• 한국식물병리학회지
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• 제11권1호
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• pp.66-72
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• 1995

The coat protein (CP) gene of cucumber mosaic virus-As (CMV-As) strain was engineered for expression in the plant by using the cauliflower mosaic virus 35S transcript regulatory sequences. The CP gene was cloned into an Agrobacterium-derived binary vector. A chimeric gene was constructed by the cDNA of CMV-As CP and plant expression vector pBI121. The clone, pCMAS66, was first introduced into the phagemid vector pSPORT1 for situating sense orientation for translation and making restriction sites in order to re-introduce plant expression vector, pHI121. The resulting subclone pCASCP02 and plant expression vector pBI121 were treated with BamHI-SacI for excising the target gene and removing GUS gene, respectively. After Agrobacterium transformation by freeze-thaw technique, the clone, pCMASCP121-123 which contains sense orientation of the target gene, was selected and confirmed by restriction endonuclease analysis. The CMV-As CP gene was introduced into A. tumefaciens. The results on tobacco plant transformation with the vector system revealed that the system could be successfully introduced and showed high frequency of selection to putative transformations.

• KSBB Journal
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• 제8권2호
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• pp.104-109
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• 1993

한 개의 pladsmid에 Gus gene과 Hygromycin resistant gene(Hpt)을 합친 vector를 개발하였다. 이 재조합된 DNA를 벼의 건조한 종자에 imbibition시켜 형질전환했을 때, hygromycin 배지에서 선별한 결과 그 발현율은 single Hpt vector와 차이 가 없었으며, 주로 뿌리의 생장점이나 자엽초에서 Gus expression을 볼 수 있였다. 또한 hygromycin 배지에서 선별되어 성체가 된 개체에서 그 genomic DNA를 뽑아 PCR을 한 결과 1Kb Hpt gene을 확인하였다. 그리고 생체에서 추출한 총 RNA에서 cDNA를 만든 후 reverse transcription PCR을 통하여, 외부 유전자의 발현을 증명하였다.

• The Plant Pathology Journal
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• 제32권4호
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• pp.371-376
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• 2016

Grapevine Algerian latent virus (GALV) is a member of the genus Tombusvirus in the Tombusviridae and infects not only woody perennial grapevine plant but also herbaceous Nicotiana benthamiana plant. In this study, we developed GALV-based gene expression and virus-induced gene silencing (VIGS) vectors in N. benthamiana. The GALV coat protein deletion vector, pGMG, was applied to express the reporter gene, green fluorescence protein (GFP), but the expression of GFP was not detected due to the necrotic cell death on the infiltrated leaves. The p19 silencing suppressor of GALV was engineered to inactivate its expression and GFP was successfully expressed with unrelated silencing suppressor, HC-Pro, from soybean mosaic virus. The pGMG vector was used to knock down magnesium chelatase (ChlH) gene in N. benthamaina and the silencing phenotype was clearly observed on systemic leaves. Altogether, the GALV-derived vector is expected to be an attractive tool for useful gene expression and VIGS vectors in grapevine as well as N. benthamiana.

• The Plant Pathology Journal
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• 제33권4호
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• pp.429-433
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• 2017

Chrysanthemums (Chrysanthemum morifolium) are susceptible to tobacco mosaic virus (TMV). TMV-based expression vectors have been used in high-throughput experiments for production of foreign protein in plants and also expressing green fluorescent protein (GFP) to allow visualization of TMV movement. Here, we used TMV expressing the GFP to examine the infection of chrysanthemum by a TMV-based expression vector. Viral replication, movement and GFP expression by TMV-GFP were verified in upper leaves of chrysanthemums up to 73 days post inoculation (dpi) by RT-PCR. Neither wild-type TMV nor TMV-GFP induced symptoms. GFP fluorescence was seen in the larger veins of the inoculated leaf, in the stem above the inoculation site and in petioles of upper leaves, although there was no consistent detection of GFP fluorescence in the lamina of upper leaves under UV. Thus, a TMV-based expression vector can infect chrysanthemum and can be used for the in vivo study of gene functions.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1987년도 식물생명공학 심포지움 논문집 Proceedings of Symposia on Plant Biotechnology
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• pp.241-260
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• 1987

The regulatory mechanisms of gene expression in higher plant were not ascertained in detail because the genome size is very large and complex. However, the above-mentioned study is remarkably progressed in parallel with development of DNA recombinant technology and plant vector system. Some research results connected with the mechanisms could be summarized as follows. 1. Many plant genes including chloroplast genes are cloned. 2. The structures of some regulatory regions of gene expression are determined, and it is confirmed that new regulatory units are made by transposable elements. 3. Plant gene expression is regulated not only at transcriptional level but also at translational level. 4. The factors that regulate plant gene expression could be divided as two categorys. One is endogenous elements including the structural change of chromatin during development stage and tissue differentiation. The other is environmental stimulations such as air, water, heat, salts and light. However, some sufficient research-aid fund is essential in order to study the regulatory mechanisms of gene expression more systematically.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
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• pp.23-33
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• 1985

