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Functional Analysis of Candidate Genes Controlling Carotenoid Contents in Capsicum Fruit : 고추 카로티노이드 색소 함량에 대한 후보 유전자의 기능 분석

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dc.contributor.advisor강병철-
dc.contributor.author장소정-
dc.date.accessioned2021-11-30T06:13:12Z-
dc.date.available2021-11-30T06:13:12Z-
dc.date.issued2021-02-
dc.identifier.other000000164712-
dc.identifier.urihttps://hdl.handle.net/10371/176439-
dc.identifier.urihttps://dcollection.snu.ac.kr/common/orgView/000000164712ko_KR
dc.description학위논문 (석사) -- 서울대학교 대학원 : 농업생명과학대학 농림생물자원학부, 2021. 2. 강병철.-
dc.description.abstractPepper (Capsicum spp.) is one of the most important horticultural crops in the world. The diverse fruit colors of peppers (Capsicum spp.) are due to variations in carotenoid composition and content. Mature fruit color in peppers is known to be regulated by three independent loci, C1, C2, and Y, which encode Arabidopsis pseudo response regulator2-like (PRR2), phytoene synthase 1 (PSY1), and capsanthin-capsorubin synthase (CCS), respectively. However, the various pepper fruit color cannot be fully explained by the combination of these three loci. Therefore, the exact mechanisms of carotenoids related genes still need to be studied. In my thesis, two studies were conducted. The first study was the analysis of two F2 pepper populations that fit the three-locus model, focusing on the function of C1. As the identity of C1 locus was only recently reported, two pepper population with different fruit colors and genotypes were used for further study: an F2 population from a cross of Capsicum frutescens AC08-045 and AC08-201, and another F2 population derived from C. annuum 'Micropep Red (MR) and 'Micropep Yellow (MY)'. Transmission electron microscopy and virus-induced gene silencing (VIGS) of PRR2 showed that C1 controls overall carotenoid content by regulating the plastid compartment. The aim of the second chapter was to identify the gene controlling yellow color. In this study, orange fruit cultivar C. annuum SNU-mini Orange (SO) and yellow fruit cultivar SNU-mini Yellow (SY) were crossed and the locus controlling yellow color was mapped using genotyping by sequencing (GBS). Zeaxanthin epoxidase (ZEP) was located in this area, thus selected as a candidate gene. To show ZEP is involved in fruit color formation, VIGS was conducted in pepper with red and yellow mature fruit color. Both fruit colors were affected by ZEP silencing. HPLC analysis showed the contents of zeaxanthin were increased in these fruits, which demonstrated that ZEP is essential in yellow and red color. In summary, I revealed the role of PRR2 and verification of the three-locus model, and the role of ZEP in red to the yellow color formation.-
dc.description.abstract고추는 세계적으로 가장 중요한 원예작물 중 하나이다. 고추의 다양한 색은 카로티노이드 조성 및 함량 차이에 의해 나타난다. 고추 성숙과색은 세 독립적 유전자좌인 C1, C2, Y에 의해 조절된다고 알려져 있는데, 이는 각각 Arabidopsis pseudo response regulator2-like (PRR2), phytoene synthase 1 (PSY1), capsanthin-capsorubin synthase (CCS)를 암호화하고 있다. 하지만 고추의 다양한 과색은 위의 세 가지 유전자좌의 조합으로 나타나는 여덟 유전형 만으로 나뉠 수 없다. 따라서 각 카로티노이드 관련 유전자들이 어떠한 기작으로 과색에 영향을 주는지에 대한 연구가 필요하다. 이 논문에는 두 가지 연구가 진행되었다. 첫번째는 C1의 기능 연구를 중심으로 세 유전자좌 모델에 적합한 두가지 F2 집단이 사용하였다. C1의 경우 매우 최근에 그 정체가 보고되었기에 추가 연구를 위해 서로 다른 색상과 유전형을 가지는 두 집단으로 PRR2 유전자에서만 차이를 가지는 Capsicum frutescens AC08-045과 AC08-201의 교배 집단과 세 유전자좌 모두에서 유전형 분리가 일어나는 C. annuum 'Micropep Red (MR) 과 'Micropep Yellow (MY)의 교배집단을 사용하였다. 투과전자전자현미경과 바이러스를 사용한 PRR2의 유전자 발현 억제 실험 결과 C1이 색소체 구조를 조절하면서 전반적인 카로티노이드 함량을 결정하는 것을 볼 수 있었다. 두 번째 연구의 목적은 세 유전자좌 모델로 설명할 수 없는 노란색이 주황색에 대해 우성으로 나타나는 유전자를 밝히는 것이었다. 이 연구에서는 주황색 품종인 C. annuum SNU-mini Orange (SO)과 노란색 과색 품종인 SNU-mini Yellow (SY)을 교배하고, GBS 방법을 사용하여 주황색 표현형과 연관된 유전자좌를 찾았다. Zeaxanthin epoxidase (ZEP)가 해당 범위에 있다는 것을 찾아 이를 후보 유전자로 선정하였다. ZEP가 과색 형성에 미치는 영향을 보기 위해 빨간색과 노란색의 성숙과색을 가지는 고추에서 VIGS 실험을 진행한 결과 두 집단 모두에서 색 변화가 나타났다. 기질인 zeaxanthin의 비율 변화를 보여준 HPLC 분석 결과와 종합해보았을 때 ZEP가 특정 색상을 결정하는데 필수적임을 보여주었다. 본 논문에서는 PRR2-
dc.description.tableofcontentsCONTENTS

