[보고서]식물 대사공학기술을 이용한 인삼 사포닌 및 사포제닌 생산 최적화 기술개발

최용의

보고서 정보

주관연구기관

강원대학교
Kangwon National University

연구책임자

최용의

참여연구자

한정연 , 안창호 , 황환수 , 권은경 , 전성원 , 구효빈 , 김규리 , 유자혜 , 김민경

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2021-02

과제시작연도

2020

주관부처

농촌진흥청
Rural Development Administration(RDA)

등록번호

TRKO202100009907

과제고유번호

1395062835

사업명

차세대바이오그린21(R&D)

DB 구축일자

2021-10-02

키워드

인삼 사포닌.대사공학.사포닌합성 증대기술.사포닌 운반단백질.전사조절인자.saponin.metabolic engineering.saponin synthesis.saponin carrier protein.transcriptional regulatory factor.

표 Ocotillol-type ginsenoside를 만드는 유전자는 squalene epoxidase로 추정됨
표 Biosynthetic pathway of triterpenes in P . ginseng. Triterpenes undergo oxidation and glycosylation and converted into triterpene sapon
표 Yeast complementation analysis of the PgSE1 and PgSE2 genes. A. Two yeast constructs harbouring the cDNA of PgSE1 or PgSE2 using the expression vector pYES2.1/V5-His-TOPO, together with the empty vector control, were transformed into erg1-deficient yeast, which does not grow without ergosterol feeding. The recombinant yeasts were plated onto medium with (A) and without (B) ergosterol. C-E. GC analysis of recombinant yeast extracts. C. GC-MS chromatogram of the methanol extract of erg1 yeast expressing PgSE1. D. GC-MS chromatogram of the methanol extract of erg1 yeast expressing PgSE2. E. GC-MS chromatogram of standard compounds (squalene, 2,3-oxidosqualene, and ergosterol)
표 Analysis of gene integration and expression of the introduced PgSE1 gene in transgenic ginseng roots. A. T-DNA regions of the binary vector (pH7WG2D) used in this experiment. Abbreviation. LB, left border sequence; HPT, hygromycin resistance gene; 35S-Pro; CaMV 35S promoter; NOS-Ter; NOS terminator; RB, right border sequence. B. Three transgenic ginseng root lines overexpressing PgSE1 and wild-type roots. C. Genomic PCR for analysis of the HPT gene in transgenic ginseng roots. D. RT-PCR analysis of HPT genes in the wild-type (Wt) line and three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgSE1
표 qPCR analysis for PgSE1 (A), PgDDS (B), and PgPNX (C) genes in wilt-type line and transgenic ginseng root lines overexpressing PgSE1. β-actin was employed as the internal control. The analysis results are presented as the means ± SEs of three independent experiments, and columns with the same letter are not significantly different (P ≤ 0.05)
표 Ginsenoside analysis in the roots of the wild-type line and three transgenic lines (Tr1-Tr3) overexpressing PgSE1 by LC analysis. A. LC chromatograms of ginsenosides extracted from wild-type roots. B-D. Ginsenoside chromatograms in the three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgSE1. E. Chromatograms of authentic ginsenosides
표 Contents of ginsenosides in wild-type and three transgenic ginseng root lines. The analysis results are presented as the means ± SEs of three independent experiments, and columns with the same letter are not significantly different (P ≤ 0.05)
표 Squalene and phytosterol analysis in the wild-type line and transgenic root lines overexpressing PgSE1 by GC analysis. A. GC-MS chromatograms of squalene and phytosterols (campesterol, stigmasterol, and β-sitosterol) extracted from wild-type roots. B-D. Squalene and phytosterol chromatograms in the three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgSE1. E. Chromatograms of authentic squalene and phytosterols
표 Contents of squalene (A) and phytosterols (B) in the wild-type line and three transgenic ginseng root lines overexpressing PgSE1 gene. The analysis results are presented as the means ± SEs of three independent experiments, and columns with the same letter are not significantly different (P ≤ 0.05)
