초록 ▼

- 신자미 고구마로부터 카로티노이드 대사관련 IbCCD4 유전자 분리 및 발현 등의 특성을 분석하였음.
- 카로티노이드 축적관련 고구마 Orange (IbOr) 단백질과 IbPSY의 결합 특성을 분석하고 IbPSY를 고온스트레스로부터 보호하는 기능을 확인하였음.
- IbOr과 결합하는 oxygen-evolving enhancer protein(PsbP) 단백질을 동정하였으며 IbOr이 고온 스트레스로부터 IbPsbP 단백질을 보호하는 기능을 확인하였음.
- 카로티노이드 생합성 유전자 (IbCHY-β, LCY-ε, 및

- 신자미 고구마로부터 카로티노이드 대사관련 IbCCD4 유전자 분리 및 발현 등의 특성을 분석하였음.
- 카로티노이드 축적관련 고구마 Orange (IbOr) 단백질과 IbPSY의 결합 특성을 분석하고 IbPSY를 고온스트레스로부터 보호하는 기능을 확인하였음.
- IbOr과 결합하는 oxygen-evolving enhancer protein(PsbP) 단백질을 동정하였으며 IbOr이 고온 스트레스로부터 IbPsbP 단백질을 보호하는 기능을 확인하였음.
- 카로티노이드 생합성 유전자 (IbCHY-β, LCY-ε, 및 LCY-β)를 각각 RNAi 벡터를 이용하여 형질전환 고구마 식물체를 개발하여 스트레스 내성특성을 분석하였음.
- 고구마 식물체 잎, 줄기, 저장뿌리 및 형질전환 배양세포의 카로티노이드 함량을 분석하였음. 율미 및 형질전환체 고구마 잎 6종과 염스트레스를 준 고구마 잎 6종의 카로티노이드와 엽록소의 함량분석을 실시하였음
- 고구마 카로티노이드 및 안토시아닌 추출물의 기능성을 규명하기 위하여 위암상피세포인 AGS cell에 Helicobacter pylori를 감염시켜 염증반응을 유도한 후 고구마의 안토시아닌 추출물과 카로티노이드 추출물을 처리하여 색소추출물의 항염증활성기전 규명 및 항비만활성을 측정하였음.

(출처 : 보고서 요약서 3p)

Abstract ▼

Purpose&Contents
In order to development of transgenic sweetpotato plants with high level of carotenoid by manipulation of the carotenoid biosynthesis related genes, the contents of in this study are as follows.
- Functional study of carotenoid metabolism-related genes in sweetpotato
- Func

Purpose&Contents
In order to development of transgenic sweetpotato plants with high level of carotenoid by manipulation of the carotenoid biosynthesis related genes, the contents of in this study are as follows.
- Functional study of carotenoid metabolism-related genes in sweetpotato
- Functional study of sweetpotato Orange and characterization of IbPsbP1 genes
- Characterization of transgenic sweetpotato plants with enhanced carotenoid contents
- Analysis of antioxidant pigments in transgenic cultured callus and storage root of sweet potato through metabolic redesign
- Establishment of background for utilization of the antioxidant pigments through functional study of anti-inflammatory, anti-cancer, anti-obesity activities of the antioxidant pigment extracts

Results
- Carotenoids content is not only regulated by biosynthesis, but also by catabolism. Carotenoid cleavage dioxygenases (CCDs) are responsible for enzymatic turnover of carotenoids into apocarotenoids. In this study, CCD1 and CCD4 were obtained from sweetpotato. The transcript levels of IbCCD4 gene were increased in leaf sweetpotato plant. IbCCD4 is localized in the chloroplasts, and interacts with IbOr, which confers accumulation of carotenoids.
In addition, purple-fleshed sweetpotato plants overexpressing IbOr contained high levels of CCD1, CCD4 and NCED transcript. The expression profiles of carotenoid metabolism-related genes in different tissues and response to various types of abiotic stresses are under study.

