농업적, 환경적, 경제적 및 사회적인 이익으로 농업생명공학에 의한 유전자변형(GM) 작물의 재배는 점차 증가되고 있다.국내에서도 주요 작물을 대상으로 유용 GM작물이 개발되고있으며, 최근 Choline kinase 유전자(OsCK1)가 도입된 병저항성 형질전환벼가 개발되었다. GMO의 안전성과 관련하여, 표시제의 시행 또는 사후 이력추적을 위해서 검정법이 필수적으로 요구되고 있다. 본 연구에서 각 134, 306, 243bp의 PCR증폭산물을 갖는 유전자 특이, 구조 특이 및 이벤트 특이primer를 병저항성(OsCK1) GM벼의 검출에 사용하였고, 다른어떤 작물에서도 반응산물을 나타내지 않았다. 이벤트 특이primer CKRB32-1/02-2를 사용한 정성 duplex PCR을 통해서OsCK1 GM벼에 대한 검출한계(LOD)가 0.05%임이 확인되었다. Real-time PCR을 이용한 정량검정을 위해서 벼 내재유전자 염기와 OsCK1 GM벼의 5’-인접염기를 갖는 pSPSCKR을표준물질로 제조하였고, 10 copies 범위까지 정량검출이 가능한 것으로 나타났다. 따라서 도출된 real-time PCR 방법의 정확성 및 정밀성을 확인하고자 0.5, 1, 3, 5 및 10%로 GM시료에 대하여 정량 분석하였으며, 표준편차 및 상대표준변이가 20% 내로 확인되었다. 이상의 결과로, 개발된 이벤트 특이정성 및 정량 PCR 방법이 OsCK1 GM벼의 사후 GMO 모니터링 및 이력추적에 효과적으로 적용 가능할 것으로 판단된다.

A variety of genetically modified (GM) crops have been developed in Korea. In these crops, the resveratrol-enriched transgenic rice plant (Agb0102) has moved ahead to generate the dossier for regulatory review process required for commercialization of GM crop. The resveratrol-enriched transgenic rice plant could be released to farmers for cultivation after national regulators have determined that it is safe for the environment and human health. Here, we developed a PCR-based DNA marker based on flanking sequences of transgene for the discrimination of resveratrol-enriched transgenic rice plant. This DNA markers will be useful for identifying of resveratrol-enriched transgenic rice plant, and can also be used to estimate transgene movement occurred by pollen transfer or seed distribution. Moreover, it is helpful for prompt screening of a homozygote-transgenic progeny in the breeding program.

Natural and artificially induced mutants have provided valuable resources for plant genetic studies and crop improvement. Some variations induced in the process of plant transformation have often been observed in regenerated plants. In this study, we investigated the insertion number of transgene and the flanking sequences of T-DNA in tall-induced line BP23, which was unexpectedly gained in the process of transformation of insect-resistant rice with cryBP1 gene, and also analyzed the whole-genome sequencing by using the NGS technologies to gain a better understanding of the sequence and structural changes between tall line or natural cultivar and rice reference. than others, was confirmed with two copies of foreign gene insertion, which was inserted in one genomic site facing each other between the position 2,430,152~2,430,151 of rice chromosome 12 without any deletion of genomic sequences. Sequencing analysis also revealed that 18bp-unknown sequences were added in the 5′ insertion site of T-DNA. This position in rice genome was confirmed with none of expressed gene sites. By the NGS analysis, we detected 86560 SNPs and 1091/1472 large insertion/deletion (indel) sites (100bp) between BP23 and rice reference, and 84743 SNPs and 1094/1451 large indels between natural cultivar Nagdong and rice reference. The possible mechanisms for the gene mutation, the developmental and tissue expression of the taller height in BP23 line may need to be scrutinized a few more.

The selectable marker-free rice plants containing mcry1Ac insecticidal gene isolated from Bacillus thuringiensis (Bt) were generated using a non-selection approach by Agrobacterium tumefaciens-mediated transformation. The nutritional composition of two lines of transgenic rice plants (RTB5 and RTB11) was compared with that of its non-transgenic counterpart. The results showed that, except for small differences in dietary fiber and some minerals, there was no significant difference between transgenic rice and conventional counterpart variety with respect to their nutrient composition. Most of measured levels of nutrients were within the range of values reported for other commercial cultivars, showing substantial equivalency. Therefore, the insertion of transgenes did not affect the nutritional composition of transgenic RTB5 and RTB11 rice grains.

