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WO2025110670A1 - Method for producing transgenic plant with increased saponin content - Google Patents

Method for producing transgenic plant with increased saponin content Download PDF

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WO2025110670A1
WO2025110670A1 PCT/KR2024/018246 KR2024018246W WO2025110670A1 WO 2025110670 A1 WO2025110670 A1 WO 2025110670A1 KR 2024018246 W KR2024018246 W KR 2024018246W WO 2025110670 A1 WO2025110670 A1 WO 2025110670A1
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ginseng
expression
nucleotide
ginsenoside
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김유진
김근기
김영훈
박찬우
이재은
황현정
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University Industry Cooperation Foundation of Pusan National University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)

Definitions

  • the present invention relates to a method for producing a transgenic plant with increased saponin content.
  • Ginseng ( Panax ginseng Meyer) is a perennial herb belonging to the genus Panax in the family Araliaceae.
  • the genus name Panax is derived from the Greek word panacea, meaning "all healing", and "ginseng” means a root that resembles the shape of a human being.
  • the root of ginseng has been used as an adaptogen to promote human health in East Asia, including Korea, China, and Japan, for thousands of years.
  • As the chemical composition of ginseng began to be disclosed research on the physiological activities of ginseng began, and various activities such as antioxidant, anticancer, anti-inflammation, anti-cardiovascular disease, and improving the immune system have been reported.
  • ginsenosides saponins
  • HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase 1
  • Isopentenyl diphosphate (IPP) and farnesyl diphosphate (FPP) are then produced from MVA, and the two FPPs are combined by SS (squalene synthase) to form squalene.
  • Squalene is epoxidized by squalene epoxidase to form 2,3-oxidosquelene, a precursor of triterpenes and sterols.
  • 2,3-oxidosquelene is cyclized by dammarenediol synthase to form dammarenediol, a precursor of various types of ginsenosides.
  • cytochrome P450 enzymes protopanaxadiol sythase (PPDS) and protopanaxatriol sythase (PPTS), convert damarendiol into protopanaxadiol (PPD) and convert PPD into protopanaxatriol (PPT), producing PPD- and PPT-type precursors of ginsenosides.
  • PPDS protopanaxadiol sythase
  • PPTS protopanaxatriol sythase
  • PPD protopanaxadiol
  • PPT protopanaxatriol
  • UGT UTP-glycosyl transferase
  • PgSE1 and PgSE2 were identified, but later, more than six SE genes were identified.
  • PgHMGR1 is known to play a pivotal role in the MVA pathway
  • the exact function of PgHMGR2 in ginsenoside biosynthesis has not yet been elucidated, and key factors responsible for regulating ginsenoside biosynthesis genes, such as transcription factors (TFs), have not been completely identified. Therefore, research is actively underway to identify substances that regulate ginsenoside biosynthesis; and to develop ginseng with increased ginsenoside content through such substances.
  • TFs transcription factors
  • jasmonic acid is one of the major plant hormones that regulates plant growth, responses to external stimuli, etc.
  • JA has been reported to mediate the regulation of secondary metabolite biosynthesis, such as nicotine, terpenoid indole alkaloid, and glucosinolate.
  • secondary metabolite biosynthesis such as nicotine, terpenoid indole alkaloid, and glucosinolate.
  • ginseng JA functions to enhance ginsenoside accumulation by inducing the expression of related genes.
  • PgLOX6 Lipoxygenase 6
  • PgLOX6 regulates JA biosynthesis.
  • PgLOX6 induces the expression of ginsenoside biosynthesis genes, such as SE1 and DDS, in ginseng to increase JA levels, which in turn enhances ginsenoside accumulation in ginseng.
  • the purpose of the present invention is to provide a method for producing a transgenic plant with increased saponin content.
  • Another object of the present invention is to provide seeds obtained from the transformed plant.
  • Another object of the present invention is to provide a composition for increasing saponin content.
  • the present invention provides a method for producing a transgenic plant with enhanced saponin content, comprising a step of transforming a plant with a recombinant vector comprising a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.
  • the present invention provides seeds obtained from the transformed plant.
  • the present invention provides a composition for increasing saponin content, comprising as an active ingredient a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.
  • a nucleotide having a base sequence represented by sequence number 1 or 2 derived from ginseng increases ginsenoside accumulation through regulation of ginsenoside biosynthesis gene expression, thereby being usefully utilized as a method for producing a transgenic plant with increased saponin content.
  • Figure 1a shows the results of phylogenetic tree analysis of Pg_S1528.8 (hereinafter referred to as Pg1) and Pg_S1229.1 (hereinafter referred to as Pg2) genes along with MYC2 TF (transcription factor) in various plants.
  • Panax quinquefolius bHLH22 (PqbHLH22, UUA80731.1) AaMYC2 ( Artemisia annua , KP119607), AtMYC2 ( Arabidopsis thaliana , AT1G32640), CrMYC2 ( Catharanthus roseus , AF283507), CsMYC2a ( Camellia sinensis , MK336383), CsMYC2b ( Camellia sinensis , MK336384), CsMYC2c ( Camellia sinensis , MK336385), FhMYC2 ( Freesia hybrida , XP_010919958), HbMYC2b ( Hevea brasiliensis , tabacum , GQ859161), OsMYC2 ( Oryza sativa , LOC_Os10g42430), PtrMYC2 ( Poncirus trifoliat
  • Figure 2b shows the results of functional domain location analysis of MYC2 TF in Pg1 and Pg2 genes.
  • Figure 2c shows the predicted protein structures of Pg1 and Pg2 genes.
  • Figure 2d shows the results of analyzing the sequence of MYC2 TF, which is identical to the sequence used for phylogenetic tree analysis, through MEME (Multiple Em for Motif Elicitation).
  • Figures 2a ⁇ 2b show the results of analyzing the predicted expression patterns of Pg1 and Pg2 genes using an RNA-sequencing-based database.
  • Figures 2c ⁇ 2d show the results of analyzing the expression of Pg1 and Pg2 genes in various tissues of ginseng.
  • Figure 3 shows the results of analyzing the intracellular location and binding potential of Pg1 and Pg2 and their ability to activate gene transcription.
  • Figure 4 shows the results of analyzing the effect of JA (Jasmonic acid) on Pg1 and Pg2 expression.
  • Figure 5 shows the results of analyzing the effects of Pg1 and Pg2 on ginsenoside accumulation.
  • Figure 6 shows the results of analyzing the gene expression levels of Pg1 and Pg2 for Pg1 and Pg2 ginseng overexpression lines constructed through transformation.
  • Figure 7 shows the results of analyzing the total ginsenoside content of Pg1 and Pg2 ginseng overexpressors and the appearance of the transformants.
  • the present invention provides a method for producing a transgenic plant with enhanced saponin content, comprising a step of transforming a plant with a recombinant vector comprising a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.
