ACC oxidase 발현 억제 식물체에서 폴리아민 생합성 증가에 의한 스트레스 저항성 증강

위수진,박기영 한국식물생명공학회 2011 식물생명공학회지 Vol.38 No.1

Antisense construct of cDNA for senescencerelated ACC oxidase (CAO) cDNA isolated from carnation flowers were introduced into tobacco by Agrobacteriummediated transformation. The decreasing expression of NtACO and the reduction of ethylene production were observed in these transgenic lines. In contrast, the SAMDC transcripts and spermidine content were increased. The findings that higher content of spermidine in the ethylene suppressed transgenic plants compared with wild-type should be directly resulted in the enhancement of SAMDC activity followed by the increased accumulation of SAMDC transcript. To investigate the pathogenic response in these transgenic plants, wild-type and transgenic plants were inoculated with Phytophthora parasitica pv. nicotianae. Transgenic plants suppressing ethylene production showed the increased resistance against fungal pathogen, comparing with wild-type plant. PR-protein genes expression in CAO-AS-2 and CAOAS- 4 were also higher at the normal growth condition and pathogenic response than in wild-type plants. The results of higher spermidine content and SAMDC activity in transgenic plants, CAO-AS-2 and CAO-AS-4, support the possibility that an increase in spermidine content might induce the higher transcripts of PR-protein genes. This results agreed with the phenomena that spermidine promoted the expression of PR1a and a SAMDC inhibitor, MGBG, decreased the expression of PR1a in leaf discs. These results suggest that the resistance against fungal pathogen in transgenic tobacco impaired in ethylene production might be caused by increasing in polyamine, especially spermidine, biosynthesis. 에틸렌 생합성 효소인 ACC oxidase 유전자인 CAO cDNA 가 antisense 방향으로 도입된 형질전환 담배 식물체는endogenous한 NtACO의 발현 및 에틸렌 생합성이 모두 억제되었다. 또한 폴리아민 생합성 효소인 SAMDC의 유전자 발현 및 효소 활성이 증가하였고 spermidine 함량이 증가하였다. 이러한 결과는 에틸렌 생합성의 감소로 인하여 세포 내 폴리아민 함량의 변화가 유도되는 경쟁적 관계로 spermidine이 증가하였고 이것은 SAMDC의 발현 및활성 증가를 유도하였다고 여겨진다. 또한 이들 형질전환 식물체는 담배 역병균에 대한 저항 효과 뿐만 아니라PR 단백질의 유전자들의 발현양이 증가하였는데 이는에틸렌 감소에 유도되는 폴리아민 중에서도 spermidine의증가가 PR 단백질의 유전자들의 발현을 촉진시키고 그결과 병원균 감염에 대한 저항성을 유도하는 기작이 작동한 것으로 여겨진다. Spermidine을 합성하는데 중요하게 작용하는 SAMDC의 활성을 억제하는 MGBG를 처리한 경우에는 PR1a 발현이 억제된 경우에서 이 기작의 가능성을 확인하였다. 결론적으로 병원균 감염에 대한 과정에서 spermidine의 생합성이 증가하게 되면 PR 단백질등 스트레스 저항성 관련 단백질들이 유도되어 스트레스저항성을 높일 수 있을 것으로 사료된다.

토마토 (Solanum lycopersicum) 과육의 숙성정도에 따른 peptide:N-glycanase 발현 분석

위수진,박기영 한국식물생명공학회 2014 식물생명공학회지 Vol.41 No.3

eukaryotes, proteins that are secreted into ERare post-translationally modified by N-glycosylation, thepatterns of which are significantly different between plantand animal cells. Biotechnology industry has already produceda number of therapeutic glycoproteins in plant cells. However,the aberrant glycosylation of therapeutic recombinant proteinsin plant systems can cause immune problems in humans. Therefore, it is important to develop strategies for producingnon-glycosylated forms to preserve biological activity andnative conformation by a peptide: N-glycanase (PNGase). Inthis study, we try to isolate PNGase T gene from tomato,which can use as a platform plant for biotechnology industry. We isolated a cDNA (GenBank Accession number KM401550)from tomato leaves with 1,767 bp, which encoded a polypeptideof 588 amino acids with a predicted molecular mass of 65.8kDa. We also investigated the expression patterns of PNGaseT during fruit ripening of tomato. The transcripts of PNGaseT, which were constitutively induced in tomato fruit fromgreen stage, were significantly increased and reached a peakat orange stage. After which, those transcripts were continuouslyreduced. The expression pattern of PNGase T was coincidedwell with transcripts profiles of metacaspase gene, LeMCA,and senescence-related gene members of ACC synthase,LeACS2, LeACS4, and LeACS6, for ethylene biosynthesisduring fruit ripening. These results suggest that PNGase T isinvolved in a de-glycosylation process associated withsenescence and fruit ripening.