- We report that IbOr plays a role in the regulation of sweetpotato phytoene synthase (IbPSY) stability and a function involved in carotenoids accumulation and abiotic stresses resistance. The transcript levels of IbOr gene were increased in stem, root and calli of sweetpotato after exposure to heat stress.
IbOr with DnaJ like cysteine-rich zinc finger domain is localized in both the nuclear and the chloroplasts, and interacts with IbPSY, one of the key enzymes in the carotenoids biosynthesis pathway. Interestingly, IbOr is predominantly localized in chloroplasts after treatment of heat shock. IbOr can also stabilize IbPSY protein with its holdase chaperone activity. Furthermore, the overexpression of IbOr in Arabidopsis exhibited enhanced resistance to heat and oxidative stresses. These results showed that holdase chaperone activity of IbOr is involved in carotenoids accumulation by regulation of IbPSY and confers enhanced heat and oxidative stresses resistance to plant. Our findings thus suggest that IbOr will be contributed to develop plants with enriched carotenoids contents and enhanced stress resistance.

- The Or protein regulates carotenoid biosynthesis and environmental stress in plants. Previously, we reported that overexpression of the sweetpotato Or gene (IbOr) in transgenic Arabidopsis (referred to as IbOr-OX/At) increased the efficiency of photosystem II (PSII) and chlorophyll content after heat shock.

However, little is known about the role of IbOr in PSII-mediated protection against abiotic stress. In this study, comparative proteomics revealed that expression of PsbP (an extrinsic subunit of PSII) is up-regulated in heat-treated IbOr-OX/At plant. We then identified and functionally characterized the PsbP-like gene (IbPSbP) from sweetpotato. IbPsbP is predominantly localized in chloroplast, and its transcripts are tissue-specifically expressed and up-regulated in response to abiotic stress. In addition, IbOr interacts with IbPsbP and protects it from heat-induced denaturation, consistent with the observation that transgenic sweetpotato overexpressing IbOr maintained higher PSII efficiency and chlorophyll content upon exposure to heat stress. These results indicate that IbOr can protect plants from environmental stress not only controlling carotenoid biosynthesis but also by directly stabilizing PSII.

- β-carotene hydroxylase (CHY-β) is a key regulatory enzyme in the beta–beta-branch of carotenoid biosynthesis. Transgenic sweetpotato plants (cv. Yulmi) by down regulation of IbCHY-β using RNAi method were developed (referred to as RC plants). The RNAi-IbCHY-β construct was introduced into sweetpotato plants using Agrobacterium-mediated transformation. As expected, dramatic reduced levels of IbCHY-β transcripts were detected in RC plants compared to NT plants. The RC plants displayed orange flesh and showed over 2.4 times and 18 times higher of total carotenoid and β-carotene content in the storage roots compared to NT plants, respectively. Antioxidative capacity of RC Plants was evaluated. The RC plants exhibited tolerance to methyl viologen (MV)-mediated oxidative stress and higher 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity compared to NT plants. These results suggest that down-regulation of IbCHY-β enhances anti-oxidative capacity of RC plants. To evaluate the salt stress tolerance of RC plants, NT and RC plants were irrigated with 250 mM NaCl solution every 3 days for 6 days and recovery for 9 days. NT plants showed severely inhibited growth after treatment, wilting and chlorosis 6 days later. This difference in wilting symptoms became even more pronounced at 9 days after re-watering. RC plants maintained higher levels of chlorophyll and higher photosystem II efficiency than that of NT plants, which is consistent with their salt-resistant phenotypes. These results indicate that down-regulation of IbCHY-β increases tolerance to salt stress in sweetpotato plants.

- Lycopene epsilon cyclase (LCY-ε) converts lycopene to α-carotene, which is the first step of α-branch synthesis pathway of carotenoids. In this study, to develop transgenic sweetpotato plants with increased carotenoids and enhanced stress tolerance using RNAi technology, we constructed RNAi-IbLCY-ε in pCAMBIA3300 and transformed it into embryogenic callus of sweetpotato cultivar by Agrobacterium tumefaciens-mediation (referred to as RLE plant).