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium -mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

A variety of genetically modified (GM) crops have been developed in Korea. In these crops, the resveratrol-enriched transgenic rice plant has moved ahead to generate the dossier for regulatory review process required for commercialization of GM crop. The resveratrol-enriched transgenic rice plant could be released to farmers for cultivation after national regulators have determined that it is safe for the environment and human health. Here we developed a PCR-based DNA marker based on flanking sequences of transgene for the discrimination of zygosity in resveratrol-enriched transgenic rice plant. This DNA marker will be useful for identifying of resveratrol-enriched transgenic rice plant, and can also be use to estimate transgene movement occurred by pollen transfer or seed distribution.

The β-carotene biofortified transgenic soybean was developed recently through Agrobacterium-mediated transformation using the recombinant PAC (Phytoene synthase-2A-Carotene desaturase) gene in Korean soybean (Glycine max L. cv. Kwangan). GM crops prior to use as food or release into the environment required risk assessments to environment and human health in Korea. Generally, transgenic plants containing a copy of T-DNA were used for stable expression of desirable trait gene in risk assessments. Also, information about integration site of T-DNA can be used to test the hypothesis that the inserted DNA does not trigger production of unintended transgenic proteins, or disrupt plant genes, which may cause the transgenic crop to be harmful. As these reasons, we selected four transgenic soybean lines expressing carotenoid biosynthesis genes with a copy of T-DNA by using Southern blot analysis, and analyzed the integration sites of their T-DNA by using flanking sequence analysis. The results showed that, T-DNA of three transgenic soybean lines (7-1-1-1, 9-1-2, 10-10-1) was inserted within intergenic region of the soybean chromosome, while T-DNA of a transgenic soybean line (10-19-1) located exon region of chromosome 13. This data of integration site and flanking sequences is useful for the biosafety assessment and for the identification of the β-carotene biofortified transgenic soybean.

본 연구는 Bacillus thuringiensis 유래의 살충성 mCry1Ac 유전자를 무선발 형질전환 방법으로 일미 벼에 도입하여 개 발된 마커프리 형질전환 Bt 벼 2계통의 일반성분 및 주요성분 (무기질, 아미노산) 함량을 확인하여 모본벼 및 다른 일반품 종과 함량차이를 비교 분석 함으로서 형질전환 벼의 영양성 분 동등성 여부를 확인하고자 수행되었다. 영양성분 분석결과 GM 벼 현미의 일반성분 조성 중 식이섬유 함량과 일부 무기 질 함량이 모본 벼인 일미와 비교하여 다소 유의적 차이가 있 었지만 일반품종에서 나타나는 함량범위 안에 포함되는 수치 이며, 아미노산 성분과 대부분의 일반성분 및 무기질의 함량 은 전반적으로 모본과 유의적 차이가 없었다. 따라서 형질전 환 벼에서 관찰된 일부 성분 차이는 Bt 유전자의 도입 효과가 아닌 재배 환경 및 토양성분의 차이에서 기인된 것으로 형질 전환에 의한 비의도적 영양성분 변화는 없는 것으로 판단된다.

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.

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

Two carotenoid biosynthetic genes, phytoene synthase (Psy) and carotene desaturase (CrtI) linked via synthetic 2A sequence under control of CaMV 35S promoter (two T0 plants 5 and 6) or β- conglycinin promoter (three T0 plants 7, 13 and 16) were transformed into soybean variety Kwangan. After agronomic and phenotypic selection at early generations, T5 progeny of PAC soybean were analyzed by Southern blot to confirm T-DNA copy numbers. A total of 27 homologous lines derived from one of three T0 plants (line 7 under the control of β- conglycinin promoter) with one copy T-DNA insertion, were separated and planted into greenhouse. Flanking sequence analysis was carried out on one of homologous line 6-2-3 and results indicated the T-DNA was intergenic inserted into chromosome 14 from 10,873,131 to 10,872,998 base of soybean chromosome. T-DNA insertion structure, flanking sequence and inserted gene expressions need to be analyzed in the further study.

Resveratrol rice Iksan526 was developed by overexpession of T-DNA (RB::P-Ubi::RS::T-NOS::P-35S::PAT::T-35S::LB) in rice variety Dongjin. To confirm one locus insertion of T-DNAs, Mendelian genetic analysis was carried out on selection marker bar gene and objective RS gene separately by using a F2 population derived from a cross of Dongjin/Iksan526 (T6). A total of 450 four-leaf-old plants from F2 population were treated by 0.3% basta, and a phenotypic separation ratio of 3:1 (321 survival: 129 dead, p>0.90) complied with Mendelian inheritance indicating one locus insertion of bar gene. Genotypic separation was analyzed by using PCR with specific primers for 300 plants, which were selected from 321 survival plants after phenotypic separation. Results revealed a ratio 1:2 of homologous to heterozygous (92:208, p>0.90), which further confirmed one locus insertion of RS gene. In addition, comparison on agronomic traits and resveratrol contents between transgenic rice and the donor variety were launched to evaluate the phenotypic performance over multi-generations (years).

