Cloning vectors for the expression of green fluorescent protein fusion proteins in transgenic plants

A series of versatile cloning vectors has been constructed that facilitate the expression of protein fusions to the Aequorea victoria green fluorescent protein (GFP) in plant cells. Amino-terminal- and carboxy-terminal protein fusions have been created and visualized by epifluorescence microscopy, both in transgenic Arabidopsis thaliana and after transient expression in onion epidermal cells. Using tandem dimers and other protein fusions to GFP, we found that the previously described localization of wild-type GFP to the cell nucleus is most likely due to diffusion of GFP across the nuclear envelope rather than to a cryptic nuclear localization signal. A fluorescence-based, quantitative assay for nuclear localization signals is described. In addition, we have employed the previously characterized mutants GFP–S65T and GFP–Y66H in order to allow for the expression of red-shifted and blue fluorescent proteins, respectively, which are suitable for double-labeling studies. Expression of GFP-fusions was controlled by a cauliflower mosaic virus 35S promoter. Using the Arabidopsis COP1 protein as a model, we confirmed a close similarity in the subcellular localization of native COP1 and the GFP-tagged COP1 protein. We demonstrated that COP1 was localized to discrete subnuclear particles and further confirmed that fusion to GFP did not compromise the activity of the wild-type COP1 protein.     

Improved binary vectors for Agrobacterium-mediated plant transformation

Improved plant transformation vectors were constructed which utilize the pRiHRI origin of replication for highly stable maintenance in Agrobacterium tumefaciens, the ColE1 origin of replication for high copy maintenance in Escherichia coli, and a gentamycin resistance gene as a strong selectable marker for bacteria. Concise T-DNA elements were engineered with border sequences from the TL-DNA of pTiA6, the Tn5 neomycin phosphotransferase gene (npt II) expressed from either CaMV 35S or mannopine synthase (mas) promoters, and the lac Z gene segment from pUC18 as a source of unique restriction sites as well as an insertional inactivation marker for cloned DNA. The order of T-DNA components in all vectors is left border, plant marker cassette, lac Z, and right border, respectively. The prototype vector, pCGN1547, was shown to be very stable in A. tumefaciens strain LBA4404 and to act as an efficient donor of T-DNA in tomato transformation experiments. Use of the other vectors is also described.     

Differential gene expression in nematode-induced feeding structures of transgenic plants harbouring promoter—gusA fusion constructs

Sedentary plant-parasitic nematodes are able to induce specialized feeding structures in the root system of their host plants by triggering a series of dramatic cellular responses. These changes presumably are accompanied by a reprogramming of gene expression. To monitor such changes, a variety of promoter— gus A fusion constructs were introduced into Arabidopsis and tobacco. Transgenic plants were analysed histochemically for GUS activity in the nematode feeding structures after infection with either Heterodera schachtii or Meloidogyne incognita . Promoters of the Cauliflower Mosaic Virus 35S gene, the bacterial nopaline synthase, rooting loci ( rol ) and T- cyt genes and the plant-derived phenylalanine ammonia-lyase I gene, which are highly active in non-infected roots, were all downregulated in the feeding structures as indicated by the strong decrease of GUS activity inside these structures. Less stringent down-regulation was observed with chimeric gus A fusion constructs harbouring truncated rol B and rol C promoter sequences. Similar observations were made with transgenic Arabidopsis lines that carried randomly integrated promoterless gus A constructs to identify regulatory sequences in the plant genome. Most of the lines that were selected for expression in the root vascular cylinder demonstrated local down-regulation in feeding structures after infection with H. schachtii . The reverse pattern of GUS activity, a blue feeding structure amidst unstained root cells, was also found in several lines. However, GUS activity that was entirely specific for the feeding structures was not observed. Our data show that the expression of a large number of genes is influenced during the development of the nematode feeding structures.     

A built-in strategy for containment of transgenic plants: creation of selectively terminable transgenic rice

Plant transgenic technology has been widely utilized for engineering crops for trait improvements and for production of high value proteins such as pharmaceuticals. However, the unintended spreading of commercial transgenic crops by pollination and seed dispersal is a major concern for environmental and food safety. Simple and reliable containment strategies for transgenes are highly desirable. Here we report a novel method for creating selectively terminable transgenic rice. In this method, the gene(s) of interest is tagged with a RNA interference cassette, which specifically suppresses the expression of the bentazon detoxification enzyme CYP81A6 and thus renders transgenic rice to be sensitive to bentazon, a herbicide used for rice weed control. We generated transgenic rice plants by this method using a new glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene from Pesudomonas putida as the gene of interest, and demonstrated that these transgenic rice plants were highly sensitive to bentazon but tolerant to glyphosate, which is exactly the opposite of conventional rice. Field trial of these transgenic rice plants further confirmed that they can be selectively killed at 100% by one spray of bentazon at a regular dose used for conventional rice weed control. Furthermore, we found that the terminable transgenic rice created in this study shows no difference in growth, development and yield compared to its non-transgenic control. Therefore, this method of creating transgenic rice constitutes a novel strategy of transgene containment, which appears simple, reliable and inexpensive for implementation.     