표 Genomic PCR in leaves of transgenic CK5 tobacco additionally overexpressing PgSE1. Confirmation of integration of HPT and PgSE1 genes in transgenic CK5 tobacco additionally expressing of PgSE1. BAR, PgDDS, CYP716A47, and UGT71A27 were also expressed in three transgenic lines of CK5 tobacco additionally expressing of PgSE1
표 RT-PCR and qPCR in leaves of transgenic CK5 tobacco additionally overexpressing PgSE1. A. RT-PCR analysis of PgSE1 genes in wild-type (Wt), CK5 (as control), and three transgenic lines (Tr1, Tr2, Tr3) overexpressing PgSE1. B. qPCR analysis of PgSE1, PgDDS, CYP716A47, and UGT71A27 genes in leaves of CK5 tobacco (as control) and three transgenic lines ((Tr1, Tr2, Tr3) additionally overexpressing PgSE1. The analysis results are presented as the means ± SEs of three independent experiments
표 Single ion mode (SIM) chromatogram of LC-IT-TOF-MS analysis in the roots of wild-type (Wt), CK5 tobacco as control, and three transgenic CK5 tobacco lines (Tr1, Tr2, and Tr3) overexpressing PgSE1. A. SIM chromatogram in wild-type roots. B. SIM chromatogram in CK5 tobacco as control. C-E. SIM chromatograms in the three transgenic lines overexpressing PgSE1 (Tr1, Tr2, and Tr3). F. SIM chromatograms of authentic CK and protopanaxadiol
표 Squalene and phytosterol analysis in CK5 tobacco as control and transgenic CK5 tobacco additionally overexpressing PgSE1 by GC-MS. A. Squalene and phytosterol chromatograms in CK5 tobacco. B-D. Squalene and phytosterol chromatograms in the three transgenic lines (Tr1, Tr2, and Tr3) of CK5 tobacco overexpressing PgSE1. E. Chromatograms of authentic squalene and phytosterols
표 Contents of ginsenoside (CK) and its precursor (protopanaxadiol), squalene and phytosterols (campesterol, stigmasterol, and β-sitosterol) in CK5 tobacco and transgenic CK5 tobacco additionally overexpressing PgSE1. A. Contents of ginsenoside (CK) and its precursor (protopanaxadiol) in CK5 tobacco and three lines (Tr1, Tr2, and Tr3) of transgenic CK5 tobacco additionally overexpressing PgSE1. B-C. Contents of squalene (B) and phytosterols (C) in CK5 tobacco and transgenic CK5 tobacco additionally overexpressing PgSE1. The analysis results are presented as the means ± SEs of three independent experiments
표 Genomic PCR in leaves of transgenic CK5 tobacco additionally overexpressing PgSS1. Confirmation of integration of NPTII and PgSS1 genes in transgenic CK5 tobacco producing CK
표 RT-PCR analysis in leaves of transgenic CK5 tobacco additionally overexpressing PgSS1. A. RT-PCR analysis of PgSS1 genes in wild-type (Wt), CK5 (as control), and three transgenic lines (Tr1, Tr2, Tr3) overexpressing PgSS1
표 Genomic PCR in leaves of transgenic CK5 tobacco additionally overexpressing PgSS1. Confirmation of integration of PgSE1. BAR, PgDDS, CYP716A47, and UGT71A27 expressed in three transgenic lines of CK5 tobacco additionally expressing of PgSS1
표 LC chromatogram of LC-MS-MS analysis in the leaves of wild-type, CK5 control tobacco (CK control) and new transgenic tobacc line additionally overespressing PgSS1 gene (PgSS1-1, PgSS1-2, and PgSS1-3). A. LC chromatogram in wild-type tobacco leaves. B. LC chromatogram in CK5 tobacco as control. C-E. LC chromatograms in the three transgenic CK tobacco lines additinally overexpressing PgSS1 (PgSS1-1, PgSS1-2, and PgSS1-3). F. LC chromatograms of authentic CK standard
표 MS spectrum of CK peaks from transformed tobacco leaves and those of authentic standards. CK: compound K
표 Potulated pathway of ocotillol-type ginsenoside biosynthesis in Panax spceis
