Non-antibiotic Selection Marker Research Articles

A novel non-antibiotic selectable marker GASA6 for plant transformation

Selectable markers help the transformed cell/tissue to survive in an otherwise lethal exposure to an antibiotic or herbicide. Unfortunately, almost all the traditional selectable markers are antibiotic and herbicide resistance genes, which are controversial on human health concerns and environmental impact. Novel plant-derived, non-antibiotic, and non-herbicide selectable markers are urgently needed in plant transformation. Our previous work showed that the seedlings of overexpression Arabidopsis lines of AtGASA6 survived on medium with a high concentration of sugar, which leads to the hypothesis that AtGASA6 could be a selectable marker on media with high or low sugar content. In this study, leaf explants of AtGASA6 overexpression tobacco lines regenerated shoots on sugar-free shooting medium while those of wild type could not. Moreover, the seeds of AtGASA6 overexpression tobacco lines germinated and grew into normal seedlings on sugar-free MS medium while those of WT could not. Attractively, no developmental defects were observed in AtGASA6 transgenic progenies. Using AtGASA6 as a selectable marker, overexpression tobacco lines of GAI, which restrains plant size, were created on sugar-free media. The GAI overexpression lines had a smaller plant size than that of control. Considering its plant-derived and non-antibiotic nature, GASA6 is promising to be used as a selectable marker in plant transformation.

Broad-specificity amino acid racemase, a novel non-antibiotic selectable marker for transgenic plants

The broad-specificity amino acid racemase (Bsar) from Pseudomonas putida catalyzes the racemization of various amino acids, offering a flexible and feasible platform to develop a new non-antibiotic selectable marker system for plant transformation. In the present study, we demonstrated that a Bsar variant, Bsar-R174K, that is useful as a selectable marker gene in Arabidopsis and rice that were susceptible to l-lysine and D-alanine. The introduction of wild-type Bsar, Bsar-R174K or Bsar-R174A into E. coli lysine or asparagine auxotrophs was able to rescue the growth of these microorganisms in minimal media supplemented with selectable amino acid enantiomers. The transformation of Arabidopsis with Bsar or Bsar variants based on d-alanine selection revealed that Bsar-R174K had the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). Whereas, l-lysine-based selection exhibited lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis revealed normal growth properties. In addition, Bsar-R174K transgenic rice was obtained on l-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice revealed morphologically normal phenotypes comparable with their wild-type counterparts. This study presents the first report of broad range amino acid racemase Bsar-R174K as a non-antibiotic selectable marker system applied in transgenic plants.

Crystal Structures of Lysine-Preferred Racemases, the Non-Antibiotic Selectable Markers for Transgenic Plants

Lysine racemase, a pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase that catalyzes the interconversion of lysine enantiomers, is valuable to serve as a novel non-antibiotic selectable marker in the generation of transgenic plants. Here, we have determined the first crystal structure of a lysine racemase (Lyr) from Proteus mirabilis BCRC10725, which shows the highest activity toward lysine and weaker activity towards arginine. In addition, we establish the first broad-specificity amino acid racemase (Bar) structure from Pseudomonas putida DSM84, which presents not only the highest activity toward lysine but also remarkably broad substrate specificity. A complex structure of Bar-lysine is also established here. These structures demonstrate the similar fold of alanine racemase, which is a head-to-tail homodimer with each protomer containing an N-terminal (α/β)8 barrel and a C-terminal β-stranded domain. The active-site residues are located at the protomer interface that is a funnel-like cavity with two catalytic bases, one from each protomer, and the PLP binding site is at the bottom of this cavity. Structural comparisons, site-directed mutagenesis, kinetic, and modeling studies identify a conserved arginine and an adjacent conserved asparagine that fix the orientation of the PLP O3 atom in both structures and assist in the enzyme activity. Furthermore, side chains of two residues in α-helix 10 have been discovered to point toward the cavity and define the substrate specificity. Our results provide a structural foundation for the design of racemases with pre-determined substrate specificity and for the development of the non-antibiotic selection system in transgenic plants.

The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP) based detection procedures.

PMI (manA) as a nonantibiotic selectable marker gene in plant biotechnology

Due to the rising public concern over the use of genes conferring antibiotic and herbicide resistance, alternative systems for selection after plant transformation are being developed. A positive selection system consists of a physiologically inert metabolite as the selection agent and a respective gene which determines a metabolic advantage via selection agent utilization. The transformed cells are able to overcome the suppressive effects of the selection, while the untransformed ones starve but are not killed. The enzyme phosphomannose isomerase (PMI, E.C. 5.3.1.8) catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate in prokaryotic and eukaryotic organisms. The PMI selection system is called positive due to the effect of “starvation” caused to the nontransformed plant tissue because of its incapability to utilize mannose as a carbon source. In this mini-review we researched the literature to obtain a more detailed view of the characteristics, specifics, problems, and advantages of applying the PMI/mannose selection system.

