• Journal of Microbiology and Biotechnology
• /
• 제24권9호
• /
• pp.1162-1169
• /
• 2014

Glyphosate is the active component of the top-selling herbicide, the phytotoxicity of which is due to its inhibition of the shikimic acid pathway. 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) is a key enzyme in the shikimic acid pathway. Glyphosate tolerance in plants can be achieved by the expression of a glyphosate-insensitive aroA gene (EPSPS). In this study, we used a PCR-based two-step DNA synthesis method to synthesize a new aroA gene ($aroA_{R.\;leguminosarum}$) from Rhizobium leguminosarum. In vitro glyphosate sensitivity assays showed that $aroA_{R.\;leguminosarum}$ is glyphosate tolerant. The new gene was then expressed in E. coli and key kinetic values of the purified enzyme were determined. Furthermore, we transformed the aroA gene into Arabidopsis thaliana by the floral dip method. Transgenic Arabidopsis with the $aroA_{R.\;leguminosarum}$ gene was obtained to prove its potential use in developing glyphosate-resistant crops.

• 식물조직배양학회지
• /
• 제27권6호
• /
• pp.435-439
• /
• 2000

겨울 상추 품종인 청치마 상추의 자엽조직을 ${\gamma}$-TMT유전자가 도입된 A. tumefaciens LBA 4404와 공동배양한 후, 50 mg/L kanamycin, 500 mg/L carbenicillin, 0.1 mg/L NAA, 0.5mg/L BA가 첨가된 MS재분화 배지에 옮겨 약4주 배양하여 재분화된 신초를 얻었다. 재분화된 신초를 50 mg/L kanamycin, 250mg/L carbenicillin이 포함된 MS 기본 배지에 옮겨 발근된 소식물체를 얻었다. 선발된 형질전환 식물체의 genomic DNA에 대한 PCR분석과 Southern분석을 수행하여 애기장대 ${\gamma}$-TMT유전자 특이적 DNA단편이 상추의 genomic DNA내에 삽입되었음을 확인하였다. 선발된 상추 형질전환체 잎의 $\alpha$-tocopherol/${\gamma}$-tocopherol 함량 비율이 대조 식물체에 비하여 약 4배 증가하여 도입된 ${\gamma}$-TMT유전자가 안정적으로 발현함을 나타내었다.

• 한국잡초학회지
• /
• 제30권3호
• /
• pp.225-232
• /
• 2010

과산화계 제초제 oxyfluorfen이 처리된 비형질전환 벼와 형질전환 벼에서 Protox 발현 위치가 제초제 저항성에 미치는 영향을 비교하였다. Arabidopsis protoporphyrinogen oxidase(Protox; AP 계통)를 색소체에만 발현하는 형질전환 벼와 Myxococcus xanthus Protox 유전자를 색소체와 미토콘드리아에 모두 발현하는 형질전환 벼(TTS 계통)가 형질전환 시스템으로 사용되었다. Oxyfluorfen이 처리된 TTS4 계통은 AP 계통이나 비형질전환 벼에 비해 낮은 수준의 세포질 누출 및 malonyldialdehyde를 보여주었고, 높은 5-aminolevulinic acid 합성 능력을 유지하였다. Oxyfluorfen 작용 동안, TTS4 계통은 AP1 계통보다 높은 제초제 저항성을 보여주었는데, 이는 색소체만에서의 Arabidopsis Protox의 발현에 비해 색소체와 미토콘드리아에서의 M. xanthus Protox의 쌍발현이 광역학적인 protoporphyrin IX의 축적을 더 효율적으로 억제하였기 때문일 것이다. 이 결과들은 미토콘드리아 내 Protox의 발현이 Protox 저해형 제초제에 대한 식물의 저항성에 크게 기여함을 의미한다.

• Molecules and Cells
• /
• 제21권3호
• /
• pp.418-427
• /
• 2006

Ionotropic glutamate receptors (iGluRs) are ligand-gated nonselective cation channels that mediate fast excitatory neurotransmission. Although homologues of the iGluRs have been identified in higher plants, their roles are largely unknown. In this work we isolated a full-length cDNA clone (RsGluR) encoding a putative glutamate receptor from small radish. An RsGluR:mGFP fusion protein was localized to the plasma membrane. In Arabidopsis thaliana overexpressing the fulllength cDNA, glutamate treatment triggered greater $Ca^{2+}$ influx in the root cells of transgenic seedlings than in those of the wild type. Transgenic plants exhibited multiple morphological changes such as necrosis at their tips and the margins of developing leaves, dwarf stature with multiple secondary inflorescences, and retarded growth, as previously observed in transgenic Arabidopsis overexpressing AtGluR3.2 [Kim et al. (2001)]. Microarray analysis showed that jasmonic acid (JA)-responsive genes including defensins and JA-biosynthetic genes were up-regulated. RsGluR overexpression also inhibited growth of a necrotic fungal pathogen Botrytis cinerea possibly due to up-regulation of the defensins. Based on these results, we suggest that RsGluR is a glutamate-gated $Ca^{2+}$ channel located in the plasma membrane of higher plants and plays a direct or indirect role in defense against pathogen infection by triggering JA biosynthesis.

