Molecular and Functional Characterization of Monocot-specific Pex5p Splicing Variants, Using OsPex5pL and OsPex5pS from Rice (Oryza sativa)

이중로,이상열,정지현,Jae Sook Kang,김종철,정인정,Min Sook Seok,Ji Hye Kim,김외연,김민갑,김재연,임채오,이균오 한국분자세포생물학회 2007 Molecules and cells Vol.23 No.2

Temporal and Spatial Regulation of Cell Cycle Genes during Maize Sex Determination

이중로,김종철,Lee, Jung-Ro,Kim, Jong-Cheol Korean Society of Life Science 2006 생명과학회지 Vol.16 No.5

옥수수 (Zea mays L.) 꽃은 암술 세포사멸과 수술 세포 성장정지 등을 통하여 양성상태에서 단성 상태로 성결정 과정을 완성한다. 본 논문에서는 옥수수 성 결정 동안 세포주기 유전자들의 시간적, 공간적 발현조절을 조사하였다. 세포주기의 양성조절 인자 즉 cyclin A, cyclin B, cyclin dependent kinase A (CDK A), Mad2 유전자들은 성장하는 암술과 수술에서 높게 발현되는 반면 죽어가는 암술과 성장이 정지되는 수술에서는 이들의 발현이 사라졌다. 이와 반대로, Wee1과 CDK inhibitor (CKI) 같은 세포주기 음성 조절유전자들은 야생형 암꽃과 tasselseed2 돌연변이 수꽃의 성장이 정지하고 있는 수술에서 발현이 증가되었지만, 흥미롭게도, 이들 유전자들은 죽어가는 암술세포에서는 발현되지 않았다. 이들 결과들을 통하여 옥수수 성 결정 과정 중에서 암술 세포사멸과 수술세포 성장정지는 세포주기조절과 밀접한 관계가 있으며, 특히 성장이 정지하는 수술과 죽어가는 암술에서의 음성 세포주기 조절 유전자들의 다른 발현양상은 이 둘의 성 결정 메커니즘이 구별 될 것이라고 사료된다. Maize (Zea mays L.) pistil cell death and stamen cell arrest are pivotal process on the sex determination, which diverges from bisexual state of floral meristem to unisexual state in staminate or pistillate floret. We investigated the temporal and spatial distribution of cell cycle gene expression during maize sex determination. The positive regulatory genes of cell cycle, cyclin A, cyclin B, cyclin dependent kinase (CDK) and Mad2 were highly expressed in the developing pistil and stamen but the expression was disappeared in the dying pistil and arresting stamens. In contrast, the negative regulatory genes of cell cycle, Wee1 and CDK inhibitor (CKI) were expressed in the arresting stamens in the wild-type ear and tasselseed2 mutant tassel, however, these genes were not detected in dying pistil although the cyclin B gene expression was disappeared. These results suggest that both the pistil cell death and stamen cell arrest process in maize sex determination are involved in cell cycle regulation, but the different expression patterns of negative regulatory cell cycle genes in the arresting stamens and aborting pistils suggest that the two processes may have distinctive modes of action.

Genetic Diversity and Population Structure of a Korean Rice Germplasm Based on DNA Profiles

이경준,이중로,신명재,조규택,마경호,이기안,정종욱 한국작물학회 2018 Korean journal of crop science Vol.63 No.1

Information on the patterns of genetic diversity and population structure is essential for the rational use and efficient management of germplasms; accurate information aids in monitoring germplasms, and can also be used to predict potential genetic gains. In this study, we assessed genetic diversity, focusing on Korean rice accessions for theand their sustainable conserved diversity. Using DNA profiling with 12 simple sequence repeat (SSR) markers, we detected a total of 333 alleles among 2,016 accessions. The number of alleles ranged from 21 to 53, with an average of 27.8. Average polymorphism information content was 0.797, with the lowest being 0.667 and the highest 0.940. CA cluster analysis and the model-based population structure revealed two main groups that could be subdivided into five subgroups. Analysis of the molecular variance study based on the SSR profile data showed 5% variance among the profiles, whereas we recorded 93% variance among individuals and 2% variance within individuals. Specifically, the utilized diversity for of the breeding program is restricted in that cultivars were located in limited clades. These results revealed that preserving the diversity of Korean landraces could be useful sources for breeding new rice cultivars, and cwould be the basis for the sustainable conservation and utilization of a Korean rice germplasm.

