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Kafe 바로가기주관연구기관 | 한국원자력연구원 Korea Atomic Energy Research Institute |
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연구책임자 | 강시용 |
참여연구자 | 권순재 , 진창현 , 안준우 , 홍민정 , 이면주 , 유재일 , 이민규 , 김동건 , 김정민 , 한아름 , 김진백 , 임승주 , 김장훈 , 남보미 , 김보람 , 김효영 , 김현미 , 김상훈 , 조영득 , 최홍일 , 류재혁 , 김세원 , 이성일 , 박재완 , 김선영 , 이상우 , 권유정 |
보고서유형 | 1단계보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2018-12 |
과제시작연도 | 2018 |
주관부처 | 과학기술정보통신부 Ministry of Science and ICT |
과제관리전문기관 | 한국연구재단 National Research Foundation of Korea |
등록번호 | TRKO201900019833 |
과제고유번호 | 1711072557 |
사업명 | 방사선기술개발사업(R&D) |
DB 구축일자 | 2020-04-11 |
키워드 | 방사선육종.돌연변이.신품종.생물소재.양성자빔.유전자원.radiation breeding.mutation.new variety.bio-materials.proton beam.genetic resources. |
○ 파이토케미컬 고함유 유망 대두 계통의 생산력 평가 및 품종보호권 출원
○ 동계 재배용 신규 도입 두류 렌틸 및 파바빈 유전자원 평가 및 M₂ 세대 변이체 선발
○ 두류 내한성 및 유용 성분 관련 유전자 변이 탐색
○ 방사선육종 두류(아피오스, 렌틸, 기능성두류) 추출물 획득 및 대사체 분리/구조분석 완료
○ 추출물 및 지표성분 활용 염증 및 대사성질환 생리활성 평가 완료
○ 방사선육종 차조기 추출물의 기술이전 완료(관절염 개선 효능) 및 신규화합물 분리 분석
○ 방사선육종 국화 품종을 이용한 대사
○ 파이토케미컬 고함유 유망 대두 계통의 생산력 평가 및 품종보호권 출원
○ 동계 재배용 신규 도입 두류 렌틸 및 파바빈 유전자원 평가 및 M₂ 세대 변이체 선발
○ 두류 내한성 및 유용 성분 관련 유전자 변이 탐색
○ 방사선육종 두류(아피오스, 렌틸, 기능성두류) 추출물 획득 및 대사체 분리/구조분석 완료
○ 추출물 및 지표성분 활용 염증 및 대사성질환 생리활성 평가 완료
○ 방사선육종 차조기 추출물의 기술이전 완료(관절염 개선 효능) 및 신규화합물 분리 분석
○ 방사선육종 국화 품종을 이용한 대사체 분리/항산화 활성 평가 및 티백 시제품 제조
○ 대표작물(14종)에 대한 양성자빔 LD50, RD50 정보 확보 및 SOBP 적용 생체영향 분석
○ 애기장대 양성자빔 조사선량별 발아율, 생존율, 종자결실율 분석 및 LD50 및 어깨선량 구명
○ 애기장대 주요 선량별 M₂ 집단 구축 및 16개체 전장 유전체 서열 확보
○ 벼 및 고추 양성자빔 생체영향 및 감수성 데이터 구축
○ 정량적 성과: 논문 15편 (SCI(E) 10건, 비SCI 5건) 게재, 국내외 학술대회 초청강연 및 논문 발표 55건, 품종보호권 등록 5건 및 출원 1건을 하였고, 제407회 학연산 연구성과 교류회를 개최.
