본 연구의 목적은 NIH3T3와 HepG2 세포에서 에탄올유도 세포독성 및 유전독성에 대하여 녹차엑기스(GTE)와 epigallocatechin-3-gallate (EGCG)의 보호작용을 평가하는데 있다. 세포생존율은 MTT assay를 실시하였으며 DNA 손상도는 Comet assay로 실시한 결과 에탄올은 농도의존적인 세포독성과 유전독성을 나타내었다. 한편 GTE와 EGCG는 에탄올 유도 세포독성 및 DNA 손상에 대하여 유의성 있는 억제효과를 나타내었으며 DPPH시험과 LDL oxidation 및 8OH-2``dG 생성시험에서 항산화효과를 나타내었다. 한편 녹차성분 함유 시판 리큐르주도 순수 에탄올에 비하여 세포독성억제 및 DNA 손상억제효과를 나타내었다. 이상의 시험결과 GTE와 함유 EGCG는 항산화성 유전독성억제기전을 통한 에탄올독성저감 물질로 판단된다.

제초제 저항성 유전자가 도입된 형질전환 토마토에서 T-DNA의 특성을 분석하였다. 형질전환 토마토의 T-DNA 도입형태를 분석하기 위한 primer쌍을 제작하여 rpPCR을 실시하 였다. T0-21(15:1, 4 copy), 26(3:1, 3 copy), 34(63:1, 4 copy)의 경우에는 5쌍의 primer를 이용하여 수행한 rpPCR 결과에서 양쪽이 절단된 불완전한 T-DNA 반복이 확인되었다. 도입된 T-DNA의 주변 서열 및 분포를 알아보기 위하여 총 52개체의 형질전환체에 대하여 TAIL-PCR을 실시하였다. 이 중 22개 형질전환체의 TAIL-PCR 산물의 염기서열을 분석하였는데, 2개의 형질전환체에서 나온 염기서열 결과는 토마토의 12번 염색체와 90%, 1개체는 토마토 clone 135006R과 98%, 5개체는 담배의 엽록체와 97% 상동성을 보였으며, 12개체는 다양한 vector의 염기서열을 나타내었고, 그 외 나머지 개체들은 토마토 유전체 정보와 는 유사성을 찾을 수 없었다.

This study was conducted to determine the agronomic characteristics of low amylose lines which were derived from induced mutants by T-DNA insertion. The agronomic and physicochemical properties of the low amylose mutants were analyzed and compared with a donor cultivar 'Dongjin' and a low amylose cultivar 'Baekjinju'. The heading date of the low amylose mutants was similar to the donor cultivar 'Dongjin' and yield index of the mutants was 78~92% compared with 'Dongjin'. The amylose content of four mutants in brown rice was ranged from 16.1 to 16.7%. Among low amylose mutants, 'P50-4-4-5' was lower 3.6%(13.3%) than those of 'Dongjin'(16.9%) in amylose content of milled rice. The grain length of 'P50-4-4-5' was similar to the donor cultivar, however, thousand grain weight(18.9g) was lighter than those of 'Dongjin'. The score of alkali digestion in brown rice of 'P50-4-4-5'(5.5) was lower than that of 'Dongjin'(6.8) and similar to 'Baekjinju'(5.8). The gel consistency of 'P50-4-4-5'(84mm) in milled rice exhibited that was longer than 'Dongjin'(76mm) and 'Baekjinju'(81mm). The result of eating quality showed that 'P50-4-4-5'(78.8) was higher than those of 'Dongjin'(60.3) and 'Baekjinju'(67.2). Thus our data suggest that 'P50-4-4-5' will facilitate the development of a new cultivar with low amylose rice.

