Genetically modified (GM) crops have never been cultivated commercially in Korea, it is necessary for a thorough assessment of the risks associated with their environmental release. We determined the frequency of pollen mediated gene flow from disease resistant GM rice (OsCK1) to non-GM rice (Nagdongbyeo) and weedy rice (R55). A total of 449,711 or 164,604 seeds were collected from non-GM and weedy rice, respectively which were planted around OsCK1. Resistance of the hybrids was determined by repeated spraying of herbicide and DNA analysis using specific primer to confirm hybrids. Though non-GM rice and weedy rice have similar flowering time, the hybrids were found only in non-GM rice and out-crossing ranged from 0.018% at 0.3 m to 0.013% at 0.6 m. All of hybrids were located within 0.6 m distance from the GM rice plot in southerly direction. The meteorological factors including temperature and relative humidity during flowering time were found to be the most important factors for determining rice out-crossing. It should be considered many factors like the local weather condition and flowering time to set up the safety management policy to prevent pollen mediated gene flow between GM and conventional crop.

It is necessary to carry out a risk assessment to determine the consequences of releasing a particular plant species containing specific transgenes before transgenic plants can be grown under filed conditions. Gene flow from transgenic plants to wild closely related species has raised concern recently. Since transgenic crops were released in 1996, the global area of transgenic crops has been increasing rapidly. The transgene introgression from transgenic crops to their wild relatives is unavoidable in some species. Transgene introgression is of concern because the crop–wild plant hybrids might be conferred with a selection advantage to increase their performance, which could result in negative ecological consequences to natural ecosystems. The genus Brassica has 159 species, including a number of wild species that are of great importance to the economy. Most transgenic Brassica gene flow research has focused on the most successful cross between transgenic oilseed rape Brassica napus and its wild relatives Brassica rapa, a widely distributed weed in the farming system in Europe and America, since the hybridization can spontaneously happen and the generations can backcross to B. rapa easily in the wild conditions. In this study, we aimed to characterize transgene introgression, segregation, and expression in backcrossed generations between tramsgenic B. napus and B. rapa. These results will contribute to the environmental risk assessment and assist in biosafety management.

β-카로틴 강화벼 도입유전자의 도입 위치와 안정성을 도입 위치 주변염기서열 분석과 서던 분석한 결과, 벼 염색체 2번 중 30,363,938번과 30,363,973번 사이의 단일 부위에 도입유 전가 도입되었으며, T5-T7 세대 동안 도입된 모든 유전자들 이 안정적으로 유지되고 있으며, 도입유전자의 운반체인 Backbone DNA (pSB11)는 β-카로틴 강화벼에 삽입되지 않 았음을 확인하였다. 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 T5-T7 세대별, 생육시기별로 안정적으로 발 현됨을 검정하였으며, 목적 형질인 β-카로틴 분석 결과에서 도 모품종인 낙동벼에 비해 9.4배 함량이 증가됨을 확인하였 다. 이상의 분석기법을 통해 복수세대에서 β-카로틴 강화벼 의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

가뭄저항성벼의 복수세대에 대한 후대안정성을 서던 분석과 PCR로 분석한 결과, 가뭄저항성벼의 Agb0103 T4～T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, T-DNA 구성요소 이외의 backbone DNA는 가뭄저항성벼에 삽입되지 않았음을 확인하였다. 목적 유전자인 CaMsrB2와 제초제 저항성 선발 마커인 bar 유전자가 가뭄저항성벼 Agb0103의 T4～T6 세대에서 안정적으로 발현됨을 검증하였다. 제초제 저항성 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 Agb0103 T4～T6의 3세대에서 생육시기별, 부위별로 안정적으로 발현됨을 입증하였다. 도입유전자의 삽입 위치를 확인한 결과, 가뭄저항성벼 Agb0103의 도입유전자가 벼 8번 염색체 내에서 intergenic한 상태로 안정적으로 유지되고 있음을 확인하였다. 이상의 분석 기법을 통해 복수세대에서 가뭄저항성벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

Genetically modified (GM) crops have never been cultivated commercially in Korea, it is necessary for a thorough assessment of the risks associated with their environmental release. We quantified the amount determined the frequency of pollen mediated gene flow from disease resistant GM rice to weedy rice (R55). A total of 164,604 seeds were collected from weedy rice, which were planted around GM rice. Resistance of the hybrids was determined by repeated spraying of herbicide and DNA analysis using specific primer to confirm hybrids. Though weedy rice has similar flowering time, the hybrids were found only in non-GM rice and out-crossing ranged from 0.018% at 0.3 m to 0.013% at 0.6 m. All of hybrids were located within 0.6 m distance from the GM rice plot in southerly direction. The meteorological factors including temperature and relative humidity during flowering time were found to be the most important factors for determining rice out-crossing. It should be considered many factors like the local weather condition and flowering time to set up the safety management policy to prevent pollen mediated gene flow between GM and conventional crop.

