• The Plant Pathology Journal
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• 제28권4호
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• pp.373-380
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• 2012

Burkholderia pyrrocinia CH-67 is a biocontrol bacterium with strong antifungal activity against several plant pathogenic fungi. Transposon mutagenesis was performed to identify the genes responsible for the antifungal activity of B. pyrrocinia CH-67. Of the 2,500 mutants tested using the Fulvia fulva spore screening method, a mutant deficient in antifungal activity, M208, was selected. DNA sequence analysis of the transposon-inserted region revealed that a gene encoding an adenylate kinase-related kinase was disrupted in M208. Antifungal activity was restored in M208 when a full-length adenylate kinase gene with its promoter was introduced in trans. The deduced amino acid sequence of adenylate kinase from CH-67 was 80% identical to that of B. cenocepacia MCO-3. Adenosine diphosphate supplementation or high levels of adenosine triphosphate and adenosine monophosphate together restored antifungal activity in M208, suggesting that adenylate kinase of B. pyrrocinia CH-67 is involved in antifungal activity expression.

• Journal of Microbiology and Biotechnology
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• 제15권3호
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• pp.466-471
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• 2005

An Escherichia coli strain with high phytase activity was screened from pig excreta. The phytase gene, appA, was amplified by PCR technique. To obtain large amounts of appA phytase, the appA gene was subcloned into the baculovirus transfer vector pVL1393 under the control of the Polyhedrin promoter. The recombinant baculovirus harboring the appA gene was obtained after co-transfection and screening. The early $5^{th}$ instar larvae of silkworm were infected with the recombinant virus. Using this system, the appA phytase was overproduced up to 7,710 U per ml hemolymph. SDS-PAGE analysis revealed the baculovirus-derived appA phytase to be approximately 47 kDa in size. The optimal temperature and pH of the expressed phytase were $60^{\circ}C$ and pH 4.5, respectively. The enzymatic activity was increased by the presence of 1 mM $Ca^{2+}$, 1 mM $Mn^{2+}$, or $0.02\%$ Triton X-100.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2001년도 The 8th International Symposium
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• pp.49-58
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• 2001

We tried to introduce two forsythia genes related in lignan biosynthesis, dirigent protein and pinoresinol/lariciresinol (P/L) reductase, into potatoes for accumulation of lignans in transgenic potatoes. We made binary vectors overexpressing dirigent protein gene and P/L reductase gene driven by a CaMV35S promoter and transformed into potatoes via Agrobacterium mediated transformation. And in order to control the metabolic flux of lignan biosynthesis pathway, we tried to inhibit chalcone synthase genes of potatoes by antisense inhibition technique also. We tried to use PCR screening method for selection of transgenic plants of different vectors. We tried to determine and compare lignan contents from different transgenic potato lines.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1459-1463
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• 2006

This study explored yeast diauxic promoters using a green fluorescent protein (GFP) reporter to screen growth phase-controlled promoters applicable for foreign protein production. Twenty-five diauxic promoters were inserted into a yeast 2-micron vector in front of the reporter GFP gene. The expressed GFP signal intensity measurements showed that 23 out of the 25 promoters produced a significant fluorescent signal when the cells were in the diauxic growth phase. Among the two strongest promoters pYDL204W and pYLR258W, the former remained constantly active after its activation at the diauxic shift, whereas the latter was only transiently activated right after the deprivation of the medium glucose.

• Food Science and Biotechnology
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• 제15권4호
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• pp.625-630
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• 2006

Polymerase chain reaction (PCR) method was developed for the specific detection of insect-resistant rice containing cry1Ac gene derived from Bacillus thuringiensis (Bt). Primers were designed from the 35S promoter, NOS terminator, cry1Ac gene, and sucrose phosphate synthase (SPS) for general screening of Bt rice. By sequencing the PCR products from the two putative kinds of Bt rice, we designed a specific primer from the junction region between the cry1Ac gene and the NOS terminator that had been inserted into Bt rice. The construct-specific primer was employed to amplify a 147 bp product in the two lines of Bt rice. No amplified products were observed from the other Bt crops with various Bt genes introduced. In qualitative PCR analysis, the limit of detection was 0.005 ng from genomic DNA of Bt rice. In addition, PCR analysis was performed on 64 kinds of rice presently available in the Korean market, and no Bt rice was detected. This method presented in this paper can be used as a highly sensitive and specific detection method of Bt rice.