The rice leaf roller, Cnaphalocroci smedinalis Guenée (Lepidoptera: Pyralidae) is a leaf-feeding pest of rice world-widely distributed. For better understanding of the pest insect, geographic sequence variation of the species were performed using the mitochondrial A+T-rich region, with the samples collected from seven Korean and six Chinese localities. A total of 94 haplotypes obtained from 187 individuals showed the length variation, ranging from 339 bp to 348 bp. The maximum divergence of 4.57% appears to evidence a substantial sequence variation, indicating the applicability of this molecular marker to the study of geographic variation. Overall, a high per generation migration ratio (Nm = 3.67742 ~ infinite), a low level of genetic fixation (FST = 0 ~ 0.11969), and no discernable isolated population were noted in the most C. medinalis populations. AMOVA analysis to find out allocation of genetic variability of C. medinalis populations has shown allocation of majority of variation to the within-population, rather than among-populations and between-region, suggesting that the C. medinalis populations in both China and Korea are largely well connected. This result is consistent with current knowledge of the dispersal ability of the species. The structure analysis of the A+T-rich region has shown that the typical structural elements found in other lepidopteran insects also is well preserved in the C. medinalis A+T-rich region (i.e., a poly-T stretch and a microsatellite-like A/T repeat).

In an effort to gain a better understanding of the nature of the population genetic structure of the pest insect, Spodoptera litura (Lepidoptera: Noctuidae), tobacco cutworms were collected from six Korean and five Chinese localities and their mitochondrial A+T-rich region and nuclear internal transcribed spacer 2 (ITS2) regions were cloned and sequenced. A total of 106 A+T-rich region haplotypes and 92 ITS2 sequence types were obtained from 158 individuals. Compared to the preliminary mitochondrial COI gene sequence data that provided all identical sequence types in all samples, the A+T-rich region and nuclear ITS2 are highly variable. Overall, a low level of genetic fixation in the A+T-rich region (FST = 0 ~ 0.02965) and nuclear ITS2 (FST = 0 ~ 0.34491), and no discernable isolated population was noted to exist among most S. litura populations. The presence of a majority of within-population variations, rather than variations among populations or between China and Korea, indicates that the S. litura populations are composed of heterogeneous individuals. The fixation index at hierarchical level of among regions shows no significance, suggesting that the S. litura populations in both Korea and China are profoundly interrelated with one another. This finding is in accord with the current knowledge that S. litura has sufficient flight capacity for dispersal.

In order to understand the nature of population genetic structure of the notorious pest insect, Spodoptera litura (Lepidoptera: Noctuidae), a total of 159 individuals of the tobacco cutworm collected from six Korean and five Chinese localities were cloned and sequenced their mitochondrial A+T-rich region. A total of 90 haplotypes ranged 324 bp ~ 372 bp in length and 0.30% ~ 4.85% in sequence divergence. Compared to the preliminary mitochondrial COI gene sequence data that provided nearly identical sequences in all samples, the A+T-rich region provided substantially high sequence variation, validating the applicability of this molecule to the study of intraspecific variation. Overall, a very high per generation migration ratio (Nm = 5.91 ~ infinite), a very low level of genetic fixation (FST = 0 ~ 0.077796), and no discernable isolated population were noted to exist among populations of S. litura, although some significant genetic differentiations were found between several pairs of populations. These results collectively suggest that the S. litura populations in both Korea and China are very well interrelated to each other. The structural analysis revealed that the S. litura A+T-rich region harbored two stretches of the [TA(A)]n sequence, the poly-A stretch, and a stem-and-loop structure that are well found in other lepidopteran A+T-rich region. Furthermore, the region harbors the 13 ~ 19-bp long T stretch and 5-bp long motif that have been suggested to function as a possible recognition site for the initiation of replication of the minor strand of mtDNA also were found.

