• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.51-62
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• 1987

Higher plant transformation vector systems are mainly developed based on the natural biosystems which infecting higher plants. Two major groups attracting much of the research are Cauliflower mosaic virus and Agrobacterium tumefaciens. Cauliflower mosaic virus has a double stranded genome, and a portion of the genome can be substituted for a foreign DNA segment without loosing the ability of infection. A. tumefaciens carries a large plasmid. Ti plasmid whose portion can be substitute and trasferred into the plant chromosome.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.2
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• pp.103-108
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• 2000

To produce of transgenic orchardgrass, the seed-derived calli of orchardgrass (Dactylis glomerata L.) co-cultivated with Agrobacterium turnefaciens EHAlOl harboring binary vector pIG121-Hm were selected with hygromycin and then transferred onto N6 regeneration medium containing 1 rngl l of NAA, 5 rngl l of kinetin, 250 rngl l of carbenicillin and 50 mg/ l of hygromycin. The efficiency of transformation was differed on cultivars, that is, 'Potomac' appeared 12% of transformation efficiency while 'Amba' did 5.5%. The addition of acetosyringone during co-cultivation was a key to successhl transformation of orchardgrass. Transgene fragments were identified by PCR analysis and the constitutive expression of GUS gene was confirmed by Northern blot analysis. (Key words : Acetosyringone, Agrobacterium tumefaciens, Orchardgrass (Dactylis glomerata L.), Transformation)

• Korean Journal of Microbiology
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• v.25 no.3
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• pp.173-179
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• 1987

Ti plasmids were isolated from three strains of Agrobacterium tumefaciens in Korea and their types and molecular weights were determined. All of these are octopine-type and their molecular weights are 44Kb (pTi 12), 180Kb (pTi 14) and 172Kb (pti 49), respectively. In order to construct physical map of pTi 12, pTi 12 was digested with restriction endonucleases Sma I and Hind III. Sma I degestion of pTi 12 produce 8 fragments and Hind III produced 10 fragments. Physical arrangements of these fragments was determined by Southern hybridization techniques.

• Korean Journal of Microbiology
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• v.21 no.4
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• pp.238-244
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• 1983

In order to obtain Ti plasmid which may be used in genetic engineering of higher plants, virulent Agrobacterium tumefaciens were isolated from soil with selective medium. For their identification, morphological and cultural characteristics were investigated in parallel with physiological and turnorigenesis test. 14 strains were isolated from various regions in Korea. 3 of them were identified as biotype 1 and the others were so heterogeneous that they are not yet conformed to any biotypes. However, Ti plasmid was isolated from A. tumefaciens KU 14 strain, one of the above mentioned 14 strains.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.145-148
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• 2006

A new functional lipolytic enzyme (AT4) has recently been found from Agrobacterium tumefaciens C58 Cereon using a genome-wide approach. The enzyme has some sequence similarity to E. coli acetyl hydrolase, Emericella nidulans lipase, Moraxella sp. lipase, Acinetobacter lwoffii esterase, and Streptomyces hygroscopicus acetyl hydrolase. However, the sequence similarities are very low (less than $25\%$), suggesting that it is a new lipase/esterase enzyme. ill the present study, intact cell of the A. tumefaciens strain was shown to have lipolytic activity on a tributyrin-LB plate. The AT4 gene was then expressed at a high level in E. coli BL21 (DE3) cells and the enzyme was purified simply by Ni-NTA column chromatography. The purified enzyme showed hydrolytic activity toward p-nitrophenyl caproate, but not toward olive oil, suggesting that the AT4 enzyme was a typical esterase rather than lipase. AT4 esterase had a maximum hydrolytic activity at $45^{\circ}C$ and pH 8.0, when p-nitrophenyl caproate was used as a substrate. It was relatively stable up to $40^{\circ}C$ and at pH 5.0-9.0. Calcium ion and EDT A did not affect the activity and thermal stability of the enzyme. As for substrate specificity, AT4 enzyme could rapidly hydrolyze acetyl and butyl groups from p-nitrophenyl esters and 1-naphthyl esters. In addition, it also released acetyl residues from acetylated glucose and xylose substrates. Therefore, this new esterase enzyme might be used as a biocatalyst in acetylation and deacetylation reactions performed in the fine chemical industry.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.118-125
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• 2006