  • nucleotide having the base sequence represented by the above sequence number 1 is named “Pg_S1528.8” or “Pg1”
  • nucleotide having the base sequence represented by the above sequence number 2 is named “Pg_S1229.1” or “Pg2”.
  • a nucleotide having a base sequence represented by the above sequence number 1 can encode a peptide having an amino acid sequence represented by the above sequence number 3, and a nucleotide having a base sequence represented by the above sequence number 2 can encode a peptide having an amino acid sequence represented by the above sequence number 4.
  • the base sequence (DNA sequence) represented by the above sequence number 1 or sequence number 2; or the amino acid sequence represented by the sequence number 3 or sequence number 4 is as follows.
  • the above saponin may be ginsenoside.
  • the above nucleotides may be derived from ginseng ( Panax ginseng ).
  • the above plant may be at least one selected from the group consisting of, but is not limited to, Arabidopsis thaliana, tobacco, rice, wheat, barley, corn, sorghum, oats and soybeans.
  • the present invention provides seeds obtained from a transgenic plant.
  • the present invention provides a composition for increasing saponin content, comprising as an active ingredient a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.
  • Ginseng callus was obtained from mature embryos cultured on Murashige and Skoog solid medium (MS, Duchefa Biochie, Netherlands) containing 1 mg/L of 2,4-dichlorophenoxyacetic acid and 0.5 mg/L of 6-benzylaminopurine (hereinafter referred to as D1Ba0.5), and the conditions of a 16-h light cycle, 22°C temperature, and 60% relative humidity were maintained during callus formation. After callus formation, the callus was transferred to the same liquid medium and cultured under the conditions of 130 RPM for proliferation.
  • MS Murashige and Skoog solid medium
  • D1Ba0.5 6-benzylaminopurine
  • Tobacco ( Nicotiana) donated by Professor Jeong Gi-hong of Kyunghee University tabacum ) seeds were sown and kept in a dormant state at 4°C for 3 days. Afterwards, they were cultured for 4 weeks under conditions of a 16-hour light cycle, 22°C, and 60% relative humidity.
  • Pg1 Pg_S1528.8
  • Pg2 Pg_S1229.1
  • SNU ginseng DB http:/ginsengdb.snu.ac.kr/
  • GenBank GenBank
  • Pg1; Pg2; and the amino acid sequences of the 18 MYC2 transcription factors were aligned with ClustalX 1.83 to confirm the similarity between Pg1 and Pg2 with other MYC2 transcription factors.
  • Phylogenetic tree analysis was performed by MEGA11 using the statistical method of neighbor-joining for the amino acid sequences, and 1,000 bootstraps were replicated.
  • a total of 20 amino acid sequences including Pg1 and Pg2 were also submitted to MEME Suite (https:/meme-suite.org/meme/), and logos of conserved motifs among the sequences were obtained.
  • the promoter region sequences of 1,800 bp upstream of DDS (Pg_Scaffold3318), PPTS (Pg_Scaffold1770), and PPDS (Pg_Scaffold4733) were obtained from SNU ginseng DB.
  • the upstream 1,300 bp of PgHMGR1 (Pg_Scaffold6083) and PgSE1 (Pg_Scaffold0129) sequences were also obtained in the same manner as above.
  • the upstream 477 bp of PgHMGR2 (Pg_Scaffold1295) sequence was also analyzed in the same manner as other sequences.
  • ginseng transgenic lines overexpressing Pg1 and Pg2 the full-length of both genes was cloned into the pCambia1301 vector behind the cauliflower mosaic virus p35S promoter. Since both genes lack introns, Pg1 and Pg2 were amplified from ginseng genomic DNA, and the myc-tag was fused behind Pg2 to construct recombinant plasmids (p35S:Pg1 and p35S:Pg2-myc), respectively.
  • Pg1 and Pg2 were cloned into pGreen-GFP containing p35S to produce Pg1-GFP and Pg2-GFP, which were used for subcellular localization analysis.
  • Pg2 was cloned into pGreen vector containing mCherry tag to produce Pg2-mCherry, and the Pg2-mCherry and Pg1-GFP were used for co-lactalization analysis.
  • Pg1 and Pg2 were cloned into PGreen-NV and PGreen-CV to produce Pg1-NV and Pg2-CV, which were used for BiFC analysis.
  • 1,281 bp and 1,626 bp upstream from the translation start site of Pg1 and Pg2 were amplified from ginseng genomic DNA.
  • the amplified products were cloned into the pCambia1301 vector carrying the GUS gene to produce pPg1-GUS and pPg2-GUS.
  • ginseng callus was transferred to 100 ml of fresh D1Ba0.5 liquid medium, subcultured for 3 weeks, and treated with hormone (JA). 100 ⁇ M JA was treated to the subculture flask containing ginseng callus for 3 days. The control group was treated with the same volume of ethanol as JA. After harvesting, the callus was frozen in liquid nitrogen and stored at -80°C.
  • wild-type ginseng callus transformant lines as a control were subcultured in fresh medium for 3 weeks and treated with 100 ⁇ M JA.
  • the control group was treated with the same volume of ethanol as JA.
  • JA and ethanol were treated for 3 days to wild type, Pg1 (OX #4, OX #10, and OX #11), and Pg2 (OX #19, OX #22, and OX #62), respectively, and then frozen in liquid nitrogen immediately after harvest.
  • ginseng callus was grown in D1Ba0.5 liquid medium, two types of plasmids (p35S:Pg1 and p35S:Pg2-myc) were introduced into the rhizome of Agrobacterium tumefaciens LBA4404) was transformed with ginseng callus.
  • Ginseng callus was cut into pieces, pretreated with 0.05 M magnesium sulfate (MgSO 4 ), and then infected with rhizome. After transformation, the infected callus was selected on D1Ba0.5 medium containing cefotaxime and hygromycin for 12 weeks. Surviving calli were analyzed, and Pg1 and Pg2 expression was confirmed, and transformant lines were selected.
  • RNA samples used for expression analysis were frozen immediately after harvest, and total RNA was obtained using an easy-spin Total RNA Extraction Kit (iNtRON), and cDNA was synthesized from RNA using a PrimeScript RT reagent Kit with a gDNA Eraser (Takara).
  • iNtRON easy-spin Total RNA Extraction Kit
  • cDNA was synthesized from RNA using a PrimeScript RT reagent Kit with a gDNA Eraser (Takara).
  • RT-PCR was performed using a Mastercycler Nexus Gradient (Ependorf) and T100 Thermal Cycler (Bio-Rad) PCR instrument.
  • the amplification cycle used for RT-PCR analysis was uniformly 35 cycles, and the amplified products were loaded onto a 1% (w/v) agarose gel.
  • qRT-PCR was performed using the CFX Connect Real-Time System (Bio-Rad) and SYBR Green Mastermix (Takara). The experimental results were expressed by Ct normalization to the internal control ⁇ -Actin by the 2 - ⁇ Ct and 2 - ⁇ Ct methods.
  • Nicotiana benthamiana, 4 weeks old benthamiana ) plants were used for subcellular localization; co-localization; and BiFC assays of Pg1 and Pg2.
  • pGreen-Pg1-GFP, pGreen-Pg2-GFP, pGreen-Pg2-mCherry, pGreen-Pg1-NV, and pGreen-Pg2-CV were isolated from Agrobacterium tumefaciens GV3101) and infiltrated into tobacco (tabacco) leaves for transient expression of genes.
  • the root nematode containing pGreen-Pg1-GFP and pGreen-Pg2-GFP was separately injected into tobacco leaves.
