Xu Jin
(Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University)
,
Mo Ming-He
(Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University)
,
Zhou Wei
(Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University)
,
Huang Xiao-Wei
(Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University)
,
Zhang Ke-Qin
(Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University)
In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants ${\mu}g^{-1}$ per linearized plasmid DNA we...
In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants ${\mu}g^{-1}$ per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. $90\%$ of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.
In this study, the nematode-trapping fungus, Monacrosporium sphaeroides, was transformed with a plasmid harboring the hygromycin B phosphotransferase gene, via restriction enzyme-mediated integration (REMI). Frequencies of up to 94 transformants ${\mu}g^{-1}$ per linearized plasmid DNA were obtained by optimizing the PEG concentration, as well as the category and quantity of the added restriction enzyme. $90\%$ of the transformants were determined to be stable for drug resistance when 20 randomly selected transformants were tested. Southern analyses revealed that the transforming DNA was integrated into the M. sphaeroides genome either with or without rearrangement. Five mitotic stable mutant strains were obtained using this approach, all of which had been altered with regard to sporulation capacity and pathogenicity toward nematodes. Southern blot analyses of the five mutants revealed that foreign plasmid DNA had integrated into the genome. Three of the mutants, Tms2316, Tms3583 and Tms1536, exhibited integration at a single location, whereas the remaining two, Tms32 and Tms1913, manifested integration at double or multiple locations. Our results suggest that the transformation of M. sphaeroides via REMI will facilitate insertional mutagenesis, the functional analysis of a variety of genes, and the tagging or cloning of genes of interest.
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제안 방법
redivivus) were introd니ced to each of these plates. After 3 more days of incubation at 28℃, the predation rate of each of the strains was calculated, by recording the proportion of nematodes trapped by the trapping-devices. This experiment was repeated once, with five replicates for each is아ate.
(1996). Probe labeling and hybridization were conducted with Digoxigenin-dUTP Kits (Boehringer Mannheim, USA) and Nylon membranes (positively charged, Cat. No, 1209299, Roche, Germany), according to the manufacturere instructions.
대상 데이터
The nematode-trapping fungus, M. sphaeroides Castaner (YMF1.00539), was used in this study. This fungal strain was originally isolated from soil, and stored in the Fungal Store Centre of sir laboratoiy.
성능/효과
3). In addition, the predation rates of Tms32, Tmsl536, and Tmsl913 decreased to 0, 4% and 12% comparing with the rate of the wild-type strain (34%, average of 10 replicates). Tms2316 and Tms3583 manifested no pathogenicity toward nematodes.
In conclusion, we have established an effective REMI protocol for the induction of increased transformation fre- q니encies in M. sphaeroides, and have successfully utilized this transformation procedure to introduce random mutations into this hinges. It is worthy of note that we obtained mutants exhibiting significant morphological deficiencies, which will make it possible to clone the genes involved in the formation of the trapping-device and conidia in nematode-trapping fungi.
In this study, the REMI method was shown to increase transfomiation frequencies from 1 ~5 transfomiaiits/eg DNA (in the absence of restriction enzyme) to 46 ~94 transfdriTiants/j.ig DNA (in the presence of the enzyme). Similar results were reported in experiments involving fungal REMI transformation.
Tms2316 was unable to form both the trapping-device and the conidia. The mycelial growth rates of Tmsl913, Tms2316 and Tms3583 on TGA were similar to that of the wild-type strain, b니i the myc이ial growth rates of 1 ms32 and Tmsl536 were observed to have decreased by more than 50%. Furthermore, the Tms32 and Tmsl536 variants exhibited signify icant folding on the s*faucie and the irregular edges of the colonies growing on TGA, as compared to the wild-type strain (Fig.
4) could be explained as the integration of two modified copies of vector DNA into two different genomic sites. The results of Southern blots of Tmsl913 (Lane 7, 8, 9 in Fig. 4) verified that the vector DNA had been inserted into the genome, but this pattern was too vague to make any confident conclusions.
In brief, during the final step of transformation, the reaction solution was mixed with the medium and hygromycin B prior to being poured into a petri dish, rather than being mixed only with medium, and then an upper layer medium containing hygromycin B was overlaid after an overnight incubation. This measure resulted in a 50% red니ction in the amo니nt of hygromycin B required, and also simplified the protocol, while still maintaining the req니ired transformation frequency.