(출처 : 보고서 요약서 4p)
Ⅳ. Results
1. Development of legume genetic resources for lifecare materials using radiation breeding
1-1. Selection of soybean bred mutant lines with high-efficiency phy tochemicals
A. Development of soybean mutant bred lines with high fatty acid and oligosaccharide
- Construction
Ⅳ. Results
1. Development of legume genetic resources for lifecare materials using radiation breeding
1-1. Selection of soybean bred mutant lines with high-efficiency phy tochemicals
A. Development of soybean mutant bred lines with high fatty acid and oligosaccharide
- Construction of mutant pools derived from Danbaek, Daepung original soybean variety by using gamma irradiation
- Selection of 10 high linolenic acid soybean mutants through investigated to agronomic traits and unsaturated fatty acid contents
- DB-HL4 (11.29%), 5 (10.88%) were significantly increased linolenic acid contents, approximately 1.7-fold higher than the Danbaek (6.53%) original variety. Also, DP-HL6 were 1.6-fold higher than the Depung original culitvar
- Construction of mutant pools derived from Paldal soybean variety
- Result of content of sucrose and oligosaccharide such as raffinose, stachyose which showed increased levels in selected mutant lines. Especially, I01 line were recorded highest sucrose content (11.81 mg/100g)
B. Development of lipoxygenase free new soybean mutant variety ‘Cheongmiwon’ and application for protection
- Development of new soybean mutant derived from hwanggum original variety by using radiation breeding, naming “Cheongmiwon” that have been application for protection (application number: 2018-118)
- new soybean mutant that exhibited lipoxygenase free phenotype
1-2. Analysis of functional compounds in high-efficiency phytochemical soybean bred mutant lines and Investigation of genetic variation in phytochemical related genes
A. Analysis of agronomic traits of high-efficiency phytochemical soybean bred mutant lines for variety registration.
- Construction of mutant diversity pool among 210 soybean mutant lines
- Construction of agronomic traits database
B. For compared fatty acid biosynthesis gene function, a collection of four selected soybean bred mutants with original culitvars, was used to investigate genetic variation of genomic DNA sequence and genes expression levels.
- Aligned sequence lengths of identified FAD genes relative to the Wm82 reference sequence were as follows: 2,151 bp (FAD2-1A); 1,830 bp(FAD2-1B); 1,409 bp (FAD2-2A); 2,112 bp (FAD2-2B); 2,092 bp (FAD2-2C); 2,088 bp (FAD2-2D); 4,250 bp (FAD3A); 4,104 bp (FAD3B); 2,724 bp (FAD3C) and 3,431 bp (FAD3D)
- On stage-3 expression levels, where dramatic changes were observed relative to original seeds. Transcript levels of FAD2-2B, FAD2-2D, FAD3C and FAD3D genes were significantly increased in all mutant seeds
- The highest statistically significant increased expression of FAD3C and FAD3D genes in four mutants was at stage 3, which suggests that genes were involved in linolenic acid synthesis in the selected mutants(Manuscript submitted)
1-3. Development of new legume cultivars for the prevention of metabolic disease through construction of mutation pool with functional foreign legume crops faba bean and lentil
A. Construction of mutation pools using radiation breeding technology and process of generation.
- Gamma-irradiated Lentil and faba bean mutant pools were generated M₂ seed pooling
- Investigation of radio-sensitivity in lentil bean to gamma-irradiation(Manuscript published)
- Selection of 10 elite faba bean individuals from 371 accessions according to agronomic traits and winter hardiness (Manuscript published)
- Faba bean mutants were exhibited 30% to 84% survival rate in winter annual
1-4. Establishment of the new conceptual molecular marker system and available component characteristic analysis
A. Development of marker-assisted selection system to screen in early generation
- Confirmation of DNA mutation rate in 552 M₁ faba bean mutants by using TRAP marker system
- Investigation of relationship between gamma-radiation dose and genotyping results (Manuscript submitted)
B. Analysis of functional compound contents and activity in soybean bred mutants
- Comparsion of L-Dopa, fatty acids and isoflavone content of compositae faba bean (Manuscript published)
- Result of starch physicochemical properties analysis in faba bean, these exhibited increased solubility under low melting temperature (30℃). Probably faba bean starch seems to be a novel starch category
- Soybean mutant (D-28-01) was increased 5.5-fold isoflavone content than Danbaek original variety
- Faba bean was showed increased tyrosinase activity than soybean, lentil seeds. On the other hand, antioxidant assay such as DPPH, ABTS free radical scavenging activity, that was recorded highest lentil bean
C. A study on processing properties and component characteristic of soybean bred mutant lines.
- Analysis of functional enzyme activity and changed component levels after differential processing method in faba bean and lentil
D. Exploring cold tolerance genes in faba bean transcriptome
- Analysis of gamma-irradiated Faba bean transcriptome by using PAC-bio sequencing
- Selected 6 cold tolerance candidate genes from EMBL-EBI database
- Faba bean transcriptome were 96 contig related to cold tolerance gene .