Experimental models for creation of transgenic plants resistant to stressors

Experimental models of primary potato transgenic plants that express the cry3aM-licBM2 hybrid gene were created. The molecular analysis and biotests of the experimental models allow a new system of cry genes expression in plants to be proposed. This system is based on the expression of hybrid genes containing the reporter lichenase gene sequence and the use of a light-induced promoter ensuring preferential expression of the regulated genes only in green plant tissues (leaves), the target tissues for pests, as a regulatory element. In is shown that the presence of lichenase in hybrid proteins facilitates selection and analysis of the level of expression of hybrid proteins in transgenic plants. Judging by the properties of the reporter protein lichenase in hybrid proteins, it seems possible to use this reporter system for transgene monitoring in agrocenosis, because this system is fairly simple and precise and does not need considerable material and time expenses.     

Construction of small binary vectors forAgrobacterium-Mediated transformation in plants

Despite the popular use of theAgrobacterium-mediated method for transforming plants, only a very limited number of binary vectors are available. In addition, those binary vectors are rather inconvenient for plasmid manipulation because of their large size. Here, we report the construction of small binary vectors, pBmin1 and pBminGFP1, forAgrobacterium-mediated transformation. The sizes of pBmin1 and pBminGFP1 are approximately 5.0 kb and 6.5 kb, respectively. These vectors contain one kanamycin resistance gene, with a double promoter for expression in both bacteria and plant cells. Vector pBminGFP1 has the 35S-GFP chimeric gene as a reporter. These small binary vectors would greatly facilitate plasmid manipulation and plant transformation.     

Experimental models for creation of transgenic plants resistant to stress factors

Experimental models of the potato primary transgenic plants which express the hybrid gene cry3aM-licBM2 have been created. Modecular analysis and the biotests of the experimental models allow proposing a new system of cry genes expression in plants. The system is based on the expression of hybrid genes possessing the sequence of reporter lichenase gene and the use as a regulator element of a light-induced promoter providing preferential expression of the controled genes only in green plant tissues (leaves)--the target tissues for pests. The lichanase presence in hybrid proteins facilitates selection and analysis of the expression level of the hybris proteins in transgenic organisms. Basing on the lichenase properties in hybrid proteins it seems possible to use this reporter system for transgene monitoring in agrocoenosis as this system is rather simple and precise and does not need large material and time expenses.     

Gateway RFP-fusion vectors for high throughput functional analysis of genes

There is an increasing demand for high throughput (HTP) methods for gene analysis on a genome-wide scale. However, the current repertoire of HTP detection methodologies allows only a limited range of cellular phenotypes to be studied. We have constructed two HTP-optimized expression vectors generated from the red fluorescent reporter protein (RFP) gene. These vectors produce RFP-tagged target proteins in a multiple expression system using gateway cloning technology (GCT). The RFP tag was fused with the cloned genes, thereby allowing us localize the expressed proteins in mammalian cells. The effectiveness of the vectors was evaluated using an HTP-screening system. Sixty representative human C2 domains were tagged with RFP and overexpressed in HiB5 neuronal progenitor cells, and we studied in detail two C2 domains that promoted the neuronal differentiation of HiB5 cells. Our results show that the two vectors developed in this study are useful for functional gene analysis using an HTP-screening system on a genome-wide scale.     

Characterization and molecular analysis of transgenic plants obtained by microprotoplast fusion in sunflower

Asymmetric somatic hybrid (ASH) plants were obtained by PEG-mediated mass fusion of microprotoplasts from perennial Helianthus species and hypocotyl protoplasts of Helianthus annuus. The formation of micronuclei in perennial sunflower cell cultures was induced, at early log phase, by addition of the herbicides amiprophos-methyl or oryzalin. Sub-diploid microprotoplasts were isolated by high-speed centrifugation and the smallest enriched by sequential filtration through nylon sieves of decreasing pore size. Fusion products were cultured and the regenerated plants phenotypically, genetically and cytologically characterized. DNA analysis using RAPD markers revealed that 28 out of 53 regenerated plants were asymmetric hybrids. Subsequent nuclear-DNA flow cytometric analysis showed that these plants had a higher DNA content than the receptor H. annuus, suggesting that they represented addition lines. Cytological investigation of the metaphase cells of 16 hybrids revealed an addition of 2–8 extra chromosomes in these plants. The phenotype of most ASH plants resembled H. annuus. These results indicate that micronuclear induction and asymmetric somatic hybridization represent a potent tool for partial genome transfer aimed at the specific transfer of economically important traits in breeding programs. Received: 21 December 1999 / Accepted: 25 March 2000<@head-com-p1a.lf>Communicated by K. Glimelius     