참고문헌 (21)
Ahman, J., E.K. Bo, L. Rask, and A. Tunlid. 1996. Sequence analysis and regulation of a cuticle degrading serine protease from the nematophagous fungus Arthrobotrys oligospora. Microbiology 142, 1605-1616
Black, M., F. Seeber, D. Soldati, K. Kim, and J.C. Boothroyd. 1995. Restriction enzyme-mediated integrating elevates transformation frequency and enables co-transfection of Toxoplasma gondii. Molecular and Biochemical Parasitology 74, 55-63
Bolker, M., H.U. Bohnert, K.H. Braun, H. Gorl, H.B. Jansson, A. Tunlid, and B. Nordbring-Hertz. 1997. Biological control: nematodes, p. 38-50. In Anke, T., (ed). Fungal Biotechnology. Chapman and Hall, Weinheim, Germany
Kuspa, A. and W.F. Loomis. 1992. Tagging developmental genes in Dictyostelium by restriction enzyme-mediated integration of plasmid DNA. Proc. Natl. Acad. Sci. USA 89, 8803-8807
Liang, P.Y., H.D. Liu, and X.F. Xiao. 1981. Some factors affecting the formation and regeneration protoplasts in two Penicilium chrysogenum auxotrophs. Acta Phytothysiologia Sinica 7, 1-10
Lu, S., L. Lyngholm, G. Yang, C. Bronson, O.C. Yoder, and B.G. Turgeon. 1994. Tagged mutations at the Tox1 locus of Cochliobolus heterostrophus by restriction enzyme-mediated integration. Proc. Natl. Acad. Sci. USA 91, 12649-12653
Persson, Y., S. Erland, and H-B. Jansson. 1996. Identification of nematode-trapping fungi using RFLP analysis of the PCRamplified ITS region of ribosomal DNA. Mycol. Res. 5, 531- 534
Riggle, P.J. and C.A. Kumamoto. 1998. Genetic analysis in fungi using restriction-mediated integration. Curr. Opin. Microbiol. 1, 395-399
Rosen, S., K. Sjollema, M. Veenhuis, and A. Tunlid. 1997. A cytoplasmic lectin produced by the fungus Arthrobotrys oligospora functions as a storage protein during saprophytic and parasitic growth. Microbiology 143, 2593-2604
Sanchez, O., R.E. Navarro, and J. Aguirre. 1997. Increased trans formation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI). Mol. Gen. Genet. 258, 89-94
Schiestl, R.H. and T.D. Petes. 1991. Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 88, 7585-7589
Scholler, M., G. Hagedorn, and A. Rubner. 1999. A reevaluation of predatory Orbiliaceous fungi. II. A new genetic concept. Sydowia 51, 89-113
Shi, Z., D. Christian, and H. Leung. 1995. Enhanced transformation in Magnaporthe grisea by restriction enzyme mediated integration of plasmid DNA. Phytopathology 85, 329-333
Stirling, G.R., L.J. Smith, K.A. Licastro, and M.E. Lois. 1998. Control of root-knot nematode with formulations of the nematodetrapping fungus Arthrobotrys dactyloides. Biol. Control 11, 224-230
Tanaka, A., H. Shiotani, M. Yamamoto, and T. Tsuge. 1999. Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-Toxin in the Japanese pear pathotype of Alternaria alternata. Mol. Plant-Microbe Interact. 12, 691-702
Tunlid, A., J. Ahman, and R.P. Oliver. 1999. Transformation of the nematode-trapping fungus Arthrobotrys oligospora. FEMS Microbiology Letters 173, 111-116
Tunlid, A. and S. Jansson. 1991. Proteases and their involvement in the infection and immobilization of nematodes by the nematophagous fungus Arthrobotrys oligospora. Appl. Environ. Microbiol. 57, 2868-2872
Zhao, M.L., M.H. Mo, and K.Q. Zhang. 2004. Characterization of a neutral serine protease and its full-length cDNA from the nematode-trapping fungus Arthrobotrys oligospora. Mycologia 96, 16-22
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