エノキタケ単核菌糸体のアグロバクテリウムを介した形質転換系における3種類のバクテリア株との親和性

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Acccepted:April 2, 2018

Corresponding author: Eiji Tanesaka ([email protected])

Compatibility of three bacterial strains in Agrobacterium-mediated transformation of

monokaryotic mycelia of Flammulina velutipes

Eiji Tanesaka, Misuzu Mori, Kenya Tsuji, Takuji Tsukiyama

Faculty of Agriculture, Kindai University（Nara 631-8505, Japan）

Summary: Compatibility of three bacterial strains in Agrobacterium tumefaciens-mediated transformation of

monokaryotic mycelia of Flammulina velutipes was compared. The bacterial strains LBA4404 and C58C1 yielded four- to ﬁ ve-fold higher transforming efﬁ ciencies than did EHA105. A selection medium containing 50 μg/ml of hygromycin B successfully selected the transformants.

Key words: AMT, enokitake mushroom, monokaryon

Introduction

Flammulina velutipes (enokitake) is an economically

important cultivated edible mushroom and has potential for use as a model fungus for molecular genetics and breeding.

Agrobacterium tumefaciens-mediated transformation (AMT) has

been employed for practical gene tagging and genetic engineering in a variety of plants and fungi, including cultivated edible fungi such as Agaricus bisporus (Chen et al., 2000), F.

velutipes (Cho et al., 2006; Okamoto et al., 2010), Hypsizigus marmoreus (Hatoh et al., 2013; Zang et al., 2014), Pleurotus ostreatus (Ding et al., 2011), and Volvariella volvaceae (Wang et

al., 2008). Hatoh et al. (2013) reported an effective AMT method for transforming mushroom species using A. tumefaciens EHA105 harboring the binary plasmid pPZP-HYG2 (Walton et al., 2005), which carries the Cryptococcus neoformans actin gene promoter driving the expression of a hygromycin B resistance gene (hph) as a selectable marker. However, the transforming efficiency of F. velutipes was not sufficient to permit the construction of large numbers of mutants as needed for a mutant library. To improve the transforming efﬁ ciency of this fungus, this study compared the transforming efﬁ ciency of three Agrobacterium strains on F. velutipes mycelia.

Materials and Methods

F. velutipes monokaryotic bmHY4, a pigmented strain capable

of fruiting, was used for the present study. This strain was selected among monokaryons obtained from basidiospores derived from the cultivated dikaryotic stock Hatsuyuki . A.

tumefaciens EHA105 (Hood et al, 1993) harboring the binary

plasmid pPZP-HYG2 (gift from Dr. Chihiro Tanaka; Hatoh et

al., 2013) was used for transformation. The pPZP-HYG2 was also transformed into A. tumefaciens strains LBA4404 (TakaraBio, Kyoto) and C58C1 (gift from Dr. Gento Tsuji; Tsuji et al., 2003) by electroporation (GenePulser, BioRad, USA).

AMT was performed according to the methods of Hatoh et al. (2013) and as follows. Agrobacterium cells harboring pPZP-HYG2 were incubated for 2 days at room temperature in LB liquid medium containing 200 μg/ml kanamycin using a rotary shaker. The culture was centrifuged and cells were resuspended in induction medium (IM) (containing 200 μM acetosyringone: Bundock et al., 1995) at an optical density at 660 nm (OD660) of

0.2. F. velutipes bmHY4 was incubated for 7 days at room temperature in complete medium (CM) (Tanaka et al., 1991) using a linear shaker. The medium was removed by centrifugation and the mycelia were resuspended in 1 ml IM medium and homogenized (Tissueruptor, Quiagen, Germany).

Agrobacterium cells and the fungal suspension were mixed in a

1:1 (v/v) ratio. Aliquots (200 μl/ﬁ lter) of the mixture were spread on cellulose acetate filters (Y008A035A, Toyo Roshi Kaisha, Ltd., Tokyo) placed on IM plates, and the plates were incubated at 25㱥 for 2-4 days. After the co-cultivation, the filters were transferred to a selective medium supplemented with 50 μg/ml cefotaxime and 50 μg/ml tetracycline hydrochloride (to counter-select Agrobacterium cells) and 50 μg/ml hygromycin B (to select fungal transformants). Fungal colonies resistant to hygromycin were transferred to MYP medium (per L: 7 g malt extract, 1 g soytone, 0.5 g yeast extract). To confirm that the fungal transformants carried the hph gene, genomic DNA was extracted from mycelia and subjected to PCR for ampliﬁ cation of the hph region according to the method of Izumitsu et al. (2012).

31 J. Crop Res. 63：31 − 33（2018）

Copyright 近畿作物・育種研究会（The Society of Crop Science and Breeding in Kinki, Japan）

Note

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Results and Discussion

PCR targeting the hph gene revealed that all isolates obtained from the selective medium carried the hph gene region (Fig. 1), suggesting that 50 μg/ml of hygromycin B successfully selected the transformants. Table 1 shows transforming efficiency, expressed as the ratio of the number of hygromycin-resistant colonies isolated to the number of filters used, following two independent experiments using each of three Agrobacterium strains. The efficiency (6/32 – 17/68) obtained with strain EHA105 was similar to the results (2/18) reported by Hatoh et al. (2013). The bacterial strains LBA4404 and C58C1 yielded four- to fi ve-fold higher transforming effi ciencies than did EHA105 (F = 348, P = 0.00028). Nevertheless, the transforming effi ciencies observed in this study are still not sufficient for practical construction of a F. velutipes mutant library. Further optimization of the AMT method for this fungus will be needed. Notably, no apparent phenotypic differences were observed among the 127 transformants obtained from 196 filters; all transformants exhibited brownish pigmentation of the mycelia and fruiting properties resembling those of the bmHY4 parent strain.

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Fig. 1 PCR analysis targeting the hph gene in genomic DNA obtained from hygromycin-resistant transformants of F. velutipes . An arrow indicates the position of the target bands. Lanes: M, 100-bp ladder; P, pPZP-HYG2 (positive control); N, untransformed bmHY4 parent strain (negative control); 1-5, individual transformants.

Table. 1 Transforming efficiency1)_{of three A. tumefaciens} strains on F. velutipes mycelia.

Experiment EHA105 LBA4404 C58C1 1st 17/68 34/32 36/32 2nd 6/32 17/16 17/16 1)_{: Transforming efficiency is the ratio of the number of hygromycin} B-resistant colonies isolated to the number of ﬁ lters tested.

Acknowledgments

This work was financially supported by Grants-in-Aid for Scientific Research (17K07612) from Japan Society for the Promotion of Science. We also are grateful to Professor C. Tanaka, Kyoto University, for his technical advice on AMT.

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