• Plant Biotechnology Reports
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• 제5권4호
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• pp.301-308
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• 2011

This study tested the relative and combined efficacy of ShPx2 and ShPAL transgenes by comparing Nicotiana tabacum hybrids with enhanced levels of $_L$-phenylalanine ammonia lyase (PAL) activity and cationic peroxidase (Prx) activity with transgenic parental lines that overexpress either transgene. The PAL/Prx hybrids expressed both transgenes driven by the 35S CaMV promoter, and leaf PAL and Prx enzyme activities were similar to those of the relevant transgenic parent and seven- to tenfold higher than nontransgenic controls. Lignin levels in the PAL/Prx hybrids were higher than the PAL parent and nontransgenic controls, but not significantly higher than the Prx parent. All transgenic plants showed increased resistance to the necrotrophs Phytophthora parasitica pv. nicotianae and Cercospora nicotianae compared to nontransgenic controls, with a preponderance of smaller lesion categories produced in Prx-expressing lines. However, the PAL/Prx hybrids showed no significant increase in resistance to either pathogen relative to the Prx parental line. These data indicate that, in tobacco, the PAL and Prx transgenes do not act additively in disease resistance. Stacking with Prx did not prevent a visible growth inhibition from PAL overexpression. Practical use of ShPAL will likely require more sophisticated developmental control, and we conclude that ShPx2 is a preferred candidate for development as a resistance transgene.

• 한국응용곤충학회지
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• 제56권1호
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• pp.19-28
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• 2017

폴리드나바이러스(polydnavirus: PDV)는 일부 내부기생봉에 공생하는 이중나선형 DNA 바이러스 분류군이다. Cotesia plutellae bracovirus (CpBV)는 프루텔고치벌(C. plutellae)에 공생하는 일종의 PDV이다. 프루텔고치벌은 어린 배추좀나방(Plutella xylostella) 유충에 기생한다. 기생 초기에 발현하는 CpBV-ELP1 유전자는 혈구세포에 세포독성을 발휘하면서 기주의 세포성 면역을 억제하여 기생에 중요한 역할을 담당하고 있다. 본 연구는 이 유전자를 담배 식물에서 발현하여 해충에 대한 경구독성을 분석하는 데 목적을 두었다. 재조합 CpBV-ELP1 단백질이 배큘로바이러스 발현시스템을 통해 합성되어 세포배양액에 분비되었다. 수거된 세포배양액은 일련의 단백질 분리과정(ammonium sulfate 단백질 분획, size exclusion 크로마토그래피, 이온교환 크로마토그래피)을 통해 CpBV-ELP1 단백질을 분리하는 데 이용되었다. 분리된 rCpBV-ELP1 단백질은 파밤나방(Spodoptera exigua) 혈구에 대한 뚜렷한 세포독성을 보였다. CpBV-ELP1은 파밤나방 5령충에 대해서 혈강 주입하여 살충력을 나타냈고, 엽침지법을 이용하여 경구독성을 갖고 있는 것을 확인하였다. CpBV-ELP1 유전자를 CaMV 35S 유전자 프로모터와 opaline synthase 유전자 전사종결신호를 갖는 pBI121 벡터에 클로닝하여 Agrobacterium tumefaciens LBA4404 세균에 형질전환을 유도하였다. 형질전환된 세균은 담배(Nicotiana tabacum Xanthi)잎에 감염하여 캘러스를 유도하게 하였고 이후 차세대(T1)를 확보하였다. T1 세대 담배는 파밤나방에 대한 해충저항성을 갖고 있음을 확인하였다. 이러한 결과는 CpBV-ELP1 유전자가 형질전환작물을 통해 해충방제에 응용될 수 있다는 것을 제시하고 있다.

• Journal of Plant Biotechnology
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• 제31권4호
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• pp.255-259
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• 2004

Agrobacterium과 자엽절 공동배양으로 대두 형질전환체를 생산하였다. 대두 배양재료는 3개의 품종과 1개의 genotype의 자엽절 절편을 사용하였으며, bar유전자와 GUS유전자로 제작된 pPTN289와 pCAMBIA3301벡터를 LBA4401, GV3101, EHA101, C58에 각각 형질전환하여 공동 배양하였고 모든 형질전환 방법은 약간 변형된 Zhang 등(1999)의 방법에 따라 수행하였다. 형질전환빈도는 아그로박테리움의 종류에 따라 현저한 차이가 있었으며, 특히 사용한 균주중 EHA101에서 3.6%로 최대치를 보였다. Glufosinate가 첨가된 선발배지에서 106개의 식물체를 얻었으며, 이중 Thorne에서 5개체, 1049에서 5개체, 백운콩에서 1개체 등 모두 11개로부터 GUS양성반응을 확인하였다. Southern분석과 basta검정법에 의하여 T1세대 식물체로부터 GUS유전자와 bar유전자가 발현되고 있음을 확인하였다.