표 Neighbor-joining phylogenetic analysis of the PgSE genes isolated from Panax ginseng and other plant squanene epoxidase genes
표 Multiple sequence alignment of PgSE1, PgSE2, and PgSE3 sequences using the ClustalW multiple alignment tool
표 TMHMM (transmembrane hidden Markov model) posterior probabilities for PgSQE1 (A), PgSQE2 (B) and PgSE3 (C) amino acid sequence are shown. Transmembrane domains were predicted by the TMHMM program http://www.cbs.dtu.dk/services/TMHMM-2.0/. The prediction showed that the four regions of the PgSQE1 (A), the three regions of the PgSQE2 (B), and the two regions of the PgSE3 (C) represented the transmembrane domains. Red bars indicate transmembrane domains, blue lines indicate intracellular loops, and magenta lines indicate extracellular loops
표 GC analysis of PgSE3 gene including PgSE1 and PgSE2 genes by heterologous expression in erg1-deficient yeast, which does not grow without ergosterol feeding. GC analysis of recombinant yeast extracts. A. GC-MS chromatogram of the methanol extract of erg1 yeast expressing PgSE1. B. GC-MS chromatogram of the methanol extract of erg1 yeast expressing PgSE2. C. GC-MS chromatogram of the methanol extract of erg1 yeast expressing PgSE3. E. GC-MS chromatogram of standard compounds (squalene, 2,3-oxidosqualene, 2,3;22,23-dioxidosqualene, and ergosterol)
표 Analysis of gene integration and expression of the introduced PgSE3 gene in transgenic ginseng roots. A. T-DNA regions of the binary vector (pB72GW7) used in this experiment. Abbreviation. LB, left border sequence; HPT, hygromycin resistance gene; 35S-Pro; CaMV 35S promoter; NOS-Ter; NOS terminator; RB, right border sequence. B. Genomic PCR for analysis of the BAR gene in transgenic ginseng roots. C. RT-PCR analysis of PgSE3 gene in the wild-type (Wt) and three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgSE3
표 Analysis of gene integration and expression of the PgSE3 gene in transgenic ginseng roots by RNAi interferrence of PgSE3 gene. A. T-DNA regions of the binary vector (pB7GWIWG) used in this experiment. Abbreviation. LB, left border sequence; HPT, hygromycin resistance gene; 35S-Pro; CaMV 35S promoter; NOS-Ter; NOS terminator; RB, right border sequence. B. Genomic PCR for analysis of the BAR gene in transgenic RNAi ginseng roots. C. RT-PCR analysis of PgSE3 gene in the wild-type (Wt) and three RNAi transgenic lines (Tr1, Tr2, and Tr3) interferring PgSE3 gene
표 Ginsenoside analysis in the roots of the wild-type line and six transgenic lines overexpressing and RNAI interferring of PgSE3 by HPLC. A. LC chromatograms of ginsenosides extracted from wild-type roots. B-D. Ginsenoside chromatograms in the three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgSE3. E-G. Ginsenoside chromatograms in the three transgenic lines (Tr1, Tr2, and Tr3) RNAi interferring PgSE3.. H. Chromatograms of authentic standard ginsenosides
표 Multiple sequence alignment of PgbHLH1 sequence with those known to bHLHs of other plants using the ClustalW multiple alignment tool
표 Neighbor-joining phylogenetic analysis of the PgbHLH1 isolated from Panax ginseng and other plant transcription factors
표 Expression of PgbHLH1 and PgbHLH2 gene in Panax ginseng roots after treatment of methyl jasmonate at various concentrations for 24 hours