2. Evaluation of functional activity of mutagenized plant resources and investigation for commercialization
2-1. Isolation of metabolite and investigation for physiological activity from radiation mutant Apio
A. Isolation and structural analysis of metabolite from radiation mutant Apio
- Isolation and structural analysis of 20 metabolites from ethyl acetate and butanol extracts of radiation mutant Apios
- Conformation of structure by NMR
B. Evaluation of physiological activity of metabolite from radiation mutant Apios
- 4 compounds among isolated 20 compounds had shown anti-inflammatory activity
- 2 compounds among isolated 20 compounds had shown inhibitory activity for alpha-glucosidase
- AP7 had 50% inhibitory activity at 50 μM for DPP-IV
C. Evaluation of mechanism for anti-inflammatory activity of lupinalbin A isolated from radiation mutant Apio
- Lupinalbin A inhibited the production of iNOS mRNA and protein in a dose dependent manner
- Lupinalbin A inhibited the production of IL-6, TNF-α, MCP-1iNOS in a dose dependent manner
- Lupinalbin A inhibited the production of IFN-beta and STAT1 protein
2-2. Acquisition of extracts and investigation of physiological activity from soybean bred mutant lines
A. Acquisition of extracts from 150 soybean bred mutant lines and development of analysis condition for LC/MS
- Development of optimal analysis condition for UPLC/ELSA and UPLC/Q-tof. MS using 11 standard compounds
- Development of preprocessing condition for LC/MS using soybean bred lines
B. Development of molecular networking condition and LC/MSMS analysis using soybean bred 45 mutant lines
- Development of optimal molecular networking condition via structural similarity uisng 11 standard compounds
- Conformation of structural information of metabolites contained into extracts by LC/MS analysis results
C. Multivariate statistical techniques
- Evaluation of difference between wild type and mutant soybean line using the PCA, PLS-DA, and HCA
D. Evaluation of regulatory activity for metabolic disease of 150 soybean bred mutant lines
- Investigation about whether the extract of 150 soybean bred mutant lines can inhibit the activity of alpha-glucosidase and DPP-IV enzyme
- There were no mutant lines showed inhibitory activity
2-3. Acquisition of extracts and investigation of physiological activity from radiation mutant lentil
A. Acquisition of extracts and isolation of compounds from lentil
- Acquisition of ethnolic extracts and solvent extracts from lentil
- Isolation of 5 metabolites from butanolic extract from lentil
B. Investigation of physiological activity of lentil extracts and components
- 4 compounds among isolated 5 compounds had shown inhibitory activity for TBK-1 enzyme in a dose dependent manner
- 3 compounds among isolated 5 compounds had shown inhibitory activity for both alpha-glucosidase and DPP-IV enzyme in a dose dependent manner
C. Analysis of molecular docking
- 3 compounds shown inhibitory activity for DPP-IV has good position for DPP-IV S1 and S2. And all flavone core was mainly positioned into S1, furthermore glycoside of that was correlated with S2
2-4. Follow-up study for development of functional food using radiation mutant Perilla extracts
A. Acquisition of supercritical carbon dioxide extracts and isolation of compounds from mutant Perilla and wild type
- Acquisition of supercritical carbon dioxide extracts from mutant and wild type Perilla dried leaves
- Isolation of 9-hydroxy isoegomaketone (New compound) from mutant Perilla extract and isolation of myristicin from wild type Perilla extract
- The contents of 4 monoterpene compounds was analyzed between mutant and wild type Perilla extracts
B. Investigation of anti-inflammatory activity of new compound (1)
- New compounds had shown inhibitory activity for NO production in LPS-induced RAW264.7 cells
C. Investigation of anti-arthritic activity of mutant Perilla extract on CIA model
- Clinical arthritis scoring and paw thickness was statistically reduced in the group treated by mutant Perilla extract
- The level of IL-1beta in blood was statistically reduced in the group treated by mutant Perilla extract
- Conformation of anti-arthritic effect of mutant Perilla extract (133 mg/kg/day) in a CIA animal model
D. Analysis of toxicology of mutant Perilla extract by single oral administration
- In the case of female rat, ALD (approximate lethal dose) was 1,000 mg/kg. And in male rat, ALD was between 1,000 mg/kg and 2,000 mg/kg
