• 한국미생물생명공학회:학술대회논문집
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• 한국미생물생명공학회 2001년도 Proceedings of 2001 International Symposium
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• pp.62-72
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• 2001

The N-carbamoyl-D-amino acid amidohydrolase (D-carbamoylase) gene (dcb) from Agrobacterium tumefaciens AM 10 has been successfully cloned and expressed in Escherichia coli. Expression of D-carbamoylase gene under the 17 promoter in different host strains showed that the optimal expression was achieved in E. coli JM109 (DE3) with a 9-fold increase in enzyme production compared to the wild-type strain. The co-expression of the GroEL/ES protein with D-carbamoylase protein caused an in vivo solubilization of D-carbamoylase in an active form. The synergistic effect of GroEL/ES at 28$^{\circ}C$ led to 60 ％ solubilization of the total expressed target protein with a 6.2-fold increase in enzyme activity in comparison to that expressed without GroEL/ES and 43-fold increase in enzyme activity compared to A. tumefaciens AM 10. Attempts to express D-carbamoylase in an altered redox cytoplasmic milieu did not improve the enzyme production in an active form. The Histidyl-tagged D-carbamoylase was purified in a single step by Nickel-affinity chromatography and was found to have a specific activity of 9.5 U/mg protein.

• 한국초지조사료학회지
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• 제18권1호
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• pp.69-76
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• 1998

This study was conducted to construct of the transgenic plants wliich are resistant to oxidative stresses including ozone with B. mpestris cytosolic glutathione reductase cDNA using the binary vector system of Agrobacterium tumefaciens. The 1.8kb B. campestris cytosolic GR cDNA was subcloned into the unique Sma I site of the plant transformation vector pBKSI- I, downstream of the constitutive CaMV 35s promoter and upstream of the nos termination sequence, in place of the uidA (GUS) reporter gene. The resulting plant transformation vector, pBKS-GRI, was introduced into A. tumefaciens LBA4404 by two cycles of tkeze-thaw method. The B. nqus cotyledonary petioles were transformed by the Agrubaferium harboring pBKS-GRI. Transformed shoots were induced and selected on regeneration medium supplemented with kanarnycin. The shoot formation was increased remarkably by addition of Ag$NO_3$, in MS media. The transgenic plants were analyzed for the presence of the B. campestris GR gene by Southern blot analysis and it was confirmed that a foregin gene was stably integrated into the genomes of B. nqus plants.

• Journal of Microbiology and Biotechnology
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• 제17권2호
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• pp.226-233
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• 2007

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NTI (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-$_{DL}$-homoserine lactone ($C_8-HSL$) and N-decanoyl-$_{DL}$-homoserine lactone ($C_{10}-HSL$).

• The Plant Pathology Journal
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• 제35권1호
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• pp.19-31
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• 2019

Bud rot (BR) is the most devastating disease affecting oil palm (Elaeis guineensis) crops in Colombia. Its causal agent, Phytophthora palmivora, initiates the infection in immature oil palm leaflets producing necrotic lesions, followed by colonization of opportunistic necrotrophs, which increases disease damage. To improve the characterization of the disease, we transformed P. palmivora using Agrobacterium tumefaciens-mediated transformation (ATMT) to include the fluorescent proteins CFP-SKL (peroxisomal localization), eGFP and mRFP1 (cytoplasmic localization). The stability of some transformants was confirmed by Southern blot analysis and single zoospore cultures; additionally, virulence and in vitro growth were compared to the wild-type isolate to select transformants with the greatest resemblance to the WT isolate. GFP-tagged P. palmivora was useful to identify all of the infective structures that are commonly formed by hemibiotrophic oomycetes, including apoplastic colonization and haustorium formation. Finally, we detected cell death responses associated with immature oil palm tissues that showed reduced susceptibility to P. palmivora infection, indicating that these tissues could exhibit age-related resistance. The aim of this research is to improve the characterization of the initial disease stages and generate cell biology tools that may be useful for developing methodologies for early identification of oil palm materials resistant or susceptible to BR.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.108.1-108
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• 2003

Soil metagenome is untapped total microbial genome including that of the majority of unculturable bacteria present in soil. We constructed soil metagenomic library in Escherichia coli using DNA directly extracted from two different soils, pine tree rhizosphere soil and forest topsoil. Metagenomic libraries constructed from pine tree rhizosphere soil and forest topsoil consisted of approximately 33,700 clones and 112,000 clones with average insert DNA size of 35-kb, respectively. Subsequently, we screened the libraries to select clones with antimicrobial activities against Saccharomyces cerevisiae and Agrobacterium tumefaciens using double agar layer method. So far, we have a clone active against S. cerevisiae and a clone active against A. tumefaciens from the forest topsoil library. In vitro mutagenesis and DNA sequence analysis of the antifungal clone revealed the genes involved in the biosynthesis of antimicrobial secondary metabolite. Metagenomic libraries constructed in this study would be subject to search for diverse genetic resources related with useful microbial products.

• 한국초지조사료학회지
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• 제33권2호
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• pp.100-104
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• 2013

포장에서 생육된 잔디 포복경을 이용한 Agrobacterium 형질전환에 있어서 큰 제한인자였던 곰팡이 오염을 제거할 목적으로 포복경 조직에 대한 새로운 살균법을 개발하고자 하였다. 여러 가지 살균방법 중에서 30% NaOCl로 15분간 처리한 다음 0.1% $HgCl_2$로 25분간 처리 했을 때 포복경 절편체의 생존율이 높았으며, 0.1% $HgCl_2$로 처리시 800 mbar의 진공처리를 5분간 실시했을 때 가장 효과적이었다. 또한 Agrobacterium과 공동배양 시 2.5 mg/l의 amphotericin B를 첨가해 준 배지에서 배양했을 때 가장 높은 생존율을 나타내었다. AmB의 처리는 Agrobacterium의 생장에 영향을 미치지 않았다. 또한 살균된 포복경으로 부터 신초의 재분화에도 영향을 미치지 않았으며 곰팡이 오염만을 효율적으로 억제하는 것으로 나타났다. 이러한 결과는 포장에서 대량으로 생육시킨 잔디 포복경을 이용한 Agrobacterium 형질전환 시 그 효율을 증가시키는데 큰 기여를 할 것으로 추측된다.

