• 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.

• Journal of Plant Biology
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• 제37권1호
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• pp.77-83
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• 1994

종자를 자연건조시킨 상태에서 ${\beta}-glucuronidase$ (GUS) 유전자와 hygromycin phosphotransferase (HPT) 유전자를 가진 plasmid DNA 용액을 imbibition시켰다. GUS 유전자의 경우 품종, vector의 종류, imbibition의 온도에 따라 표지유전자의 발현율에 차이가 있었으며 약 30-50%의 transient expression을 나타내었다. Hygromycin B (HmB)배지에서 선별된 개체의 genomic DNA를 뽑아 외부유전자의 존재를 dot 분석을 통하여 확인하였다. Inverse polymerase chain reaction 결과 만들어지는 생성물을 cloning하고 sequencing한 결과 CaMV35S promoter sequence를 찾았다. Hygromycin이 첨가된 배지에서 선별된 개체들에서 GUS 유전자의 primer를 이용하여 PCR를 수행한 결과 20개체 중 18개체에서 GUS 유전자가 안정되게 존재하여 HmB 배지에서 GUS 유전자의 존재비율은 90%였다. 본 연구의 결과로부터 두 개의 유전자를 소유한 pYJH vector system이 고등식물의 형질전환에 유용하게 이용될 수 있음을 알 수 있었다.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2022년도 추계학술대회
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• pp.221-221
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• 2022

Chromosome breakage occurred by DNA methylation inhibitor. Zebularine is known as DNA methylation inhibitor and suitable for water solubility among different DNA methylation inhibitors as 5-Azacytidine and 5-aza-2'-deoxycytidine. We used zebularine as mutagen according to different methods by roots absorption and seed imbibition. After zebularine treatment, DNA methylation inhibitor, we observed mitotic chromosome behavior what is different according to two different treatment methods. First, seed imbibition treatment in 1,000 μM of zebularine solution for 72 hours in dark conditions. The second treatment to seedlings of Keumkang was also treated in 1,000 μM of zebularine solution for 72 hours after germination. Root and shoot showed different elongations in each treatment. Root absorption treatment(3.01±0.48, 2.00±0.26) showed the shortest elongation in root and shoot than control(8.16±0.61, 4.03±0.48) and seed imbibition treatment(4.33±0.80, 2.48±0.36). It can be explained root tip meristematic cell activity was damaged by DNA methylation inhibitor. Primary root tips were collected in DW for 24 hours at low temperature(0℃) and fixed in fixation solution for 3 days to chromosome observation in mitosis. Mitotic index, chromosome structure and chromosome aberration were observed by phase-contrast microscope. Mitotic index of the control(0.29) showed twice mitotic cells as the treated groups(imbibition 0.15, absorption 0.14). Observation of chromosomes showed some short chromosomes and loosen chromosomes affected by zebularine. It is considered because of zebularine damage DNA in mitosis. We observed "gap by chromosome breakage" in chromosomes that have loose parts between centromere and telomere. It seems demethylation of zebularine occurs chromosome breakage.

• 생명과학회지
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• 제25권2호
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• pp.158-163
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• 2015

쌀의 쌀눈은 배유에 비해 단백질, 지방, 비타민 B1 등의 영양분을 더 많이 함유하고 있다. 본 연구에서는 식물에서 발현 할 수 있는 p53 플라스미드를 제작하였으며, 여러가지의 배지에서 쌀눈 발아의 최적조건을 확립하였다. p53 플라스미드는 pcDNA-p53 플라스미드에서 p53을 얻어내어 TA 벡터에 subcloning을 한 후 식물 플라스미드인 pGEM-CaMV에 p53을 삽입하여 식물에서 발현 가능한 pGEM-CaMV-p53 플라스미드를 제작하였다. 그리고 효율적인 p53 유전자의 도입을 위하여 최적의 팽윤버퍼의 조성 및 배지의 조건을 확립하였다. 팽윤방법을 통한 유전자의 도입에서 팽윤버퍼는 염과 detergent의 서로 다른 농도로 조성하였지만, 이 버퍼조성 사이에서의 쌀눈 발아율의 유의한 차이는 확인되지 않았다. 또한 팽윤된 쌀눈의 발아를 극대화 시키기 위하여 고체한천배지, 액체 배지, 페이퍼 타올 배지의 3가지 조건에 대해서 발아실험을 진행하였다. 그 결과 고체배지에서의 발아율은 70% 정도로 가장 높고 액체배지에서의 발아율은 20% 정도로 가장 낮았으며, 페이퍼 타올배지에서의 발아율은 60% 정도였다. 앞서 확인한 최적의 발아조건에서 쌀눈에 p53 플라스미드를 도입하였고, 그 결과 쌀눈에서의 human p53 발현을 확인 할 수 있었다. 따라서, 팽윤방법에 의한 쌀눈에서의 효율적인 유전자 발현은 쌀의 새로운 부가가 치를 창출할 수 있을 것이다.