Antisense Expression of Carnation cDNA Encoding ACC Synthase or ACC Oxidase Enhances Polyamine Content and Abiotic Stress Tolerance in Transgenic Tobacco Plants

위수진,Ky Young Park 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.2

The amount of polyamines (such as putrescine, spermidine, and spermine) increased under environmental stress conditions. We used transgenic technology in an attempt to evaluate their potential for mitigating the adverse effects of several abiotic stresses in plants. Because there is a metabolic competition for S-adenosylmethionine as a precursor between polyamine (PA) and ethylene biosyntheses, it was expected that the antisense-expression of ethylene biosynthetic genes could result in an increase in PA biosynthesis. Antisense constructs of cDNAs for senescence-related 1- aminocyclopropane-1-carboxylic acid (ACC) synthase (CAS) and ACC oxidase (CAO) were isolated from carnation flowers that were introduced into tobacco by Agrobacterium-mediated transformation. Several transgenic lines showed higher PA contents than wild-type plants. The number and weight of seeds also increased. Stress-induced senescence was attenuated in these transgenic plants in terms of total chlorophyll loss and phenotypic changes after oxidative stress with hydrogen peroxide (H2O2), high salinity, acid stress (pH 3.0), and ABA treatment. These results suggest that the transgenic plants with antisense CAS and CAO cDNAs are more tolerant to abiotic stresses than wild-type plants. This shows a positive correlation between PA content and stress tolerance in plants.

Inhibition of Biphasic Ethylene Production Enhances Tolerance to Abiotic Stress by Reducing the Accumulation of Reactive Oxygen Species in Nicotiana tabacum

위수진,Su Jin Jang,Ky Young Park 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.1

Reactive oxygen species (ROS), such as H2O2, are impor-tant plant cell signaling molecules involved in responses to biotic and abiotic stresses and in developmental and physiological processes. Despite the well-known physio-logical functions of ethylene production and stress signal-ing via ROS during stresses, whether ethylene acts alone or in conjunction with ROS has not yet been fully eluci-dated. Therefore, we investigated the relationship between ethylene production and ROS accumulation during the response to abiotic stress. We used three independent transgenic tobacco lines, CAS-AS-2, -3 and -4, in which an antisense transcript of the senescence-related ACC syn-thase (ACS) gene from carnation flower (CARACC, Gen-Bank accession No. M66619) was expressed heterolo-gously. Biphasic ethylene biosynthesis was reduced sig-nificantly in these transgenic plants, with or without H2O2 treatment. These plants exhibited significantly reduced H2O2-induced gene-specific expression of ACS members, which were regulated in a time-dependent manner. The higher levels of NtACS1 expression in wild-type plants led to a second peak in ethylene production, which resulted in a more severe level of necrosis and cell death, as deter-mined by trypan blue staining. In the transgenic lines, upregulated transcription of CAB, POR1 and RbcS re-sulted in increased photosynthetic performance following salt stress. This stress tolerance of H2O2-treated trans-genic plants resulted from reduced ethylene biosynthesis, which decreased ROS accumulation via increased gene expression and activity of ROS-detoxifying enzymes, in-cluding MnSOD, CuZnSOD, and catalase. Therefore, it is suggested that ethylene plays a potentially critical role as an amplifier for ROS accumulation, implying a synergistic effect between biosynthesis of ROS and ethylene.