E. Analysis of toxicology of mutant Perilla extract by 14-day repeated oral administration
- In the case of female rat, MTD (maximum tolerated dose) was above 150 mg/kg. And in male rat, MTD was above 300 mg/kg/day
- The target organ of mutant Perilla extracts was supposed the thymus and spleen
F. Carry out the technological transfer using mutant Perilla extract
- Completion of technological trasnfer
- Company: SFC Bio
- Fixed amount: 200,000,000(KW, containing milestone 170,000,000)
2-5. Development of method for isolation of new compound from mutant Perilla
A. Isolation of new compounds using centrifugal partition chromatography(CPC)
- Isolation of three compounds (9-hydroxy-isoegomaketone (1), isoegomaketone (2), Perillaketone (3)) from mutant Perilla extract
B. Determination of distribution coefficient (K), isolation coefficient (α), and solvent system for the CPC isolation condition
- Determination of K and α using various solvent condition
- Descending mode-> n-hexane:ethyl acetate:ethanol:water = 5:5:5:5
- Ascending mode-> n-hexane:ethyl acetate:ethanol:water = 8:2:8:2
C. Isolation of compounds using CPC
- Isolation of 9-hydroxy-isoegomaketone (2.4mg, 96.7% purity) using descending mode
- Isolation of isoegomaketone (56.1mg, 97.6% purity) and perillaketone(78.6mg, 96.1% purity) using ascending mode
2-6. Follow-up study for development of anti-oxidant food using radiation mutant Chrysanthemum cultiva
A. Acquisition of extract from ARTI-dark chocolate, nobel wine, and natural one
- Acquisition of ethanolic extract from three
- Acquistion of solvent extract from ARTI-dark chocolate
B. Investigation of anti-oxidant activity using the extracts of ARTI-dark chocolate, nobel wine, and natural one
- ARTI-dark chocolate has highest anti-oxidant activity among three using ABTS and DPPH assay
C. Isolation of compounds from ARTI-dark chocolate
- Isolation 7 compounds from ethylacetate extracts of ARTI-dark chocolate
D. Comparison of the compounds among the extracts from ARTI-dark chocolate, noble wine, and natural one
- ARTI-dark chocolate contained more flavonoid and phenolic compounds than noble wine and natural one, however contained less flavonoid aglycone than those
2-7. Comparative study between commercial Chrysanthemum tea and ARTI-dark chocolate tea via leaching temperature and time
A. Production of tea prototype using ARTI-dark chocolate
- Production of tea prototype using ARTI-dark chocolate using low-temp method (Temp 15℃, moisture 20%)
B. Acquisition of extracts via leaching temperature and time, comparative study for anti-oxidant activity
- Tea (1g) was leached by 75℃, 100℃ for 2 min, 4 min respectively
- The tea made by ARTI-dark chocolate had shown more anti-oxidant activity that commercial Chrysanthemum tea in DPPH assay
3. Development of base technology on crop breeding using proton beams
3-1. Elucidation and comparison of variation mechanism according to different radiation types (gamma rays and proton beams) using model plant
A. Construction of database for biological effect and sensitivity of model plant according to conditions for proton beam-irradiation
○ Analysis of biological effect of proton beam and gamma-rays on M₁ plants
- Development of database on germination rate, survival rate, and weight of seeds for Arabidopsis groups treated by each radiation source at each dose
- Determination of doses important in radiation breeding: LD50 (proton beams: 1,051 Gy, gamma-rays: 1,087 Gy), shoulder dose Dq (proton beams: 787 Gy, gamma-rays: 900 Gy), Dq 50-75% (proton beams: 394-590 Gy, gamma-rays: 450-675 Gy)
B. Analysis of mutation frequency and spectrum in proton beam mutation populations of model plant
- Development of 400 lines for each LD50, Dq, and Dq 60% dose in each radiation source
- Analysis of frequency and spectrum of phenotypic mutations in each M₂ population: detection of 16 and 9 phenotypic mutations in proton beam and gamma-ray populations, respectively
C. Analysis of relationship between biological effect and phenotypic mutation frequency
- Comparative analysis of frequencies of phenotypic mutations among populations for important doses that were determined based on survival rate in M₁ generation: detection of the highest mutation rate in 787 Gy(Dq dose)-irradiated population in proton-beams (mutation rate: 5.25%) and 1,200 Gy(dose nearby LD50)-irradiated population in gammairradiated population (mutation rate: 4.5%), respectively.