  • pGreen-Pg1-GFP and pGreen-Pg2-mCherry were co-injected into Nicotiana benthamiana leaves. Since the expression sites of Pg1 and Pg2 were identical in the co-localization assay, the BiFC assay was performed to confirm the possibility of their binding, and pGreen-Pg1-NV and pGreen-Pg2-CV were also co-injected into tobacco leaves.
  • the tobacco was kept in a dart state at 22°C for 24 hours, and 3 days after infiltration, fluorescence signals were observed at 488 nm for GFP and 530 nm for mCherry using a confocal laser scanning microscope (K1-Fluo, Nanoscope systems, Korea).
  • ginsenoside extraction was performed. Transformed calli subcultured for 4 weeks in 100 ml of D1Ba0.5 liquid medium of each transformant line; and wild-type calli as a control were obtained, and frozen in liquid nitrogen immediately after harvest. All frozen samples were finely ground and then freeze-dried for one week. 0.2 g of each sample was quantified, and 2 ml of 70% methanol was added to the quantified sample. Thereafter, the extract was extracted using an ultrasonic extractor at 50°C for 30 minutes, and centrifuged at 15,000 RPM for 3 minutes to obtain the supernatant of the extract.
  • RNA of Pg1 OX #4 and Pg2 OX #19 was obtained, and total RNA of wild-type callus was prepared as a control. Three RNA lines were extracted through three repetitions, and the sequences of the lines were read through Macrogen (https:/dna.macrogen.com/main.do#). Compared to the wild-type control, the gene expression changes of Pg1 OX #4 and Pg2 OX #19 were confirmed, and genes with low expression levels were normalized by log2fold values to identify DEGs.
  • PgLOX6 increases JA (Jasmonic acid) and ginsenoside biosynthesis
  • MYC2 TF candidate increases gene expression levels in PgLOX6 overexpression lines.
  • Two MYC2s were identified among the previously known ginseng genome sequences. The genomic and coding sequences corresponding to Pg_S1528.8 and Pg_S1229.1 were obtained, and the two JA response factors were confirmed to correspond to MYC2 through sequence blast. Therefore, the two JA response factors were named Pg1 (Pg_S1528.8) and Pg2 (Pg_S1229.1), respectively.
  • Pg1 and Pg2 are closely related to CsMYC2a, which has been reported to regulate indole biosynthesis in Camellia sinensis ; and TwMYC2s (TwMYC2a and TwMYC2b), which are known to regulate diterpene biosynthesis in Tripterygium wilfordii (Fig. 1a).
  • CsMYC2a showed 67% similarity to Pg1 and Pg2
  • TwMYC2a showed 63% and 67% similarity to Pg1 and Pg2, respectively
  • TwMYC2b showed 62% similarity to Pg1 and Pg2, respectively (Fig. 1a).
  • Pg1 and Pg2 showed approximately 60% similarity to WsMYC2 and SlMYC2.
  • phylogenetic tree analysis results showed that Pg1 and Pg2 belong to the MYC2 TF family, which indicates that they have the function of regulating the expression of genes involved in the biosynthesis of secondary metabolites such as triterpenes. From the above results, it was confirmed that Pg1 and Pg2 have the function of regulating triterpene biosynthesis within the Araliaceae family.
  • amino acid sequence analysis results confirmed that Pg1 and Pg2 belong to the MYC2 TF family taxonomically, similar to 18 other MYC2 TFs previously reported (Fig. 1b).
  • the predicted protein structures of Pg1 and Pg2 were predicted, and JID, bHLH, and BIF/ACT-like domains were displayed in both Pg1 and Pg2 (Fig. 1c).
  • the predicted structures of Pg1 and Pg2 were both similar to NtMYC2a.
  • Pg1 and Pg2 showed a structure similar to BIF/ACT-like domain mediated by two strands of ⁇ -helix and ⁇ strand, including double helix strand representing bHLH.
  • three conserved motifs in PgMYC and MYC of other plants were analyzed using MEME tool (Fig. 1d), and it was confirmed that DNA binding domain was the most highly conserved (Fig. 1d).
  • a bimolecular-fluorescence complementation (BiFC) assay was performed to confirm the physical interaction between Pg1 and Pg2.
  • the combination of Pg1-N-fragment of venus (NV) and Pg2-C-fragment of venus (CV) showed positive signals in the nucleus of tobacco cells (Fig. 3c), confirming that Pg1 and Pg2 interact with each other in the nucleus.
  • overexpression lines of Pg1 and Pg2, respectively were generated through Agrobacterium-mediated transformation.
  • the Pg1 and Pg2 transformant lines were obtained with an efficiency of approximately 20%. Due to the expression effect of the transgene, the expression levels of the transformant lines varied depending on each line.
  • the Pg1 overexpression lines commonly showed an expression level that was approximately 2-fold higher than that of the wild type. However, there were also lines in which the expression did not significantly increase compared to the wild type.
  • the Pg2 overexpression lines showed an expression level that was approximately 3-fold higher than that of the wild type, and there were also lines in which the expression was lower than that of the wild type.
  • Pg1 OX #4, OX #10, and OX #11
  • Pg2 OX #19, OX #22, and OX #62
  • HPLC high performance liquid chromatography
  • ginsenoside accumulation was significantly increased in the overexpression lines compared to the control group (Fig. 5a).
  • the overexpression lines contained more than 500 mg/L of ginsenoside
  • the control group contained about 200 mg/L of ginsenoside (Fig. 7).
  • the content of each ginsenoside, including ginsenosides Re, Rg1, Rf, Rg1, Rg2, Rh1, Rc, Rb2, Rb3, and Rd increased in the overexpression lines, but in some lines, no increase in Rb2, Rb3, Rd, and Rh1 was observed (Fig. 5a).
  • ginsenoside biosynthetic genes Pg1 and Pg2 contribute to the enhanced ginsenosides
  • QRT-PCR was performed to analyze the expression of HMGR1, HMGR2, SE1, and SE2 (Fig. 5b-5e).
  • HMR1 was significantly increased by Pg1 (OX #4, OX #11) and Pg2 (OX #19 and OX #22) (Fig. 5b).
  • HMGR2 expression was also increased by six transgenic lines (Pg1 OX #4, #10, #11; Pg2 OX #19, #22, and #62) (Fig. 5c) compared to the control (wild-type callus).
  • RNA-seq analysis was performed.
  • HMGR2 Pg_S1295.30
  • PPTS Pg_S1770.12
  • SE1 Pg_S0129.28
  • Pg_S0245.36 was confirmed in DEGs (differentially expressed genes).
  • HMGR2 was confirmed in both Pg1 OX #4 and Pg2 OX #19.
  • Pg_S0245.36 in DEGs was identified as a candidate UGT that can be classified into the UGT71 family involved in the biosynthesis of compound K and ginsenoside Rd.
  • RNA-seq analysis the expression of HMGR2 was decreased in Pg1 OX #4, whereas it was increased in Pg2 OX #19.
  • the results were also confirmed by qRT-PCR analysis (Fig. 5c), and from the results, it was confirmed that Pg2 significantly affects HMGR2 expression.
  • MYC2 is known as a JA response TF
  • the expression of Pg1 and Pg2 was analyzed after JA treatment.
  • Pg1 and Pg2 expression increased (Fig. 4a).
  • Pg1 expression increased 2-fold compared to the control group (Fig. 4)
  • Pg2 expression increased about 3-fold compared to the control group.
  • Pg1 and Pg2 expression was also increased by JA in each of the overexpression lines of Pg1 and Pg2. Specifically, the expression of Pg1 and Pg2 increased about 3-4 times in the overexpression lines compared to the control group (Fig. 4). From the above results, it was confirmed that JA promotes the expression of Pg1 and Pg2.

