Recently, in Korea various kinds of genetically modified (GM) crops have been imported and used as a raw material to manufacture foods and livestock feeds, but the different social concerns about the benefits and the potential risks of GM crops are being shown with a different reaction from the public. Thus a persistent management is required for the safe utilization of genetically modified organism (GMO). PCR analysis of transgene into crop is generally performed for the efficient post management of GMOs. The most important prerequisite for the application of nucleic acid detections is to decide the effective DNA-extraction methods. Particularly, in the case of processed feeds, the nucleic acids of which may be damaged by heating, high pressure, pH treatments, fermentation, etc. in processing, DNA must be extracted with high sensitivity from the samples to perform the PCR successfully. In this study, seven of DNA-extraction methods used commercially and non-commercially were compared with respect to the yields and quality of DNA extracted from livestock feeds and those crop materials. Amounts of genomic DNA obtained from the extraction methods varies according to feed configurations and crop materials. The DNA yield and uniformity of samples extracted with PG, CTAB, and QF method is greater than that obtained from other extraction methods. In the DNA integrity of the selected extraction methods, PCR analysis showed distinct amplifications and similar patterns in detecting crop endogenous genes and GMO genes. These results would be applicable for the selection of an adequate DNA-extraction method in extracting processed feeds and/or crop materials.

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.

본 연구에서는 제초제 저항성인 Ab벼와 해충저항성 Bt벼, 이들의 모본인 동진벼의 현미에서 일반성분, 아미노산, 무기물, 지방산, 비타민 및 항영양소를 분석하여 유전자변형 벼(Ab, Bt)의 영양성분 조성 차이를 비교하고자 수행하였다. 그 결과 무기물의 철분이 모본인 동진벼와 유전자 변형 벼(Ab, Bt) 사이에 약 2배의 차이를 나타내었지만 codex 범위 내에 존재하였고, 비타민 류에서는 Bt벼와 동진벼 간의 유의적인 차이가 나타나지 않은 반면 Ab벼의 비타민 B1, B7, E에서 유의적인 차이를 보였다. 또한 트립신 저해제는 시료 모두에서 0.1 TIU/mg 미만으로 극소량 검출되었다. 총 46가지 분석 성분 중 17가지가 함량 및 조성에 유의적인 차이를 보였으나 이러한 차이는 이들의 모본인 동진벼, OECD 표준기술서, 그리고 일반 상업화 품종의 범위 내에 존재해 영양학적 측면에서 실질적으로 차이가 없는 것으로 판단된다.

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.

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.

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.

본 연구는 내충성 독소 발현 유전자(CryIIIA)를 벼에 형질 전환하여 해충에 대한 저항성을 갖도록 국내에서 개발한 해충저항성 GM벼(Btt12R)와 그 모본인 낙동벼의 주요영양성분과 항영양소를 분석하여 각 성분의 함량에 차이가 있는지를 비교하기 위해서 수행하였다. 이를 위해, 낙동벼와 Btt12R 뿐만 아니라 국내 상업화 품종인 영안벼와 화성벼를 수원 GMO 격리포장에서 동일한 조건하에 재배하여 수확한 현미를 사용하였다. 47가지 주요영양성분(8가지 일반성분, 17가지 아미노산, 8가지 지방산, 9가지 미네랄, 5가지 비타민) 중에 16가지 성분의 함량이 모본과 Btt12R 간에 차이를 보였지만, Btt12R의 이 16가지 성분은 함께 재배한 일반벼와 OECD에 명기된 함량 범위 내에 있었다. 2가지 항영양소 중 트립신 저해제는 모든 시료에 0.1 TIU/mg 미만의 극미량으로 존재했으며, Btt12R의 피트산 함량은 낙동벼와 일반벼의 피트산 범위에 포함되었다. 이상의 결과를 종합해보면, 분석한 Btt12R의 모든 주요영양성분 및 항영소의 함량이 모본 및 상업화 품종의 함량 범위에 포함되었으며, 이를 통해 CryIIIA 유전자를 벼 게놈에 삽입하는 것이 현미의 영양학적 품질에 영향을 미치지 않음을 확인하였다.

