고추역병균(Phytophthora capsici)은 고추 생육 전반에 걸쳐 병을 발생시켜 농가 소득에 큰 손실을 일으키고 있다. 고추 역병균 저항성은 양적 형질 유전자좌(Quantitative Trait Loci, QTL)에 의해 조절되며 주동 유전자는 고추의 5번 염색체에 존재한다고 보고 되었지만, 후보 유전자의 선발 및 저항성 유전자 규명 연구는 아직 초기 단계이다. 특히, 고추는 형질전환이 어려운 작물로써 병원균과의 상호작용 연구를 통한 저항성 유전자 동정에 제한이 많다. 반면 고추와 같은 가지과 작물인 담배(Nicotiana benthamiana)는 병원균 상호작용 모델로 알려져 형질전환을 통해 저항성 유전자 규명에 활용된다. 본 연구에서는 고추 역병 저항성 기작 규명을 위한 기초 연구로써, 식물 저항성 유사 유전자(Resistance Gene Analog, RGA)를 선발하고, 이들 유전자들에 대한 담배 형질전환 기법 최적화 연구를 수행하였다. 고추 5번 염색체에 존재하는 고추 역병 저항성 분자표지들을 분석하여 RGA 후보 유전자인 CaNBARC105, CaNBARC112 유전자를 동정하였다. 이들 유전자들에 대해 Agrobacterium tumefaciens를 매개체로 하여 고추 RGA가 삽입된 담배 형질전환체를 개발하였다. 형질전환 여부는 유전자 특이적인 서열을 이용한 genomic PCR과 RT-PCR 검증을 통해 이들 형질전환 된 담배들의 생육 및 발달에 영향이 없다는 것을 확인하였다. 본 연구는 향후 고추 병 저항성 후보 유전자들이 삽입된 담배 형질전환체는 고추 역병 저항성 유전자 규명 및 기작 연구에 기반이 될 것이다.

Bemisia tabaci is a serious pest in various horticultural crops in the world. Due to use of chemical pesticide for their management they develop pesticide resistance and environmental contamination. It is necessary to develop alternative bio-pesticides using natural products from plants and natural enemies. Nicotiana benthamiana is a variety of wild tobacco plants and produce acyl sugars from glandular trichomes in the leaves. When adult whiteflies were reared with fresh N. benthamiana leaves, they were completely dead within 84 h. Oral feeding of 20% N. benthamiana extracts using ethanol and water showed complete mortality of whiteflies within 48 hours. Spray of N. benthamiana extracts into the leaves was lethal to eggs but not to nymphs of whiteflies. Further, tomato plants sprayed with N. benthamiana extracts were highly repellent to adult whiteflies. Quantitative real-time PCR indicated that the expression of various genes of B. tabaci was changed by oral feeding of N. benthamiana extract. This study suggests N. benthamiana extract is a useful for the control of whiteflies and can be used as an alternative natural pesticide for the whitefly management.

Bemisia tabaci is a serious pest in various horticultural crops in the world. The management of B. tabaci has been typically carried out by chemical pesticides. Due to the development of pesticide resistance and environmental contamination, however, it is necessary to develop alternative biopesticides using natural products from plants and natural enemies. Nicotiana benthamiana is a variety of wild tobacco plants and produce acyl sugars from glandular trichomes in the leaves. Acyl sugars are known to be highly toxic to various plant sapping insects such as whiteflies, aphids and thrips. Here, we extracted acyl sugars in two different ways from the leaves. At first, collected leaves were simply washed with water. Otherwise, collected leaves firstly dried and homogenized into the powder, then extracted with ethanol. Spray of 10% water-extracted solution into adult whiteflies showed 80% mortality. Otherwise, spray of 10% ethanol-extracted solutions showed complete mortality at 48 h after treatment and also strong repellency of adult whiteflies into the treated tomato plants. Our results suggest N. benthamiana is a useful for the control of whiteflies and can be used as an alternative natural pesticide for the whitefly management.

Background : Recently, wild ginseng cultured ginseng cultured in a bioreactor is mass produced using biotechnological tissue culture technology. PgTRx1 gene which is involved in the production of useful substances in fermented wild ginseng cultured root was selected and introduced into a model plant (Nicotiana benthamiana) to investigate transformation useful gene expression and possible production of useful substances. Methods and Results : The PgTRx1 gene was amplified and isolated from fermented wild ginseng cultured root. Isolated PgTRx1 gene was ligated to the plant expression vector pMBP1. Overexpression genes were recombined and cloned into E. coli. Agrobacterium tumefaciens LBA4404 was transformed, cultured A. tumefaciens LBA4404 was agro-infiltrated into a model plant for transient assay. Agro-infiltration model plants were sampled on days 0, 1, 2, and 3, and cDNA synthesis was performed after total RNA extraction. The expression level of PgTRx1 gene increased with time, and NbNR, NbHSR, NbAPx, NbSIP, NbPAL, NbPR1a and NbNOA1 genes showed a decrease in the expression level. The samples were taken to determine antioxidant activity, acetylcholine hydrolase inhibitory activity and glutamate content at 0 h, 12 h, 14 h, and 36 h. The highest antioxidant activity was observed at 24 h of sample, acetylcholine hydrolase inhibitory activity at 12 h, and glutamate at 36 h. Conclusion : The possibility of introducing the model plant of the PgTRx1 gene derived from fermented wild ginseng cultured root was confirmed. The results showed that various activities were increased with time of agro-infiltration.

Background : Temperature is major factor for growth plant. Recently, because of global warming, abnormal temperature included drought, deluge, sudden temperature change and heavy snow damaged crops in the world. In Korea, crops have been sensitive to low temperature on early growth stage, e.g. fruit tree and ginseng, were damaged owing to sudden heavy snow and cold on Spring. Therefore, recently interest in cold resistance crops were increased in demand rapidly. This study was performed to establish transgenic Nicotiana benthamiana by transforming cold resistant gene related to cold tolerance S-adenosylmethionine synthetase (SAMS) isolated from Miscanthus sinensis. Methods and Results : Total RNA was extracted from leaves of M. sinensis using Trizol assay and isolated MsSAMS. Isolated MsSAMS was insert into SacⅠ- XbaⅠ sites of pMBP1 vector. The vector was transformed to Agrobacterium tumefaciens strain LBA4404 by DH5α. A. tumefaciens with binary plasmid were selected at YEP medium supplemented with kanamycin. Cut leaves of tobacco were co-cultured with selected A. tumefaciens. Co-cultured leaves was grown on regeneration medium for a month at dark condition, and transferred to at light condition. Regeneration shoot from callus were excised and transferred to root-induction medium. Approximately, 58% of leaves explant produced callus. Nearly, 30% of callus had shoot and approximately, 94% of shoots were rooted in root-induction medium. Conclusion : We established an efficient transformation system of N. benthamiana transformed by using MsSAMS gene related to cold tolerance isolated from M. sinensis. We may use the produced transgenic plants to prevent damages carried by cold.

The full-length cDNA encoding Perilla frutescens limonene synthase (PFLS) (603 amino acids, GenBank accession no. D49368) was cloned. To elucidate the role of PFLS in gene regulation, we transiently transformed full-length PFLS into tobacco plants. PFLS mRNA was first detected in the intact leaves of the plants at 6 h, and the LS transcript level increased after 12 h in leaves treated with oxidative stress-related chemicals. The transient overexpression of PFLS resulted in increased transcription of NbPR1 and NbSIP in Nicotiana benthamiana leaves. Thus, our result confirmed that the infiltration of PFLS gene act as a transcriptional regulator of NbPR1 or NbSIP genes in the tobacco.