• 식물조직배양학회지
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• 제24권5호
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• pp.305-311
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• 1997

Bacillus thuringiensis는 그람 양성 토양 세균으로 포자형성시 결정화된 내포체를 형성하는데, 이 내포체를 구성하는 결정단백질은 각 곤충에 대하여 특이적인 독성을 나타낸다. 살충성 결정단백질 중 Cry II A 결정단백질은 인시류와 쌍시류 곤충에 모두 특이적으로 작용한다. 결정단백질은 살충제로서 불안정하고 포장에서 지속성이 낮은 단점을 가지고 있으므로, 본 연구에서는 Cry II A 결정단백질 유전자가 형질전환된 담배 식물을 제작하고자 하였다. cry IIA 유전자가 삽입된 벡터를 대장균에 형질전환한 후, 알칼리 용액에 대한 용해도의 차이를 이용하여 Cry II A 결정단백질(70 kDa)을 분리하였고, 분리한 Cry II A 결정단백질을 trypsin 처리하여 활성화된 Cry II A (50 kDa)를 확인하였다. 식물 형질전환을 위하여 두 개의 CaMV 35S promoters에 의해 발현이 조절되는 식물 발현 벡터에 cry II A 유전자를 클로닝 하였다. 이 식물 발현 벡터를 Agrobacterium을 이용한 엽편형질 전환을 통해 담배(N. tabacum var. Petit Havana SRI)에 형질전환 시켰으며, 재분화 과정을 거쳐 여섯 개체의 형질전환 식물체를 얻었다. Southern blot을 통하여 분석한 결과 세 개체 내에 cry II A 유전자가 존재하였는데, 한 개체에는 하나의 cry II A 유전자가, 또 한 개체에는 두 개의 cry II A 유전자가, 다른 한 개체에는 잘려진 형태의 cry II A 유전자가 존재함을 확인할 수 있었다. 본 연구를 통하여 얻어진 cry II A 형질전환 식물체는 살충성 검정 등을 거친 후 내충성 식물 생산 및 후대 유전 양상 분석을 위한 기초 자료로 이용될 수 있을 것이다.

• BMB Reports
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• 제40권3호
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• pp.404-411
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• 2007

Transformation of cantaloupes with the coat protein (cp) gene of papaya ringspot virus type W (PRSV-W), Thai isolate, was used to introduce virus resistance. Binary vectors containing either the full length coat protein coding region under control of the 35S CaMV promoter(pSA1175), or the inverted-repeat of a coat protein coding region (pSA1304), were constructed and used for Agrobacteriummediated transformation of cotyledonary explants of the cantaloupe cultivar Sun Lady. Four independent transgenic lines were obtained using pSA1304 and one using pSA1175. Integration of the PRSV-W cp gene into the genome of these transgenic lines was verified by PCR amplification, GUS assays and Southern blot hybridization. In vitro inoculation of these lines with PRSV-W revealed that whereas the line containing pSA1175 remained sensitive, the four lines containing pSA1304 were resistant. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral cp gene in transgenic lines resistant to PRSV-W supports the involvement of post-transcriptional gene silencing in the establishment of resistance.

• 한국작물학회:학술대회논문집
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.164-164
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• 2017

High throughput transcriptome investigations of immunity in plants highlight the complexity of gene networks leading to incompatible interaction. To identify genes crucial to resistance against Xanthomonas oryzae pv oryzae, functional genetic analysis of selected differentially expressed genes from our microarray data set was carried out. A total of 13 overexpression vector constructs were made using 35S CaMV promoter which drive constitutive expression in rice. Most of the genes are developmentally expressed especially during maximum tillering stage and are commonly highly expressed in the leaves. When screened against Xoo strain K2, the transgenic plants displayed shorter lesion length compared with wild type Dongjin which indicates partial resistance. The levels of ROS continuously magnified after inoculation which indicates robust cellular sensing necessary to initiate cell death. Elevated transcripts levels of several defense-related genes at the downstream of defense signal network also corroborate the phenotype reaction of the transgenic plants. Moreover, expression assays revealed regulation of these genes by cross-communicating signal-transductions pathways mediated by salicylic and jasmonic acid. These collective findings revealed the key immune signaling conduits critical to mount full defense against Xoo.