• Molecules and Cells
• /
• 제28권4호
• /
• pp.375-382
• /
• 2009

The 26S proteasome is a 2-MDa complex with a central role in protein turn over. The 26S proteasome is comprised of one 20S core particle and two 19S regulatory particles (RPs). The RPN12a protein, a non-ATPase subunit of the 19S RP, was previously shown to be involved in cytokinin signaling in Arabidopsis. To further investigate cellular roles of RPN12a, RNAi transgenic plants of RPN12a were constructed. As expected, the 35S:RNAi-RPN12a plants showed cytokinin signaling defective phenotypes, including abnormal formation of leaves and inflorescences. Furthermore, RNAi knock-down transgenic plants exhibited additional unique phenotypes, including concave and heart-shape cotyledons, triple cotyledons, irregular and clustered guard cells, and defects in phyllotaxy, all of which are typical for defective cytokinin signaling. We next examined the mRNA level of cytokinin signaling components, including type-A ARRs, type-B ARRs, and CRFs. The expression of type-A ARRs, encoding negative regulators of cytokinin signaling, was markedly reduced in 35S:RNAi-RPN12a transgenic plants relative to that in wild type plants, while type-B ARRs and CRFs were unaffected. Our results also indicate that in vivo stability of the ARR5 protein, a negative regulator of cytokinin signaling, is mediated by the 26S proteasome complex. These results suggest that RPN12a participates in feedback inhibitory mechanism of cytokinin signaling through modulation of the abundance of ARR5 protein in Arabidopsis.

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

• BMB Reports
• /
• 제41권5호
• /
• pp.382-387
• /
• 2008

It is known that the stable protein 1 (SP1) detected in aspen plants remains soluble upon boiling and that sp1 expression in transgenic aspen is resistant to salt stress. Presently, we analyzed the effect of expression of SP1 in Arabidopsis thaliana plants and their response to high temperature stress. After $45^{\circ}C$ for 16 h, relative to wild type plants, sp1 transgenic plants exhibited stronger growth and were better in several physiological properties including chlorophyII, chlorophyII fluorescence, water content, proline content, and malondialdehyde content. These preliminarily results suggest that the over-expression of SP1 may notably enhance heat-tolerant level of transgenic A. thaliana plants.

• BMB Reports
• /
• 제42권11호
• /
• pp.752-757
• /
• 2009

Copper is essential but toxic in excess for aerobic organisms. Yeast transcription factor ACE1 functions as a sensor for copper and an inducer for the transcription of CUP1. In addition, ACE1 can activate the transcription of superoxide dismutase gene (sod1) in response to copper. In this study, we introduced the yeast ACE1 into Arabidopsis and analyzed its function in plant. Under high copper stress, the transgenic plants over-expressing ACE1 showed higher survival rate than the wild-type. We also found that over-expression of ACE1 in Arabidopsis increased the activities of SOD and POD, which were beneficial to the cell in copper buffering. Excess copper would suppress the expression of chlorophyll biosynthetic genes in Arabidopsis, RT-PCR analysis revealed that over-expression of ACE1 decrease the suppression. Together, our results indicate that ACE1 may play an important role in response to copper stress in Arabidopsis.

• Journal of Plant Biotechnology
• /
• 제47권4호
• /
• pp.283-288
• /
• 2020

Although the evolution of Arabidopsis thaliana and humans diverged approximately 1.6 billion years ago, recent studies have demonstrated that protein function and cellular processes involved in disease response remain remarkably conserved. Particularly, γ-secretase, a multisubunit protein complex that participates in intramembrane proteolysis (RIP) regulation, is also known to mediate the cleavage of more than 80 substrates including the amyloid precursor protein (APP) and the Notch receptor. Although the genes (PS1/2, APH-1, PEN-2, and NCT) coding for the γ-secretase complex components are present in plant genomes, their function remains largely uncharacterized. Given that the deposition of 42 amino acid long amyloid-β peptides (hAβ₄₂) is thought to be one of the main causes of Alzheimer's disease, we aimed to examine the physiological effects of hAβ₄₂ peptides on plants. Interestingly, we found that Arabidopsis protoplast death increased after 24 h of exposure to 3 or 5 µM hAβ₄₂ peptides. Furthermore, transgenic Arabidopsis plants overexpressing the hAβ₄₂ gene exhibited changes in primary root length and silique phyllotaxy. Taken together, our results demonstrate that hAβ₄₂ peptides, a metazoan protein, significantly affect Arabidopsis protoplast viability and plant morphology.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
• /
• pp.77.2-78
• /
• 2003

An EREBP/AP2-type transcription factor (CaPFl) was isolated by DDRT-PCR following inoculation of soybean pustule pathogen Xanthomonas axonopodis pv. glycines Bra which induces HR on pepper leaves. Genomic Southern blot analysis revealed that the CaPFl gene is present as a single copy within the hot pepper genome. The deduced amino acid sequence of CaPFl has two potential nuclear localization signals, a possible acidic activation domain, and an EREBP/AP2 motif that could bind to a conserved cis- element present in promoter region of many stress-induced genes. The mRNA level of CaPFl was induced by both biotic and abiotic stresses. We observed higher-level transcripts in resistance-induced pepper tissues than diseased tissues. Expression of CaPFl is also induced upon various abiotic stresses including ethephon, MeJA, cold stress, drought stress and salt stress treatments. To study the role of CPFI in plant, transgenic Arabidopsis and tobacco plants which express higher level of pepper CaPFl were generated. Global gene expression analysis of transgenic Arabidopsis by cDNA microarray indicated that expression of CaPFl in transgenic plants affect the expression of quite a few GCC box and DRE/CRT box-containing genes. Furthermore, the transgenic Arabidopsis and tobacco plant, expressing CaPFl showed tolerance against freezing temperature and enhanced resistance to Pseudomonas syrnigae pv. tabaci. Taken together, these results indicated that CaPFl is a novel EREBP/AP2 transcription factor in hot pepper plant and it may has a significant role(s) in regulation of biotic and abiotic stresses in plant.