Characterizations of a novel glucosyltransferase and pro-angiogenic components in Citrus.
- Abstract
- PART1
bGT612 당전이효소는 한국 재래감귤인 병귤에서 클로닝하였다. 병귤의 genomic DNA 서열을 통해 bGT612 서열의 특이적인 프라이머를 합성하고 병귤잎에서 뽑은 total RNA 에서 PCR 수행하였다. bGT612 cDNA 의 전사해석틀은 1446 개의 염기쌍으로 이루어졌고 PSPG BOX 를 포함하였다. 복제한 유전자를 대장균에서 발현하였고 당전이효소 생성물은 크로마토그래피 방법으로 분석하였다. apigenin, riodictyol, kaempferol, luteolin, naringenin, quercetin 의 6 가지 flavonoid 로 bGT612 의 활성을 검정하였다. bGT612 당전이효소 생성물은 5 가지 기질에서 당전달하였고 또한 특이적으로 kaempferol-5-O-glucoside 생성하였다. 이러한 결과는 다양한 기질과 kaempferol-5-O-glucoside 에 특이적인 당전이효소를 이해하는데 도움이 될 수 있다
PART2
감귤껍질의 수용성 추출액은 신생혈관형성 효과물질의 헤스페리딘과 나리루틴으로 구성되었고 이번 연구는 감귤껍질에서 헤스페리딘과 나리루틴의 추출수율을 높이기 위해 적절한 용매를 찾는 것이었다. Methanol 과 dimethyl sulfoxide 를 사용했을 때 아세토니트릴과 에틸아세테이트, 에탄올, 아이소프로판올 비교했을 때 훨씬 높아진 추출율을 얻을 수 있었고 특히, DMSO 의 헤스페리딘 추출율이 메탄올보다도 훨씬 높았다. 또한 DMSO 로 추출한 헤스페리딘은 인간 제대정맥혈관내피세포의 신생혈관형성 유도를 하였고 ERK1/2 신호전달의 인산화를 upregulate 하였다. 이러한 결과는 추출된 헤스페리딘의 신생혈관형성 효과와 감귤껍질에서 헤스페리딘과 나리루틴을 간단하고 쉽게 그리고 효과적으로 추출하는 방법을 보여준다.
PART1
A flavonoid glucosyltransferase, bGT612, was cloned from Byungkyool (Citrus platymamma Hort.et Tanaka) that is a Korean native citrus species. Using a genomic sequence data, specific primers for bGT612 were designed and PCR was performed with total RNA prepared from leaves. The open reading frame of bGT612 cDNA consists of 1446 base pairs and contains PSPG (Plant Secondary Product Glucosyltransferase) box. The cloned gene was expressed in E.coli and gene product was analyzed by chromatography methods. Six kinds of flavonoids such as apigenin, eriodictyol, kaempferol, luteolin, naringenin, and qucertin were used to confirm the specific activity of bGT612. Gene product of bGT612 can transfer glucose to all of tested substrates. Interestingly, bGT612 shows specific activity for Kaempferol, producing kaempferol-5-O-glucoside (K-5). These results help understanding specificity of GTs for various substrates and K-5 characterization.
PART2
Aqueous extraction of Citrus unshiu peels (AECUP) is mainly comprised with pro-angiogenic hesperidin and narirutin. In this study, we report approaches to increasing the yields of extracted hesperidin and narirutin from Citrus unshiu peels using proper solvents. Significantly improved yields of both compounds were obtained using methanol and dimethyl sulfoxide (DMSO) compared to acetonitrile, ethyl acetate, ethanol, and isopropyl alcohol. Especially, effect of DMSO was by far the better of the two solvents in extraction of hesperidin. In addition, the DMSO extracted hesperidin significantly induced the pro-angiogenic effects of human umbilical vein endothelial cells (HUVECs) and markedly up-regulated phosphorylation of the ERK1/2 signaling pathway. These results demonstrate that pro-angiogenic inducer; hesperidin and narirutin can be simply, easily, and effectively extracted from Citrus unshiu peels.
