Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.

ptxD/Phi as alternative selectable marker system for genetic transformation for bio-safety concerns: a review

Antibiotic and herbicide resistance genes are the most common marker genes for plant transformation to improve crop yield and food quality. However, there is public concern about the use of resistance marker genes in food crops due to the risk of potential gene flow from transgenic plants to compatible weedy relatives, leading to the possible development of “superweeds” and antibiotic resistance. Several selectable marker genes such as aph, nptII, aaC3, aadA, pat, bar, epsp and gat, which have been synthesized to generate transgenic plants by genetic transformation, have shown some limitations. These marker genes, which confer antibiotic or herbicide resistance and are introduced into crops along with economically valuable genes, have three main problems: selective agents have negative effects on plant cell proliferation and differentiation, uncertainty about the environmental effects of many selectable marker genes, and difficulty in performing recurrent transformations with the same selectable marker to pyramid desired genes. Recently, a simple, novel, and affordable method was presented for plant cells to convert non-metabolizable phosphite (Phi) to an important phosphate (Pi) for developing cells by gene expression encoding a phosphite oxidoreductase (PTXD) enzyme. The ptxD gene, in combination with a selection medium containing Phi as the sole phosphorus (P) source, can serve as an effective and efficient system for selecting transformed cells. The selection system adds nutrients to transgenic plants without potential risks to the environment. The ptxD/Phi system has been shown to be a promising transgenic selection system with several advantages in cost and safety compared to other antibiotic-based selection systems. In this review, we have summarized the development of selection markers for genetic transformation and the potential use of the ptxD/Phi scheme as an alternative selection marker system to minimize the future use of antibiotic and herbicide marker genes.

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

Pigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti-Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and –tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

Phenotypic Examination of Camelina sativa (L.) Crantz Accessions from the USDA-ARS National Genetics Resource Program

Camelina sativa (L.) Crntz. is a hardy self-pollinated oilseed plant that belongs to the Brassicaceae family; widely grown throughout the northern hemisphere until the 1940s for production of vegetable oil but was later displaced by higher-yielding rapeseed and sunflower crops. However, interest in camelina as an alternative oil source has been renewed due to its high oil content that is rich in polyunsaturated fatty acids, antioxidants as well as its ability to grow on marginal lands with minimal requirements. For this reason, our group decided to screen the existing (2011) National Genetic Resources Program (NGRP) center collection of camelina for its genetic diversity and provide a phenotypic evaluation of the cultivars available. Properties evaluated include seed and oil traits, developmental and mature morphologies, as well as chromosome content. Selectable marker genes were also evaluated for potential use in biotech manipulation. Data is provided in a raw uncompiled format to allow other researchers to analyze the unbiased information for their own studies. Our evaluation has determined that the NGRP collection has a wide range of genetic potential for both breeding and biotechnological manipulation purposes. Accessions were identified within the NGRP collection that appear to have desirable seed harvest weight (5.06 g/plant) and oil content (44.1%). Other cultivars were identified as having fatty acid characteristics that may be suitable for meal and/or food use, such as low (<2%) erucic acid content, which is often considered for healthy consumption and ranged from a high of 4.79% to a low of 1.83%. Descriptive statistics are provided for a breadth of traits from 41 accessions, as well as raw data, and key seed traits are further explored. Data presented is available for public use.

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi

The rodent parasite Plasmodium chabaudi is an important in vivo model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti-Plasmodium immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection. P. chabaudi also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable in vivo to in vitro model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for P. chabaudi and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear PlasmoGEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded P. chabaudi gene-deletion and –tagging vectors for transfection in our fluorescent P. chabaudi mother-lines. This produces a tool-kit of P. chabaudi lines, vectors and transfection approaches that will be of broad utility to the research community.

Construction of Marker-Free Genetically Modified Maize Using a Heat-Inducible Auto-Excision Vector

Gene modification is a promising tool for plant breeding, and gradual application from the laboratory to the field. Selectable marker genes (SMG) are required in the transformation process to simplify the identification of transgenic plants; however, it is more desirable to obtain transgenic plants without selection markers. Transgene integration mediated by site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for repeated transformation or sexual crossing. The vector combines an inducible site-specific recombinase (hsp70::Cre) that allows for the precise elimination of the selectable marker gene egfp upon heating. This marker gene is used for the initial positive selection of transgenic tissue. The egfp also functions as a visual marker to demonstrate the effectiveness of the heat-inducible Cre. A second marker gene for anthocyanin pigmentation (Rsc) is located outside of the region eliminated by Cre and is used for the identification of transgenic offspring in future generations. Using the heat-inducible auto-excision vector, marker-free transgenic maize plants were obtained in a precisely controlled genetic modification process. Genetic and molecular analyses indicated that the inducible auto-excision system was tightly controlled, with highly efficient DNA excision, and provided a highly reliable method to generate marker-free transgenic maize.