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Metabolite Profiling of Herbicide Tolerant Rice, Iksan483 and Milyang204
제초제 저항성 벼 익산483과 밀양204의 대사체 연구

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Authors
최훈
Advisor
김정한
Major
농생명공학부
Issue Date
2012-02
Publisher
서울대학교 대학원
Abstract
This study aimed to investigate the substantial equivalence of two genetically modified rice varieties, Iksan483 and Milyang204, developed as glufosinate-tolerant rice in Korea and the corresponding parental lines. In order to assess the compositional changes and identify potential unintended effects of the genetic modification, different strategies such as the non-targeted and targeted (compound-specific, nutritional composition) metabolite profiling approaches were applied. The volatile metabolic constituents in leaf and root were also investigated for possible environmental effect.
The metabolite profiling in non-targeted approach was carried out using two kinds of sample preparation method (Method I and II). Moreover, the mass spectral library of expected metabolites in rice was produced including both mass spectra and retention index. The retention indices of total 191 metabolites (59 lipid and 132 polar metabolites) were calculated and used for identification of unknown components in this study. It was possible for simpler and faster preparation by method II than by method I. By method I, 64, 34, and 42 metabolites were identified in rice grain, leaf and root, respectively, whereas, 96, 98 and 51 metabolites were identified by method II. In principle component analysis, examination of the scores and loadings plots showed effects on the metabolite profile resulting from genetic modification of Iksan483 and Milyang204 were of lower magnitude than the effects of the genetic diversity or difference between conventional varieties.
Comparative assessment of nutritional composition as the targeted approach was conducted with genetically modified rice grains and its conventional counterparts. Nutrients including proximates, fatty acids, amino acids, minerals, vitamins, and antinutrients were investigated using several statistical comparisons. The results showed that, except for small differences in a few fatty acids, minerals, and trypsin inhibitor, there was no significant difference between genetically modified rice and conventional counterpart variety with respect to most of their nutrient composition. Most of levels of nutrients were in good compliance with the literature ranges, showing substantial equivalency. The results of principle component analysis demonstrated that any differences between the genetically modified and conventional rice varieties are in range of the differences observed among conventional varieties grown in different years.
The comparison between results of nutritional analysis and metabolite profiling suggested that metabolite profiling could be a valuable tool for qualitative assessment and appropriate for screening of the difference of metabolites in a brief time. The quantitative analysis of target components and cross-check must be conducted if the difference between treatments was observed in metabolite profiling.
The volatile constituents in leaf and root of genetically modified rice and conventional rice were isolated by solvent-assisted flavor evaporation (SAFE). Various compounds (15~17) were identified in rice leaves, while 10~12 volatiles were identified in rice roots. The major volatiles were phenylacetaldehyde, 3-methylbutanoic acid, 2,3-butanediol, 2-hexenal, benzyl alcohol and hexanal. The PCA results indicated no evidence of any significant differences between the conventional and transgenic rice.
Therefore, it was concluded that the herbicide tolerant rice varieties, Iksan483 and Milyang204, are substantially equivalent to their parental lines including Anjung, Shindongjin, Dongjin and Junam in aspects of metabolite, nutritional and volatile composition.
본 연구에서는 국내에서 개발된 제초제 저항성 벼인 익산483과 밀양204이 이들 모품종과 실질적으로 동등하다는 것을 규명하고자 하였다. 이를 위해 유전자재조합 벼와 모품종인 안중벼, 신동진벼, 동진벼 그리고 주남벼의 현미, 잎, 뿌리를 대상으로 metabolite profiling을 이용하여 식물체내의 다양한 대사체를 비교해석하였으며, 중요한 영양성분 및 반영양성분 등을 정량분석하였다. 또한, 환경영향평가의 일환으로 잎과 뿌리의 휘발성 대사물질을 비교분석하였다.
제초제 저항성 벼의 metabolite profiling는 두 가지 전처리법(Method I, II)을 활용해 수행하였다. 보다 편리하고 정확한 대사체 동정을 위해 벼에서 존재할 것이라 예상되는 191개 대사체에 대한 mass spectra와 retention index 정보가 담긴 library를 자체 제작하였으며 본 연구에서 대사체를 동정하는데 사용하였다. 두 가지 전처리방법 중 Method II에 따른 전처리가 휠씬 빠르고 간단하였다. Method I를 이용한 metabolite profiling 결과, 현미, 잎, 뿌리에서 각각 64개, 34개, 42개의 대사체를 확인하였으며 Method II를 이용한 경우는 각각 96개, 98개, 51개 대사체가 동정되었다. 다변량 분석결과, 익산483과 밀양204의 대사체 조성은 일부 대사체를 제외하고 4개 모품종과 큰 유의적 차이를 보이지 않았다.
영양성분 비교분석은 일반조성(수분, 조단백, 조지방, 조섬유, 회분), 지방산, 미네랄, 비타민 그리고 phytic acid, lectine, trypsin inhibitor와 같은 반영양성분을 대상으로 수행하였다. 유전자재조합 벼와 모품종간의 동등성은 일부 지방산, 미네랄, trypsin inhibitor 성분을 제외한 대부분의 영양성분에서 증명되었다. 또한, 조사된 영양성분 함량은 기존 문헌에서 보고된 결과내에 존재하였다. 다변량분석결과, 계통보다는 재배환경에 의해 영양성분 조성이 더 큰 영향을 받았음을 확인하였다. 따라서, 익산483과 밀양204의 영양성분 조성은 유전자재조합에 의해 영향을 받지 않았다.
Metabolite profiling 결과와 영양성분 분석결과 간의 비교를 통해, metabolite profiling은 정량분석보다는 정성분석에 더 적합하며 단시간내에 두 개체간의 대사체 차이를 확인하는데 더 큰 효용성이 있음을 확인하였다. Metabolite profiling에서 개체간의 대사체 차이를 확인하였다면, 해당 대사체의 정량분석을 통해 교차검정이 반드시 이루어져야 한다.
SAFE 전처리방법을 통해 유전자재조합 벼와 모품종의 잎과 뿌리에서 휘발성 물질을 분석, 비교하였다. 잎에서는 15~17개의 휘발성분이 확인되었으며 뿌리에서는 10~12개 휘발성분이 검출되었다. 주요 휘발성분은 phenylacetaldehyde, 3-methylbutanoic acid, 2,3-butanediol, 2-hexenal, benzyl alcohol, hexanal 이었다. 다변량 분석결과, 제초제 저항성 벼와 모품종 사이에서 휘발성분 조성의 유의적 차이를 확인할 수 없었다.
결론적으로, 제초제 저항성 벼인 익산483과 밀양204는 모품종과 대사체, 영양성분, 휘발물질 조성측면에서 실질적으로 동등하였다.
Language
eng
URI
http://hdl.handle.net/10371/156295

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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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