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Abstract

The present invention relates to a method for producing a transgenic plant having an increased saponin content. It is confirmed that a nucleotide having a base sequence represented by SEQ ID NO: 1 or 2 derived from ginseng Panax ginseng increases ginsenoside accumulation by regulating the expression of genes involved in ginsenoside biosynthesis. Therefore, the method can be advantageously applied to the production of transgenic plants with increased saponin content.

Description

사포닌 함량이 증진된 형질전환 식물체 제조방법Method for producing transgenic plants with increased saponin content

본 발명은 사포닌 함량이 증진된 형질전환 식물체 제조방법에 관한 것이다.The present invention relates to a method for producing a transgenic plant with increased saponin content.

인삼(Panax ginseng Meyer)은 Araliaceae 과(family) Panax 속(genus)에 속하는 여러해살이풀로서, 속명 Panax는 그리스어 panacea에서 유래한 것으로 "모든 치유"를 의미하며, “ginseng”은 인간의 형태를 닮은 뿌리를 의미한다. 모든 약초의 왕으로 알려진 인삼의 뿌리는 수천년 전부터 한국, 중국, 일본 등 동아시아에서 인류의 건강 증진을 위한 강장제(adaptogen)로 사용되어 왔다. 인삼의 화학적 조성이 공개되기 시작하면서, 인삼의 생리 활성에 대한 연구가 시작되었고, 현재 항산화, 항암, 항염증, 항심혈관질환, 면역체계 개선 등 다양한 활성이 보고되었다. Ginseng ( Panax ginseng Meyer) is a perennial herb belonging to the genus Panax in the family Araliaceae. The genus name Panax is derived from the Greek word panacea, meaning "all healing", and "ginseng" means a root that resembles the shape of a human being. Known as the king of all medicinal herbs, the root of ginseng has been used as an adaptogen to promote human health in East Asia, including Korea, China, and Japan, for thousands of years. As the chemical composition of ginseng began to be disclosed, research on the physiological activities of ginseng began, and various activities such as antioxidant, anticancer, anti-inflammation, anti-cardiovascular disease, and improving the immune system have been reported.

