Characterization of genes controlling ginsenoside biosynthesis and application to metabolic engineering in Panax ginseng = 인삼 (Panax ginseng) 진세노사이드 생합성 조절 유전자 발굴 및 대사공학적 응용

국문 초록 (Abstract)

인삼 (Panax ginseng)은 예로부터 건강을 증진시키는 약재로 가장 널리 알려진 약용식물이다. 인삼 뿌리에는 트리터페노이드 사포닌이라고 불리는 진세노사이드가 함유되어 있는데 이는 인삼 생리활성 성분을 대표하는 이차대사 산물이다. 인삼 사포닌은 메발론산으로부터 이스프레노이드 생합성 사이클을 거친다. 스쿠알렌 에폭시다제는 스쿠알렌을 옥시도스쿠알렌으로 바꾸게 하는데 이과정으로부터 사포닌 및 식물스테롤의 생합성과정이 분지된다. 한편 인삼 진세노사이드의 생합성에 결정적으로 관여하는 옥시도스쿠알렌 사이클라제는 담마란다이올 II 합성 유전자이며 이로부터 담마린다이올이라는 인삼 사포닌의 전형적인 골격이 만들어진다.
본 학위논문에서는 스쿠알렌 에폭시다제와 담마란다이올 합성 유전자를 분리하고 기능을 규명하였다. 또한 3국의 삼 (wild-grown P. ginseng, P. quinquefolium, 및 P. japonicum)으로부터 부정근을 유도하고 증식하기 위한 배양조건 개발하였고 특히 메틸 자스몬 산을 처리하였을 경우 인삼 진세노사이드 생합성에 관여하는 유전자의 발현을 비교하였다.
이 결과 스쿠알렌 에폭시다제(PgSE1)는 1,609-bp로 된 유전자이며 59.1 kDa 아미노산으로 구성되어 있었다. 인삼 진세노사이드 합성 전구물질 및 메틸 자스몬산을 처리한 경우 PgSE1 mRNA의 발현이 증가함을 확인하였으며 아울러 진세노사이드 생합성이 증가되었다. 스쿠알렌 에폭시다제를 post-transcriptional gene silancing (RNAi)라는 기법으로 발현을 억제시킨 형질전환 뿌리를 유도한 결과 인삼 진세노사이드의 함량이 50% 줄어들어서 스쿠알렌 에폭시다제는 인삼 진세노사이드 생합성에 중요한 유전자임을 밝혔다.
한편 인삼 진세노사이드의 기본 골격은 담마란다이올이라는 아글리콘인데 이는 옥시도스쿠알렌 사이클라제 일종인 담마란다이올 합성 유전자가 관여한다. 담마란다이올이라고 추정되는 유전자를 인삼의 화아에서 분리한 유전자한 다음, DDS라고 명명하였다. 이 유전자는 화아에서 가장 발현율이 높았다. 메틸 자스몬산 처리시 강하게 DDS발현이 증가되었다. 또한 이 유전자를 이스트뮤탄트 (erg7)에 발현시켰을 경우 이스트에서 담마란다이올 및 하이드록시담마라논이 생성됨을 LC/APCIMS로 확인하였다. 한편 DDS 유전자를 RNA interference (RNAi) 방법을 이용하여 형질전환 인삼을 제작한 결과 형질전환체의 뿌리에 진세노사이드 함량이 84.5%가 감소됨을 확인하였다.
한편 산삼 뿌리로부터 부정근을 유도 하기위한 조건을 조사한 결과 배지에 NH4NO3를 제거해주는 것이 매우 효과적이었으며 이러한 경향은 미국삼 (P. quinquefolium) 및 죽절삼 (P. japonicum)에서도 같은 경향을 보였다. 산삼이 나이가 들수록 부정근 유도율이 현저히 감소 되었다. 산삼의 부정근을 효과적으로 증식시키기 위해서는 부정근 절편을 NH4NO3가 제거된 배지에서 2주간 배양한 후 NH4NO3가 첨가된 배지에서 배양하는 2단계 배양방법을 이용하는 경우 매우 효과적이었다. NH4NO3가 제거된 부정근의 유도를 촉진시키는 조건에서는 사포닌 생합성 유전자의 발현이 높게 나타났으며 진세노사이드 또한 생합성능이 증가되었다.

다국어 초록 (Multilingual Abstract)