표 Analysis of gene integration and expression of the introduced PgbHLH1 gene in transgenic ginseng roots. A. T-DNA regions of the binary vector (pB2G2W7) used in this experiment. Abbreviation. LB, left border sequence; HPT, hygromycin resistance gene; 35S-Pro; CaMV 35S promoter; NOS-Ter; NOS terminator; RB, right border sequence. B. Three transgenic ginseng root lines overexpressing PgbHLH1 gene and wild-type roots. C. Genomic PCR for analysis of the BAR gene in transgenic ginseng roots. D. RT-PCR analysis of PgbHLH1 and other genes involved in ginsenoside biosynthesis in the wild-type (Wt) line and three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgbHLH1
표 Analysis of gene integration and expression of the introduced PgbHLH1 gene in transgenic ginseng roots. A. T-DNA regions of the binary vector (pB2G2W7) used in this experiment. Abbreviation. LB, left border sequence; HPT, hygromycin resistance gene; 35S-Pro; CaMV 35S promoter; NOS-Ter; NOS terminator; RB, right border sequence. B. Three transgenic ginseng root lines overexpressing PgbHLH1 gene and wild-type roots. D. Genomic PCR for analysis of the BAR gene in transgenic ginseng roots. E. RT-PCR analysis of PgbHLH1 and other genes involved in ginsenoside biosynthesis in the wild-type (Wt) line and three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgbHLH1
표 Ginsenoside analysis in the roots of the wild-type and three transgenic lines (Tr1-Tr3) overexpressing PgbHLH1 by LC analysis. A. LC chromatograms of ginsenosides extracted from wild-type roots. B-D. Ginsenoside chromatograms in the three transgenic lines (Tr1, Tr2, and Tr3) overexpressing PgbHLH1. E. Chromatograms of authentic seven ginsenos
표 Ginsenoside analysis in the roots of the wild-type and three transgenic RNAi lines (Tr1-Tr3) interferring PgbHLH1 gene by LC analysis. A. LC chromatograms of ginsenosides extracted from wild-type roots. B-D. Ginsenoside chromatograms in the three transgenic RNAi lines (Tr1, Tr2, and Tr3). E. Chromatograms of authentic seven ginsenosides
표 Contents of ginsenosides in wild-type and transgenic ginseng root lines overexpressing (A) and interferring of PgbHLH1 gene (B)
표 Multiple sequence alignment of PgPDR3 sequence with those known to other PDR genes involved in diterpene transporter using the ClustalW multiple alignment tool
표 Expression of PgPDR3 gene in Panax ginseng roots after treatment of methyl jasmonate at various concentrations for 24 hours
표 Analysis of gene introduction and expression of PgPDR3 gene in RNAi transgenic ginseng root lines. A. T-DNA regions of the binary vector (pB7GWIWGII) used in this experiment. Abbreviation. LB, left border sequence; HPT, hygromycin resistance gene; 35S-Pro; CaMV 35S promoter; NOS-Ter; NOS terminator; RB, right border sequence. B. Genomic PCR of BAR gene in eight transgenic ginseng root RNAi lines and wild-type roots. C. RT-PCR analysis of PgPDR3 gene in the wild-type (Wt) line and eight RNAi transgenic lines
표 LC analysis in the adventitious roots of wild-type and transgenic PgPDR3 RNAi lines of Panax ginseng. A. LC chromatogram in wild-type ginseng roots. B-D. LC chromatogram of transgenic PgPDR3 RNAi lines. E. LC chromatograms of seven authentic ginsenosides

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식물 대사공학기술을 이용한 인삼 사포닌 및 사포제닌 생산 최적화 기술개발
Development of optimazation technology for the production of saponin and sapogenin using plant metabolic engineering 원문보기

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식물 대사공학기술을 이용한 인삼 사포닌 및 사포제닌 생산 최적화 기술개발 Development of optimazation technology for the production of saponin and sapogenin using plant metabolic engineering 원문보기

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식물 대사공학기술을 이용한 인삼 사포닌 및 사포제닌 생산 최적화 기술개발
Development of optimazation technology for the production of saponin and sapogenin using plant metabolic engineering 원문보기

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