인삼의 효능은 주로 진세노사이드(ginsenosides)라고 불리는 사포닌(saponin)에서 유래하는데, 진세노사이드는 Panax 속에서만 생산될 수 있는 트리테르페노이드(triterpenoid) 2차 대사산물이다. 인삼은 아세틸-CoA(acetyl-CoA)를 MVA(mevalonate)로 변환하는 역할을 하는 HMGR(3-hydroxy-3-methylglutaryl coenzyme A reductase 1)에 기여되는 MVA pathway를 통해 진세노사이드를 생합성한다. 이후, IPP(Isopentenyl diphosphate) 및 FPP(farnesyl diphosphate)가 MVA로부터 생산되고, 2개의 FPP는 SS(squalene synthase)에 의해 결합되어, 스쿠알렌(squalene)을 형성한다. 스쿠알렌은 SE(squalene epoxidase)에 의해 에폭시화되어 트리테르펜(triterpene) 및 스테롤(sterol)의 전구체인 2,3-옥시도스쿠알렌(2,3-oxidosquelene)로 변환된다. 2,3-옥시도스쿠알렌은 DDS(dammarenediol-synthase)에 의해 고리화되어(cyclized) 다양한 유형의 진세노사이드의 전구체인 다마렌디올(dammarenediol)을 형성한다. 그 다음, 사이토크롬(cytochrome) P450 효소인 프로토파낙사디올 합성효소(Protopanaxadiol sythase; 이하 PPDS라 함) 및 프로토파낙사트리올(Protopanaxatriol sythase; 이하 PPTS라 함)이 다마렌디올을 PPD(Protopanaxadiol)로 변환하고, PPD를 PPT(Protopanaxatriol)로 변환하여 진세노사이드의 PPD- 및 PPT- 타입 전구체를 생산한다. The efficacy of ginseng is mainly derived from saponins called ginsenosides, which are triterpenoid secondary metabolites that can only be produced in the genus Panax. Ginseng biosynthesizes ginsenosides through the MVA pathway contributed by HMGR (3-hydroxy-3-methylglutaryl coenzyme A reductase 1), which converts acetyl-CoA to MVA (mevalonate). Isopentenyl diphosphate (IPP) and farnesyl diphosphate (FPP) are then produced from MVA, and the two FPPs are combined by SS (squalene synthase) to form squalene. Squalene is epoxidized by squalene epoxidase to form 2,3-oxidosquelene, a precursor of triterpenes and sterols. 2,3-oxidosquelene is cyclized by dammarenediol synthase to form dammarenediol, a precursor of various types of ginsenosides. Next, cytochrome P450 enzymes, protopanaxadiol sythase (PPDS) and protopanaxatriol sythase (PPTS), convert damarendiol into protopanaxadiol (PPD) and convert PPD into protopanaxatriol (PPT), producing PPD- and PPT-type precursors of ginsenosides.

다양한 진세노사이드를 생산하는 물질로는 UGT(UDP-glycosyl transferase)가 확인되었다. UGTPg1이 compound K를 합성하고, PgUGT74AE2 및 PgUGT74Q2는 진세노사이드 Rg3 및 Rd 생산에 관여하고, UGTPg101 및 UGTPg102 진세노사이드 F1 및 Rg1 합성에 관여하며, PgUGT8 및 PgUGT18은 진세노사이드 Ro을 합성한다고 보고되었다. 하지만, 인삼 유전자에 대해 아직 명확하게 알려진 내용도 많이 남아있다. 인삼 내에는 HMGR1 뿐만 아니라, PgHMGR1과 상당히 유사한 PgHMGR2도 존재한다. 처음에는 PgSE1 및 PgSE2로 명명된 2개의 SE 유전자가 확인되었으나, 이후 6개 이상의 SE 유전자가 확인되었다. 또한, PgHMGR1은 MVA 경로에서 중추적인 역할을 하는 것으로 알려져 있으나, 진세노사이드 생합성에서 PgHMGR2의 정확한 기능은 아직 밝혀지지 않았고, 전사인자(transcription factor; 이하 TF라 함)와 같은 진세노사이드 생합성 유전자의 조절을 담당하는 핵심 인자들은 완전히 밝혀지지 않았다. 따라서, 진세노사이드 생합성을 조절하는 물질 규명; 및 상기 물질을 통한 진세노사이드 함량을 높인 인삼 개발을 위한 연구가 활발히 진행 중인 상황이다.UGT (UDP-glycosyl transferase) has been identified as a substance that produces various ginsenosides. It has been reported that UGTPg1 synthesizes compound K, PgUGT74AE2 and PgUGT74Q2 are involved in the production of ginsenosides Rg3 and Rd, UGTPg101 and UGTPg102 are involved in the synthesis of ginsenosides F1 and Rg1, and PgUGT8 and PgUGT18 synthesize ginsenoside Ro. However, there are still many things that are clearly known about ginseng genes. In ginseng, not only HMGR1 but also PgHMGR2, which is quite similar to PgHMGR1, exists. Initially, two SE genes named PgSE1 and PgSE2 were identified, but later, more than six SE genes were identified. In addition, although PgHMGR1 is known to play a pivotal role in the MVA pathway, the exact function of PgHMGR2 in ginsenoside biosynthesis has not yet been elucidated, and key factors responsible for regulating ginsenoside biosynthesis genes, such as transcription factors (TFs), have not been completely identified. Therefore, research is actively underway to identify substances that regulate ginsenoside biosynthesis; and to develop ginseng with increased ginsenoside content through such substances.

한편, 자스민산(Jasmonic acid; 이하 JA라 함)은 식물 생장, 외부 자극에 대한 반응 등을 조절하는 주요 식물 호르몬 중 하나이다. 또한, JA는 니코틴(nicotine), 테르페노이드 인돌 알칼로이드(terpenoid indole alkaloid) 및 글루코시놀레이트(glucosinolate)와 같은 2차 대사산물 생합성 조절을 매개하는 것으로 보고되었다. 인삼에서는 JA가 관련 유전자의 발현 유도를 통해 진세노사이드 축적을 향상시키는 기능을 한다. 또한, PgLOX6(Lipoxygenase 6)가 JA 생합성을 조절한다고 보고되었는데, PgLOX6은 인삼에서 SE1 및 DDS와 같은 진세노사이드 생합성 유전자의 발현을 유도하여 JA 수치를 증가시켜, 결과적으로 인삼 내 진세노사이드 축적을 향상시킨다.Meanwhile, jasmonic acid (JA) is one of the major plant hormones that regulates plant growth, responses to external stimuli, etc. In addition, JA has been reported to mediate the regulation of secondary metabolite biosynthesis, such as nicotine, terpenoid indole alkaloid, and glucosinolate. In ginseng, JA functions to enhance ginsenoside accumulation by inducing the expression of related genes. In addition, it has been reported that PgLOX6 (Lipoxygenase 6) regulates JA biosynthesis. PgLOX6 induces the expression of ginsenoside biosynthesis genes, such as SE1 and DDS, in ginseng to increase JA levels, which in turn enhances ginsenoside accumulation in ginseng.