Rice (Oryza sativa) is the most important staple food of over half the world’s population. This study was conducted to evaluate the possible impact of transgenic rice cultivation on the soil microbial community. Microorganisms were isolated from the rhizosphere of GM and non-GM rice cultivation soils. Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with GM and non-GM rice were similar to each other, and there was no significant difference between GM and non-GM rice. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM rice were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in GM and non-GM rice cultivated soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed similar patterns, but didn’t show significant difference to each other. DNAs were isolated from soils cultivating GM and non-GM rice and analyzed for persistence of inserted gene in the soil by using PCR. The PCR analysis revealed that there were no amplified protox gene in soil DNA. These data suggest that transgenic rice does not have a significant impact on soil microbial communities, although continued research may be necessary.

Genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnology companies. Commercialization of GM crops will be required the assessment of risk associated with the release of GM crops. The purpose of this research is a molecular characterization of introduced T-DNA in transgenic rice T4 ∼ T6 generation lines harboring a pepper MsrB2 gene under the control of stress inducible Rab21 promoter, as a part of biosafety evaluation for drought-tolerant transgenic rice (Agb0103). We identified the structure and sequence of transformation vector of T-DNA and analyzed insertion sites, flanking sequences, and generational stability of inserted T-DNA in transgenic rice lines. The transformation vector was consisted of right border, a drought-tolerant CaMsrB2 gene unit (Rab21 promoter::CaMsrB2::PinII terminator), a selectable marker herbicide resistance unit (CaMV 35S promoter::bar::Nos terminator), and left border in sequential order. Based on the adaptor-ligation PCR and whole genome sequence database, we confirmed that T-DNA was introduced 2 copies (head to head type) at the position of 2,471,957 ∼ 2,472,049 bp of chromosome No. 8. From the generational stability study, T-DNAs were stably inherited through the T4 to T6 generations, and also stable expression of bar gene from T-DNA was confirmed. It was also confirmed that the backbone DNA of transformation vector containing antibacterial gene (aadA) was not present in Agb0103 rice genome. These results will be filed to biosafety assessment document of Agb0103

The perturbation of the steady state of reactive oxygen species due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 promoter with/without a selection marker, the bar gene. A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol. An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice

본 연구는 대규모 GMO 격리포장에서 고추 유래 가뭄저항성CaMsrB2유전자가 도입된 가뭄저항성벼(Agb0103)의 안정성 평가에 대한 가이드라인 및 프로토콜을 개발하고자 수행하였다. 경북대학교 농업생명과학대학 GMO 실습격리포장에서 총 사용면적 4,700 m²에서 Agb0103와 모품종인 일미벼에 대해 거미류를 포함한 곤충류 다양성을 조사하였다. 조사기간 동안 채집된 개체들은 기능별로 해충군, 천적군, 거미군으로 크게 구분하여 계수하였으며, 조사된 개체군의 해충군, 천적군, 거미류 등의 밀도는 기능군 별로 Agb0103와 일미벼간에 유의성을 보이지 않았다. 해충의 발생양상을 비교 분석한 결과, 전반적으로 일미벼에서 발생량이 다소 많은 경향으로 나타났으나, 대부분의 시기별 발생량 평균 간의 차이는 없는 것으로 나타나서 Agb0103와 일미벼과의 해충 발생양상에는 유의성을 찾기가 어렵다고 판단된다. 또한 천적류 발생양상은 해충 발생양상과는 달리 Agb0103에서 발생량이 다소 많은 경향으로 나타났으나, 시기별 평균 간 차이는 없는 것으로 분석되어 천적 발생양상에도 특이한 유의성을 없다고 판단된다. 따라서 가뭄저항성 CaMsrB2유전자가 도입된 Agb0103에서의 해충 및 천적의 개체군 밀도 및 발생양상은 모본인 일미벼에서와 차이가 없는 것으로 조사되었다.

국내에서는 아직까지 유전자변형 작물이 재배되고 있지 않으나 유전자변형 작물의 환경방출을 위해서는 반드시 환경위해성 평가가 수행되어야 한다. 본 연구에서는 병저항성 유전자변형 벼(OsCK1)로부터 비 형질전환 벼(낙동벼)와 잡초성벼(R55)로의 화분 매개에 의한 유전자 이동성을 평가하였다. 비 형질전환 벼로부터 449,711립의 종자를 얻었으며 잡초성벼로부터는 164,604립의 종자를 수확하였다. 교잡개체는 2회의 제초제 살포와 병저항성 벼에 특이적인 프라이머를 이용한 분자생물학적 방법을 통해 저항성 여부를 검증하였다. 개화기간이 서로 일치하였음에도 불구하고 비 형질전환 벼에서만 교잡개체를 확인할 수 있었으며, 교잡율은 교잡개체가 발생한 거리에 따라 0.013～0.018%로 나타났다. 모든 교잡개체들은 병저항성 유전자변형 벼에 근접한 0.6 m내에서 발견되었다. 화분 매개에 의한 병저항성 유전자변형 벼의 유전자 이동성은 앞서 연구된 결과와 유사한 것으로 나타나 농업환경에 방출되더라도 환경에 미치는 영향은 다른 벼들과 비슷할 것으로 추정되었다. 개화기간 중 온도와 습도 등 기상요인이 벼의 교잡율을 결정하는데 중요한 요소이었다. 따라서 유전자변형 벼와 주변 재배종 및 잡초성벼로의 유전자 이동에 의한 안전관리 대책을 마련하기 위해서 지역의 기상요인과 개화기 중복 등의 요인들이 충분히 고려되어야 할 것이다.