Panax ginseng is one of the most highly regarded of herbal medicines in the Orient, where it has gained an almost magical reputation for being able to maintain the quality of life. Ginseng roots containing tetracyclic triterpenenoid saponins, called to ginsenosides, those are the major effective ingredients in P. ginseng.
Mevalonate is the preferential precursor for triterpene biosynthesis. The enzyme squalene synthase catalyzes the first step from the central isoprenoid pathway towards triterpenoid biosynthesis. Squalene synthase catalyzes the reductive condensation of two molecules of FPP to squalene, via the intermediate presqualene diphosphate. Squalene epoxidase catalyzes the conversion of squalene to 2,3(S)-oxidosqualene which is the first oxygenation step in the sterol biosynthetic pathway. Both phytosterols and triterpenes in plants are synthesized from the product of cyclization of 2,3-oxidosqualene. The 2,3-oxidosqualene cyclases compose a family of biocatalysts that convert 2,3-oxidosqualene to polycyclic triterpenes. The step in ginsenoside synthesis involves cyclization of 2,3-oxidosqualene to oleanane and a dammarene-type triterpene skeleton. The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by the oxidosqualene cyclase (dammarenediol synthase).
In this thesis, two genes such as squalene epoxidase and dammarenediol II synthase were isolated and characterized their functions in thebiosynthesis of ginsenosides in ginseng roots. Moreover, adventitious root culture system in three Panax species (wild-grown P. ginseng, P. quinquefolium, and P. japonicum) were extablished, and expression of the genes involved in triterpene biosynthesis in the ginseng advnetitious roots was analyzed by RT-PCR after treatment with methyl jasmonate.
Squalene epoxidase, catalyzes the epoxidation of squalene to 2,3-oxidosqualene, is branch points for the committed step for triterpene and phytosterol biosynthesis. In this thesis, squalene epoxidase gene (PgSE1) was isolated and the role of PgSE1 on the biosynthesis of triterpene and phytosterol was investigated. A ORF cDNA coding PgSE1 was a 1,609-bp with a predicted molecular mass of 59.1 kDa amino acids. Methyl jasmonate treatment resulted in obvious accumulations of PgSE1 mRNA in root compared with the response by the control. Three precursor treatments (mevalonate, farnesol and squalene) resulted in enhanced expression of squalene synthase (PgSS1), squalene epoxidase (PgSE1) and dammarenediol II synthase (PgDDS) but no change in the expression of cycloartenol synthase (PNX). Ginsenoside analysis revealed that squalene treatment was the most effective for accumulation of ginsenoside compared to other two precursor treatments. Post-transcriptional gene silancing (RNAi) of PgSE1 gene in transgenic P. ginseng resulted in silencing of PgSE1 expression, which resulted in reduced ginsenosides accumulation at 50% in roots. These results indicate that expression of PgSE1 is important regulator for the biosynthesis of ginsenosides in P. ginseng.
The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by the oxidosqualene cyclase (dammarenediol synthase). The expression of dammarenediol synthase gene (DDS) together with the genes involved in ginsenoside biosynthesis (SS, SE, PNX, PNY, PNY2 and PNZ) was analysed. Expression of DDS mRNA was higher in flower buds compared to root, leaf and petiole of ginseng plants. Elicitor (methyl jasmonate) treatment upregulated the expression of DDS mRNA. Ectopic expression of DDS in yeast mutant (erg7) lacking lanosterol synthase resulted in the production of dammarenediol and hydroxydammarenone, which were confirmed by LC/APCIMS. RNA interference (RNAi) of DDS in transgenic P. ginseng resulted in silencing of DDS expression, which leads reduction of ginsenosides production to 84.5% in roots. These results indicate that expression of DDS played a vital role in the biosynthesis of ginsenosides in P. ginseng.
Adventitious roots were produced directly from root segments of Panax ginseng seedlings when cultured on an MS solid medium containing 3.0 mg L-1 IBA. Omitting NH4NO3 from this medium greatly enhanced both the frequency of adventitious root formation and the number of roots per explants. This frequency declined markedly with the age of the root, but could be increased through repeated sub-culturing events. A two-step procedure that included NH4NO3-free media for the first two weeks of culture, followed by transfer onto media containing NH4NO3 for another four weeks, greatly improved total fresh weights of these adventitious roots compared with a method of continuous culture over six weeks in media that always contained NH4NO3. Expression of the genes involved in triterpene biosynthesis was analyzed by RT-PCR. Ginsenoside contents were enhanced by the omission of NH4NO3 and were also greatly increased by treatment with methyl jasmonate.
Adventitious root culture system in three Panax species (wild-grown P. ginseng, P. quinquefolium, and P. japonicum) was established to analyze their ginsenoside productivity. Adventitious roots were induced directly from segments of seedlings after cultured on MS solid medium containing 3.0 mg/l IBA. Omission of NH4NO3 from the medium greatly enhanced both the frequency of adventitious root formation and number of roots per explants in all the three Panax species. However, elongation of post-induced adventitious roots was enhanced on medium with NH4NO3. Two-step culture protocol: NH4NO3-free medium for first two weeks of culture, followed by NH4NO3 containing medium for further 4 weeks, greatly enhanced the fresh weight increase of adventitious roots in all the three ginseng species. The fresh weight of adventitious roots was high in P. quinquefolium and low in P. ginseng, followed by P. japonicum regardless of the composition of medium. Pattern and content of ginsenosides in adventitious roots differed among the three Panax species. Total ginsenoside content of adventitious roots in P. quinquefolium, P. ginseng, and P. japonicum was 8.03, 15.7 and 1.2 mg/g dry weight, respectively. Among the three speices, adventitious roots in P. quinquefolium produced high amount of ginsenosides. The pattern of ginsenoside fractions between P. ginseng and P. quinquefolium was similar but the amount of ginsenoside differed between the two. While, in P. japonicum, total ginsenoside content was very low and some ginsenosides such as ginsenoside Rb2 and Rf were not detected.