본 발명의 목적은 사포닌 함량이 증진된 형질전환 식물체 제조방법을 제공하는 것이다.The purpose of the present invention is to provide a method for producing a transgenic plant with increased saponin content.

본 발명의 다른 목적은 상기 형질전환 식물체로부터 수득한 종자를 제공하는 것이다.Another object of the present invention is to provide seeds obtained from the transformed plant.

본 발명의 또 다른 목적은 사포닌(saponin) 함량 증진용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for increasing saponin content.

상기 목적을 달성하기 위해, 본 발명은 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드; 또는 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드를 포함하는 재조합 벡터로 식물체를 형질전환하는 단계를 포함하는, 사포닌 함량이 증진된 형질전환 식물체 제조방법을 제공한다.To achieve the above object, the present invention provides a method for producing a transgenic plant with enhanced saponin content, comprising a step of transforming a plant with a recombinant vector comprising a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.

또한, 본 발명은 상기 형질전환 식물체로부터 수득한 종자를 제공한다.In addition, the present invention provides seeds obtained from the transformed plant.

또한, 본 발명은 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드; 또는 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드를 유효성분으로 포함하는 사포닌(saponin) 함량 증진용 조성물을 제공한다.In addition, the present invention provides a composition for increasing saponin content, comprising as an active ingredient a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.

본 발명에 따르면, 인삼(Panax ginseng)으로부터 유래한 서열번호 1 또는 2로 표시되는 염기서열을 가지는 뉴클레오타이드가 진세노사이드(ginsenoside) 생합성 유전자 발현 조절을 통해 진세노사이드 축적을 증가시키는 것을 확인함으로써, 사포닌(saponin) 함량이 증진된 형질전환 식물체 제조방법으로써 유용하게 활용될 수 있다.According to the present invention, it is confirmed that a nucleotide having a base sequence represented by sequence number 1 or 2 derived from ginseng ( Panax ginseng ) increases ginsenoside accumulation through regulation of ginsenoside biosynthesis gene expression, thereby being usefully utilized as a method for producing a transgenic plant with increased saponin content.

도 1a는 다양한 식물 내 MYC2 TF(transcription factor)와 함께, Pg_S1528.8 (이하 Pg1이라 함) 및 Pg_S1229.1(이하 Pg2라 함) 유전자의 계통발생 트리(Phylogenetic tree analysis) 분석 결과이다. Panax quinquefolius bHLH22(PqbHLH22, UUA80731.1) AaMYC2(Artemisia annua, KP119607), AtMYC2(Arabidopsis thaliana, AT1G32640), CrMYC2(Catharanthus roseus, AF283507), CsMYC2a(Camellia sinensis, MK336383), CsMYC2b(Camellia sinensis, MK336384), CsMYC2c(Camellia sinensis, MK336385), FhMYC2(Freesia hybrida, XP_010919958), HbMYC2b(Hevea brasiliensis, XM021809140.2), MdMYC2(Malus domestica, MDP0000029168), NtMYC2b(Nicotiana tabacum, GQ859161), OsMYC2(Oryza sativa, LOC_Os10g42430), PtrMYC2(Poncirus trifoliata, MT919252), SlMYC2(Solanum lycopersicum, Solyc08g076930), SmMYC2(Salvia miltiorrhiza, KJ945636), TaMYC2(Triticum aestivum, MF679157), TcMYC2(Taxus chinensis, MG494378), TwMYC2a(Tripterygium wilfordii, MN836714), TwMYC2b(Tripterygium wilfordii, MN836715), WsMYC2(Withania somnifera, MG434696). 도 2b는 Pg1 및 Pg2 유전자 내 MYC2 TF의 기능적 도메인 위치 분석 결과이다. 도 2c는 Pg1 및 Pg2 유전자의 예측 단백질 구조를 나타낸다. 도 2d는 계통발생 트리 분석에 사용한 서열과 동일한 MYC2 TF의 서열을 MEME(Multiple Em for Motif Elicitation)을 통해 분석한 결과이다.Figure 1a shows the results of phylogenetic tree analysis of Pg_S1528.8 (hereinafter referred to as Pg1) and Pg_S1229.1 (hereinafter referred to as Pg2) genes along with MYC2 TF (transcription factor) in various plants. Panax quinquefolius bHLH22 (PqbHLH22, UUA80731.1) AaMYC2 ( Artemisia annua , KP119607), AtMYC2 ( Arabidopsis thaliana , AT1G32640), CrMYC2 ( Catharanthus roseus , AF283507), CsMYC2a ( Camellia sinensis , MK336383), CsMYC2b ( Camellia sinensis , MK336384), CsMYC2c ( Camellia sinensis , MK336385), FhMYC2 ( Freesia hybrida , XP_010919958), HbMYC2b ( Hevea brasiliensis , ​ ​ tabacum , GQ859161), OsMYC2 ( Oryza sativa , LOC_Os10g42430), PtrMYC2 ( Poncirus trifoliata , MT919252), SlMYC2 ( Solanum lycopersicum , Solyc08g076930), SmMYC2 ( Salvia miltiorrhiza , KJ945636), TaMYC2 ( Triticum aestivum , MF679157), TcMYC2 ( Taxus chinensis , MG494378), TwMYC2a ( Tripterygium wilfordii , MN836714), TwMYC2b ( Tripterygium wilfordii , MN836715), WsMYC2 ( Withania somnifera , MG434696). Figure 2b shows the results of functional domain location analysis of MYC2 TF in Pg1 and Pg2 genes. Figure 2c shows the predicted protein structures of Pg1 and Pg2 genes. Figure 2d shows the results of analyzing the sequence of MYC2 TF, which is identical to the sequence used for phylogenetic tree analysis, through MEME (Multiple Em for Motif Elicitation).