Among the 10 resultant transgenic sweetpotato plants, three lines were selected for further characterization on the basis of IbLCY-ε transcript levels. As expected, dramatic reduced levels of LCY-ε transcripts were detected in RLE plants compared to non-transformed (NT) plants. Transgenic sweetpotato plants showed salt stress tolerance and drought stress compared to NT plants. In the further study, transgenic plants will be characterized in terms of carotenoid contents and abiotic stress tolerance.

- Lycopene beta-cyclase (LCY-Β) is a key enzyme involved directly in the synthesis of α- and β-branch carotenoids such as α-carotene or β-carotene through the cyclization of lycopene. In this work, we have constructed IbLCY-β-RNAi vector and introduced into sweetpotato embryogenic calli. Among the 10 resultant transgenic sweetpotato plants (referred to as RLB plants), three lines were selected for further characterization on the basis of IbLCY-β transcript levels. Further characterization of RLB plants, specifically regarding gene expression and abiotic stress tolerance, is underway. Taken together biotechnological development of transgenic sweetpotato plants that accumulate increased carotenoid contents on marginal agricultural lands.

- Analytical method by HPLC-DAD was established for anthocyanin components in sweet potato root, and variation of anthocyanin and carotenoid contents in the cultivar was determined. Carotenoid analysis was done for the nine transgenc Jayoung potato leaves and the pigment analysis was accomplished with the transgenic sweet potato samples through six times. The effect of IbOr gene on carotenoid cleavage dioxygenase 4 (CCD4) was also determined.

- While carotenoid extract showed the higher anti-cancer activity in vitro assay with AGS cells than anthocyanin extract, the anthocyanin extract displayed antioxidant activity in AGS cells infected with Helicobacter pylori and inhibited the activation of MAPK pathway. Meanwhile anthocyanin and carotenoid extracts failed to show anti-H. pylori activity. Polygonatum cuspidatum showed potent anti-H. pylori and two potent reponsible compounds were isolated from the root of this plant. In order to evaluate the in vivo anti-obesity activity of carotenoid and anthocyanin extracts, eight week-high fat diet program was on going with C57BL/6N mice.

Expected Contribution
- Transgenic sweetpotato plants with increased carotenoid contents will be expected to contribute to produce the valuable biomaterial and would be beneficial for sustainable agriculture on marginal agricultural lands around the world.
- Metabolic engineering of carotenoid biosynthesis-related genes could be used to produce not only valuable nutrients but also enhanced stress tolerant crops.
- Mass production of safe natural pigments through transgenic sweetpotato which can be used as functional foods, medicines, etc
- Establishment of background for utilization of the antioxidant pigments with anti-inflammatory and other activities

(출처 : Summary 7p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 국 문 요 약 문 ... 4
• Summary ... 7
• 목차 ... 10
• 제 1 장 연구 개발 과제의 개요 ... 11
• 제1절 연구 개발 목적 ... 11
• 제2절 연구 개발의 필요성 ... 12
• 제3절 연구 개발 범위 ... 13
• 제 2 장 연구 수행 내용 및 결과 ... 15
• 제 1 절 고구마 카로티노이드 대사관련 유전자 기능 및 대사공학 연구 ... 15
• 제 2 절 고구마 색소항산화화합물의 성분분석 및 기능성 확보 ... 41
• 제 3 장 목표달성도 및 관련분야 기여도 ... 65
• 제 1 절 목표대비 달성도 ... 65
• 제 2 절 : 정량적 성과 (논문게재, 특허출원, 기타)를 기술 ... 68
• 제 4 장 연구 결과의 활용계획 ... 75
• 제 5 장 연구 개발 결과의 보안 등급 ... 76
• 제 6 장 국가과학기술지식정보서비스에 등록한 연구시설·장비 현황 ... 76
• 제 7 장 연구개발과제의 대표적 연구실적 ... 77
• 제 8 장 기타사항 ... 78
• 제 9 장 참고문헌 ... 79
• 끝페이지 ... 81