ABSTRACT ...............................................................................................................i
CONTENTS ...........................................................................................................iii
LIST OF TABLES ..................................................................................................vii
LIST OF FIGURES ...............................................................................................viii
LIST OF ABBREVIATIONS ...................................................................................x
GENERAL INTRODUCTION .................................................................................1


CHAPTER I. Arabidopsis pseudo response regulator2-like (PRR2) is Related to Color Intensity of Pepper Fruit

ABSTRACT ............................................................................................................10
INTRODUCTION ..................................................................................................12
MATERIALS AND METHODS ............................................................................18
Plant materials ..............................................................................................18
Nucleic acid extraction .................................................................................21
PCR amplification and sequencing…...........................................................22
Transmission election microscopy (TEM) ...................................................24
Virus-induced gene silencing of PRR2........................................................25
Carotenoid extraction and saponification of pepper fruits...........................28
RESULTS ................................................................................................................29
White color of AC08-201 was controlled by a single recessive gene.......29
PRR2 is expressed in immature fruit.............................................................31
PRR2 mutant contained less developed plastid compartment.....................33
Silencing of PRR2 in yellow pepper resulted in lighter color.......................36
The fruit color of MR × MY F2 population was segregated to eight group..39
Peppers of PSY1/ccs with less carotenoid contents had PRR2 mutation .....43
White pepper cultivars had PSY1, CCS, and PRR2 mutations in common .45
DISCUSSION .........................................................................................................47
REFERENCES .......................................................................................................51

CHAPTER II. Mutation in ZEP Controls Orange Fruit Color in Pepper (Capsicum annuum)

ABSTRACT ............................................................................................................54
INTRODUCTION ..................................................................................................56
MATERIALS AND METHODS ............................................................................59
Plant materials ..............................................................................................59
Nucleic acid extraction .................................................................................61
SNP detection by Genotyping-by-sequencing (GBS) and SNP calling........62
Bin map construction.............................................................................63
PCR amplification and sequencing ..............................................................64
Genotyping using HRM marker..............................................................65
Carotenoid extraction and saponification of fruit pericarp...........................66
Virus-induced gene silencing of ZEP.......................................................67
Expression analysis of ZEP in VIGS-treated fruit using RT-PCR...............69
RESULTS ................................................................................................................70
Bin map was constructed in SO × SY F2 population....................................70
ZEP was selected using BLAST search against a reference genome...........71
SO had a splicing mutation in ZEP......................................................77
Orange color was controlled by a single recessive gene......................81
SO had high zeaxanthin and total carotenoid contents.........................83
Silencing of ZEP in MY led to orange color.................................................85
Silencing of ZEP in MR led to red-orange color..................................88
DISCUSSION .........................................................................................................91
REFERENCES .......................................................................................................95

ABSTRACT IN KOREAN .....................................................................................99
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dc.format.extentx, 100-
dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subjectPepper-
dc.subjectMature fruit color-
dc.subjectCarotenoid-
dc.subjectZeaxanthin epoxidase (ZEP)-
dc.subjectPhytoene synthase (PSY)-
dc.subjectCapsanthin-capsorubin synthase (CCS)-
dc.subjectArabidopsis pseudo response regulator2-like (PRR2)-
dc.subjectPlastid development-
dc.subjectThree-locus model-
dc.subject고추-
dc.subject완숙과색-
dc.subject카로티노이드-
dc.subject.ddc631.52-
dc.titleFunctional Analysis of Candidate Genes Controlling Carotenoid Contents in Capsicum Fruit-
dc.title.alternative고추 카로티노이드 색소 함량에 대한 후보 유전자의 기능 분석-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthorSo-Jeong Jang-
dc.contributor.department농업생명과학대학 농림생물자원학부-
dc.description.degreeMaster-
dc.date.awarded2021-02-
dc.contributor.major원예생명공학전공-
dc.identifier.uciI804:11032-000000164712-
dc.identifier.holdings000000000044▲000000000050▲000000164712▲-
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