A chimaeric hygromycin resistance gene as a selectable marker in plant cells

Summary We show here that plant cells are sensitive to the antibiotic hygromycin-B⁴. We also show that a chimaeric gene consisting of the nopaline synthase (nos) gene regulatory elements and the E. coli derived hygromycin phosphotransferase (hpt) gene, when transferred to plants' cells, confers resistance to hygromycin B. The chimaeric nos-hpt gene enables efficient selection of DNA transfer to plant cells when used in conjunction with Ti plasmid-derived binary vectors in cocultivation experiments.     

Streptothricin resistance as a novel selectable marker for transgenic plant cells

 Streptothricins are known as antimicrobial agents produced by Streptomyces spp. Bacterial resistance to streptothricin is mediated by specific enzymes exhibiting an acetyltransferase activity which renders the drug non-toxic for bacteria. The nucleotide sequence of several streptothricin resistance genes from bacteria have been described. Certain cells of eukaryotic parasites (such as Ustilago maydis or Leishmania spp.) are sensitive to streptothricin and the introduction of the bacterial resistance gene sat2 renders them resistant. We show that numerous species of plants are sensitive to low concentrations of streptothricin. Moreover, introduction of the bacterial resistance gene sat3 under the control of the 35S cauliflower mosaic virus promoter protects these cells from the toxic action of streptothricin. Therefore, sat3-mediated streptothricin resistance appears to be a promising selective marker for genetic manipulation of plant cells. Received: 6 November 1996 / Revision received: 9 January 1997 / Accepted: 22 March 1999     

Molecular characterization of two alanine-rich Lea genes abundantly expressed in the resurrection plant C. plantagineum in response to osmotic stress and ABA

Expression of many genes is induced during dehydration in vegetative tissues of the desiccation tolerant resurrection plantCraterostigma plantagineum. The most abundant group of desiccation-related gene products belong to the LEA (= Late Embryogenesis Abundant) proteins. Here we describe structures and expression patterns of members of group 3 and group 4Lea genes fromC. plantagineum. The most intriguing observation is the strong conservation of repeat motifs inLea genes found across divers plant species includingC. plantagineum and non-desiccation tolerant plants. This conservation of structural elements leads to speculations about evolution of desiccation tolerance in the resurrection plant.     

Efficacy of an intron-containing kanamycin resistance gene as a selectable marker in plant transformation

In this project we have analysed the use of an intron-containing neomycin phosphotransferase II - nptII - gene. The advantage of this construct is that only eukaryotic organisms will be able to process this gene. Accordingly, the theoretical risk of horizontal gene flow of antibiotic resistance genes from transgenic plants to enteric bacteria is eliminated. The ST-LS1 intron IV2 from potato was inserted into the coding region of nptII. Transformation of Solanum tuberosum (potato) and Nicotiana tabacum (tobacco) with constructs containing the intron nptII showed similar transformation frequencies to transformation with constructs containing the normal nptII. Analysis of total DNA and RNA confirmed that the intron-containing nptII gene was present in the plants and that the mRNA was processed correctly.     

The salt-tolerance gene rstB can be used as a selectable marker in plant genetic transformation

The salt-tolerance gene rstB under the control of the cauliflower mosaic virus 35S promoter was used as a selectable marker gene in the Agrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum cv. Xanthi). The selective agent for plant regeneration was tolerance to 170 mM sodium chloride. The highest selection efficiency was 83.3%. No obvious differences in selection efficiencies were observed when those obtained using the standard selectable marker gene hpt and a selection regime of 10 mg l⁻¹ hygromycin. Transgenic events were confirmed by PCR, Southern blot, RT-PCR and green fluorescent protein studies. The rstB transgenic plants showed improved salt tolerance and a normal phenotype. Based on these results, we suggest that the rstB gene may be used as a promising selectable marker and an alternative to the antibiotic- or herbicide-resistance genes in plant transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis

The dhlA gene of Xanthobacter autotrophicus GJ10 encodes a dehalogenase which hydrolyzes dihalo- alkanes, such as 1, 2-dichloroethane (DCE), to a halogenated alcohol and an inorganic halide (Janssen et al. 1994, Annu. Rev. Microbiol. 48, 163-191). In Xanthobacter, these alcohols are further catabolized by alcohol and aldehyde dehydrogenase activities, and by the product of the dhlB gene to a second halide and a hydroxyacid. The intermediate halogenated alcohols and, in particular, the aldehydes are more toxic than the haloalkane substrates or the pathway products. We show here that plants, including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild-type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhlA. The dhlA marker therefore provides haloalkane dehalogenase reporter activity and substrate dependent negative selection in transgenic plants.     