Genetically modified (GM) crops have been developed worldwide through the recombinant DNA technology and commercialized by various agricultural biotechnological companies. Commercialization of GM crops will be required the assessment of risk associated with the release of GM crops. In this study, we carried out to provide the molecular characterization of introduced T-DNA in transgenic rice T4 ~ T6 generation lines harboring a pepper MsrB2 gene under the control of stress inducible Rab21 promoter, as a part of biosafety evaluation for drought-tolerant transgenic rice (CaMsrB2). We identified the structure and sequence of transformation vector of T-DNA and analyzed insertion sites, flanking sequences, and generational stability of inserted T-DNA in transgenic rice lines. The transformation vector was consisted of right border, a drought-tolerant CaMsrB2 gene unit, a selectable marker herbicide resistance unit, and left border in a sequential order. Based on the adaptor-ligation PCR and whole genome sequence database, we confirmed that T-DNA was introduced at the position of 41,737,284 bp of chromosome No. 1. From the generational stability study, T-DNAs were stably inherited through the T4 to T6 generations, and also stable expression of bar gene from T-DNA was confirmed. These results will be filed to biosafety assessment document of CaMsrB2 rice.

Rice (Oryza sativa) is the most important staple food of over half the world’s population. This study was conducted to evaluate the possible impact of transgenic rice cultivation on the soil microbial community. Microorganisms were isolated from the rhizosphere of GM and non-GM rice cultivation soils. Microbial community was identified based on the culture-dependent and molecular biology methods. The total numbers of bacteria, fungi, and actinomycete in the rhizosphere soils cultivated with GM and non-GM rice were similar to each other, and there was no significant difference between GM and non-GM rice. Dominant bacterial phyla in the rhizosphere soils cultivated with GM and non-GM rice were Actinobacteria, Firmicutes, and Proteobacteria. The microbial communities in GM and non-GM rice cultivated soils were characterized using the denaturing gradient gel electrophoresis (DGGE). The DGGE profiles showed similar patterns, but didn’t show significant difference to each other. DNAs were isolated from soils cultivating GM and non-GM rice and analyzed for persistence of inserted gene in the soil by using PCR. The PCR analysis revealed that there were no amplified protox gene in soil DNA. These data suggest that transgenic rice does not have a significant impact on soil microbial communities, although continued research may be necessary.

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.

가뭄저항성벼의 복수세대에 대한 후대안정성을 서던 블롯과 PCR로 분석한 결과, 가뭄저항성벼의 CaMsrB2-8 T4 ~ T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, T-DNA 구성요소 이외의 backbone DNA는 가뭄저항성벼에 삽입되지 않았음을 확인하였다. 목적 유전자인 CaMsrB2와 제초제 저항성 선발 마커인 bar가 가뭄저항성벼의 CaMsrB2-8 T4 ~ T6 세대에서 안정적으로 발현됨을 검증하였다. 제초제 저항성 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 CaMsrB2-8 T4 ~ T6의 3세대에서 생육시기별, 부위별로 안정적으로 발현됨을 입증하였다. 도입유전자의 삽입 위치를 확인한 결과, 가뭄저항성벼 CaMsrB2-8의 도입유전자가 벼 1번 염색체 내에서 intergenic한 상태로 안정적으로 유지되고있음을 확인하였다. 이상의 분석 기법을 통해 복수세대에서 가뭄저항성벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.

비타민A 강화벼의 복수세대에 대한 후대안정성을 Southern blot과 PCR로 분석한 결과, 비타민A 강화벼의 PAC T3～T6 세대에서는 도입된 모든 유전자들이 안정적으로 도입되어 있으며, backbone DNA는 비타민A 강화벼에 삽입되지 않았음을 확인하였다. 선발 마커로 도입된 PAT 단백질의 발현 분석 결과에서도 PAC T3～T6 복수세대에서 생육시기별 부위별로 안정적으로 발현됨을 입증하였으며, 최종 목적 산물인 카로티노이드 분석 결과에서도 모품종인 낙동벼에 비해 비타민A 강화벼에서 β-carotene은 10.6배 함량이 증가되고, zeaxanthin과 α-carotene는 생성되었음을 확인하였다. 이상의 분석기법을 통해 복수세대에서 비타민A 강화벼의 도입 유전자들이 안정적으로 유지되고 목적 단백질들이 안정적으로 발현되고 있음을 확인하였다.