The creation of sugar beet transgenic plants expressing bar gene

The parameters of transformation using Agrobacterium tumefaciens EHA 105 for 5 domestic sorts and lines of sugar beet (Beta vulgaris L. var. saccharifera (Alef) Krass) were optimized. The system of transgenic tissue selection based on resistance to phosphinothricin, allowing to avoid the appearing of chimeric shoots among initial transformants was developed. The transgenic plants of sugar beet sorts Ramonskaya single seed 47, L’govskaya single seed 52 and RMS 73, and LBO 17 and LBO 19 lines expressing the gene of phosphinothricin acetyl transferase bar have been obtained. The resistance of these sorts and lines to the effect of phosphinothricin in vitro has been shown.     

A set of novel Ti plasmid-derived vectors for the production of transgenic plants

Summary We describe in this paper the construction and use of a set of novel Ti plasmid-derived vectors that can be used to produce transgenic plants. These vectors are based on one of two strategies: 1) double recombination into the wild-type Ti plasmid of genetic information flanked by two T-DNA fragments on a wide-host range plasmid; 2) the binary vector strategy. The vector based on the double recombination principle contains a kanamycin resistance gene for use as a plant selectable marker, a polylinker for the insertion of foreign genes, and a nopaline synthase gene. The vector was constructed such that a disarmed T-DNA results from the double recombination event. The binary vector combines several advantageous features including an origin of replication that is stable in Agrobacterium in the absence of selection, six unique sites for insertion of foreign genes, an intact nopaline synthase gene, and a kanamycin resistance marker for selection of transformed plant cells. All of these vectors have been used to produce tobacco plants transformed with a variety of foreign genes.     

可剪切多拷贝抗菌肽融合表达载体的构建

抗菌肽是生物体防御系统产生的一类对外源病菌具有高效杀灭活性的小分子多肽, 在植物抗病基因工程中具有重要的应用价值。Thanatin是刺肩蝽(Podisus maculiventris)成虫经诱导产生的一种抗菌肽, 由21个氨基酸残基组成, 该抗菌肽对革兰阳性、革兰阴性菌以及真菌都有很强的抗菌活性。为研究该抗菌肽转入油菜对菌核病抗性提高的效果, 采用同尾酶反复酶切连接的方法构建了分别含1~5拷贝的Thanatin串联融合表达载体, 并导入农杆菌用于油菜的遗传转化。研究采用引物重叠法扩增并克隆了抗菌肽基因, 并采用了一种在植物体内可被特异性切割的短肽作为连接肽, 使多拷贝融合表达的抗菌肽在植物体内可自动剪切为有功能活性的单个抗菌肽单元, 以增加抗菌肽表达丰度和抗菌肽的稳定性。研究还采用了大豆几丁质酶的信号肽作为引导肽引导多拷贝融合表达的抗菌肽分泌到细胞间隙, 以增强抗菌肽作用效果。     

Quantitative analysis of dynamic protein-protein interactions in planta by a floated-leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.     

Construction of two Gateway vectors for gene expression in fungi

We report the construction of two Gateway fungal expression vectors pCBGW and pGWBF. The pCBGW was generated by introducing an expression cassette, which consists of a Gateway recombinant cassette (attR1-Cmr-ccdB-attR2) under the control of fungal promoter PgpdA and a terminator TtrpC, into the multiple cloning site of fungal vector pCB1004. The pGWBF is a binary vector, which was generated from the plant expression vector pGWB2 by replacing the CaMV35S promoter with PgpdA. The pGWBF can be transformed into fungi efficiently with Agrobacterium-mediated transformation. The applicability of two newly constructed vectors was tested by generating the destination vectors pGWBF-GFP and pCBGW-GFP and examining the expression of GFP gene in Trichoderma viride and Gibberella fujikuroi, respectively. Combining with the advantage of Gateway cloning technology, pCBGW and pGWBF will be useful in fungi for large-scale investigation of gene functions by constructing the interested gene destination/expression vectors in a high-throughput way.     

Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli

Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.