- Issued Date
- 2017
- Awarded Date
- 2017. 2
- Type
- Dissertation
- Alternative Author(s)
- Myung-Seung, KIM
- Department
- 대학원 생명공학과
- Advisor
- 김재훈
- Table Of Contents
- PART1 CONTENTS........................................................................................... i
LIST OF FIGURES..............................................................................iii
ABSTRACT...........................................................................................1
1. INTRODUCTION.............................................................................2
2. MATERIALS AND METHODS......................................................4
2.1. Cloning and expression of bGT612.…………………….……….....…….…4
2.2. Purification of bGT612 ………………………………………....….…..…...4
2.3. Alignment of glucosyltransferase…...............................................................5
2.4. Phylogenetic tree………………………………………….....…….……......5
2.5. Enzyme assay that using bio-transformation……….….……...…...….........5
2.6. Purified bGT612 protein activity………………………....…………..…….6
2.7. HPLC analysis……………….………...…...……….....................................6
2.8. Production of kaempferol-5-O-glucoside ...……………….………...….......6
2.9. Preparative-HPLC analysis……………….…......……...…...……................7
2.10. LC-MS analysis……………….………...…...……........................................7
2.11. Measurement of Fluorescent……………….……........................................7
3. RESULTS …………………………………………….………...…..8
3.1. Phylogenetic tree and Alignment…………………………….…...................8
3.2. Cloning and gene expression at protein level……………………................11
3.3. Characterization of glucosyltransferase………...………………......….......12
3.4. Preparative-HPLC analysis...........................................................................19
3.5. LC-MS analysis...............................................................................................20
3.6. Fluorescent of Kaempferol-5-O-glucoside…..............................….............21
4. DISCUSSION...................................................................................22
5. CONCLUSION................................................................................24
6. REFERENCES................................................................................25
ABSTRACT IN KOREAN……………………………………..…....27
PART2 CONTENTS.......................................................................................... 28
LIST OF FIGURES.............................................................................30
ABSTRACT...........................................................................................31
1. INTRODUCTION.............................................................................32
2. MATERIALS AND METHODS......................................................34
2.1. Preparation of Citrus Unshiu peel extraction........…………….…….……….34
2.2. Cell culture and reagents ……………………………………..…….……......34
2.3. Measurement of cell viability ….....................................................................35
2.4. Migration assay……………………………………………..……….…….....35
2.5. Tube formation……………….………...…...……...........................................35
2.6. High-performance liquid chromatography (HPLC) analysis……………….....36
2.7. Preparative-HPLC……………….………...…...…….......................................36
2.8. Western blot analysis……………….………...…...……..................................37
2.9. Statistical analysis……………….………...….........…….................................37
3. RESULTS AND DISCUSSIONS ...……….…….....……….…...…38
3.1. Chemical structures of hesperidin and narirutin..............................................39
3.2. Characterization of constituent elements using different solvents. Citrus Unshiu peel was extracted with sodium acetate buffer (A), MeOH (B), or DMSO (C).........................................................................................................40
3.3. Quantification of extracted narirutin and hesperidin. Extracted hesperidin and narirutin were quantified using HPLC analysis..….......…....................….…..42
3.4. Purification of DMSO extracted hesperidin from Citrus Unshiu peel. .…......44
3.5. Pro-angiogenic effects of DMSO extracted hesperidin. ….…........................45
4. REFERENCES................................................................................48
ABSTRACT IN KOREAN…………………………....…...………...50
ACKNOWLEDGEMENT...................................................................51
- Degree
- Master
- Publisher
- 제주대학교 일반대학원
- Citation
- 김명승. (2017). Characterizations of a novel glucosyltransferase and pro-angiogenic components in Citrus.
- Appears in Collections:
- Faculty of Biotechnology > Molecular Biotechnology
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