A novel light‐dependent selection marker system in plants

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants.

Lysine racemase: a novel non-antibiotic selectable marker for plant transformation

A non-antibiotic based selection system using L-lysine as selection agent and the lysine racemase (lyr) as selectable marker gene for plant transformation was established in this study. L-lysine was toxic to plants, and converted by Lyr into D-lysine which would subsequently be used by the transgenic plants as nitrogen source. Transgenic tobacco and Arabidopsis plants were successfully recovered on L-lysine medium at efficiencies of 23 and 2.4%, respectively. Phenotypic characterization of transgenic plants clearly revealed the expression of normal growth and developmental characteristics as that of wild-type plants, suggesting no pleiotropic effects associated with the lyr gene. The specific activity of Lyr in transgenic tobacco plants selected on L: -lysine ranged from 0.77 to 1.06 mU/mg protein, whereas no activity was virtually detectable in the wild-type plants. In addition, the composition of the free amino acids, except aspartic acid, was not affected by the expression of the lyr gene in the transgenic tobacco plants suggesting very limited interference with endogenous amino acid metabolism. Interestingly, our findings also suggested that the plant aspartate kinases may possess an ability to distinguish the enantiomers of lysine for feedback regulation. To our knowledge, this is the first report to demonstrate that the lysine racemase selectable marker system is novel, less controversial and inexpensive than the traditional selection systems.

Plant native tryptophan synthase beta 1 gene is a non-antibiotic selection marker for plant transformation

Gene transformation is an integral tool for plant genetic engineering. All antibiotic resistant genes currently employed are of bacterial origin and their presence in the field is undesirable. Therefore, we developed a novel and efficient plant native non-antibiotic selection system for the selection of transgenic plants in the model system Arabidopsis. This new system is based on the enhanced expression of Arabidopsis tryptophan synthase beta 1 (AtTSB1) and the use of 5-methyl-tryptophan (5MT, a tryptophan [Trp] analog) and/or CdCl2 as selection agent(s). We successfully integrated an expression cassette containing an AtT-SB1 cDNA driven by a cauliflower mosaic virus 35S promoter into Arabidopsis by floral dip transformation. Transgenic plants were efficiently selected on MS medium supplemented with 75 microM 5MT or 300 microM CdCl2 devoid of antibiotics. TSB1 selection was as efficient as the conventional hygromycin selection system. Northern blot analysis of transgenic plants selected by 5MT and CdCl2 revealed increased TSB1 mRNA transcript whereas uneven transcript levels of hygromycin phosphotransferase II (hpt) (control) was observed. Gas chromatography-mass spectrometry revealed 10-15 fold greater free Trp content in AtT-SB1 transgenic plants than in wild-type plants grown with or without 5MT or CdCl2. Taken together, the TSB1 system provides a novel selection system distinct from conventional antibiotic selection systems.

Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase

Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase

Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase

A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l(-1) sucrose with 10 g l(-1) mannose in the first selection to only 10 g l(-1) mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization.

Use of a herbicide or lysine plus threonine for non-antibiotic selection of transgenic chickpea.

A desensitized aspartate kinase (AK) gene has been developed as a non-antibiotic selection marker for use in the production of transgenic chickpea ( Cicer arietinum L.). Transgenic shoots regenerated from embryo explants bombarded with the desensitized AK gene were selected on media containing two amino acids, lysine and threonine (LT). Approximately 15% of the putative transgenic shoots of vars. P-362 and P-1042 survived after 4 weeks of growth on MSB5 medium (MS mineral salts and B5 vitamins) containing 2 microM thidiazuron (TDZ) and 2 mM lysine and 2 m M threonine. These shoots were subsequently grown on MSB5 medium supplemented with 2 micro M TDZ and 5 mM lysine and 5 mM threonine, and nearly 1% continued to grow after 16 weeks of selection. A phosphinothricin (PPT) selection system for Agrobacterium-mediated chickpea transformation was also developed. Three varieties of chickpea, P-362, P-1042 and P-1043, were successfully used for Agrobacterium transformation. Following Agrobacterium infection, 3-8% of the regenerated shoots remained green and continued to grow on MSB5 medium supplemented with 2.5 mg l(-1 )PPT. Increasing the concentrations of PPT to 15 mg l(-1) reduced transgenic shoot production in P-362, P-1042 and P-1043 to 0.7%, 1.2% and 1.1%, respectively. Selected putatively transformed shoots of all three varieties were rooted and grown to maturity. Southern hybridization analysis revealed single as well as multiple integration of genes in selected transgenic lines. The level of AK activity detected in LT-selected plants was higher than that detected in the non-transformed control.

Construction of K88- and K99-expressing clones of Salmonella typhimurium G30: immunogenicity following oral administration to pigs

Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 10(11) vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.