• Journal of Microbiology and Biotechnology
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• 제10권3호
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• pp.415-418
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• 2000

Productivity of a rice plant is greatly influenced by salt stress. One of the ways to achieve tolerance to salinity is to transfer genes encoding protective enzymes from other organisms, such as microorganisms. The bacterial gene, mtlD, which encodes mannitol-1-phosphate dehydrogenase (Mtl-DH), was introduced to the cytosol of a rice plant by an imbibition technique to overproduce mannitol. The germination and survival rate of the imbibed rice seeds were markedly increased by transferring the mtlD gene when it was delivered in either a pBIN19 or pBmin binary vector. When a polymerase chain reaction was performed with the genomic DNAs of the imbibed rice leaves as a template and with mtlD-specific primers, several lines were shown to contain an exogenous mtlD DNA. However, a reverse transcription (RT)-PCR analysis revealed that not all of them showed an expression of this foreign gene. This paper demonstrates that the growth and germination of rice plants transiently transformed with the bacterial gene, mtlD, are enhanced and these enhancements may have resulted from the experssion of the mtlD gene. The imbibition method empolyed in this study fulfills the requirements for testing the function of such a putative gene in vivo prior to the production of a stable transgenic plant.

• 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.63-67
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• 1999

We have isolated more than a dozen cDNA clones corresponding to genes that were expressed in Arabidopsis leaves when they were kept in the dark. The nucleotide sequence analysis showed that some of the clones encoded proteins with significant homology to $\beta$-glucosidase (din2), branched-chain $\alpha$-keto acid dehydrogenase subunit E1$\beta$(din3), and another subunit E2 (din4), yeast RAD23 (din5), asparagine synthetase (din6), pre-mRNA splicing factor SRp35 (din7), phosphomannose isomerase (din9), seed imbibition protein (din10), and 2-oxoacid-dependent oxidase (din11). Accumulation of transcripts from din3,4,6 and 10 occurred rapidly after the plants were transferred to darkness. Transcripts from din2,9, and 11 could be detected only after 24 h of dark treatment. Inhibition of photo-synthesis by DCMU strongly induced the accumulation of transcripts from those genes, and application of sucrose to detached leaves suppressed the accumulation both in the dark and by DCMU. These observations indicate that expression of the genes is caused by sugar starvation resulted from the cessation of photosynthesis. We further showed that din2-encoded protein also accumulated in senescing leaves. Given these results, possible roles of din genes in leaves in the dark and senescing leaves are discussed.

• Journal of Plant Biotechnology
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• 제36권3호
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• pp.281-288
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• 2009

Seed germination is the important stage to express many genes for regulation of energy metabolism, starch degradation and cell division from seed dormancy state. For the functional analysis of seed germination mechanisms, we were analyzed the rice cDNA clones (Oryzasativa cultivar Ilpum) obtained from seed imbibition during 48 hours. Total number of 18,101 Expressed Sequence Tags (ESTs) were clustered using SeqMan program. Among them, 8,836 clones were identified as unique clones. We identified the chitinase gene specifically expressed in seed germination and amylase gene involved to starch degradation from the full length cDNA analysis, and several genes were registered to NCBI GeneBank. To analyzed the commonly expressed genes between inmature seed and germinated seed, 25,66 inmature ESTs and 18,101 germinated ESTs were clustered using SeqMan program and identified 2,514 clones as commonly expressed unigene. Among them, alpha-glubulin and alcohol dehydrogenase I were supposed to LEA genes only expressed in the immature and germinated seed stages. For the clustering of orthologous group genes, we further analyzed the 8,836 EST clones from germinating seeds using NCBI clusters of orthologous groups database. Among the clones, 5,076 clones were categorized into information storage and processing, cellular processes and signaling, metabolism and poorly characterized genes, proportioning 783 (14.29%), 1,484 (27%), 1,363 (24.8%) and 1,869 (34%) clones to the previous four categories, respectively.