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.

Agrobacterium-Mediated Genetic Transformation of the Medicinal Plant Veratrum dahuricum

Veratrum dahuricum L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of Veratrum dahuricum mediated with Agrobacterium tumefaciens is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. A. tumefaciens LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. A. tumefaciens cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of Veratrum.

Effect of Phosphinothricin on Transgenic Downy Birch (Betula pubescens Ehrh.) Containing bar or GS1 Genes

Weeds are a big problem in agriculture and forestry, and herbicides are the main tools to control them. Phosphinotricin (ammonium glufosinate, PPT) is one of the most effective non-selective herbicides, to which weeds hardly gain resistance, but the reasons for its effect and toxicity to plants are still unclear, and especially, it is little studied in trees, including transgenic ones. We studied the physiological responses of downy birch (Betula pubescens Ehrh.) containing the herbicide resistance bar gene or the cytosol glutamine synthetase GS1 gene (the target enzyme of the herbicide) to PPT-based Basta herbicide treatment in various doses under open-air conditions during two years. Birch saplings with the bar gene were resistant to a double field dose (10 L/ha), but the expression of the GS1 gene only slightly increased resistance compared to the control. Herbicide treatment increased the ammonium level in leaf tissue by 3–8 times, but this, apparently, was not the main cause of plant death. Among leaf pigments, chlorophyll B was the most resistant to PPT, and carotenoids were the most sensitive. Responses of birch trees with the GS1 gene (accumulation of ammonium, pigment content, and dehydration) during treatment with a low dose of herbicide were less pronounced than in control plants. One-year-old control and transgenic plants with the GS gene died after 2.5 L/ha treatment, and two-year-old plants lost foliage after such treatment but remained alive and developed buds four weeks after treatment. Herbicide treatment of plants with the bar gene did not cause significant deviations in height (first year) or the accumulation of aboveground biomass (second year). The obtained results improve our understanding of the effect of PPT on woody plants and can be used both to clarify mechanisms of herbicide action and in plantation forestry.