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a significant disease in most rice cultivation areas. The present study was performed to identify new BB R-gene conferring resistance to Korea Xoo isolates, derived from IR65482-7-216-1-2 and to construct a physical map of the candidate gene. An F2 population derived from a cross between 11325 and Anda was used to determine the exact position of the nearest recombination event to the target region. The position of the R-gene was delimited by flanking markers, RM1233 and RM5766, on chromosome 11. Of the 56 markers designed in the flanking region, 20 were selected as anchor markers and the R-gene was mapped to a 295kb region on chromosome 11. To narrow down the interval spanning the R-gene, an additionally SSR marker, 20 STS markers, and CAPS marker between RM27320 and ID55.05-79 were developed using rice reference genome information. From the result the gene was defined by RM27320 and ID55.WA18-5 located in the BAC clone OSJNBa0036K13. The physical distance between these two markers is approximately 80kb. In a further study, gene expression analysis against listed candidate genes was investigated using semi-quantitative transcription PCR. These results will useful for future disease breeding as well as gene function studies regarding resistance genes.

A Transgenic Kimch cabbage has been developed harboring T-DNAs expressing delta-endotoxin insecticidal protein, herbicide (basta) resistant protein, and antisense transcript of AsMADS2 gene. Three transgenic lines, #24, #45, and #51, originated from the same T0 plant were analyzed in terms of molecular characterization, phenotype, and agronomic traits. Flanking sequence analysis confirmed that T-DNA, with 7132 bp intact structure, was inserted onto the pseudochromosome A10 of B. rapa and all the genes in T-DNA were functionally active. Three of GM cabbage showed 69.2～75.3% of plant height and 81.8～89.7% of diameter to those of the isogenic variety ‘Nowon’, respectively. Curving upward leaf lamina attitude was observed on GM cabbage, while straight or slight concave on non-GM cabbage. In addition, an average range of 86～91.5% of head height and 87.4～94.8% of head diameter were observed on GM cabbage to those of the isogenic variety ‘Nowon’, respectively Moreover, curled inwards or slight overlap of head-forming leaf overlap at terminal region was observed on GM cabbage, but curled outwards or erect on non-GM cabbage. AsMADS2, a transcription factor reported to be involved in early flowering, was stably expressed to RNA in the GM cabbage, but it was not shown the significant influences to flowering time.

Regulations in the EU, Japan, Korea, etc. require that foods and feeds made of or derived from genetically modified organisms (GMOs) should be approved and labeled according to a threshold. Recently, disease resistant transgenic rice was developed in Korea, which resulted from the transformation events involving choline kinase gene, OsCK1. In order to monitor unintended release of the developed GM rice in the near future, as well as to meet GM-labeling requirements, the development of a reliable method for detection of disease resistant GM rice is requisite. Here, specific primer pairs for the detection of GMO was designed on the basis of a introduced gene and the flanking junction sequences between a plant DNA and a integrated gene construct, and also SPS gene was used as an endogenous reference material. Specificities of all designed primers were tested through qualitative PCRs. Clearly, target specific amplicons could be detected from disease resistant GM rice event. In addition, the limits of detection (LOD) using the event-specific primers were approximately 0.1% for the disease resistant GM rice line. This result indicated that the developed detection method is suitable for the traceability of disease resistant GM rice, because of using the primer specifically corresponded to the junction site between plant genomic DNA and inserted DNA. Keywords: genetically modified organisms, disease resistant GM rice, PCR detection, event-specific primer

The rice water weevil (RWW), Lissorhoptrus oryzophilus are major pests of aquatic rice plant in Korea as well as throughout the country. Larvae of RWW sucking the nourishment on roots, causes a stunted root system and reduces grain yields. To prevent these damages, we constructed various plant expression vectors, which were harbored by insecticidal genes, cryBP1 and cryIIIa, and fused with the actin promoter and/or the modified RCg2 root-preferential promoters for expressing the insect-toxic genes in leaves and roots. A cryBP1 was cloned from Bacillus popilliae, producing crystal toxin against Japanese beetle, and CryIIIa was modified from the δ-endotoxin gene of Bacillus thuringiensis ssp. tenebrionis, encoding the coleoptera-specific toxin. The vectors containing the insecticidal genes were transferred into Oryza sativa japonica cultivar, Nakdong, by Agrobacterium -mediated transformation method. Several independent transgenic lines were selected by Southern blotting and Western blotting, confirming that cryBP1 and cryIIIa genes were stably integrated into the plant genomes and were expressed in transgenic plants. Upon insect bioassay using RWW, the mortality of insect larvae on cryBP1 and cryIIIa transgenic rice lines recorded up to 41% and 34%, respectively. These results suggested that the transgenic lines can be used to develop Coleoptera-resistant cultivars and could be valuable for later application in crop breeding for insect resistance.

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.