도 2a~2b는 Pg1 및 Pg2 유전자의 예측 발현 패턴을 RNA-sequencing 기반 데이터베이스로 분석한 결과이다. 도 2c~2d는 인삼의 다양한 조직 내 Pg1 및 Pg2 유전자 발현을 분석한 결과이다.Figures 2a~2b show the results of analyzing the predicted expression patterns of Pg1 and Pg2 genes using an RNA-sequencing-based database. Figures 2c~2d show the results of analyzing the expression of Pg1 and Pg2 genes in various tissues of ginseng.

도 3은 Pg1 및 Pg2의 세포 내 위치와 결합 가능성 및 유전자 전사 활성화 능력을 분석한 결과이다.Figure 3 shows the results of analyzing the intracellular location and binding potential of Pg1 and Pg2 and their ability to activate gene transcription.

도 4는 JA(Jasmonic acid)가 Pg1 및 Pg2 발현에 미치는 영향을 분석한 결과이다.Figure 4 shows the results of analyzing the effect of JA (Jasmonic acid) on Pg1 and Pg2 expression.

도 5는 Pg1 및 Pg2가 진세노사이드(ginsenoside) 축적에 미치는 영향을 분석한 결과이다.Figure 5 shows the results of analyzing the effects of Pg1 and Pg2 on ginsenoside accumulation.

도 6은 형질전환을 통해 구축한 Pg1 및 Pg2 인삼 과발현체 라인에 대한 Pg1 및 Pg2의 유전자 발현량을 분석한 결과이다.Figure 6 shows the results of analyzing the gene expression levels of Pg1 and Pg2 for Pg1 and Pg2 ginseng overexpression lines constructed through transformation.

도 7은 Pg1 및 Pg2 인삼 과발현체의 총 진세노사이드 함량을 분석한 결과 및 형질전환체의 모습을 나타낸 것이다.Figure 7 shows the results of analyzing the total ginsenoside content of Pg1 and Pg2 ginseng overexpressors and the appearance of the transformants.

이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.

본 발명은 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드; 또는 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드를 포함하는 재조합 벡터로 식물체를 형질전환하는 단계를 포함하는, 사포닌 함량이 증진된 형질전환 식물체 제조방법을 제공한다.The present invention provides a method for producing a transgenic plant with enhanced saponin content, comprising a step of transforming a plant with a recombinant vector comprising a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.

본 명세서에서, 상기 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드는 “Pg_S1528.8” 또는 “Pg1”라고 명명하였고, 상기 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드는 “Pg_S1229.1” 또는 “Pg2”라고 명명하였다.In this specification, the nucleotide having the base sequence represented by the above sequence number 1 is named “Pg_S1528.8” or “Pg1”, and the nucleotide having the base sequence represented by the above sequence number 2 is named “Pg_S1229.1” or “Pg2”.

상기 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드는 서열번호 3으로 표시되는 아미노산 서열을 가지는 펩타이드를 암호화할 수 있고, 상기 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드는 서열번호 4로 표시되는 아미노산 서열을 가지는 펩타이드를 암호화할 수 있다.A nucleotide having a base sequence represented by the above sequence number 1 can encode a peptide having an amino acid sequence represented by the above sequence number 3, and a nucleotide having a base sequence represented by the above sequence number 2 can encode a peptide having an amino acid sequence represented by the above sequence number 4.

상기 서열번호 1 또는 서열번호 2로 표시되는 염기서열(DNA 서열); 또는 서열번호 3 또는 서열번호 4로 표시되는 아미노산 서열은 하기와 같다.The base sequence (DNA sequence) represented by the above sequence number 1 or sequence number 2; or the amino acid sequence represented by the sequence number 3 or sequence number 4 is as follows.

[서열번호 1] (2013bp)[Sequence number 1] (2013bp)

ATATAACTAGGGAATTGAATTTCTCTGGATTCGGGTTTGATGGAATAAGTAGTACTGTTAGGAAAGGGAATTCGAATTCACATGTTTGTAAGCCTGAATCCGGAGAAAAACTGAATTTTGGGGAAAGTAAGAGGAGTTGTAGTGGAAATGGGACTTTGTTTGCAGGACATTCACAATTTGTGGGAATTGTTGAGGATAGCAAGAAGAAGAGATCGTCGAGTTCGAGAGGTAGTTATCATGAAGAGGGTGGTATACTTTCCTTCAGTTCTGGTATGATTTTGCCGTCTTCTGGTATCGTAAAATCCAGCGGTGGTGGTGGGGATTCTGACCATTCGGATCTTGAACCCTCTGTAGTTAAAGAGGCTATTGTTAGTCAAGTTGTGGATCCAGAAAGAAAACCACGAAAGCGGGGAAGAAAGCCGGCTAATGGGAGAGAAGAGCCATTGAATCATGTTGAAGCAGAACGACAGAGGAGGGAGAAGCTTAACCAGAAGTTCTATGCCCTCCGAGTAGTGGTACCTAATGTATCAAAAATGGACAAGGCTTCACTTCTCGGAGATGCTATTGTTTTCATCAATGAGTTGAAAGCCAAACTCCAGACATCGGATTCAGAAAAGGATGAGTTGCGAAGCCAATTGGAGTCTTTGAAAAAGGAGTTGGCTAGTAAAGAATCGCAATATTCAAGTCAGATGGCAGCTGATAAAGATCTCAAAATATCAAATGACCATGGAAATAAGTTTATTAATTTGGACATAGATGTGAAGATTATTGGTTGGGACGCGATGATCCGAATCCAATGTAGTAAGAAGAACCACCCTGCAGCTAGATTGATGGCGGCTTTGGAAGAGATGGACCTAGAAGTCAGCCACGCGAGTATATCCGTCGTCAATGATTTGATGATTCAACAGGCTACGGTGAAGATGGGTAGTCGATTTTACACCCAAGAACAGCTTAGAGTAGCATTGGCAGCCAAAGTCTCAGAAACAAGATAAATATAACTAGGGAATTGAATTTCTCTGGATTCGGGTTTGATGGAATAAGTAGTACTGTTAGGAAAGGGAATTCGAATTCACATGTTTGTAAGCCTGAATCCGGAGAAAAACTGAATTTTGGGGAAAGTAAGAGGAGTTGTAGTGGAAATGGGACTTTGTTTGCAGGACATTCACAATTTGTGGGAATTGTTGAGGATAGCAAGAAGAAGAGATCGTCGAGTTCGAGAGGTAGTTATCATGAAGAGGGT GGTATACTTTCCTTCAGTTCTGGGTATGATTTTGCCGTCTTCTGGTATCGTAAAATCCAGCGGTGGTGGTGGGATTCTGACCATTCGGATCTTGAACCCTCTGTAGTTAAAGAGGCTATTGTTA GTCAAGTTGTGGATCCAGAAAGAAAACCACGAAAGCGGGGAAGAAAGCCGGCTAATGGGAGAGAAGAGCCATTGAATCATGTTGAAGCAGAACGACAGAGGAGGGAGAAGCTTAACCAGAAGTT CTATGCCCTCCGAGTAGTGGTACCTAATGTATCAAAAATGGACAAGGCTTCACTTCTCGGAGATGCTATTGTTTTCATCAATGAGTTGAAAGCCAAACTCCAGACATCGGATTCAGAAAAGGATGAGTTGCGAAGCCAATTGGAGGTCTTTGAAAAAGGAGTTGGCTAGTAAAGAATCGCAATATTCAAGTCAGATGGCAGCTGATAAAGATCTCAAAATATCAAATGACCATGGAAATAAGTTTTATTA ATTTGGACATAGATGTGAAGATTATTGGTTGGGACGCGATGATCCGAATCCAATGTAGTAAGAAGAACCACCCTGCAGCTAGATTGATGGCGGCTTTGGAAGAGATGGACCTAGAAGTCAGCCACGCGAGTATATCCGTCGTCAATGATTTGATGATTCAACAGGCTACGGTGAAGATGGGTAGTCGATTTTACACCCAAGAACAGCTTAGAGTAGCATTGGCAGCCAAAGTCTCAGAAACAAGATAA