Hormones play a crucial role in controlling physiological processes, and thus plants grow and develop in response to environmental cues through the interlocked actions of the hormones. Brassinosteroids (BRs) were found as growth-promoting steroid hormones. Rice, as a monocotyledonous model plants and the major staple crop, has been used to study BR action mechanisms. However, many components of BR pathways and the mechanisms of their molecular interactions have yet to be fully understood. Because the use of the BR biosynthetic inhibitor, Brassinazole (Brz), allowed us to identify important components of BR signaling such as the transcription factor BZR1, we decided to employ a similar strategy to identify novel signaling factors using propiconazole (Pcz), a new potent BR inhibitor. We screened a rice T-DNA mutant population which belongs to Dongjin variety and were developed by the Gene An’s group using pGA2715 T-DNA vector. Using Pcz treatments we searched for resistant plants, which were reflected on their lengths of roots and/or leaves. We isolated a total of 17 mutant lines, which are being analyzed phenotypically and at molecular level. So far, we have been able to found various lines presenting high or low yield compared to their wild type counterparts. We have found differences in panicle organization of these mutants. Our current experiments include the confirmation of Pcz resistance of these lines and molecular studies involving BR marker genes to understand the relation among yield and BR action in rice.

We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes over-expression constructs under the control of the constitutive (CaMV 35S) promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also flanking sequence tags (FSTs) analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 157 flanking sequence tags (FSTs), among which 58 (36%) were integrated into genic regions and 97 (62%) into intergenic regions. About 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants were displayed higher levels of transgene protein. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.

GM벼 OsCK는 벼 유래의 OsCK1 유전자를 벼에 형질전환 하여 벼흰잎마름병 및 벼도열병에 대한 저항성을 높게 한벼로 농촌진흥청에서 개발하였다. 형질전환 벡터의 구성은 양쪽 border (LB, RB) 상간에 2개의 MAR 염기서열이 서로 마주보는 형태로 위치하고 있으며, 제초제 저항성 유전자 PAT는 CaMV 35S promoter에 의하여 발현이 유도되고, 목표 유전자인 choline kinase (OsCK)는 actin promoter에 의하여 발현이 조절되며 left border 기준으로 역방향으로 배치되었다. 도입유전자 확인을 위하여 adaptor ligation PCR을 수행하였는데, MAR 영역에 위치하는 제한효소로 GM벼 genomic DNA를 절단한 후 adaptor를 붙였다. 염기서열 분석을 위하여 T-DNA의 양 말단에서 primer를 제작한 후 sequence 분석을 하였다. 분석한 결과, T-DNA의 right border 인근의 MAR sequence가 벼 genome의 10번 염색체 129971번 염기와 연결되어 있음을 확인하였다. Left 영역의 삽입위치는 이후 실시한 Illumina NGS 시스템을 이용하여 확인할 수 있었으며, GM 벼에는 2개의 T-DNA가 도입되었음을 알 수 있고, 첫 번째 T-DNA는 벼 10번 염색체 BAC클론 OSJNBa0014J14의 128947번째 염기와 129970째 염기에 위치하고 벼 genome 염기 1024 bp가 결실됨을 확인하였다. 이 과정에서 첫 번째 T-DNA left border와 첫 번째 MAR sequence 일부(370 bp)가 결실되었고 right border와 두 번째 MAR 영역 199 bp가 결실되었음도 확인하였다. 두 번째 T-DNA는 right border가 결실된 형태로 첫번째 T-DNA의 35S promoter 중간에 삽입되었음을 확인하였다.

We have generated 383 independent transgenic lines for genetically modified (GM) rice that contained GPD, UtrCSP, BrTSR15 and BrTSR53 genes overexpression constructs under the control of the constitutive CaMV 35S promoter. TaqMan copy number assay was determined inserted T-DNA copy number. Also FSTs analysis was isolated from 203 single copy T-DNA lines of transgenic plants and sequence mapped to the rice chromosomes. In analyzing single copy lines, we identified 95 FSTs, among which 37 (38.9%) were integrated into genic regions and 58 (61.1%) into intergenic regions. About 27 homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assay. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNAs from leaf tissue of single copy, intergenic region of T-DNA insertion and homozygous T2 plants. The mRNA expression levels of the examined transgenic rice were significantly increased in all of the transgenic plants. In addition, myc-tagged 35S::BrTSR15 and 35S::BrTSR53 transgenic plants were displayed higher levels of transgene protein than WT plants. These results may be useful for producing of large-scale transgenic plants or T-DNA inserted mutants in rice.