국내 승인 유전자변형 작물의 검출 기법 확립

설민아,이중로,최원균,조범호,문정찬,신수영,엄순재,김일룡,송해룡 한국식물생명공학회 2015 식물생명공학회지 Vol.42 No.3

AbstractLiving modified organisms (LMO) are one of the most widespread products of modern biotechnology after DNA discovery. Due to the decline of grain selfsufficiency rate and the increase of reliance on LMO imports in Korea, a series of concerns with regard to safety of living modified(LM) crops has been raised. The aim of this study is to establish the detection methods for unintentional release or growing of LMO plants in environmental conditions. To detect LM crop events, general concepts of specific primer design and PCR conditions were provided by the Joint Research Centre (JRC). The certified reference materials of seven LM events (4 soybean, 2 cotton and 1 corn) were obtained from the Institute for Reference Materials and Measurements (IRMM) and the American Oil Chemists’ Society (AOCS). Genomic DNA from seven LM events were purified and PCR amplifications were carried out by using individual event-specific primer sets. LM-specific PCR products of all seven events were efficiently amplified by our methods. The results indicate that the established detection method for LMOs is suitable as a scientific tool to monitor whether the crops found in natural environments are LMOs.

Multiplex PCR 방법을 이용한 국내 승인 5개 LM 유채의 검출법 개발

조범호,이중로,최원균,문정찬,신수영,엄순재,설민아,김일룡,송해룡 한국식물생명공학회 2015 식물생명공학회지 Vol.42 No.2

Canola is a crop globally used for production of oil and biofuel. Cultivation area and import volume of living modified (LM) canola have been increasing every year. As canola import dependence has reached 100% in Korea, efforts have been made for safety management of LM canola and ecological risk assessment. We developed a set of multiplex PCR method for simultaneous detection of 5 LM canola events (Topas 19/2, Rf3, Ms8, RT73 and T45) approved in Korea. The multiplex PCR assay developed allows amplification of estimated products of 5 LM canolas from event specific primer sets. Primer extension time was skipped for a time-consuming process and two annealing steps (20 cycles at 55°C and 20 cycles at 60°C) were performed for yielding the best result which was sufficient to distinguish five LM canolas. Our results suggest that multiplex PCR method provides a cost and time-effective approach for LM canola detection.

Bacillus thuringiensis 유래 Vip3Aa 단백질 순수분리 및 꿀벌(Apis mellifera)에 대한 위해성평가

정영준,유수향,이중로 한국환경생물학회 2019 환경생물 : 환경생물학회지 Vol.37 No.4

Most insect-resistant LMOs have been produced by applying Cry and Vip3Aa proteins. Vip3Aa protein is activated during the vegetative stage of Bacillus thuringensis (Bt) and the inhibitory activity of the Vip3Aa protein against pathogenic attacks from lepidopteran insect species is well known. However, a risk assessment of the Vip3Aa protein compared to the Cry protein has not been conducted in South Korea. This study demonstrates a possible risk assessment method for Vip3Aa protein against honeybees (Apis mellifera). For the risk assessment of the protein, we purified the recombinant Vip3Aa protein in Escherichia coli. The survival rate and symptoms of general intoxication of 4 months honeybees were measured after Vip3Aa exposure. These results indicated that there was no significant difference in the survival rate and the symptom between Vip3Aa and the control buffer. In this study, we established standard methods of Vip3Aa protein purification and oral adult toxicity test using A. mellifera as an LMO risk assessment technique for preserving the natural ecosystem of South Korea. 본 연구는 LMO 유전산물의 위해성평가를 위해 바실러스로부터 증폭된 Vip3Aa 유전자를 이용하여 대장균에서 단백질 순수분리 하였으며, MALDI-TOP 분석법을 통해 기존의 알려진 살충성 Vip3Aa 단백질과 동등한 단백질임을 증명하였다. 순수 분리한 Vip3Aa 단백질을 이용하여 꿀벌 과독성 급성섭식독성평가를 수행하였다. 그 결과 무처리군, Hepes buffer, Vip3Aa 단백질 처리군 모두 치사 및 일반중독증상을 보이는 개체는 발견되지 않았다. 이 결과를 통해 Vip3Aa 단백질은 꿀벌에 위해성을 나타내지 않는다는 결론을 얻을 수 있었다. 본 연구 결과는 향후 국내 LMO 유전자 산물 위해성평가에 유용하게 활용될 것이라 사료된다.