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Development of transformation vectors based upon a modified plant alpha-tubulin gene as the selectable marker

A plant transformation and selection system has been developed utilizing a modified tubulin gene as a selectable marker. The vector constructs carrying a mutant alpha-tubulin gene from goosegrass conferring resistance to dinitroaniline herbicides were created for transformation of monocotyledonous and dicotyledonous plants. These constructs contained beta- and/or mutant alpha-tubulin genes driven either by ubiquitin or CaMV 35S promoter. The constructs were used for biolistic transformation of finger millet and soybean or for Agrobacterium-mediated transformation of flax and tobacco. Trifluralin, the main representative of dinitroaniline herbicides, was used as a selective agent in experiments to select transgenic cells, tissues and plantlets. Selective concentrations of trifluralin estimated for each species were as follows: 10 microM for Eleusine coracana, Glycine max, Nicotiana plumbaginifolia and Nicotiana sylvestris; 3 microM for Linum usitatissimum. PCR and Southern blotting analyses of transformed lines with a specific probe to nptII, alpha-tubulin or beta-tubulin genes were performed to confirm the transgenic nature of regenerated plants. Band specific for the mutant alpha-tubulin gene was identified in transformed plant lines. Results confirmed the stable integration of the mutant tubulin gene into the plant genomes. The present study clearly demonstrates the use of a plant mutant tubulin as a selective gene for plant transformation.     

Effects of chloramphenicol on plant cells: Potential as a selectable marker for transformation studies

The growth of tobacco and carrot cells in suspension cultures and on solidified medium was strongly inhibited by the antibiotic chloramphenicol. This effect was irreversible. The drug had no effect on [³H]-uridine incorporation but inhibited protein synthesis and induced striking morphological alterations in intoxicated cells. O₂ consumption was also reduced in treated cells. Tobacco and carrot cells were unable to inactivate chloramphenicol by enzymatic acetylation.     

Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells.

Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells.     

Unusual sequences of group 3 LEA mRNA inducible by maturation or drying in soybean seeds

Two cDNA clones, pGmPM8 and pGmPM10, which correspond to two mRNA species in mature or dry soybean seeds, were characterized. The deduced proteins, based on DNA sequence analysis, have a molecular mass of 49 and 51 kDa for pGmPM8 and pGmPM10, respectively. These two cDNA clones share a high homology with an amino acid identity of about 90% between the two deduced proteins. Both proteins appear to be extremely hydrophilic except at their N-termini that contain a 29 amino acid hydrophobic region at the N-terminus and the sizes of proteins decrease after co-incubating with ER membranes. These two proteins contain more than 30 similar, contiguous repeats of 11 amino acids, which is characteristic of group 3 LEA proteins. The mRNAs corresponding to pGmPM8 and pGmPM10 were expressed at high levels in dried or mature soybean seeds, but not in fresh immature seeds. The RNAs were also present in abscisic acid (ABA) treated leaves or cultured cells, and in tissues subjected to water stress or low temperatures.     

脱落酸调节植物抵御水分胁迫的机制研究

干旱、盐渍、低温等均可导致植物可利用水分的亏缺,表现为水分胁迫。植物感受到水分胁迫,诱导脱落酸（abscisic acid,ABA）生物合成。ABA可通过促使气孔关闭或抑制气孔开放,使作物尽可能地降低蒸腾失水,以抵御水分胁迫。该文就植物激素ABA及其下游信号过氧化氢（hydrogenperoxide,H2O2）、一氧化氮（nitric oxide,NO）以及Ca2＋等在植物气孔运动调节方面的研究进展进行概述,以构建水分胁迫下ABA调节植物气孔运动的可能模式。     

A point mutation in the Medicago sativa GSA gene provides a novel, efficient, selectable marker for plant genetic engineering

Bacterial selectable marker genes (SMG) conferring antibiotic resistance are valuable tools in plant genetic engineering, but public concern and regulatory requirements have stimulated the development of alternative selection systems. We have previously demonstrated that a mutated Synechococcus elongatus HemL gene encoding glutamate 1-semialdehyde aminotransferase (GSA) is an efficient SMG in alfalfa. In fact, GSA is irreversibly inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid), but the mutated enzyme is gabaculine insensitive. With the aim to develop a plant derived SMG, we cloned and sequenced the Medicago sativa GSA cDNA and reproduced one of the two mutations associated with gabaculine resistance in Synechococcus, a transversion resulting in a methionine to isoleucine (M → I) substitution. This mutated gene was assessed as a SMG in tobacco and alfalfa Agrobacterium transformation, in comparison with the wild type gene. In tobacco, about 43% of the leaf explants produced green shoots, whereas in alfalfa 47% of the explants produced green embryos in the presence of 30 μM gabaculine when the M → I GSA was introduced. Escapes were absent in tobacco and only 6% in alfalfa. No effect on the plant phenotype was noticed. We propose this new SMG as a widely acceptable alternative to those currently used.     

