Agrobacterium-mediated transformation of the ascomycete mushroom Morchella importuna using polyubiquitin and glyceraldehyde-3-phosphate dehydrogenase promoter-based binary vectors

Abstract

Morchella importuna is a worldwide distributed edible mushroom with high ecological and economic values, but the molecular and genetic research about this mushroom has been hindered due to lack of an efficient transformation method. Here, we report for the first time the successful transformation of M. importuna by using a hypervirulent Agrobacterium tumefaciens strain bearing the constructed binary plasmid p1391-U-GUS. The selectable markers used were the genes for hygromycin resistance under the control of the polyubquitin promoter from M. importuna. The reporter genes were those for enhanced green fluorescent protein (EGFP) and the β-Glucuronidase (GUS) under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and polyubquitin promoter respectively. The presence of the reporter gene EGFP in the transformants was confirmed by the fluorescence and confocal microscope and molecular analysis and that of the reporter gene GUS was verified by enzyme activity and molecular analysis. The analysis results of both reporter genes indicated that Agrobacterium-mediated transformation was successfully performed in M. importuna.

Functional Analysis of a Novel ABL (Abnormal Browning Related to Light) Gene in Mycelial Brown Film Formation of Lentinula edodes

“…are commonly used as vectors for the generation of transgenic plants, yeasts, and filamentous fungi [21,22]. Agrobacterium tumefaciens-mediated transformation has been used in diverse mushroom species such as Agaricus bisporus [23][24][25], Flammulina velutipes [26,27], Hebeloma cylindrosporum [28], Hypsizygus marmoreus [29], L. edodes [30], Morchella importuna [31], Pleurotus eryngii [32], Pleurotus ostreatus [33], and Volvariella volvacea [34]. Furthermore, T-DNA-mediated genetic disruption for screening genes related to mycelial development was reported in Ophiocordyceps sinensis [35].…”

Mating-Type Genes Play an Important Role in Fruiting Body Development in Morchella sextelata

“…AC1010S, Weidi Biotechnology Co., Ltd., Shanghai, China) was used for fungal transformation. The media used for Agrobacterium-mediated transformation, including a complete yeast medium (CYM), a minimal medium (MM), an induction medium (IM), and a cocultivation medium (CoM), were as previously presented [ 19 ].…”

“…Transformation was performed according to the procedure used by Lv et al [ 19 ]. Briefly, strains pregrown in the dark at 20 °C for 5 days on CYM were used as transformation materials.…”

Effects of Medium Composition and Genetic Background on Agrobacterium-Mediated Transformation Efficiency of Lentinula edodes

“…Nevertheless, this method was not widely used, considering that electroporation is highly toxic to the cells and basidiospores are more difficult to distinguish the phenotypic variations caused by genetic differentiations or transformation mutants [22]. The Agrobacterium -mediated transformation system was applied successfully to transform L. edodes by using the mycelium of a monokaryon strain as a recipient, while it failed when using the mycelium of a dikaryon strain [23]. Genetic transformation systems were established in several fungi: The basidiomycete fungi A. bisporus and G. lucidum , the cucurbit powdery mildew pathogen Podosphaera xanthii , the ascomycete Morchella importuna [23,24,25,26].…”

“…The Agrobacterium -mediated transformation system was applied successfully to transform L. edodes by using the mycelium of a monokaryon strain as a recipient, while it failed when using the mycelium of a dikaryon strain [23]. Genetic transformation systems were established in several fungi: The basidiomycete fungi A. bisporus and G. lucidum , the cucurbit powdery mildew pathogen Podosphaera xanthii , the ascomycete Morchella importuna [23,24,25,26]. Regardless of the feasibility of existing transformation systems, most fungi still have no genetic transformation systems available.…”