Partial Purification and Properties of a Phosphatidylinositol 4,5-Bisphosphate Hydrolyzing Phospholipase C from the Soluble Fraction of Soybean Sprouts

Soo Kwon Park,이중로,이승식,손효진,Ji Young Yoo,문정찬,Heun Young Kwon,임채오,박정동,조무제,이상열 한국분자세포생물학회 2002 Molecules and cells Vol.13 No.3

Three soluble enzyme fractions (F-I, F-II, and F-III) that hydrolyze phophoinositides were separated from soybean sprouts by using Matrex green gel column chromatography. Among the three phosphatidylinositol (PI)-specific phopholipsase C (PLC) enzymes, only the third fraction (F-III) was able to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) as well as phosphatidylinositol (PI) and phosphatidylinositol phosphate (PIP) as substrates. The F-I and F-II fractions only showed enzymatic activities for PI and PIP. The PIP2-hydrolyzing PLC protein, F-III, was partially purified using the chromatographic steps of the Matrex green gel, phenyl Toyopearl, Matrex orange gel, Mono S cation exchange, and superose 6 gel filtration columns. The molecular weight of the F-III protein was estimated to be about 64 kDa on SDS-PAGE. The protein showed immunocross-reactivity with a polyclonal antibody that was prepared against the X and Y motifs of animal PLC enzymes, the conserved catalytic domains. Ca2+ ion critically affected the PIP2- hydrolyzing PLC activity of the F-III protein, representing maximal activity at 10 M Ca2+ concentration. The PIP2-hydrolyzing PLC activity of the protein was also significantly increased by sodium deoxycholate (SDC) from 0.05 to 0.08%. However, the activity was greatly reduced above the concentration, and no activity was detected at 0.3% SDC. In addition, the protein exhibited maximal PIP2-hydrolyzing PLC activity at pH, in the range of 6.5−7.5.

Safety management regulation and practice standards on living modified organism (LMO) facilities under the Ministry of Environment

Nam Kyong-Hee,이중로 한국식물생명공학회 2023 Plant biotechnology reports Vol.17 No.6

Living modified organisms (LMOs) in South Korea are managed under the Transboundary Movement, Etc. of LMOs Act, the domestic implementation law of the Cartagena Protocol on Biosafety. The Ministry of Environment (MOE) oversees the risk review and safety management of LMOs for environmental remediation, and consultative review on the risk posed to the natural ecosystem by LMOs for other purposes. In particular, the MOE operates a risk assessment institute to promote scientific and systematic safety management by standardizing an evaluation criterion for the relevant LMOs. These must be established according to the standards of the LMOs Act. In this review, a new technique for reliable management of the LMO facility, including a confined field, was proposed to comply with the safety standards of the MOE. Based on the analysis of the LMOs Act and the practices of established LMO facilities, potential facility management directions under the MOE were discussed from the perspectives of the facility, cultivation, waste, record, and education. These suggestions can help physically isolate LMO facilities and prevent the unintentional release of LMOs, thus helping avoid hybridization between LMOs and non-LMOs. In addition, a monitoring system was proposed to prevent transgene escape and subsequent hybridization with the wild relatives of LMOs. These proposals can be widely used for safety management when researching the development and commercialization of LMOs at facilities under the MOE.

자연생태계 모니터링을 통한 glyphosate와 glufosinate-ammonium에 저항성을 가지는 유전자변형 캐놀라의 발견

신수영,조범호,문정찬,이중로,최원균,설민아,김미정,송해룡 한국식물생명공학회 2016 JOURNAL OF PLANT BIOTECHNOLOGY Vol.43 No.4