목차 (Table of Contents)

• INTRODUCTION = 1
• Chapter 1. RNAi silencing of squanene epoxidase (PgSE1) resulted in decreased ginsenoside biosynthesis in Panax ginseng = 7
• Abstract = 7
• 1.1 Introduction = 8
• 1.2 Materials and Methods = 9
• 1.2.1 Isolation and sequencing analysis of squalene epoxidse (PgSE1) of P. ginseng = 9
• 1.2.2 Sequence and phylogenetic analyses = 10
• 1.2.3 Methyl jasmonate treatment = 10
• 1.2.4 Precursor treatment = 11
• 1.2.5 Generation of the RNAi silencing construct = 11
• 1.2.6 Genetic transformation and adventitious root Induction from transgenic lines = 12
• 1.2.7 RT-PCR analysis = 13
• 1.2.8 Ginsenoside analyses by HPLC = 13
• 1.3 Results and Discusssion = 14
• 1.3.1 Isolation, sequence analysis and genomic organization of PgSE1 gene = 14
• 1.3.2 Expression of PgSE1 on the different parts of plants = 15
• 1.3.3 Response of PgSE1 and other genes by ginsenoside precursor treatment = 15
• 1.3.4 RNAi-mediated silencing of PgSE1 results in ginsenoside deficiency in P. ginseng plants = 15
• Chapter 2. Expression and RNA Interference Induced Silencing of Dammarenediol Synthase Gene in Panax ginseng = 28
• 2.1 Introduction = 28
• 2.2 Materials and Methods = 31
• 2.2.1 Isolation and sequencing analysis of oxidosqualene cyclase (DDS) of P. ginseng = 31
• 2.2.2 Sequence and phylogenetic analysis = 32
• 2.2.3 RT-PCR Analysis = 32
• 2.2.4 Expression of DDS by methyl jasmonate treatment = 33
• 2.2.5 Northern blot analysis = 34
• 2.2.6 Functional expression in yeast mutant ERG7 and analysis by TLC = 34
• 2.2.7 LC/APCIMS analysis of yeast mutant = 35
• 2.2.8 Generation of the RNAi silencing construct = 36
• 2.2.9 Construction of DDS-RNAi transgenic P. ginseng and adventitious root induction from transgenic lines = 36
• 2.2.10 DDS expression in RNAi transgenic ginseng plants = 37
• 2.2.11 Ginsenoside analyses by HPLC = 37
• 2.3 Results and Discussion = 38
• 2.3.1 Isolation, sequence analysis and genomic organization of DDS gene = 38
• 2.3.2 Expression of DDS and other OSCs on the different parts of plant = 39
• 2.3.3 Response of ginseng OSCs by methyl jasmonate treatment = 40
• 2.3.4 Ectopic expression of the DDS Cdna in ERG7-deficient yeast mutant = 40
• 2.3.5 RNAi-mediated silencing of DDS results in ginsenoside deficiency in P. ginseng plants = 42
• Chapter 3. Induction of Adventitious Roots and Analysis of Ginsenoside Content and the Genes Involved in Triterpene Biosynthesis in Panax ginseng = 55
• 3.1 Introduction = 55
• 3.2 Materials and methods = 56
• 3.2.1 Plant Materials = 57
• 3.2.2 Influence of NH4NO3 on Adventitious Root Induction = 57
• 3.2.3 Adventitious Root Induction from Wild-Cultivated and True-Wild Ginseng Roots = 58
• 3.2.4 Shake-Flask Cultures and Methyl Jasmonate Treatment = 58
• 3.2.5 Ginsenoside Analysis by HPLC = 59
• 3.2.6 RT-PCR Analysis = 60
• 3.3 Results and discussion = 61
• 3.3.1 Direct Adventitious Root Induction = 61
• 3.3.2 Adventitious Root Induction from Wild-Cultivated Ginseng and True-Wild Ginseng = 62
• 3.3.3 Two-Step Shake-Flask Cultures = 63
• 3.3.4 Influence of NH4NO3 on the Expression of Genes Involved in Triterpene Synthesis and Ginsenoside Accumulation = 64
• 3.3.5 Influence of Methyl Jasmonate on Expression of Genes Involved in Triterpene Synthesis and Ginsenoside Accumulation = 65
• Chapter 4. Adventitious root development and ginsenoside production in Panax ginseng, Panax quinquefolium and Panax japonicum = 75
• Introduction = 76
• 4.2 Materials and Methods = 77
• 4.2.1 Plant materials = 77
• 4.2.2 Direct adventitious root induction from three ginseng species = 78
• 4.2.3 Shake flask culture = 78
• 4.2.4 Two step culture for active growth of adventitious roots = 79
• 4.2.5 Ginsenoside analyses by HPLC = 79
• 4.3 Results and Discussions = 80
• 4.3.1 Direct adventitious root induction among three Panax species = 80
• 4.3.2 Shake flask culture and two step culture for active growth of adventitious roots = 81
• 4.3.3 Comparison of ginsenoside production among three Panax species = 83
• Literature cited = 90