• Journal of Microbiology and Biotechnology
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• 제29권7호
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• pp.1137-1143
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• 2019

Hepatitis E virus (HEV) accounts for 20 million infections in humans worldwide. In most cases, the infections are self-limiting while HEV genotype 1 infection cases may lead to lethal infections in pregnant women (~ 20% fatality). The lack of small animal models has hampered detailed analysis of virus-host interactions and HEV-induced pathology. Here, by employing a recently developed culture-adapted HEV, we demonstrated that methyltransferase, a non-structural protein, strongly inhibits melanoma differentiation-associated gene 5 (MDA5)-mediated activation of type I interferon responses. Compared to uninfected controls, HEV-infected cells display significantly lower levels of $IFN-{\beta}$ promoter activation when assessed by luciferase assay and RT-PCR. HEV genome-wide screening showed that HEV-encoded methyltransferase (MeT) strongly inhibits MDA5-mediated transcriptional activation of $IFN-{\beta}$ and $NF-{\kappa}B$ in a dose-responsive manner whether or not it is expressed in the presence/absence of a tag fused to it. Taken together, current studies clearly demonstrated that HEV MeT is a novel antagonist of MDA5-mediated induction of $IFN-{\beta}$ signaling.

• 한국어병학회지
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• 제34권1호
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• pp.17-22
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• 2021

Eukaryotic translation is initiated by either cap-dependent or cap-independent way, and the cap-independent translation can be initiated by the internal ribosomal entry site (IRES). In this study, to know whether the 5'UTR leader sequence of marine birnavirus (MABV) segment A and segment B can act as IRES, bicistronic vectors harboring a CMV promoter-driven red fluorescent gene (mCherry) and poliovirus IRES- or MABV's leader sequence-driven green fluorescent gene (eGFP) were constructed, then, transfected into a mammalian cell line (BHK-21 cells) and a fish cell line (CHSE-214 cells). The results showed that the poliovirus IRES worked well in BHK-21 cells, but did not work in CHSE-214 cells. In the evaluation of MABV's leader sequences, the reporter eGFP gene under the 5'UTR leader sequence of MABV's segment A was well-translated in CHSE-214 cells, indicating 5'UTR of MABV's segment A initiates translation in the cap-independent way and can be used as a fish-specific IRES system. However, the 5'UTR leader sequence of MABV's segment B did not initiate translation in CHSE-214 cells. As the precise mechanism of birnavirid IRES-mediated translation is not known, more elaborate investigations are needed to uncover why the leader sequence of segment B could not initiate translation in the present study. In addition, further studies on the host species range of MABV's segment A IRES and on the screening of other fish-specific IRESs are needed.

• 한국초지조사료학회지
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• 제41권4호
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• pp.242-249
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• 2021

Forage crop management is severely challenged by global warming-induced climate changes representing diverse a/biotic stresses. Thus, screening of valuable genetic resources would be applied to develop stress-tolerant forage crops. We isolated two NAC (NAM, ATAF1, ATAF2, CUC2) transcription factors (ANAC032 and ANAC083) transcriptionally activated by multi-abiotic stresses (salt, drought, and cold stresses) from Arabidopsis by microarray analysis. The NAC family is one of the most prominent transcription factor families in plants and functions in various biological processes. The enhanced expressions of two ANACs by multi-abiotic stresses were validated by quantitative RT-PCR analysis. We also confirmed that both ANACs were localized in the nucleus, suggesting that ANAC032 and ANAC083 act as transcription factors to regulate the expression of downstream target genes. Promoter activities of ANAC032 and ANAC083 through histochemical GUS staining again suggested that various abiotic stresses strongly drive both ANACs expressions. Our data suggest that ANAC032 and ANAC083 would be valuable genetic candidates for breeding multi-abiotic stress-tolerant forage crops via the genetic modification of a single gene.

