초록 ▼

Ⅳ. 연구개발결과
베타카로틴 벼 생성 기작을 이용한 코돈 최적화 합성과 복합다중발현을 통한 카로티노이드 대사 기작 연장 운반체 제작 및 벼 형질전환체 기능 분석을 통해 카로티노이드의 생성과 증진 효과를 확인하였다. 또한 카로티노이드 생성 개체간의 교배를 통해 새로운 천연색소 생산식물 소재 개발하여 최종적으로 카로티노이드 다단계 대사공학을 통한 기능성 컬러쌀 6종의 개발을 완료하여 형질전환 쌀에서 확실한 배유색 증진을 확인함으로써 벼에서의 최적화 코돈 합성에 따른 발현 효율 증진을 가시적·과학적으로 증명하였을 뿐만 아니라 국내·

Ⅳ. 연구개발결과
베타카로틴 벼 생성 기작을 이용한 코돈 최적화 합성과 복합다중발현을 통한 카로티노이드 대사 기작 연장 운반체 제작 및 벼 형질전환체 기능 분석을 통해 카로티노이드의 생성과 증진 효과를 확인하였다. 또한 카로티노이드 생성 개체간의 교배를 통해 새로운 천연색소 생산식물 소재 개발하여 최종적으로 카로티노이드 다단계 대사공학을 통한 기능성 컬러쌀 6종의 개발을 완료하여 형질전환 쌀에서 확실한 배유색 증진을 확인함으로써 벼에서의 최적화 코돈 합성에 따른 발현 효율 증진을 가시적·과학적으로 증명하였을 뿐만 아니라 국내·외 특허 출원 및 등록 완료하였다. 기능성 카로티노이드 생성 컬러쌀의 실용화를 위한 형질전환체 분석을 통해 최종 실용화용 운반체 요소 개량 연구와 벼 형질전환 구축을 완성하였다. 벼에서의 MEP경로 관련 유전자를 분리하였고, 벼 전신발현 프로모터를 장착한 운반체를 이용하여 최종 과발현 운반체 4종의 제작을 완료하였다. 안토시아닌 생성기작과 관련하여 일반벼는 유색벼와는 달리 구조유전자 및 전사인자의 발현도가 낮아짐과 더불어서 유전자의 구조적변이가관찰되었다. 이러한 결과를 바탕으로 조기에 종자의 종피색을 판별하는 마커를 개발하였다.
안토시아닌 생성증대를 위해 전사인자 3종을 분리하고 이를 발현시킨 결과 벼 형질전환체의 종자배유조직내에서 플라보노이드계 물질이 안정적으로 축적되는 것이 관찰되었다. 유색미에서 플라보노이드, 토코페롤, 스테롤 등 친유성 화합물과, 플라보노이드 생합성 전구물질인 phenolic acid 프로파일링과 카로티노이드 생합성 경로의 추적을 위한 카로티노이드 프로파일링 기술을 개발하여, 형질전환 벼의 라인별 유전자 도입에 의한 생합성 경로의 변화를 추적하였다.

Abstract ▼

Carotenoid is a well known group of pigments, widely distributed in nature with the color ranges from yellow to red, and has taken steady concern as a target for genetic engineering because of the nutritional and economic importance of compounds belonging to this class. Carotenoids which are essenti

