Plant selection principle based on xylose isomerase

Summary The xylose isomerase genes (xylA) from Thermoanaerobacterium thermosulfurogenes and Streptomyces rubiginosus were introduced and expressed in three plant species (potato, tobacco and tomato) and transgenic plants were selected on xylose-containing medium. The xylose isomerase genes were transferred to explants of the target plant by Agrobacterium-mediated transformation. The xylose isomerase genes were expressed under the control of the enhanced cauliflower mosaic virus 35S promoter and the Ω′ translation enhancer sequence from tobacco mosaic virus. In potato and tomato, xylose isomerase selection was more efficient than the established kanamycin selection. The level of enzyme activity in the regenerated transgenic plants selected on xylose was 5–25-fold higher than the enzyme activity in control plants selected on kanamycin. The xylose isomerase system enables transgenic cells to utilize xylose as a carbohydrate source. In contrast to antibiotic or herbicide resistance-based system where transgenic cells survive on a selective medium but nontransgenic cells are killed, the xylose system is an example of a positive selection system where transgenic cells proliferate while non-transgenic cells are starved but still survive. The results show that a new selection method, is established. The xylose system is devoid of the disadvantages of antibiotic or herbicide selection, and depends on an enzyme which is already being widely utilized in specific food processes and that is generally recognized as safe for use in the starch industry.     

E.coli木糖异构酶基因的克隆及表达条件的优化

采用PCR技术以大肠杆菌JM109基因组DNA为模板扩增得到木糖异构酶基因xylA,连接到载体pET-22b( ),得到重组质粒pET-22b( )-xylA。将此重组质粒转化到大肠杆菌菌株BL21(DE3)中,重组菌株经IPTG诱导后,通过半胱氨酸-咔唑法测得木糖异构酶活力。每mL发酵液中重组菌株显示出酶活力约为0.84 U。SDS-PAGE电泳结果显示出明显的5×104(相对分子质量)特异性蛋白质条带。     

An efficient method for organic acetylation and use of dl-phosphinothricin as a negative selection agent in argE transgenic rice

We present an efficient method for the production of N-acetyl-l-phosphinothricin (N-AcPt) from commercial dl-phosphinothricin (DL-PPT) by organic acetylation for use as a negative selection agent (NSA) that induces cell death in argE transgenic rice. DL-PPT was efficiently converted into N-AcPt with tetrahydrofuran (THF) and acetic anhydride (Ac₂O). Chemical changes were confirmed using NMR and ATR-FTIR analyses. DL-PPT was toxic but N-AcPt did not show cytotoxic effects on leaf discs or seed germination of wild-type rice. Conversely, in argE–hpt transgenic rice, non-toxic N-AcPt showed the negative selection (NS) effect by inducing cell destruction in leaf discs and restricting seed germination. For inducing NS, ?0.1 mg ml⁻¹ and ?0.5 mg ml⁻¹ of N-AcPt were effective in leaf and seed assays, respectively. Further, the NS effect occurred faster in the leaf assay compared with the seed germination assay, again indicating the leaf assay was a more sensitive indicator of N-AcPt as an NSA to argE transgenic rice than the seed germination assay. This negative selection approach could be useful for the development of selectable marker free transgenic plants in the economically important monocot species and its commercialization for multiple gene transformation.     

Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry

A new method for the selection of transgenic plants has been developed. It is based upon selection of transgenic plant cells expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose. The xylose isomerase selection system was tested in potato and the transformation frequency was found to be approximately ten fold higher than with kanamycin selection. The level of enzyme activity in the transgenic plants selected on xylose was 5- to 25-fold higher than the enzyme activity in control plants. Potato transformants were stable over two generations in Southern blotting analysis. This novel selection system is more efficient than the traditionally used kanamycin-based selection systems. In addition, the xylose isomerase system is independent of antibiotic or herbicide resistance genes, but depends on an enzyme that is generally recognized as safe for use in the starch industry and which is already being widely utilized in specific food processes. Received: 13 August 1997 / Revision received: 26 November 1997 / Accepted: 15 December 1997     