• 생명과학회지
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• 제23권2호
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• pp.167-174
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• 2013

Peroxiredoxin 6 (Prx 6)는 티올-특이적 항산화 단백질에 속하는 효소로서 산화적 스트레스로부터 세포를 보호할 뿐만 아니라 과산화물의 환원작용을 촉매한다. 본 연구에서는 인간 Prx 6의 promoter를 이용하여 항산화반응을 유발하는 추출물을 효과적으로 스크리닝하는 새로운 형질전환마우스를 개발하는 최종목적을 달성하기 위한 중간단계로서, hPrx 6/Luc 벡터를 개발하고, 이들 벡터의 안정적 발현과 성공적 반응성을 세포주를 이용하여 확인하고자 하였다. 이를 위해, 인간 Prx 6 promoter를 증폭하여 luciferase cDNA와 결합한 hPrx 6/Luc 벡터를 제조하였으며, 제조된 벡터를 제한효소 절단과 염기서열분석을 통해 확인하였다. hPrx 6/Luc 벡터는 NCI-H460 세포에 transfection한 후 인삼(KWG), 홍삼(KRG), 맥문동(LP), 홍문동(RLP)의 4가지 추출물을 처리하여 luciferase activity를 측정하였다. 그 결과, luciferase activity는 4가지 추출물에 의해 효과적으로 증가하였고, 특히 KRG과 LP를 처리한 그룹이 KWG과 RLP를 처리한 그룹보다 높았다. 또한, luciferase activity는 RLP 농도에 의존적으로 증가하였다. hPrx 6/Luc 벡터와 hPrx 6 mRNA반응의 차이를 비교하기 위해, 4가지 추출물을 처리한 후 hPrx 6 mRNA의 양을 RT-PCR로 분석하였다. 그러나, hPrx 6 mRNA의 양은 비록 고농도의 RLP 추출물에서는 약간의 증가가 관찰되었지만, 대조군에 비하여 4가지 추출물에서 유의적인 차이가 없었다. 한편, 4가지 추출물에 의한 superoxide dismutase (SOD) 활성의 변화는 비록 일부 차이는 있었지만 hPrx 6/Luc 벡터와 유사한 반응을 나타내었다. 따라서, 이러한 결과는 hPrx 6/Luc 벡터는 성공적으로 제조되었고, 세포내에서 안정적으로 발현하면서 항상화물질에 민감하고 정량적으로 반응할 수 있음을 제시하고 있다. 더불어, 이러한 세포주에서 확인결과를 바탕으로 형질전환마우스가 개발된다면, 항산화물질을 정량적으로 스크리닝하는 시스템으로 적용가능성이 매우 높음을 보여주고 있다.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7467-7472
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• 2013

Background: Polymorphisms of genes encoding cytokines could be potential biomarkers to predict risk of gastric cancer (GC). Here, we investigated the association between the IL-6 -6331 (T/C, rs10499563) polymorphism in its promoter region and GC risk. Methods: In this case-control study of 215 GC cases and 518 non-cancer controls, the IL-6 -6331 (T/C, rs10499563) polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: Individuals with the TC or CC genotype were associated with a significantly decreased risk of GC (OR=0.710, 95%CI: 0.504-0.999, P=0.049) compared with TT wild-type carriers. Ther C allele was also associated with significantly decreased risk of GC (OR=0.715, 95%CI: 0.536-0.954, P=0.023) compared with the T allele. In the stratification analysis, TC or CC genotypes were associated with significantly decreased GC risk in subgroups of males, people older than 60, and H. pylori-positive cases. However, no significant interaction was observed for TC or CC genotypes with H. pylori infection. On stratification with the Lauren classification, TC or CC genotypes were associated with significantly decreased risk of diffuse-type GC (OR=0.497, 95%CI: 0.266-0.925, P=0.027), also in subgroups of males, people older than 60, and H. pylori-positive cases. Conclusions: The IL-6 -6331 (T/C, rs10499563) polymorphism is associated with genetic susceptibility of GC and may have the potential to predict GC risk.