A drought-stress-inducible histone gene in Arabidopsis thaliana is a member of a distinct class of plant linker histone variants

We have isolated and characterized a gene, His1-3, encoding a structurally divergent linker histone in Arabidopsis thaliana. Southern and northern hybridization data indicate that A. thaliana expresses three single-copy linker histone genes, each encoding a structurally distinct variant. H1-3 is a considerably smaller protein (167 amino acids with a mass of 19.0 kDa) than any other described linker histone from higher eukaryotes. We examined the expression of His1-3 at the RNA and protein levels and found that it is induced specifically by water stress. In contrast, expression of His1-1, His1-2 and His4 appear unaffected by water stress. Furthermore, the primary structure of the protein possesses distinct characteristics that are shared with another drought-inducible linker histone, H1-D, isolated from Lycopersicon pennellii. Based on structural characteristics of the deduced protein and its inducible expression, we hypothesize that H1-3 and H1-D are linker histone variants that have specialized roles in the structure and function of plant chromatin and therefore they can be considered to be members of a unique subclass of plant histones. Immunoblotting with an antibody produced against a short polypeptide in the conserved domain of this subtype indicates that similar proteins may exist in other plants.     

Effectiveness of the bacterial gene codA encoding cytosine deaminase as a negative selectable marker in Agrobacterium-mediated plant transformation

The usefulness of the E. coli codA gene encoding cytosine deaminase as a conditional toxic gene was explored during various stages of plant development and in different Agrobacterium -mediated transformation protocols. To this end, several independent tobacco lines transgenic for codA were isolated and these were tested for their sensitivity to 5-fluorocytosine (5-FC) at different developmental stages. On media supplemented with 5-FC, seedling proliferation was inhibited. Leaves failed completely to regenerate sprouts on 5-FC-containing medium. However, 40% of the shoots regenerated on non-selective medium still formed roots on rooting medium with 5-FC. In all these assays, control plants were unaffected by up to 1 mg m1⁻¹ 5-FC. Transformation of a codA and nptll -harbouring T-DNA to tobacco leaf discs did not result in any regenerant using a combined 5-FC and kanamycin selection, indicating that codA does not behave as a cell-autonomous marker here. Nevertheless, transformation of the same T-DNA to tobacco protoplasts resulted in some enrichment of codA ⁻ nptll ⁺ calluses using the proper combination of 5-FC and kanamycin for selection. Mixing of codA -containing and codA -lacking tobacco protoplasts revealed that the codA gene may behave as a cell autonomous marker under certain, appropriately chosen conditions, which seems to be in paradox with the total absence of escapes in tissue explant transformation. In all these experiments, 250 µg ml⁻¹ 5-FC was found to be the most optimal for selection. Our results suggest that codA can be successfully used as a negative selectable marker in Agrobacterium -mediated gene targeting protocols of tobacco whereby selection at the shoot regeneration level is the most effective.     

Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh.

A cDNA clone corresponding to a novel low-temperature-induced Arabidopsis thaliana gene, named lti140, was employed for studies of the environmental signals and the signal pathways involved in cold-induced gene expression. The single-copy lti140 gene encodes a 140 kDa cold acclimation-related polypeptide. The lti140 mRNA accumulates rapidly in both leaves and roots when plants are subject to low temperature or water stress or are treated with the plant hormone abscisic acid (ABA), but not by heat-shock treatment. The low-temperature induction of lti140 is not mediated by ABA, as shown by normal induction of the lti140 mRNA in both ABA-deficient and ABA-insensitive mutants and after treatment with the ABA biosynthesis inhibitor fluridone. The effects of low temperature and exogenously added ABA are not cumulative suggesting that these two pathways converge. The induction by ABA is abolished in the ABA-insensitive mutant abi-1 indicating that the abi-1 mutation defines a component in the ABA response pathway. Accumulation of the lti140 mRNA in plants exposed to water stress was somewhat reduced by treatment with fluridone and in the ABA-insensitive mutant abi-1 suggesting that the water stress induction of lti140 could be partly mediated by ABA. It is concluded that three separate but converging signal pathways regulate the expression of the lti140 gene.     

Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker

A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells. Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein. Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media. This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.