- The mutation spectrum of proton beams were broader that of gamma-rays in that the frequencies of leaf-color and leaf-shape variations were similar in proton beam irradiation while frequency of leaf-color variation was about seven times higher than that of leaf-shape variation in gamma-irradiation.
D. Analysis of whole genome sequences of mutant lines developed by proton beam-irradiation
- Analysis of whole genome sequences of each four plants for important doses in proton beam irradiation (LD50, Dq, Dq 60%) and the Dq dose in gamma-irradiation: the sequences covering 97.78% of reference genome in average were obtained.
3-2. Establishment of optimal conditions for mutation induction in 14 representative crop species and construction of database
A. Construction of database on LD50 and RD50 according to MeV and dose in proton beam irradiation in 14 representative crop species
- Construction of proton beam database on LD50 and RD50 for gamma-rays and proton beams in seeds (cultured tissues) of representative 14 crop species
- Determination of 45 MeV and without-SOBP condition as the optimal condition for mutation induction based on comparative analysis of biological effects of proton beams from TR103 accelerator to orchids according to acceleration energies (MeV) and application of SOBP
- RFT30 20 MeV (500nA) was shown not to be applicable for mutation breeding based on applicability tests.
3-3. Construction of proton beam-induced large-scale mutant population and development of useful genetic resources in two representative crop species
A. Evaluation of biological effects and sensitivity of proton beam irradiation in rice
○ Proton beam and gamma-ray irradiation to rice seeds and growth test
- Rice seeds irradiated with 100 MeV proton beams and gamma rays. Evaluation of germination rates, survival rates, and growth revealed that the biological damage of proton beams is more severe than that of gamma rays at the same dose. Doses from 100 to 150 Gy seem to be optimal to induce mutations using PBs in rice.
○ Large scale irradiation and early growth test for construction of rice proton beam mutant population
- A large amount of seeds of an elite rice variety were irradiated with proton beams at the proper doses for mutation. The seedlings were used for early growth test and transplanted to paddy field.
B. Evaluation of biological effects and sensitivity of proton beam irradiation in pepper
- Rice seeds irradiated with 100 MeV proton beams and gamma rays. Evaluation of germination rates, survival rates, and growth revealed that the biological damage of proton beams is more severe than that of gamma rays at the same dose. Doses from 300 Gy seem to be optimal to induce mutations using PBs in rice.
C. Comparative analysis on genetic variations according to gamma-ray and proton beam irradiation in Cymbidium
- Frequencies of SNP and transversion were shown to be higher in the proton beam-irradiated populations than the gamma-irradiated in comparative analysis of SNPs in orchid populations treated by gamma-rays and proton beams in various conditions
3-4. Development of next generation mutation breeding technology in legume crops (commited research)
A. Biological effect of proton beam and gamma ray irradiation in cowpea
- Okdang; Cowpea (Vigna unguiculata L. Walp.) seeds were irradiated with gamma rays and proton beams. Evaluation of germination and survival rates of the seeds revealed LD50 of 200 to 300 Gy for both two radiation.
- Measurement of plant growth revealed plant height RD50 of 300 to 400 Gy for proton beams and 200 to 300 Gy for gamma rays, and expected plant fresh weight RD50 of > 500 Gy for both two radiation.
B. Effect of proton beam and gamma ray irradiation on activity of antioxidant enzymes in cowpea
- Measurement of chlorophyll and MDA contents, and enzyme activity of APX and POD revealed difference between plants from proton beam and gamma ray irradiated seeds.
(출처 : SUMMARY 22p)
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