Living modified (LM) crops are imported each year to South Korea as food and feeds, LM canola being one of the imported crops. The cultivation of LM crops is not permitted in South Korea but the import of these crops is increasing. In this study, we surveyed the environmental risk of imported LM canola at 9 provinces, from March 2009 to June 2013. Monitoring of canola was conducted around feed factories, roadsides, harbors, farmhouses, and flower festival regions. From the total of 595 canola samples collected from 1850 monitoring sites, we identified 6 LM canola samples. The LM canola samples were subjected to protein and DNA based analysis. PCR analyses using approved 5 single event primers (T45, MS8, RT73, Rf3 and Topas 19-2) revealed that two crops were glyphosate-resistant LM canolas, and four were glufosinate-resistant LM canolas. This study suggested that environmental monitoring is a useful research tool to manage LM crops unintentionally introduced into the environment in South Korea. This result can be used as a basis for future post-management of canola crops. 식품용 및 사료용 유전자변형작물은 매년 국내에 수입이되고 있으며 유전자변형 캐놀라는 중요한 수입 작물 중의하나이다. 유전자변형작물의 국내 재배는 허용되고 있지않지만 수입양은 매년 증가하고 있는 실정이다. 본 연구에서는 2009년부터 2013년까지 전국 9개 도를 대상으로 국내수입된 유전자변형 캐놀라의 자연생태계 내 유출 정도를파악하고자 모니터링을 실시하였다. 모니터링 조사는 항만, 사료공장, 축산농가, 운송로, 축제지 주변을 대상으로실시하였다. 채집된 유전자변형 캐놀라 의심시료는 DNA 에 기반을 둔 방법과 단백질에 기반을 둔 방법으로 분석하였다. 총 1850개 지점 조사에서 총 595개의 의심시료를 발견하였으며 6개의 유전자변형 캐놀라를 확인하였다. 국내 수입 승인된 단일이벤트 T45, MS8, RT73, Rf3, Topas 19-2의primer를 사용한 PCR반응을 통해 2개의 glyphosate 저항성을가지는 이벤트와 4 개의 glufosinate-ammonium에 저항성을가지는 이벤트를 확인하였다. 본 연구를 통해 자연환경 모니터링 연구가 자연생태계 내의 유전자변형작물의 비의도적 유출을 예방하기 위해 매우 유용하다는 것을 알 수 있으며 사후관리를 수행하기 위한 기초자료를 구축하는데 활용될 것으로 사료된다.

유전자변형 면화 MON757, MON88702, COT67B, GHB811의 동시검출법 개발

김일룡,설민아,윤아미,이중로,최원균 한국환경생물학회 2021 환경생물 : 환경생물학회지 Vol.39 No.4

면화는 중요한 섬유 작물로 종자는 가축의 사료로 사용 된다. 작물 생명공학은 농업 분야에서 농업적 형질과 질을 향상시키기 위해 활용되어져 왔다. 국내 식품, 사료, 가 공 제품에 유전자변형 (LM) 면화의 사용이 증가함에 따라 환경으로의 LM 면화의 비의도적 유출 또한 증가하고 있다. LMO 모니터링 사업에서 수집된 LM 면화를 검정하기 위하여 국내 수입 승인된 LM 면화의 검출법 개발이 필요하다. 본 연구에서는 LM 면화 MON757, MON88792, COT67B, GHB811 4종을 대상으로 동시검출법을 개발하였다. 이벤트에 대한 유전 정보는 유럽 JRC와 농림축산검역본부에서 확보하였다. LM 면화의 동시검출법 개발을 위해 이벤트 특 이적인 프라이머를 설계하였으며 특이적인 증폭을 확인하였다. 특이도 검정, 무작위 표준물질 혼합물 분석, 검출한계 분석을 통하여 동시검출법의 정확도와 특이도를 검증하였다. 그 결과 본 동시검출법은 각각의 이벤트를 검출할 수 있으며 LM 표준물질을 활용하여 특이도를 검정하였다. 또한 무작위 표준물질 조합도 정확하게 검출할 수 있다. 검출한 계 분석에서는 25 ng의 미량의 주형 DNA로 단회 분석으로 검출이 가능하다. 결론적으로 4종의 LM 면화 동시검출법을 개발하였으며 LM 면화 자생체 분석에 활용될 것으로 사료 된다. Cotton is an important fiber crop, and its seeds are used as feed for dairy cattle. Crop biotechnology has been used to improve agronomic traits and quality in the agricultural industry. The frequent unintentional release of LM cotton into the environment in South Korea is attributed to the increased application of living modified (LM) cotton in food, feed, and processing industries. To identify and monitor the LM cotton, a method for detecting the approved LM cotton in South Korea is required. In this study, we developed a method for the simultaneous detection of four LM cotton varieties, MON757, MON88702, COT67B, and GHB811. The genetic information of each LM event was obtained from the European Commission-Joint Research Centre and Animal and Plant Quarantine Agency. We designed event-specific primers to develop a multiplex PCR method for LM cotton and confirmed the specific amplification. Using specificity assay, random reference material (RM) mixture analysis and limit of detection (LOD), we verified the accuracy and specificity of the multiplex PCR method. Our results demonstrate that the method enabled the detection of each event and validation of the specificity using other LM RMs. The efficiency of multiplex PCR was further verified using a random RM mixture. Based on the LOD, the method identified 25 ng of template DNA in a single reaction. In summary, we developed a multiplex PCR method for simultaneous detection of four LM cotton varieties, for possible application in LM volunteer analysis.