Four transgenic rice lines harboring insect-resistant gene cry3A showed ideal field performances characterized by high considerable resistance to rice water weevil (Lissorhoptrus oryzophilus Kuschel). In this study, we estimated the insert number of foreign genes, and analyzed the flanking sequences of T-DNA in rice genome. As a result, The T-DNA of Btt12R 3-1-1-1 line was inserted in exon region of rice chromosome 10 and Btt12R 6-1-1-1 line was inserted in two copies of foreign gene. Btt12R 9-1-1-1 line was analyzed at only left border flanking sequence. The T-DNA of Btt12R 13-1-1-1 line was inserted one copy of foreign gene between position 24,516,607~24,516,636 of rice chromosome 5 and 30bp known genomic sequences were deleted. The Btt12R 13-1-1-1 line confirmed to be inserted in intergenic region having not any expressed gene and no any deletion/addition of T-DNA sequence. From these results, we demonstrated that the molecular data of rice water weevil resistant Bt rice could be acceptable to conduct the biosafety and environment risk assessment for GM crop commercialization

This study was conducted to determine the physicochemical properties of a giant embryo rice 'P47JB-4-B-5-B' derived from the cross between 'P47', a mutant of 'Hwayoung' induced by T-DNA insertion, and 'Junam'. The grain appearance and chemical components of the embryo were analyzed and compared with a donor cultivar, 'Hwayoung'. The proportion of embryo weight to grain weight of 'P47 JB-4-B-5-B' was 2.2 times heavier (6.7%) than that (3.1%) of 'Hwayoung'. Total free amino acid content (75.81 mg/100 g) of 'P47JB-4-B-5-B' was 2.1 times higher than that of 'Hwayoung'. The GABA content in brown rice was 14.06 mg/100 g in 'P47JB-4-B-5-B' and 6.8 mg/100 g in 'Hwayoung'. Especially, the GABA content in brown rice of 'P47JB-4-B-5-B' remarkably increased (about 33 times from 1.48 mg to 44.81 mg/100 g) 2 days after germination. Continuous frequency distributions and transgressive segregation in embryo length and width were observed in the F2 population of the cross between 'P47' and 'Cheongcheong', indicating that the giant embryo was controlled by quantitative trait loci. However, embryo length and width demonstrated high broad sense heritability, implying that giant embryonic traits could be selected in earlier generations in comparison with other quantitative traits.

Recently, giant embryonic rice and functional rice food are preferred by more consumers, which are attributed to the fact that the embryo has high concentrations of essential amino acids, fatty acids, and vitamins relative to other parts of rice grains. In this report, the heredity and stability of giant embryo mutations in successive generations were analyzed regarding a giant embryonic line, 'P47', induced by T-DNA insertion and a F2 population from a cross between 'P47' and 'Junam'. The mutant lines with increases of 1.5, 1.7 and 1.8 times on embryo length, width and 100-embryo weight to those of the control showed stable inheritance across three generations. The continuous frequency distributions of embryo size in the F2 population showed that the embryo size is a quantitative trait of polygene controlled. In addition, wide range of transgressive segregations of six traits affecting embryo size confirmed exchange of genetic materials and recombination between genes controlling embryo size. Five giant embryo mutant lines selected from the F2 population will be used for artificial selection and improvement of giant embryonic varieties.

With the development of diverse agricultures worldwide, biofortified rice noted for its preferable marketability and palatability plays an important role in the world's agricultural economics and rice breeding programs. In this report, several M5 of T-DNA inserted lines derived from the donor cultivars, 'Hwayong' and 'Dongjin', were selected for high or low protein, high lipid and low amylose content, respectively. The coefficients and ranges of variation for the chemical constituents between M4 and M5 T-DNA inserted lines were evaluated in comparison with those of the donor varieties. Results indicated that T-DNA insertion might be an effective way to generate useful variations for chemical composition of rice grains which could be used for the development of biofortified rice cultivars.