대장균과 식물시스템에서 재조합 인간 prominiinsulin 생합성 분석

최유진,위수진,박기영,박수현,김지수 한국식물생명공학회 2013 식물생명공학회지 Vol.40 No.3

Recently, the number of people with diabetes is rapidly increasing, coupled with the fact that the insulin market is remarkably increasing. Therefore, molecular farming for plant-derived pharmaceutical protein production is reported as becoming more attractive than ever. In this study, we carried out experiments step by step for development of recombinant insulin constructs, which were transformed into E. coli system, in vitro transcription and translation system, and tobacco cells. At first, recombinant proinsulin protein was successfully produced in in vitro transcription and translation system with wheat germ extract. After which, recombinant construct of prominiinsulin encoded a fusion protein of 7.8 kDa with trypsin cleavage sites at N terminus and C terminus of minimized C-peptide was tried to in vitro expression using E.coli culture. After purification with His-tag column, the resulting recombinant prominiinsulin protein was processed with trypsin, and then checked insulin biosynthesis by SDS-PAGE and western blot analysis with anti-insulin monoclonal antibody. The immunoreactive product of trypsin-treated miniinsulin was identical to the predicted insulin hexamer. The construct of 35S promoter-driven preprominiinsulin recombinant gene with signal peptide region for ER-targeting and red fluorescence protein gene [N terminus → tobacco E2 signal peptide → B-peptide (1-29 AA) → AAK → A-peptide (1-21 AA) → RR → His6 → KDEL → C terminus] was transformed into BY-2 tobacco cells. A polypeptide corresponding to the 38-kDa molecular mass predicted for fusion protein was detected in total protein profiles from transgenic BY-2 cells by western analysis. Therefore, this recombinant preprominiinsulin construct can be used for generation of transgenic tobacco plants producing therapeutic recombinant insulin.

Potential of plants to produce recombinant protein products

박기영,위수진 한국식물학회 2016 Journal of Plant Biology Vol.59 No.6

Plants have great potential as photosynthetic factories to produce pharmaceutically important and commercially valuable biomedicines and industrial proteins at low cost. The U.S. Food and Drug Administration (U.S. FDA) has approved the drug Elelyso (taliglucerase alfa) produced by carrot cells for treatment of type 1 Gaucher’s disease in 2012. The commercial potential of biomedicines produced by molecular farming has dramatically improved due to the success of an experimental drug called ZMapp, which has immunological activity in Ebola patients. A cocktail of three monoclonal antibodies was produced in tobacco (Nicotiana benthamiana) plants (Chen and Davis 2016). At present, very few drugs made by this technology have been approved by worldwide authorities such as the U.S. FDA. However, plants have been proposed as a novel paradigm for commercial production of proteins over the next decade. In recent years, leading researchers on molecular farming have given more priority to the area of animal-free therapeutic proteins such as parenteral and oral vaccines. Although plant-based platforms have considerable advantages over traditional systems such as bacterial and animal systems, there are several obstacles to commercial-scale production, especially with regards to improving the quality and quantity of plant-produced biologics and industrial materials. One of the biggest barriers to commercialization of this technology is the intense scrutiny of these new plant varieties by regulatory agencies and the public as well as the high costs associated with their regulatory approval.

Increased Polyamine Biosynthesis Enhances Stress Tolerance by Preventing the Accumulation of Reactive Oxygen Species: T-DNA Mutational Analysis of Oryza sativa Lysine Decarboxylase-like Protein 1

Su Jin Jang,박기영,위수진,Yoo Jin Choi,안진흥 한국분자세포생물학회 2012 Molecules and cells Vol.34 No.3

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screen-ing in the presence of 50 mM H2O2. The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenously-applied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H2O2. Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.

Effects of phenotypic and RAPD variations on ecophysiological characterization of reeds (Phragmites australis) in Suncheon Bay

전인선,권다솜,신훈,김원복,강나루,왕선미,김미선,위수진,박기영 한국식물학회 2015 Journal of Plant Biology Vol.58 No.3

Genomic DNA of young reed leaves was subjected to PCR-based random amplified polymorphic DNA (RAPD) technique using random primers. Banding patterns from RAPD showed three major clusters. Group I showing vigorous growth was composed of plants from recently emerging reed patches on the coast shore and was established after the 2000s. Group II formed by reed patches was established at around the 1990s and showed non-vigorous growth along with typical symptoms of reed decline. Eutrophication could be the responsible factor for this reed decline, as the mudflat contained high concentrations of organic nitrogen and phosphorous. Group III reeds showed the most vigorous growth, demonstrating high values for shoot height, biomass, and panicle length along with moderate stem density. The state of Group III can be described as sub-optimal due to their old age on terrestrial habitat, as a minimum level of seawater is important for vigorous growth of reeds such as sufficient shoot height and photosynthetic capability. Reed height and panicle length have been shown to be recently declining in Suncheon Bay. These results indicate that old and rotting parts of reeds inhibit reed growth and that removal of these parts can have positive effects on shoot height and biomass.