• The Plant Pathology Journal
• /
• v.19 no.3
• /
• pp.162-165
• /
• 2003

Transformation of Fuji apple (Malus domestica 'Fuji') was performed using Agrobacterium tumefaciens harboring a coat protein (CP) gene of Cucumber mosaic virus (CMV). A plasmid DNA containing the virus CP and NPT II genes was introduced into the loaves of apple by th e Agrobacterium - mediated transformation procedure. Regenerated transformants of the apple were obtained by kanamycin resistance conferred by the introduced NPT II gene. PCR analysis showed that 3 out of 20 putatively selected R0 plant lines contain the CMV-CP gene. Nine putative transgenic lines out of 20 lines were investigated with the PCR analysis; 5 regenerants produced a 450 bp DNA band and 3 regenerants showed a 671 bp DNA band for the NPT II and CMV-CP genes, respectively. Southern hybyidization results demonstrate the successful integration of the CMV-CP gene into the genome of the apple. This is the first report on the generation of useful vius resistance source of transgenic apple for molecular breeding program.

• The Journal of Korean Society of Virology
• /
• v.27 no.2
• /
• pp.115-128
• /
• 1997

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and pRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-${\beta}$-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETAS-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.

• KSBB Journal
• /
• v.11 no.1
• /
• pp.37-45
• /
• 1996

Marine bacterial strain, highly effective agar degrading, was isolated from south sea of Korea and was identified as Pseudomonas sp. This strain was named Halophilic Pseudomonas sp. W7 and accumulated an extracellular agarase which showed a high level of enzyme activity in the presence of agar and agarose. This extracellular agarase was purified by anion-exchange chromatography and gel filtration. Purified agarase showed a single protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated to be about 89KDa. The agarase gene was cloned into Escherichia coli JM83 using the plasmid vector pUC19. DNA fragments(3.7, 3.0Kb) of Hind III-digested chromosomal DNA of Pseudomonas sp. W7 was inserted into the Hind III site of pUC19. Selected transformants, E. coli JM83/pSWl 000000and E. coli JM83/pSW3, produced agarase and this agarase was accumulated In the cytoplasmic space.

• Korean Journal of Microbiology
• /
• v.30 no.1
• /
• pp.1-7
• /
• 1992

We cloned a gene cluster encoding phenanthrene-degrading enzymes on a 6.8-kb Xhol fragment from the Pseudomonas sp. DJ77 chromosomal DNA into the vector pBLUESCRIPT SIC(+). The resultant clone, containing the recombinant plilsmid pHENX7, was able to convert 3-methylcatechol to a yellow mela-cleavage compound. Since the pHENX7R in which the DNA insert was cloned in the opposite orientation lacked extradiol dioxygenase activity. the direction of transcription was established. Four polypeptides, PhnC (24 kDa). PhnD (31 kDa), PhnE (34 kDa). and PhnF (15 kDa), were identified in E coli JM101 transformed with several pHENX7-derived plasmids. The locations and extents of ~ndividual genes were determined by subcloning. The gene order was phnC-phnD-phnE-phnF-phnG, and phnC, phnD, phnE, and phnG genes encoded glutathione S-transferase, mrta-cleavage compound hydrolase, extradiol dioxygenase, mera-cleavage compound dehydrogenase, respectively.

• Korean Journal of Microbiology
• /
• v.30 no.4
• /
• pp.246-251
• /
• 1992

Molccular cloning of nod genes from Bradvrhizobium sp. SNU001, a nitrogen-fixing symbiont isolated from thc root nodules of soybean (Clycine trim) . was carried out. nod genes were found to be located on thc genome of the symbiont by gcnomic hybridization with 4.5 kb EcoRI/HndIII fragment (nod DABC) of Rhizohium meliloti as probe. Genomic library of this symbiont was constructed using h phage EMBL3-BanlHI vector. from which five nod positive clones were sclectcd by primary and secondary screening methods. The partial restriction map of inserted genomic DNA of h CNS-l(c1one 2) was constructed. and 3.9 kh Bun7HI fragment. which showed strong hybridization signal to the probe, was subcloned into pBS KS(+) plasmid vector. Partial restriction inap ot' a selected subclone (pBjCNS-I) was constructed and nod DABC was found to be located on the 1.8 kb KpnI/Sacl fragment of this subclone.

• Korean Journal of Pharmacognosy
• /
• v.28 no.3
• /
• pp.149-155
• /
• 1997

Antaeum(ATE) has been used as a prescription for threatened abortion, associated with pregnancy in traditional medicine. Because gravida could be administered ATE for a long period, its administration might cause a harmful effect on fetus and gravida during the pregnancy. This study aimed to determine whether exposure to ATE caused mutagenicity or hepatotoxicity during the pregnant period. For mutagenicity test of ATE, Salmonella typhimurium and Bacillus subtilis were used as indications for DNA damage. In the Ames test, Samonella typhimurium TA98 and TA100 were used for mutagenicity testing, and the number of histidine revertants was measured. In Rec-assay, Bacillus subtilis H $17(Rec^+)$ and $M-45(Rec^-)$ strains were used to clarify the DNA damage property. In the SOS umu test, Salmonella typhimurium TA15335 containing plasmid pSK1002 was used as a tester strain, and we monitored the levels of umu operon expression by measuring the ${\beta}-galactosidase$ activity. From the tested results, ATE did not show DNA damage and mutagenicity. On the other hand, hepatotoxicity of ATE to female ICR mice was monitored by the measurements of s-GOT, s-GPT and LDH activities after oral feeding for 15 days. ATE did not show significant change of s-GOT, s-GPT and LDH activities in mice sera.