Carotenoid is a well known group of pigments, widely distributed in nature with the color ranges from yellow to red, and has taken steady concern as a target for genetic engineering because of the nutritional and economic importance of compounds belonging to this class. Carotenoids which are essential as secondary antenna components of the reaction center complexes in photosynthesis are pigments are synthesized by the plastidic methylerythritol phosphate (MEP) pathway that forms part of the isoprenoid biosynthetic system in plants. Thus, the genes involved in this MEP pathway might be important to regulate the level of carotenoids in plant tissue. Rice (Oryza sativa L.) is the most important staple food for a large part of the world' s human population. Several studies have reported that these pigmented rice had more phytochemicals such as phenolics, flavonoids, carotenoids, tocol and γ-oryzanols than white rice. Therefore, rice breeder and consumers have been interested in pigmented rice due to their beneficial properties in human health. As efforts to develop new functional rice crops with diverse colors of yellow, orange, and red, we constructed several single vector systems to induce multiple gene expression by the combination of bicistronic system and multi cassette system using a endosperm-specific promoter. It resulted to built new carotenoid pathways in steps from b-carotene, zeaxanthin and astaxanthin into rice endosperm. Additionally, we enhanced the level of b-carotene using a synthetic gene that was further codon optimized for rice. Very recently, the new red color rice plant was obtained by crossing a zeaxanthin rice with a Ccs rice, which a capsanthin-capsorubin synthase gene from pepper had been previously transformed into rice plant. This result proved that the combination of genetic engineering and conventional crossing could be very effective method for multi-step metabolic engineering. Anthocyanins and proanthocyanins have been received an attention for their positive attribution for human health because of long-established biomedicinal properties, including inhibition of cell proliferation and antimutagenic, antimicrobial, anti-inflammatory, antioxidant, and antihypertensive properties. To understand mechanism of anthocyanins and proanthocyanins in rice seed, the expression of five genes encoding CHS, CHI, F3H, DFR and ANS were examined their expression in seeds among nonpigmented (white) and pigmented (black and red) rice cultivars. The sequence variation of biosynthetic genes revealed that DFR gene possesses one nucleotide substitution to generate premature stop codon in nonpigmented rice. Additional sequence variation of two regulatory genes, OSB1 and Rc, was also compared among the same cultivars. The sequence of OSB1 gene in black rice was differing from red and white rice. The sequence of Rc gene in red rice was differing from white and black rice. Based on these variations, we developed two CAPS markers for DFR and OSB1 genes and an Indel marker for Rc gene. The use of three markers together discriminated the expected seed colors of white, black and red and the results were validated in the 34 rice cultivars. Hence, the combined application of the three markers might be useful to screen the seed color before seed set in rice breeding program. To increase anthocyanins and proanthocyanins contents in rice seed, we generated the basic backbone vector with seed endosperm-specific GluB1 promoter. We developed the anthocyanin enhancing system by transcription factor (MYB and bHLH) and metabolic control system that can produce delphinidin pigments through RNAi and gene expression. Transgenic rice plant with MYB gene showed different anthocyanin accumulation pattern according to rice cultivar. Transgenic rice plant by transcriptional regulator showed the flavonoid accumulation, which can be visualized using diphenyl boric acid 2-amino ethyl ester (DPBA), a flavonoid-specific dye. Nontransgenic rice plant showed a small amount of the flavonoid accumulation in embryo and aleurone layer by staining with DPBA, but transgenic rice plant exhibited strong staining in embryo, aleurone layer, and endosperm. In the future, we will analyze the compound and amount of flavonoid in transgenic rice, and develope production system of particular flavonoids using this platform. Some pigmented rice cultivars contain phytochemicals that are responsible for their colour. Generally, these coloured compounds or pigments fall into everal large groups such as chlorophylls, carotenoids and flavonoids. Flavonoids and carotenoids of pigmented rice, including five black cultivars and two red cultivars, from Korea were characterised to determine the diversity among the phytochemicals and to analyse the relationships among their contents. Black cultivars were higher in flavonoids and carotenoids than the red and white cultivars. The profiles of eight phytochemicals identified from the rice grains were subjected to principal component analysis (PCA) to evaluate the differences among cultivars. Flavonoid contents had positive correlation with carotenoid contents among all rice grains. Furthermore, an accurate and sensitive method for determining 14 lipophilic compounds, including tocopherols, tocotrienols, phytosterols, and policosanols, from rice grain was developed using gas chromatography (GC) coupled to time-of-flight mass spectrometry (TOFMS). The method is fast, requiring only 13 min for the GC-TOFMS run. During the validation procedure, the method proved to be sufficiently precise and accurate with respect to the degree of endogenous biological variability found in rice samples. In applying the profiling concept, it is crucial to perform an unbiased multi-targeted metabolite analysis in order to precisely elucidate the biochemical functions in plant metabolism. Using HPLC analysis, the peaks of 24 carotenoids were detected within 42 min. We measured the content and composition of the carotenoids in the seed of transgenic rice plants. The transgenic seeds accumulated the carotenoids by genetic engineering.

목차 Contents

• 표 지 ... 1
• 제출문 ... 2
• 요 약 문 ... 3
• SUMMARY ... 5
• 목 차 ... 7
• 제 1 장 서 론 ... 8
• 제 2 장 국내외 기술개발 현황 ... 9
• 제 1 절 국외 기술개발 현황 ... 9
• 제 2 절 국내 기술개발 현황 ... 11
• 제 3 장 연구개발수행 내용 및 결과 ... 12
• 제 1 절 제1세부과제: 베타카로틴 벼를 이용한 기능성 카로티노이드 기작 연장 연구 ... 12
• 제 2 절 제2세부과제: MEP경로 조절을 통한 베타카로틴 증진 연구 ... 25
• 제 3 절 제3세부과제: 식물항산화성 천연색소 대사체 분석 연구 ... 28
• 제 4 절 제4세부과제: 안토시아닌 생산작물을 이용한 대사조절기작연구 ... 36
• 제 4 장 연구개발 목표 달성도 및 대외 기여도 ... 48
• 제 1 절 목표대비 대외달성도 ... 48
• 제 2 절 정량적 성과 ... 50
• 제 5 장 연구개발결과의 활용계획 ... 61
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 62
• 제 1 절 국외개발 황금쌀 특허요소 분석 사례 ... 62
• 제 2 절 국외개발 황금쌀의 국내 진입 사례 ... 62
• 제 7 장 기타 중요 변동사항 ... 63
• 제 8 장 국가과학기술종합정보시스템에 등록한 연구장비 현황 ... 64
• 제 9 장 참고문헌 ... 64
• 끝페이지 ... 71