Cloning,expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a V_max of 0.142 U/mg purified rXI, and a K_M for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol K_I values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low K_M of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca²⁺ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher V_max at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016     

Effective selection and regeneration of transgenic rice plants with mannose as selective agent

A new method for the selection of transgenic rice plants without the use of antibiotics or herbicides has been developed. The phosphomannose isomerase (PMI) gene from Escherichia coli has been cloned and consitutively expressed in japonica rice variety TP 309. The PMI gene was transferred to immature rice embryos by Agrobacterium-mediated transformation, which allowed the selection of transgenic plants with mannose as selective agent. The integration and expression of the transgene was confirmed by Southern and northern blot analysis and the activity of PMI indirectly proved with the chlorophenol red assay. The results of genetic analysis showed that the transgenes were segregated in a Mendelian fashion in the T₁ generation. The establishment of this selection system in rice provides an efficient way for producing transgenic plants without using antibiotics or herbicides with a transformation frequency of up to 41%.     

Molecular cloning and expression of the glucose/xylose isomerase gene from Streptomyces sp. NCIM 2730 in Escherichia coli

Abstract A partial genomic library of Streptomyces sp. NCIM 2730 was constructed in Escherichia coli using pUC8 vector and screened for the presence of the d-glucose/xylose isomerase (GXI) gene using an 18-mer mixed oligonucleotide probe complementary to a highly conserved six-amino acid sequence of GXI from actinomycetes. Eight clones which hybridized with the radiolabelled oligoprobe showed the ability to complement xylose isomerase-defective E. coli mutants. The restriction map of the insert from one (pMSG27) of the eight GXI-positive clones showing detectable GXI activity was constructed. GXI-deficient strains of E. coli were able to utilize xylose as the sole carbon source for their growth upon transformation with pMSG27. E. coli JM105 (pMSG27) and E. coli JC1553 (pMSG27) were inducible by IPTG suggesting that the expression of the cloned gene was under the control of the lacZ promoter. Western blot analysis revealed that the cloned gene is expressed as a fusion protein of M _r 110. This is the first report of expression of a catalytically active GXI from Streptomyces in Escherichia coli .     

Relationship between promoter strength and transformation frequencies using mannose selection for the production of transgenic sugar beet

The relationship between the expression level of the selectable marker gene and transformation frequency was investigated in transgenic sugar beets with five different promoters, a modified cauliflower mosaic virus 35S RNA promoter (E35S), an enhanced nopaline synthase promoter (ENOS), a modified mannopine synthase promoter (SMAS), a heat shock protein promoter (HSP80) and a chlorophyll a/b-binding protein promoter (CAB3), to drive the expression of the selectable marker gene. The selection system employed was based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable marker gene and mannose as selective agent. The selected transgenic shoots were analysed for PMI activity and the average activity for each promoter was found to be 5.9 (HSP80), 31 (SMAS), 38 (E35S), 49 (ENOS) and 61 (CAB3) mU/mg. The weakest promoter, HSP80, resulted in the lowest transformation frequency (0.30%), suggesting that this promoter was too weak to confer sufficient resistance to mannose. On the other hand, the strongest promoters, ENOS and CAB3, only gave intermediate transformation frequencies, 0.44% and 0.47% respectively, while the somewhat weaker SMAS promoter produced the highest transformation frequency, 0.89%. Thus, these data suggest that the activity of the selectable PMI gene should be above a certain threshold level; however, above this level, no simple correlation between the PMI activities, calculated as averages, and transformation frequencies could be deduced. However, extended data analysis by dividing the transgenic shoots into 4 groups according to their PMI activities (low(<10 mU/mg), medium (10–50 mU/mg), high (50–100 mU/mg) and very high (>100 mU/mg) expressers) revealed a significant positive correlation between the relative number of shoots having medium levels of expression and transformation frequency. This indicated that promoters that predominanthly give rise to intermediate expression levels of the selectable PMI gene result in high transformation frequencies.     