• The Korean Journal of Food And Nutrition
• /
• v.9 no.4
• /
• pp.459-465
• /
• 1996

Bacillus thuringiensis serovar. darmstadiensis produced bipyramidal endo-toxin. The toxin protein was purified by Renografin-76 step gradient centrifugation and investigated by electron microscope. Analysis of total plasmid DNA patterns showed that four different size of plasmids existed in wild type B. thuringiensis serovar. darmstadiensis. Total plasmids DNA was isolated and transformed into pst I site of pBR322 cloning vector. Ten clones containing crystal toxin gene were forst screened colony hybridization by using PUYBT 9044 probe ontained B. thuringiensis kurskaki HD 1 toxin gene. Cloned-DNA was digested with EcoR1 and HindIII and transformed to pIBI30 sequencing vector. Finally, 2.6kb and 3.6kb size fragments contatined toxin-gene were cloned with restriction analysis.

• 한국균학회소식:학술대회논문집
• /
• 2014.10a
• /
• pp.49-49
• /
• 2014

In order to find indigenous beneficial fungal species from crop field soils of Nigeria, 23 soil samples were collected from various places of Nigeria in June, 2013 and fungi were isolated through serial dilution technique. Isolated fungi were purified and differentiated according to their morphological and microscopic characteristics. In total, 38 different representative isolates were recovered and the genomic DNA of each isolates was extracted using QIAGEN$^{(R)}$ Plasmid Mini Kit (QIAGEN Sciences, USA) and the identification of fungi was carried out by sequence analysis of internal transcribed spacer (ITS) region of the 18S ribosomal DNA (18S rDNA). Recovered isolates belonged to 9 fungal genera comprising Fusarium, Aspergillus, Chaetomium, Coniothyrium, Dipodascaceae, Myrothecium, Neosartorya, Penicillium and Trichoderma. Aspergillus spp., Penicillium spp. and Trichoderma spp. were the most dominant taxa in this study. The antagonistic potentiality of species belonged to Trichoderma against 10 phytopathogenic fungi (F. oxysporum, C. gloesporoides, P. cytrophthora, A. alternata, A. solani, S. rolfsii, F. solani, R. solani, S. sclerotiorum and P. nicotiana) was assessed in vitro using dual culture assay. The dual culture assay results showed varied degree of antagonism against the tested phytopathogens. The potential Trichoderma spp. will be further evaluated for their antagonistic and plant growth promotion potentiality under in vivo conditions.

• The Korean Journal of Mycology
• /
• v.17 no.4
• /
• pp.209-213
• /
• 1989

Transformation of an auxotrophic requirement for uracil in Pleurotus florida P101 has been achieved using chimeric vector containing Aspergillus nidulans ans 1, and Neurospora crassa pyr 4 DNA. Protoplasts of $Ura^-$strains of P. florida were incubated with plasmid pDJB3 containing the cloned pyr 4 gene in the presence of polyethylene glycol and $CaCl_2$. Transformants could grow on MMM showing mitotical stability. Southern hybridization analysis of DNA isolated from transformants showed that the Neurospora pyr 4 gene and vector sequence might be integrated into the P. florida chromosomes. As the transformants were monokaryon, each transformant was mated with the other monokaryon. Fruitbody shape of untransformant was eroded type but those of transformants were eroded type, funnel type, plane type and ungrowing cap type.

• Journal of Microbiology
• /
• v.33 no.4
• /
• pp.339-343
• /
• 1995

We isolated a 10 kb Bam HI fragment originated from the chromosome of a $H_2O$$^2$-resistant mutant strain of Streptomyces coelicolor, which confer $H_2O$$^2$-resistance to S. lividance upon transformation. Among various subclones ot 10kb Bam HI fragment tested for their $H_2O$$^2$-resistant phenotype in S. lividans, a subclone containing 5.2 kb Bam HI-BglII fragment was found to be responsible for $H_2O$$^2$-resistance. The plasmid containing this 5.2 kb fragment was then transformed into S. coellicolor A3(2) at early and tested for their phenotype of $H_2O$$^2$-resistance and the change in various enzymes whose activity can be stained in the gel. We found out that the 5.2 kb insert DNA conferred $H_2O$$^2$-resisstance in S. coelicolor A3(2) at early phase of cell growth. The presence of this DNA also resulted in higher level of peroxidase compared with the wild type cell containing parental vector (pIJ702) only. Esterase activity was also higher in this clone. However, alcohol dehydrogenase activity decreased compared with the wild type. These results suggest that the presence of a gene in 5.2 kb BamHI-BglII DNA fragment causes multiple changes in S. coelicolor related to its response against hydrogen peroxide. The result also implies that not only peroxidase but also esterase may function in the defencse meahsnism agianst $H_2O$$^2$-.