• 식물병연구
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• 제16권3호
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• pp.312-315
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• 2010

본 연구는 애기장대에서 무름병에 대한 저항성 유전자를 탐색하고자 2만여개의 T-DNA 삽입 돌연변이군을 이용하여 Pcc에 대한 스크리닝을 수행하고 이 방법을 소개한 연구다. 1차 선발을 통하여 15개의 저항성 line과 20개의 감수성 line을 선발하였으며, 이로부터 2차 선발하여 3개의 저항성 line과 4개의 감수성 line을 선발하였고, 최종적으로 3차 선발을 통하여 1개의 감수성 line (Atstp1)을 선발할 수 있었다. 현재 Atstp1을 이용해 flanking sequencing 하여 유전자를 탐색하고 있으며, 앞으로 클로닝을 통하여 다양한 무름병 저항성 식물 개발에 유용하게 이용될 것으로 기대한다.

• Journal of Plant Biotechnology
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• 제41권3호
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• pp.127-133
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• 2014

Bacillus thuringiensis 균주로부터 분리한 cry3A 유전자는 딱정벌레목 해충에 살충성을 가지고 있어서 효율적으로 딱정벌레목 해충을 방제 할 수 있다고 알려져 있다. 국립농업과학원에서는 cry3A 유전자 형질전환벼를 육성하였고, GMO 포장 생물검정 연구를 통해 벼물바구미에 대한 내충성을 확인하였다. 본 연구에서는 벼물바구미 내충성 벼 4 계통에 대한 도입유전자 특성을 분자생물학적 방법으로 검정하여 유전자의 도입 사본 수와 도입유전자의 염기서열 등의 분석결과를 제시하였다. BT12R1 계통은 염색체 10번의 exon 부위에 도입유전자가 단일 사본으로 삽입되었으며, BT12R2 계통은 두 개의 사본으로 삽입되었고, BT12R3 계통은 LB 인근의 염기서열만 확인되었다. BT12R4 계통의 경우 단일 사본의 도입유전자가 염색체 5번의 24,516,607 ~ 24,516,636 위치에 30개의 염색체 염기서열이 결실된 상태로 삽입되었다. 이 도입위치는 벼의 내재 유전자가 발현하지 않는 intergenic 부위로 판명되었으며, 도입유전자 이외의 벡터 염기서열이 삽입되지 않음을 확인하였다. 이러한 벼물바구미 내충성벼 BT12R4 계통에 대한 분자생물학적 분석 결과는 차후 GM 작물 실용화의 환경 위해성 및 안전성 평가에 대한 기초자료로 활용할 수 있을 것으로 생각된다.

• The Plant Pathology Journal
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• 제27권2호
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• pp.170-182
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• 2011

Plants possess multiple resistance mechanisms that protect themselves against pathogen attack. To identify unknown components of the defense machinery in Arabidopsis, gene-expression changes were monitored in Arabidopsis thaliana under 18 different biotic or abiotic conditions using a DNA microarray representing approximately 25% of all Arabidopsis thaliana genes (www.genevestigator.com). Seventeen genes which are early responsive to salicylic acid (SA) treatment as well as pathogen infection were selected and their T-DNA insertion mutants were obtained from SALK institute. To elucidate the role of each gene in defense response, bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was inoculated onto individual T-DNA insertion mutants. Four mutants exhibited decreased resistance and five mutants displayed significantly enhanced resistance against Pst DC3000-infection as measured by change in symptom development as compared to wild-type plants. Among them, member of uridin diphosphate (UDP)-glycosyltransferase (UGT) was of particular interest, since a UGT mutant (At1g05680) showed enhanced resistance to Pst-infection in Arabidopsis. In systemic acquired resistance (SAR) assay, this mutant showed enhanced activation of SAR. Also, the enhanced SAR correlated with increased expression of defense-related gene, AtPR1. These results emphasize that the glycosylation of UGT74E2 is a part of the SA-mediated disease-resistance mechanism.

• 원예과학기술지
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• 제34권1호
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• pp.112-121
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• 2016

GM작물의 개발은 병 저항성, 제초제 저항성, 스트레스 저항성, 기능성 물질의 증진 등의 목적으로 기존 작물에 존재하지 않은 새로운 형질을 도입시킬 수 있게 하였다. 이렇게 개발된 GM작물의 상업화를 위해서는 형질전환 식물체에 도입된 T-DNA가 여러 세대에 걸쳐 안정적인 유전과 표현형의 발현이 요구된다. 본 연구는 암 예방 효과가 있는 PEITC 물질이 증가된 형질전환체, IGA 계통의 $T_1$ 및 $T_2$ 세대에서 도입된 T-DNA의 안정성을 확인하였다. 이를 위해 $T_1$ 세대의 IGA 1-3번 계통과 $T_2$ 세대의 IGA 1-3-5번 계통을 PCR로 선발하였으며 선발된 $T_1$ 세대의 IGA 1-3번 계통에서 도입된 T-DNA의 삽입위치를 VA-TAIL PCR로 확인한 결과 배추 게놈 내 intergenic 부위에 삽입되었음을 확인하였다. 또한, 도입 유전자의 세대별 안정성은 IGA 1-3번 계통과 IGA 1-3-5번 계통에서 T-DNA의 내부구조와 flanking 지역을 PCR 분석으로 비교함으로써 확인하였다. 결과적으로 본 연구에서 IGA1번 계통으로 도입된 T-DNA는 구조의 변이 없이 안정적으로 후대로 유전 되었으며, IGA 1번 계통의 $T_1$ 및 $T_2$ 세대의 PEITC 함량의 증가로 표현형적 형질 또한 안정적으로 유전 되는 것으로 확인하였다. 마지막으로 IGA 1번 계통의 $T_1$ 및 $T_2$ 세대에서 QR 활성을 확인하여 암 예방 효과 또한 확인하였다.