• Journal of Microbiology and Biotechnology
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• 제16권2호
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• pp.205-211
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• 2006

A quantitative analytical method to detect new lines of genetically modified (GM) maize, NK603 and TC1507, has been developed by using a real-time polymerase chain reaction (PCR). To detect these GM lines, two specific primer pairs and probes were designed. A plasmid as a reference molecule was constructed from an endogenous DNA sequence of maize, a universal sequence of a cauliflower mosaic virus (CaMV) 35S promoter used in most GMOs, and each DNA sequence specific to the NK603 and TC1507 lines. For the validation of this method, the test samples of 0, 0.1, 0.5, 1.0, 3.0, 5.0, and 10.0% each of the NK603 and TC1507 GM maize were quantitated. At the 3.0% level, the biases (mean vs. true value) for the NK603 and TC1507 lines were 3.3% and 15.7%, respectively, and their relative standard deviations were 7.2% and 5.5%, respectively. These results indicate that the PCR method developed in this study can be used to quantitatively detect the NK603 and TC1507 lines of GM maize.

• Journal of Applied Biological Chemistry
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• 제49권3호
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• pp.75-81
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• 2006

A full-length cDNA encoding dihydroflavonol 4-reductase (st-dfr) of potato was isolated by rapid amplification of cDNA ends, and their expression was investigated from purple-fleshed potato (Solanum tuberosum L. cv. Jashim). The st-dfr exists as a member of a small gene family and its transcripts was abundant in the order of tuber flesh, stem, leaf, and root. The expressions of st-dfr gene were light inducible and cultivar dependant. Transgenic potato plants harboring antisense st-dfr (AS-DFR) sequences were analyzed. The accumulation of mRNA was nearly completely inhibited as a result of introducing an AS-DFR gene under the control of the 35S CaMV promoter into the red tuber skin Solanum tuberosum L. cv. Desiree. The anthocyanin content of the tuber peels of the transgenic lines was dramatically decreased by up to 70%. The possible production of flavonols in the peels of AS-DFR transgenic potatoes was discussed.

• 한국초지조사료학회지
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• 제18권1호
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• pp.69-76
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• 1998

This study was conducted to construct of the transgenic plants wliich are resistant to oxidative stresses including ozone with B. mpestris cytosolic glutathione reductase cDNA using the binary vector system of Agrobacterium tumefaciens. The 1.8kb B. campestris cytosolic GR cDNA was subcloned into the unique Sma I site of the plant transformation vector pBKSI- I, downstream of the constitutive CaMV 35s promoter and upstream of the nos termination sequence, in place of the uidA (GUS) reporter gene. The resulting plant transformation vector, pBKS-GRI, was introduced into A. tumefaciens LBA4404 by two cycles of tkeze-thaw method. The B. nqus cotyledonary petioles were transformed by the Agrubaferium harboring pBKS-GRI. Transformed shoots were induced and selected on regeneration medium supplemented with kanarnycin. The shoot formation was increased remarkably by addition of Ag$NO_3$, in MS media. The transgenic plants were analyzed for the presence of the B. campestris GR gene by Southern blot analysis and it was confirmed that a foregin gene was stably integrated into the genomes of B. nqus plants.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1985년도 워크샵 및 심포지엄 북한산국립공원의 식생
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• pp.59-71
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• 1985

In rice, limited efforts have been made to identify genes by the use of insertional mutagens, especially heterologous transposons such as the maize Ac/Ds. We constructed Ac and gene trap Ds vectors and introduced them into the rice genome by Agrobacterium-mediated transformation. In this report, rice plants that contained single and simple insertions of T-DNA were analyzed in order to evaluate the gene-tagging efficiency. The 3'end of Ds was examined for putative splicing donor sites. As observed in maize, three splice donor sites were identified at the 3'end of the Ds in rice. Nearly 80% of Ds elements wered excised from the original T-DNA sites, when Ac cDNA was expressed under a CaMV 35S promoter. Repetitive ratoon culturing was performed to induce new transpositions of Ds in new plants derived from cuttings. About 30% of the plants carried at least one Ds that underwent secondary transposition in the later cultures. 8% of transposed Ds elements expressed GUS in various tissues of rice panicles. With cloned DNA adjacent to Ds, the genomic complexities of the insertion sites were examined by Southern hybridization. Half of the Ds insertion sites showed simple hybriodization patterns which could be easily utilized to locate the Ds. Our data demonstrate that the Ac/Ds mediated gene trap system could prove an excellent tool for the analysis of functions of genes in rice. We discuss genetic strategies that could be employed in a largee scale mutagenesis using a heterologous Ac/Ds family in rice.