• Journal of Microbiology and Biotechnology
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• 제18권2호
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• pp.234-241
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• 2008

Agrobacterum tumefaciens-mediated transformation (ATMT) is becoming an effective system as an insertional mutagenesis tool in filamentous fungi. We developed and optimized ATMT for two Colletotrichum species, C. falcatum and C. acutatum, which are the causal agents of sugarcane red rot and pepper anthracnose, respectively. A. tumefaciens strain SK1044, carrying a hygromycin phosphotransferase gene (hph) and a green fluorescent protein (GFP) gene, was used to transform the conidia of these two Colletotrichum species. Transformation efficiency was correlated with co-cultivation time and bacterial cell concentration and was higher in C. falcatum than in C. acutatum. Southern blot analysis indicated that about 65% of the transformants had a single copy of the T-DNA in both C. falcatum and C. acutatum and that T-DNA integrated randomly in both fungal genomes. T-DNA insertions were identified in transformants through thermal asymmetrical interlaced PCR (TAIL-PCR) followed by sequencing. Our results suggested that ATMT can be used as a molecular tool to identify and characterize pathogenicity-related genes in these two economically important Colletotrichum species.

• KSBB Journal
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• 제5권3호
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• pp.247-253
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• 1990

고등식물의 형질전환용 유전자운반체로써 가장 많이 사용되고 있는 Ti-plasmid를 이용해서 당근세포를 형질 전환시키기 위한 연구의 일환으로 Agrobacterium spp를 helper로 이용하여 NPT II gene를 함유하고 있는 binary vector GA472를 당근세포에 삽입시켜 kanamycin에 대해 저항성을 나타내는 세포주를 선발하고자 본 연구를 수행하였다. 국내 토양에서 선발한 A.tumefaciens 2종과 disarmes된 PC2760 그리고 hypervirulent균주인 A281에 tri-parental mating 방법에 의해서 binary vector인 pGA472을 도입하여 transconjungants인 A. tumefaciens c-23-1/pGA472,K29-1/pGA472, PC2760/PGA472 그리고 A281/pGA472를 획득하였다. Transconjungants는 plasmid의 분리, 정제방법에 의해서 추출한 후 0.7% agarose gel 상에서 관찰해 본 결과 4균주 공히 NTPII gene이 삽입된 pGA472와 Ti-plasmid를 함유하고 있는 것을 확인 하였다. 확인된 conjugant와 당근정상조직을 동시배양방법에 의해서 형질전환을 유도한 후 정상조직은 전혀 생존이 되지않은 kanamycin에 대해서 저항을 나타내는 callus를 선발할 수 있었다.

• 식물조직배양학회지
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• 제25권4호
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• pp.225-229
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• 1998

상추의 종자 무균발아후 4일된 자엽조직을 GUS 유전자가 도입된 A. tumefaciens LBA 4404와 2일간 공동배양한 다음 0.1mg/L NAA, 1.0mg/L 2ip, 50mg/L kanamycin, 500mg/L carbenicillin이 첨가된 MS 배지에 배양하여 식물체를 재분화시켰다. PCR 분석결과 GUS 유전자가 형질전환된 식물체의 게놈상에 삽입되어 있음을 확인하였다. 해부학적 GUS 활성을 분석하여 형질전환된 식물체의 줄기, 잎 그리고 뿌리에서 GUS 유전자의 발현을 확인하였다. 형질전환체로 확인된 식물체를 자가수정시켜 얻어진 종자의 GUS 활성을 분석하여 GUS 유전자가 발현되는 것을 확인하였다.

• 식물조직배양학회지
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• 제22권2호
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• pp.95-99
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• 1995

본 연구는 이미 확립되어 있는 고려인삼의 체세포배발생을 통한 식물체 재분화와 Agrobacterium을 매개로 한 형질전환 시스템을 이용하여 항곰팡이성 인삼을 개발하고자 염기성인 강낭콩 키틴가수분해효소 유전자를 인삼으로 도입하였다. CaMV 35S promoter-강낭콩 키틴가수분해효소 유전자와 선발표지로서의 neomycin phosphotransferase II (NPT II) 유전자를 가진 pChi/748 binary 벡터를 pGA748로부터 제조하여 이를 도입한 A. tumefacience LBA4404와 인삼 접합배의 자엽절편을 1 mg/L 24-D, 0.1 mg/L kinetin이 첨가된 MS 액체배지에서 48시간 동안 공동배양한 후 동일배지에 100 mg/L kanamycine 500 mg/L carbenicillin을 첨가한 고체 배지에 옮겨 배양하였다. 배양 한달 후부터 절편의 절단면 부근으로부터 캘러스가 유도되기 시작하였으며 이어서 수많은 체세포배가 형성되었다. 이들 체세포배를 BA와 GA3가 각각 1 mg/L 첨가된 배지로 옮겨서 5주 경과되었을 때 식물체로 전환되었다. 재분화된 개체 중 선발된 8개의 식물체로부터 PCR과 이 산물의 Southern분석 결과 6개의 재분화 개체에서 강낭콩 키틴가수분해효소 유전자가 도입되었음을 확인하였다.