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large‐scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods.     

Effective selection of transgenic papaya plants with the PMI/Man selection system

The selectable marker gene phospho-mannose isomerase (pmi), which encodes the enzyme phospho-mannose isomerase (PMI) to enable selection of transformed cell lines on media containing mannose (Man), was evaluated for genetic transformation of papaya (Carica papaya L.). We found that papaya embryogenic calli have little or no PMI activity and cannot utilize Man as a carbon source; however, when calli were transformed with a pmi gene, the PMI activity was greatly increased and they could utilize Man as efficiently as sucrose. Plants regenerated from selected callus lines also exhibited PMI activity but at a lower specific activity level. Our transformation efficiency with Man selection was higher than that reported using antibiotic selection or with a visual marker. For papaya, the PMI/Man selection system for producing transgenic plants is a highly efficient addition to previously published methods for selection and may facilitate the stacking of multiple transgenes of interest. Additionally, since the PMI/Man selection system does not involve antibiotic or herbicide resistance genes, its use might reduce environmental concerns about the potential flow of those genes into related plant populations.     

Analysis of the isopentenyl diphosphate isomerase gene family from Arabidopsis thaliana

Two Arabidopsis thaliana cDNAs (IPP1 and IPP2) encoding isopentenyl diphosphate isomerase (IPP isomerase) were isolated by complementation of an IPP isomerase mutant strain of Saccharomyces cerevisiae. Both cDNAs encode enzymes with an amino terminus that may function as a transit peptide for localization in plastids. At least 31 amino acids from the amino terminus of the IPP1 protein and 56 amino acids from the amino terminus of the IPP2 protein are not essential for enzymatic activity. Genomic DNA blot analysis confirmed that IPP1 and IPP2 are derived from a small gene family in A. thaliana. Based on northern analysis expression of both cDNAs occurs predominantly in roots of mature A. thaliana plants grown to the pre-flowering stage.     

The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Efficient selection of transgenic mouse embryos using EGFP as a marker gene

We have established a reliable method that uses the EGFP (Enhanced Green Fluorescent Protein) gene as a marker for selecting transgenic embryos from preimplantation embryos. Embryos that were subjected to the pronuclear microinjection of the CMV/β‐actin/EGFP fusion gene were cultured in vitro until they developed into the morulae‐ or blastocyst‐stage. The expression of EGFP was easily observed by a fluorescent microscopy. There appeared to be no damage to the in vivo developmental ability of the embryos in response to the EGFP excitation light, which utilized an IB filter for a period of 30 min. Modified PCR analysis using Dpn I and Bal 31 digestion of the embryonic DNA showed that all of the embryos expressing EGFP in all their cells were transgenic, while more than half with mosaic expression of EGFP were not transgenic. Approximately 77% of pups born from the embryos that uniformly expressed the EGFP gene were transgenic, while 21.4% of pups from the embryos with mosaic expression were transgenics. The results showed that the use of EGFP as a marker is very useful and reliable for selecting transgenic embryos, and that it is important to transfer the embryos expressing EGFP in all their cells to obtain truly transgenic animals. Mol. Reprod. Dev. 54:43–48, 1999. © 1999 Wiley‐Liss, Inc.     

A highly efficient sulfadiazine selection system for the generation of transgenic plants and algae

