• The Plant Pathology Journal
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• 제25권3호
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• pp.286-290
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• 2009

The stability of three different kinds of pUC-derived plasmids, pDsRed, pZsYellow, and pGFPuv, was investigated in Pectobacterium strains to utilize those plasmids as tracers. All three plasmids pDsRed, pZsYellow and pGFPuv showed their specific colors in Pectobacterium strains. Especially, the plasmid pDsRed conferred bright pink colonies on the Pectobacterium strains. When the bacteria lost the plasmid pDsRed, the colonies turned white, suggesting that the plasmid could be a good marker system for Pectobacterium strains on different environmental conditions. The effect of the antibiotic pressure on the stability of the plasmid was different depending on the host bacteria. P. carotovorum subsp. betavasculorum was more sensitive to the antibiotic pressure than P. carotovorum subsp. carotovorum Pcc21. However, temperature change significantly affected plasmid stability on both Pectobacterium strains. Almost all strains lost the plasmids with the shift in temperature from $28^{\circ}C$ to $37^{\circ}C$. Presence of the plasmids did not affect bacterial pathogenicity on their own host plants. Among three plasmids, pZsYellow was not useful as a marker because the yellow fluorescent proteins from pZs Yellow were interfered with the yellow natural fluorescence of the plant tissues induced by the defense system. Since the red color of DsRed can be seen with naked eyes, plasmid pDsRed was applicable as a marker. However, the color change was slow so that additional manipulation to increase the expression speed was necessary. Plasmid pGFPuv could serve as a perfect marker without any problem, tracing the reproduction and spread of the plant pathogens perfectly.

• 한국응용곤충학회지
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• 제36권1호
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• pp.96-104
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• 1997

DNA 다형성을 이용한 누에 유전자 해석기술을 개발하기 위하여 광식성 누에 계통 J111과 비광식성계통 $C_3$의 DNA를 분리하여 유전자 은행을 제작하였다. 누에 유전자 은행은 genomic DNA를 EcoRI로 절단한후 pUC18에 ligation 시켜 DH5$\alpha$ E. coli에 형질전환 시켰다. 형질전환 후 얻어진 colony는 15개 누에 품종의 genomic DNA에 hybridization하였을 때 누에의 품종에 관계없이 highly repetitive, moderately repetitive 및 single 혹은 low copy number 로 구분되었다. RFLP마커에 적합한 single 및 low-copy number band만을 형성하는 colony probe을 신속하게 선발하고자 colony또는 genomic DNA로 hybridization하였다. Single 및 low-copy number의 특성을 가진 219개의 clone을 선발하여 Hind III등 8종의 제한효소별로 처리한 genomic DNA를 이용하여 다형성을 검정하여 J111과 $C_3$ 계통간 다형성을 보인 46개의 clone을 선발하였다. 선발된 clone의 일부를 J111과 $C_3$를 교배하여 얻은 $F_2$의 blot에 hybridization 결과 RFLP clone들이 양친검정에 이용가능하여 누에 RFLP 연관 지도 작성의 기반을 조성하게 되었다.

• Journal of Microbiology and Biotechnology
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• 제23권4호
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• pp.545-554
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• 2013

Plasmid isolation of kimchi-derived Weissella cibaria KLC140 revealed six different plasmids. The smallest plasmid, pKW2124, was DNA sequenced and characterized, showing 2,126 bp with a GC content of 36.39% and five putative open reading frames (ORFs). In silico analysis of these ORFs showed ORF1 encodes a putative replication protein similar to rolling circular replication proteins from other lactic acid bacteria. However, a single-stranded intermediate was not detected when S1 nuclease was treated, suggesting it may follow theta replication. Interestingly, the replication initiation site of this plasmid is 100% identical to other plasmids from lactic acid bacteria, suggesting it may function for replication initiation. To construct a surface layer expression vector, pTSLGFP, slpA encoding the surface layer protein from Lactobacillus acidophilus was PCR amplified and fused with the gfp gene, forming a SLGFP fused gene. The plasmid pKW2124 was cloned into the XbaI site of pUC19, forming an Weissella-E. coli shuttle vector pKUW22. NheI-linearized pTSLGFP was ligated into pKUWCAT containing pKUW22 and the chloramphenicol acetyltransferase gene from pEK104, resulting in an 8.6 kb pKWCSLGFP surface layer expression vector. After transformation of this vector into W. cibaria KLC140, a GFP fluorescence signal was detected on the surface of the transformant, substantiating production of SLGFP fused protein and its secretion. This is the first report for construction of a Weissella surface layer expression vector, which may be useful for surface layer production of beneficial proteins in Weissella.