기존의 유전자변형식물은 외래의 도입유전자를 갖고 있으며 이들로부터 기인한 단백질 또는 합성물질에 의한 의도적/비의도적 영향에 대한 안전성 논란이 사회적 이슈가 되어 왔다. 최근의 기술적 진보에 의하여 이른바 식물육종의 신기술이 발달하게 되었고 이들 기술로 만든 신규식물에 대한 안전성평가에 GMO 관련 규제의 적용 여부 문제가 대두되게 되었다. 이들 NPBTs 기술로 만든 신규식물의 특징은 SDN이나 ODM과 같이 염색체상의 정확한 위치에 짧은 염기서열의 indel(s)이나 단일염기 돌연변이를 도입하여 자연적인 돌연변이와의 구별이 거의 불가능하거나, cisgenesis와 같이 성적교잡이 가능한 종 유래의 유전자를 구조변형 없이 도입하여 근연종과 동질적인 식물을 만들거나, heterozygous 형질전환체 후대세대의 null-segregant 선발이나 epigenetic를 이용하여 도입유전자가 존재하지 않지만 목적 형질을 갖는 식물체를 만드는 장점이 있다. 또한 grafting이나 Agro-infiltration 등의 방법으로 안전성평가를 회피하거나 경감할 수 있는 가능성을 높이게 되었다. OECD를 비롯한 주요 GMO 개발국의 관련 학회에서는 SDN, ODM 및 cisgenesis 또는 intragenesis 기술로 만든 식물에 대하여 non-GM 식물과 동일한 위해성평가 규정을 적용하거나 상황에 따라 완화된 규정을 적용할 수 있다고 판단하고 있다. 현 시점에서 이들 NPBTs 기술을 이용하여 개발된 식물이 상업화된 예는 없으나 많은 국가에서 상업화를 목적으로 개발 중이며 일부에서는 안전성평가를 완료한 단계이다. 이러한 현실에서 NPBTs 기술의 개념 정립, 신규식물의 안전성평가의 방향 설정 및 현실성 있는 작물개발 방안을 마련하여야 한다. 이를 통하여 GMO에 대한 안전성 논란과 사회적 거부감을 우회하는 동시에 답보 상태에 있 는 분자육종 분야의 발전을 도모할 계기를 마련하여야 한다.

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

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).

The cultivation of genetically modified (GM) crops has increased due to their economic and agronomic advantages. Before commercialization of GM crops, however, we must assess the potential risks of GM crops on human health and environment. The aim of this study was to investigate the possible impact of Bt rice on the soil microbial community. Microbial communities were isolated from the rhizosphere soil cultivated with Bt rice and Nakdong, parental cultivar and were subjected to be analyzed using both culture-dependent and molecular methods. The total counts of bacteria, fungi, and actinomycetes in the rhizosphere of transgenic and conventional rice were not significantly different. Denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rRNA genes revealed that the bacterial community structures during cultural periods were very similar each other. Analysis of dominant isolates in the rhizosphere cultivated with Bt and Nakdong rice showed that the dominant isolates from the soil of Bt rice and Nakdong belonged to the Proteobacteria, Cloroflexi, Actinobacteria, Firmicutes, and Acidobacteria. These results indicate that the Bt rice has no significant impact on the soil microbial communities during cultivation period. Further study remains to be investigated whether the residue of Bt rice effect on the soil environment.

Tocopherols (α-, β-, γ- and δ-tocopherols) represent a group of lipophilic antioxidants which are synthesized only by photosynthetic organisms. It is widely believed that protection of pigments and proteins of photosynthetic system and polyunsaturated fatty acids from oxidative damage caused by reactive oxygen species (ROS) is the main function of tocopherols. In the present study, NtTC, which encodes a tobacco tocopherol cyclase ortholog, was cloned and characterized. Compared with control plants, NtTC transgenic rice showed higher tolerance to drought stress, and total tocopherol content increased by 52 % in leaf. Additionally, total antioxitant activity of NtTC transgenic lines was increased significantly by 19%. These results demonstrate that over-expressing NtTC could improve the tolerance to abiotic stress in rice, and tocopherols play a crucial role in the protection of oxidative stress.

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.