Plant cell culture is emerging to express bioactive foreign proteins because it has several advantages in that it is safe, economical, genetically stable and eukaryotic expression system comparing with other expression systems. However several limitations such as slow growth rate, low expression level and lack of well established down stream process need to be answered. As a preliminary approach to produce the immunologically interested molecules through the plant cell culture, we tested if granulocyte-macrophage colony stimulating factors (GM-CSFs) from both murine (mGM-CSF) and human (hGM-CSF) are produced as a biologically active form through plant cell culture. The murine and human GM-CSF genes were cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring both recombinant GM-CSF (rGM-CSF) genes. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activities of both murine and human GM-CSF from plant cell culture were confirmed by measuring the proliferation of the GM-CSF dependent FDC-PI and TF-1 cells, respectively.

• The Plant Pathology Journal
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• 제21권2호
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• pp.158-163
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• 2005

Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. However, efficient VIGS has only been studied in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on TRV would produce recognizable phenotypes in soybean. Here, we report that VIGS using the Tobacco rattle virus (TRV) viral vector can be used in several soybean cultivars employing various agro-inoculation methods including leaf infiltration, spray inoculation, and agrodrench. cDNA fragments of the soybean phytoene desaturase(PDS) was inserted into TRV RNA-2 vector. By agrodrench, we successfully silenced the expression of PDS encoding gene in soybean. The silenced phenotype of PDS was invariably obvious 3 weeks after inoculation with the TRV-based vector. Real-time RT-PCR analyses showed that the endogenous level of GmPDS transcripts was dramatically reduced in the silenced leaf tissues. These observations confirm that the silenced phenotype is closely correlated with the pattern of tissue expression. The TRV-based VIGS using agrodrench can be applied to functional genomics in a soybean plants to study genes involved in a wide range of biological processes. To our knowledge, this is the first high frequency VIGS method in soybean plants.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.65.1-65
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• 2003

In addition to the five well-characterized genes of Tobacco mosaic virus (TMV), this virus contains a sixth open reading frame (ORF6) that encodes a 4.8 kDa protein. TMV ORF6 overlaps the ORFs encoding the 30 kDa movement protein and the adjacent 17.5 kDa capsid protein. Although the 4.8 kDa protein could not be detected in vivo, alteration of the AUG codons of this ORF resulted in a mutant virus that attenuated the virulence of the mutated TMV in Nicotiana benthamiana, but not N. tabacum (tobacco). These sequence changes did not affect either the replication or movement of the mutated TMV. Expression of TMV ORF6 from the virus expression vector Potato virus X (PVX) intensified the virulence of this virus in N. benthmiana, but not tobacco, while expression of TMV ORF6 from the virus expression vector Tobacco rattle virus enhanced the pathogenicity observed in both N. benthamima and tobacco. Thus, the TMV ORF6 is a host- and virus-specific. virulence factor. However, two separate assays indicated that the TMV 4.8 kDa protein was not a suppression of RNA silencing. A fusion protein formed between the TMV 4.8 kDa protein and the green fluorescent protein was expressed from the PVX vector and localized to plasmodesmata. Possible roles of the 4.8 kDa protein in pathogenicity will be discussed

• 한국식물학회:학술대회논문집
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• 한국식물학회 1999년도 제13회 식물생명공학심포지움 New Approaches to Understand Gene Function in Plants and Application to Plant Biotechnology
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• pp.45-49
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• 1999

Plant cell culture is emerging to express bioactive foreign proteins because it has several advantages in that it is safe, economical, genetically stable and eukaryotic expression system comparing with other expression systems. However several limitations such as slow growth rate, low expression level and lack of well established down stream process need to be answered. As a preliminary approach to produce the immunologically interested molecules through the plant cell culture, we tested if granulocyte-macrophage colony stimulating factors (GM-CSFs) from both murine (mGM-CSF) and human (hGM-CSF) are produced as a biologically active form through plant cell culture. The murine and human GM-CSF genes were cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring both recombinant GM-CSF (rGM-CSF) genes. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plant. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activities of both murine and human GM-CSF from plant cell culture were confirmed by measuring the proliferation of the GM-CSF dependent FDC-PI and TF-1 cells, respectively.

• Journal of Plant Biology
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• 제38권4호
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• pp.365-371
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• 1995

Binary vectors, pBI-ActR1, pBI-ActF1 and pBSH-ActR1, were constructed using pGA642, the replication origin of pTi12 and the rice actin promoter. The sizes of pBI-ActR1, pBI-ActF1 and pBSH-ActR1 were 12.9 kb, 13.2 kb and 11.95 kb, respectively. These vectors containing a rice actin promoter followed by a GUS structural gene could induce stronly the expression of GUS gene in transformed rice cells. Rice explants from 3-4 day old seedlings after germinatin were cocultured with A. tumefaceins harboring pBI-ActR1, pBI-ActF1 or pBSH-ActR1, and then GUS expression in the explants was assayed. Transformation of rice explants by these binary vectors was tissue-specific, such that the meristematic regions of shoot apex, root and hypocotyl were transformed by these binary vectors.