Selectable Marker For Genetic Transformation Research Articles

Establishment of a new and efficient Agrobacterium-mediated transformation system in the nematicidal fungus Purpureocillium lilacinum

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 106 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.

Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants.

Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6-9%. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16-17 and 14-15% for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.

Development of a stable genetic system for Chlorella vulgaris—A promising green alga for CO2 biomitigation

Chlorella vulgaris has been proposed as a promising green alga for CO2 biomitigation due to its attractive traits of high growth rate, excellent CO2 fixation ability and broad industrial applications. Nevertheless, the genetic engineering of this alga is still in its infancy. In the present study, a stable and efficient transformation system was established for C. vulgaris CBS 15-2075, a robust strain with great potential for CO2 biomitigation. The antibiotic sensitivity spectrum of this organism was evaluated, and the nptII gene was selected as a dominant selectable marker for genetic transformation. The selectable marker, together with an enhanced green fluorescent protein (EGFP) gene, was delivered into C. vulgaris protoplasts by using a PEG-mediated method, giving a transformation efficiency of 356±30cfu per μg vector DNA. Molecular characterization and live-cell fluorescence microscopy demonstrated that the EGFP gene was stably integrated into C. vulgaris genome and expressed in the cytoplasm of transformed cells. Taken together, we for the first time established a stable and useful genetic toolkit for the industrially important microalga C. vulgaris, which will facilitate to a great extent the future rational genetic manipulation for strain improvement to increase CO2 fixation capacity and may as well provide valuable insights into other Chlorella species. Besides, the success in EGFP expression and live-cell fluorescence detection in C. vulgaris will be useful in molecular and cell biology for protein subcellular localization.

The usage of snapdragon Delila (Del) gene as a visible selection marker for the antibiotic-free transformation system

This study was conducted to assess the suitability of the Delila gene (Del) from snapdragon for use as a visible selectable marker, under the control of the cauliflower mosaic virus (CaMV) 35S promoter, to develop a plant genetic transformation system that helps to avoid using antibiotic- or herbicide-resistance genes, such as the gene for resistance against kanamycin or PPT. Following transformation, tobacco shoots showing red coloration always contained the Del gene, which was confirmed by PCR analysis. No chimerism or ploidy variation was observed during genetic transformation. In addition, the integration ratio of the transgene to the T1 progeny was 3:1, following typical Mendelian fashion. By anthocyanin analysis, the plants containing the Del gene were shown to have 80 times higher anthocyanin content than the control plants. Thus, we conclude that strong anthocyanin accumulation, as a result of the snapdragon Del gene, can be used as a visible selectable marker for genetic transformation in the tobacco plant, replacing the use of antibiotic- or herbicide-resistance genes.

Genetic transformation of the dermatophyte, Trichophyton mentagrophytes, based on the use of G418 resistance as a dominant selectable marker

Dermatophytes are closely related keratinophilic fungal pathogens and are the causative agents of a superficial cutaneous infection called dermatophytosis (ringworm). A lack of gene manipulation techniques has prevented detailed analyses of the mechanisms of host invasion by dermatophytes. We have introduced the tetracycline-regulatable (TR) gene expression system into dermatophytes to facilitate functional analyses of genes essential for growth and virulence. As the TR gene expression system consists of two plasmid vector components, two dominant selectable markers are required for genetic transformation. In dermatophytes, only the hygromycin B phosphotransferase gene (hph) is available as a selectable marker. We investigated the possibility of G418 resistance as a secondary selectable marker for genetic transformation in dermatophytes. A series of plasmid vectors carrying the neomycin phosphotransferase gene (nptII) were introduced into the protoplasts of Trichophyton mentagrophytes, one of the most clinically important dermatophyte species, by polyethylene glycol (PEG)-mediated transformation. Transformants were selected on selective medium containing G418 at 300-500 microg/ml. Molecular biological analyses indicated that colonies appearing on the selective medium harbored nptII in their chromosomes. Colonies produced from protoplasts transformed with the enhanced green fluorescent protein (eGFP) gene-T. mentagrophytes cyclophilin cDNA (TmcypB) fusion vector also exhibited GFP fluorescence throughout their mycelia, but accumulation of the GFP-TmCYPB fusion protein in specific intracellular compartments was not observed. This study has provided a new selectable marker for genetic transformation in dermatophytes.