The region responsible for replication of the megaplasmid pAtC58 in the nopaline-type Agrobacterium tumefaciens strain C58 was determined. A derivative ofa Co1E1 vector, pBluscript SK-, incapable of autonomous replication in Agrobacterium spp, was cloned with a 7.6-kb Bg1II-HindIII fragment from a cosmid clone of pAtC58, which contains a region adjacent to the operon for the utilization of deoxyfructosyl glutamine (DFG). The resulting plasmid conferred resistance to carbenicillin on the A. tumefaciens strain UIA5 that is a plasmidfree derivative of C58. The plasmid was stably maintained in the strain even after consecutive cultures for generations. Analysis of nested deletions of the 7.6-kb fragment showed that a 4.3-kb BglII-XhoI region sufficiently confers replication of the derivative of the ColE1 vector on UIA5. The region comprises three ORFs, which have high homologies with repA, repB, and repC of plasm ids in virulent Agrobacterium spp. including pTiC58, pTiB6S3, pTi-SAKURA, and pRiA4b as well as those of symbiotic plasmids from Rhizobium spp. Phylogenie analysis showed that rep genes in pAtC58 are more closely related to those in pRiA4 than to pTi plasmids including pTiC58, suggesting that the two inborn plasmids, pTiC58 and pAtC58, harbored in C58 evolved from distinct origins.

• Horticultural Science & Technology
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• v.17 no.6
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• pp.770-772
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• 1999

This study was conducted to provide a basic system to develop a molecular marker for plant cultivar protection using a recombinant DNA technology. Using Nicotiana tabacum L. plants, the potentiality in the utilization of the developed marker was examined. After homology test with several plant genomes, mouse adenosine deaminase (ADA) gene was selected as DNA source of a molecular marker for cultivar protection. As a result of the digestion of ADA gene with BamHI and Pst I, six DNA fragments were obtained, and 513 bp DNA fragment among them was selected as a possible DNA marker for cultivar protection. Selected 513 bp DNA fragment was efficiently inserted into pBI101 plasmid vector for plant transformation by using phagemid vector pBluescript II SK (+/-) as an intermediate vector. The recombinant pBI101, carrying 513 bp DNA fragment, possible markers for cultivar protection, was transformed into A. tumefaciens LBA4404. Nicotiana tabacum was transformed with A. tumefaciens LBA4404 having the recombinant pBI101 and was confirmed the transfer of 513 bp DNA fragment, a possible molecular marker for cultivar protection.

• Mycobiology
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• v.49 no.5
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• pp.491-497
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• 2021

An endolichenic fungus Xylaria grammica EL000614 produces grammicin, a potent nematicidal pyrone derivative that can serve as a new control option for root-knot nematodes. We optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for X. grammica to support genetic studies. Transformants were successfully generated after co-cultivation of homogenized young mycelia of X. grammica with A. tumefaciens strain AGL-1 carrying a binary vector that contains the bacterial hygromycin B phosphotransferase (hph) gene and the eGFP gene in T-DNA. The resulting transformants were mitotically stable, and PCR analysis showed the integratin of both genes in the genome of transformants. Expression of eGFP was confirmed via fluorescence microscopy. Southern analysis showed that 131 (78.9%) out of 166 transformants contained a single T-DNA insertion. Crucial factors for producing predominantly single T-DNA transformants include 48 h of co-cultivation, pretreatment of A. tumefaciens cells with acetosyringone before co-cultivation, and using freshly prepared mycelia. The established ATMT protocol offers an efficient tool for random insertional mutagenesis and gene transfer in studying the biology and ecology of X. grammica.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.322-328
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• 2005

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the $\beta$-galactosidase activity ($\beta$-GAL) of a recombinant Agrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing $\beta$-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration­dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of $\beta$-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.

• Journal of Plant Biology
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• v.34 no.4
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• pp.331-339
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• 1991

To elucidate the regulatory mechanism of virE operon from vir regions (virA, virB, virC, virD, virG, virE) of pTiA6 which have been known to be essential for efficient crown gall tumorigenesis in plants, the activity of the truncated virE, promoter was analyzed. pSM358cd, a recombinant plasmid in which virE :: Tn3-HoHo1 (Tn3-promoterless lacZ) was cloned into SalI site of pVK102, was digested with SalI, and virE :: Tn3-HoHo1 was seperated from pVK102. To construct the truncted virE recombinant plasmids (pJS031, pJS051, pJS102, pJS201, pJS301), 5'-end of vireE promoter was deleted with BAL31 and cloned into pVK102 and then transferred into a. tumefaciens A348(pTiA6). According to the activity of the truncated virE promoter in recombinant plasmids, they were classified into two groups, pJS031, pJS051, pJS101 and pJS201 belong to a functional group and pJS301 is a non-functional. The size of deleted nucleotides of pJS201 and pJS301 seemed to be about 130 nucleotides and about 250 nucleotides from 5'-end of virE promoter, respectively. Hence it was thought that the essential site of the virE promoter was located between about 130th nucleotide and 250th nucleotide from 5'-end of the virE promoter.