blot hybridization and transmission to progeny.     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants

The expression of green fluorescent protein (GFP) and its inheritance were studied in transgenic barley (Hordeum vulgare L.) plants transformed with a synthetic green fluorescent protein gene [sgfp(S65T)] driven by either a rice actin promoter or a barley endosperm-specific d-hordein promoter. The gene encoding phosphinothricin acetyltransferase (bar), driven by the maize ubiquitin promoter and intron, was used as a selectable marker to identify transgenic tissues. Strong GFP expression driven by the rice actin promoter was observed in callus cells and in a variety of tissues of T0 plants transformed with the sgfp(S65T)-containing construct. GFP expression, driven by the rice actin promoter, was observed in 14 out of 17 independent regenerable transgenic callus lines; however, expression was gradually lost in T0 and later generation progeny of diploid lines. Stable GFP expression was observed in T2 progeny from only 6 out of the 14 (43%) independent GFP-expressing callus lines. Four of the 8 lines not expressing GFP in T2 progeny, lost GFP expression during T0 plant regeneration from calli; one lost GFP expression in the transition from the T0 to T1 generations and three lines were sterile. Similarly, expression of bar driven by the maize ubiquitin promoter was lost in T1 progeny; only 21 out of 26 (81%) independent lines were Basta-resistant. In contrast to actin-driven expression, GFP expression driven by the d-hordein promoter exhibited endosperm-specificity. All seven lines transformed with d-hordein-driven GFP (100%) expressed GFP in the T1 and T2 generations, regardless of ploidy levels, and expression segregated in a Mendelian fashion. We conclude that the sgfp(S65T) gene was successfully transformed into barley and that GFP expression driven by the d-hordein promoter was more stable in its inheritance pattern in T1 and T2 progeny than that driven by the rice actin promoter or the bar gene driven by the maize ubiquitin promoter.     

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。     

Relaxed chromatin induced by histone deacetylase inhibitors improves the oligonucleotide-directed gene editing in plant cells

Improving efficiency of oligonucleotide-directed mutagenesis (ODM) is a prerequisite for wide application of this gene-editing approach in plant science and breeding. Here we have tested histone deacetylase inhibitor treatments for induction of relaxed chromatin and for increasing the efficiency of ODM in cultured maize cells. For phenotypic assay we produced transgenic maize cell lines expressing the non-functional Green Fluorescent Protein (mGFP) gene carrying a TAG stop codon. These transgenic cells were bombarded with corrective oligonucleotide as editing reagent to recover GFP expression. Repair of green fluorescent protein function was monitored by confocal fluorescence microscopy and flow cytometry was used for quantification of correction events. Sequencing PCR fragments of the GFP gene from corrected cells indicated a nucleotide exchange in the stop codon (TAG) from T to G nucleotide that resulted in the restoration of GFP function. We show that pretreatment of maize cells with sodium butyrate (5–10 mM) and nicotinamide (1–5 mM) as known inhibitors of histone deacetylases can cause elevated chromatin sensitivity to DNase I that was visualized in agarose gels and confirmed by the reduced presence of intact PCR template for the inserted exogenous mGFP gene. Maize cells with more relaxed chromatin could serve as an improved recipient for targeted nucleotide exchange as indicated by an average of 2.67- to 3.62-fold increase in GFP-positive cells. Our results stimulate further studies on the role of the condition of the recipient cells in ODM and testing the application of chromatin modifying agents in other, programmable nuclease-based genome-editing techniques in higher plants.     

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.     

Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity

Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).     

Vacuolar membrane localization of the Arabidopsis 'two-pore' K+ channel KCO1

Potassium (K+) channels play multiple roles in higher plants, and have been characterized electrophysiologically in various subcellular membranes. The K+ channel AtKCO1 from Arabidopsis thaliana is the prototype of a new family of plant K+ channels. In a previous study the protein has been functionally characterized after heterologous expression in Baculovirus-infected insect cells. In order to obtain further information on the physiological function of AtKCO1, the gene expression pattern and subcellular localization of the protein in plants were investigated. The regulatory function of the 5' region of the AtKCO1 gene was examined in transgenic A. thaliana plants carrying beta-glucuronidase (GUS) fusion constructs. Our analysis demonstrates that the AtKCO1 promoter is active in various tissues and cell types, and the highest GUS activity could be detected in mitotically active tissues of the plant. Promoter activity was strongly dependent on the presence of a 5' leader intron. The same overall structure was identified in two genes encoding AtKCO1-like K+ channels from Solanum tuberosum (StKCO1alpha and StKCO1beta). To investigate the subcellular localization of AtKCO1, the channel protein, as well as a fusion protein of AtKCO1 with green fluorescence protein (GFP), were expressed in transgenic tobacco BY2 cells. In sucrose density gradients, both proteins co-fractionate with tonoplast markers (Nt-TIPa, vATPase). In fluorescence images from transgenic AtKCO1-GFP BY2 cells fluorescence was exclusively detected in the tonoplast. Thus AtKCO1 is the first cloned K+ channel demonstrated to be a vacuolar K+ channel.