The genetic transformation of plant cells is critically dependent on the availability of efficient selectable marker gene. Sulfonamides are herbicides that, by inhibiting the folic acid biosynthetic pathway, suppress the growth of untransformed cells. Sulfonamide resistance genes that were previously developed as selectable markers for plant transformation were based on the assumption that, in plants, the folic acid biosynthetic pathway resides in the chloroplast compartment. Consequently, the Sul resistance protein, a herbicide‐insensitive dihydropteroate synthase, was targeted to the chloroplast. Although these vectors produce transgenic plants, the transformation efficiencies are low compared to other markers. Here, we show that this inefficiency is due to the erroneous assumption that the folic acid pathway is located in chloroplasts. When the RbcS transit peptide was replaced by a transit peptide for protein import into mitochondria, the compartment where folic acid biosynthesis takes place in yeast, much higher resistance to sulfonamide and much higher transformation efficiencies are obtained, suggesting that current sul vectors are likely to function due to low‐level mistargeting of the resistance protein to mitochondria. We constructed a series of optimized transformation vectors and demonstrate that they produce transgenic events at very high frequency in both the seed plant tobacco and the green alga Chlamydomonas reinhardtii. Co‐transformation experiments in tobacco revealed that sul is even superior to nptII, the currently most efficient selectable marker gene, and thus provides an attractive marker for the high‐throughput genetic transformation of plants and algae.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Molecular analysis of the acetolactate synthase gene of Chlamydomonas reinhardtii and development of a genetically engineered gene as a dominant selectable marker for genetic transformation

Genomic and cDNA clones of the acetolactate synthase (ALS) gene of Chlamydomonas reinhardtii have been isolated from a mutant, c85-20 (Hartnett et al., 1987), that is resistant to high concentrations of sulfometuron methyl (SMM) and related sulfonylurea herbicides. Comparison of the ALS gene sequences from the wild-type and the SMM resistant (SMMr) strains revealed two amino acid differences in the mature enzyme, a lysine to threonine change at position 257 (K257T) and a leucine to valine change at position 294 (L294V). Transformation of wild-type C. reinhardtii with the mutant ALS gene produced no transformants with ability to grow in the presence of a minimum toxic concentration of SMM (3 microm). Substitution of the ALS promoter with the promoter of the C. reinhardtii Rubisco small subunit gene (RbcS2) permitted recovery of SMMr colonies. In vitro mutagenesis of the wild-type ALS gene to produce various combinations of mutations (K257T, L294V and W580L) indicated that the K257T mutation was necessary and sufficient to confer the SMMr phenotype. Optimum transformation rates were obtained with two constructs (pJK7 and pRP-ALS) in which all introns in the coding region were present. Rates of transformation with construct pJK7 were approximately 2.5 x 10-4 transformants/cell (i.e. one transformant for each of 4000 initial cells) using electroporation and 8.5 x 10-6 transformants/cell using the glass bead vortexing method. These results suggest that pJK7 and pRP-ALS can serve as important additional dominant selectable markers for the genetic transformation of C. reinhardtii.     

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup^® herbicide. Glyphosate, the active ingredient in Roundup^® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup^®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.     

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.     

The use of the phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum)

In order to meet the future requirement of using non-antibiotic resistance genes for the production of transgenic plants, we have adapted the selectable marker system PMI/mannose to be used in Agrobacterium-mediated transformation of flax (Linum usitatissimum L.) cv. Barbara. The Escherichia coli pmi gene encodes a phosphomannose isomerase (E.C. 5.1.3.8) that converts mannose-6-phosphate, an inhibitor of glycolysis, into fructose-6-phosphate (glycolysis intermediate). Its expression in transformed cells allows them to grow on mannose-selective medium. The Agrobacterium tumefaciens strain GV3101 (pGV2260) harbouring the binary vector pNOV2819 that carries the pmi gene under the control of the Cestrum yellow leaf curling virus constitutive promoter was used for transformation experiments. Transgenic flax plants able to root on mannose-containing medium were obtained from hypocotyl-derived calli that had been selected on a combination of 20 g L⁻¹ sucrose and 10 g L⁻¹ mannose. Their transgenic state was confirmed by PCR and Southern blotting. Transgene expression was detected by RT-PCR in leaves, stems and roots of in vitro grown primary transformants. The mean transformation efficiency of 3.6%, that reached 6.4% in one experiment was comparable to that obtained when using the nptII selectable marker on the same cultivar. The ability of T1 seeds to germinate on mannose-containing medium confirmed the Mendelian inheritance of the pmi gene in the progeny of primary transformants. These results indicate that the PMI/mannose selection system can be successfully used for the recovery of flax transgenic plants under safe conditions for human health and the environment.