Improved protocols for functional analysis in the pathogenic fungus Aspergillus flavus

BackgroundAn available whole genome sequence for Aspergillus flavus provides the opportunity to characterize factors involved in pathogenicity and to elucidate the regulatory networks involved in aflatoxin biosynthesis. Functional analysis of genes within the genome is greatly facilitated by the ability to disrupt or mis-express target genes and then evaluate their result on the phenotype of the fungus. Large-scale functional analysis requires an efficient genetic transformation system and the ability to readily select transformants with altered expression, and usually requires generation of double (or multi) gene deletion strains or the use of prototrophic strains. However, dominant selectable markers, an efficient transformation system and an efficient screening system for transformants in A. flavus are absent.ResultsThe efficiency of the genetic transformation system for A. flavus based on uracil auxotrophy was improved. In addition, A. flavus was shown to be sensitive to the antibiotic, phleomycin. Transformation of A. flavus with the ble gene for resistance to phleomycin resulted in stable transformants when selected on 100 μg/ml phleomycin. We also compared the phleomycin system with one based on complementation for uracil auxotrophy which was confirmed by uracil and 5-fluoroorotic acid selection and via transformation with the pyr4 gene from Neurospora crassa and pyrG gene from A. nidulans in A. flavus NRRL 3357. A transformation protocol using pyr4 as a selectable marker resulted in site specific disruption of a target gene. A rapid and convenient colony PCR method for screening genetically altered transformants was also developed in this study.ConclusionWe employed phleomycin resistance as a new positive selectable marker for genetic transformation of A. flavus. The experiments outlined herein constitute the first report of the use of the antibiotic phleomycin for transformation of A. flavus. Further, we demonstrated that this transformation protocol could be used for directed gene disruption in A. flavus. The significance of this is twofold. First, it allows strains to be transformed without having to generate an auxotrophic mutation, which is time consuming and may result in undesirable mutations. Second, this protocol allows for double gene knockouts when used in conjunction with existing strains with auxotrophic mutations.To further facilitate functional analysis in this strain we developed a colony PCR-based method that is a rapid and convenient method for screening genetically altered transformants. This work will be of interest to those working on molecular biology of aflatoxin metabolism in A. flavus, especially for functional analysis using gene deletion and gene expression.

Functional analysis of transgenic rice plants expressing a novel mutated ALS gene of rice

We performed functional analysis of transgenic plants expressing the W548L/S627I mutated ALS gene from rice as well as studies on inheritance of the mutated gene, phenotype and fertility of transgenic plants to ensure availability of the mutated gene as a selectable marker for plant genetic transformation. Expression levels of the ALS (endogenous+mutated ALS) gene of transgenic rice plants were correlated with the resistance of transgenic plants to bispyribac-sodium (BS). The BS-resistant trait of a transgenic plant was stably inherited by the progeny in a Mendelian manner. A homozygote of transgenic plants harboring the mutated gene was normal in its growth and fertility compared with the wild type. These results ensured that the W548L/S627I mutated gene from rice can be efficiently used as a selectable marker for genetic transformation of rice in combination with BS.

Potential Selectable Marker for Genetic Transformation in Banana

Potential Selectable Marker for Genetic Transformation in Banana

The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.

Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria.

Mycobacteriophage L5 is a temperate phage of the mycobacteria that forms stable lysogens in Mycobacterium smegmatis. We show here that the 183-amino-acid product of L5 gene 71 confers immunity to L5 superinfection, is required for maintenance of the lysogenic state and contains a helix-turn-helix DNA-binding motif--properties associated with repressors of temperate phages. We have utilized these observations to demonstrate the use of L5 gene 71 as a selectable marker for genetic transformation of the mycobacteria. Significantly, the use of L5 gene 71 as a selectable gene avoids the requirement for antibiotic-resistance genes providing an important tool for manipulation of the pathogens Mycobacterium tuberculosis and Mycobacterium avium, and for the construction of recombinant BCG vaccines.