| Restriction Enzyme-Mediated Integration 방법으로 확보한 Fusarium oxysporum 형질전환체의 후자리산 생성능 분석 |
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이데레사 ( Theresa Lee ) , 신진영 ( Jean Young Shin ) , 손승완 ( Seung Wan Son ) , 이수형 ( Soo Hyung Lee ) , 류재기 ( Jae Gee Ryu ) |
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| Fusaric Acid Production in Fusarium oxysporum Transformants Generated by Restriction Enzyme-Mediated Integration Procedure |
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Theresa Lee*, Jean Young Shin, Seung Wan Son, Soohyung Lee and Jae-Gee Ryu |
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Microbial Safety Team, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-707, Korea |
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Received: October 31, 2013 Revised: November 20, 2013 Accepted: November 22, 2013 |
| Abstract |
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Fusaric acid (FA) is a mycotoxin produced by Fusarium species. Its toxicity is relatively low but often associated with other mycotoxins, thus enhancing total toxicity. To date, biosynthetic genes or enzymes for FA have not been identified in F. oxysporum. In order to explore the genetic element(s) for FA biosynthesis, restriction enzyme mediated integration (REMI) procedure as an insertional mutagenesis was employed using FA producing-F. oxysporum strains. Genetic transformation of two F. oxysporum strains by REMI yielded more than 7,100 transformants with efficiency of average 3.2 transformants/μg DNA. To develop a screening system using phytotoxicity of FA, eleven various grains and vegetable seeds were tested for germination in cultures containing FA: Kimchi cabbage seed was selected as the most sensitive host. Screening for FA non-producer of F. oxysporum was done by growing each fungal REMI transformant in Czapek-Dox broth for 3 weeks at 25oC then observing if the Kimchi cabbage seeds germinated in the culture filtrate. Of more than 5,000 REMI transformants screened, fifty-three made the seeds germinated, indicating that they produced little or fewer FA. Among them, twenty-six were analyzed for FA production by HPLC and two turned out to produce less than 1% of FA produced by a wild type strain. Sequencing of genomic DNA regions (252 bp) flanking the vector insertion site revealed an uncharacterized genomic region homologous (93%) to the F. fujikuroi genome. Further study is necessary to determine if the vector insertion sites in FA-deficient mutants are associated with FA production. |
| Key Words:
Fusaric acid, Fusarium oxysporum, REMI, Transformation |
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