• 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.11-15
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• 1999

In order to evlauate feasibility of the gene tagging by the maize transposable element Ac in heterologous plant systems, we have investigated physical distances and directions of transposition of the element in Arabidopsis thaliana and tobacco cultured cell line BY-2. We prepared a T-DNA construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-Scel (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. A number of transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. To examine the pattern of transposition, three out of these transgenic plants were crossed with the Arabidopsis plant that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with I-SceI, sizes of segment of DNA were determined byd pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results with three transgenic lines showed that 50% of all transposition events had occurred within 1,700 kilo-base pairs (kb) on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites. In the present paper, we report typical examples of such gene isolation studies.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
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• pp.51-57
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• 1985

In order to evlauate feasibility of the gene tagging by the maize transposable element Ac in heterologous plant systems, we have investigated physical distances and directions of transposition of the element in Arabidopsis thaliana and tobacco cultured cell line BY-2. We prepared a T-DNA construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-Scel (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. A number of transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. To examine the pattern of transposition, three out of these transgenic plants were crossed with the Arabidopsis plant that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with I-SceI, sizes of segment of DNA were determined byd pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results with three transgenic lines showed that 50% of all transposition events had occurred within 1, 700 kilo-base pairs (kb) on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites. In the present paper, we report typical examples of such gene isolation studies.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2005년도 추계학술대회 및 한일 식물생명공학 심포지엄
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• pp.383-383
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• 2005

• Journal of Microbiology
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• 제45권6호
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• 통합자연과학논문집
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• 제4권2호
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• pp.113-120
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• 2011

Brassinosteroids (BRs) are plant steroid hormones that play essential roles in growth and development. Mutations in BR-signaling pathways cause defective in growth and development like dwarfism, male sterility, abnormal vascular development and photomorphogenesis. Transition from vegetative to reproductive growth is a critical phase change in the development of a flowering plant. In a screen of activation-tagged Arabidopsis, we identified a mutant named abz126 that displayed longer hypocotyls when grown in the dark on MS media containing brassinazole (Brz), an inhibitor of BRs biosynthesis. We have cloned the mutant locus using adapter ligation PCR walking and identified that a single T-DNA had been integrated into the ninth exon of the GIGANTEA (GI) gene, involved in controling flowering time. This insertion resulted in loss-of-function of the GI gene and caused the following phenotypes: long petioles, tall plant height, many rosette leaves and late flowering. RT-PCR assays on abz126 mutant showed that the T-DNA insertion in GIGANTEA led to the loss of mRNA expression of the GI gene. In the hormone dose response assay, abz126 mutant showed: 1) an insensitivity to paclobutrazole (PAC), 2) an altered response with 6-benzylaminopurine (BAP) and 3) insensitive to Brassinolide (BL). Based on these results, we propose that the late flowering and tall phenotypes displayed by the abz126 mutant are caused by a loss-of-function of the GI gene associated with brassinosteroid hormone signaling.

• Food Science and Biotechnology
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• 제15권3호
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• pp.447-452
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• 2006

To determine the functions of specific cell wall polysaccharides, polysaccharides of three mutants, mur3-1, mur3-2, and mur3-3, obtained from Arabidopsis wild type, underwent structural characterization. Upon sequential separation of pectins (RG-I and RG-II) and cross-linking glycans (xyloglucan, XG), only XG was affected by the mud mutation. Wild-type XG contained a considerable amount of fucose, whereas the fucose level in mur3 XGs was less than 20% that of wild type. Further analysis of XGs by matrix-assisted laser-induced/ionization time-of-flight (MALDI-TOF) mass spectrometry indicated that mud lines considerably or completely lost the fucosylated XG oligosaccharides such as XXFG and XLFG and the double-galactosylated oligosaccharide XLLG $^1H$-NMR spectroscopic analyses of the XG oligosaccharides from mur3-3 plant revealed the absence of fucose and a galactose level in the galactosylated side chain that was reduced by 40% compared to that of Arabidopsis wild-type plant. In contrast, 85% less fucose and a slight loss of galactose were observed in the mur3-1 and mur3-2 lines which show normal growth habit. Of the three Arabidopsis mur3 lines studied here, mur3-3 is disrupted by a T-DNA insertion in the exon of MUR3 which encodes XG-specific galactosyltransferase, and exhibits slight dwarfism. These results indicated that the T-DNA insertion at the MUR3 locus did not induce the complete loss of galactose in XG, and that galactose, rather than fucose, in the XG side chains made a major contribution to overall wall strength.