[서열번호 2] (1,994bp)[Sequence number 2] (1,994 bp)

ATTTGGGTTTGATGGAATTAGTAGTACTAATGTTAGGAATGGAAATTTGAATTCACACGCATGTAAGCCTGAATCCGAGGAAATACTGAATTTTGGGGAAAGTAAGAGGAGTTCTTGTAGTGGAAATGGGAATTTGTTTTCGGGAAATTCACCATTTGGTGGAATTGTTGAAGACAATAAAAAGAAGAAATCTCCGAATTCCAGGGGTAGTCATGAGGAGGGTATGCTATCGTTTTCTTCCGGTGTGATTTTACCGTCTTCCGGTGTGGTAAAATCGAGTGGTGGTGGTGGGGATTCGGATCACTCGGATCTTGAAGCCTCGGTTGTTAAAGAGGTTGAGAGTAGTCGAGTTGTAGACCCGGAAAAGAAACCACGAAAAAGGGGGAGAAAGCCGGCAAATGGGAGGGAAGAGCCATTGAATCATGTTGAGGCGGAGCGACAGAGGAGGGAGAAGCTCAACCAGAGGTTCTACGCTCTTCGCGCAGTGGTACCTAATGTATCGAAAATGGACAAGGCCTCACTTCTTGGGGATGCTATTTCTTACATTAATGAGCTTAAATCCAAGCTCCAGGAATCGGATTCAGAGAAAGATGAGTTGAGAAGCCAATTGGATTCATTGAGAAAGGAATTAGCTAAAAAAGTATCACAATATCCAACTCAAGCAACTCAAGCAGCAGTCGAGCAAGATCTCAAAATGTCAAACCACCATGGGAGCAAGTTGCTTGATTTGGATATAGATGTGAAAATCATTGGTTGGGACGCCATGATCCGATTCCAATCCACTAAGAAAAACCACCCTGCAGCAAGATTGATGGCGGCTTTGAAAGAGCTGGACCTAGATGTTCATCACGCAAGCGTATCTGTTGTCAATGATTTGATGATTCAACAAGCTACGGTGAAGATGGTCAGTCGGTTTTACACCCAAGATCAGCTTAGAGTAGCGTTGACAGCCCGAGTTTCCGAAACAAGATAAATTGGGTTTGATGGAATTAGTAGTACTAATGTTAGGAATGGAAATTTGAATTCACACGCATGTAAGCCTGAATCCGAGGAAATACTGAATTTTGGGGAAAGTAAGAGGAGTTCTTGTAGTGGAAATGGGAATTTGTTTTCGGGAAATTCACCATTTGGTGGAATTGTTGAAGACAATAAAAAGAAGAAATCTCCGAATTCCAGGGGTAGTCATGAGGAGGGTATGCTATCGTTTTCTTCCGG TGTGATTTTACCGTCTTCCGGTGTGGTAAAATCGAGTGGTGGTGGTGGGGATTCGGATCACTCGGATCTTGAAGCCTCGGTTGTTAAAGAGGTTGAGAGTAGTCGAGTTGTAGACCCGGAAAAGAAACCACGAAAAAGGGGGAGAAAGCCGGCAAATGGGAGGGAAGAGCCATTGAATCATGTTGAGGCGGAGCGACAGAGGAGGGAGAAGCTCAACCAGAGGTTCTACGCTCTTCGCGCAGT GGTACCTAATGTATCGAAAATGGACAAGGCCTCACTTCTTGGGGATGCTATTTCTTACATTAATGAGCTTAAATCCAAGCTCAGGGAATCGGATTCAGAGAAAGATGAGTTGAGAAGCCAATTGGATTCATTGAGAAAGGAATTAGCTAAAAAAGTATCACAATATCCAACTCAAGCAACTCAAGCAGCAGTCGAGCAAGATCTCAAAATGTCAAACCACCACCATGGGAGCAAGTTGCTTGATTT GGATATAGATGTGAAAATCATTGGTTGGGACGCCATGATCCGATTCCAATCCACTAAGAAAACCACCCTGCAGCAAGATTGATGGCGGCTTTGAAAGAGCTGGACCTAGATGTTCATCACGCAAGCGTATCTGTTGTCAATGATTTGATGATTCAACAAGCTACGGTGAAGATGGTCAGTCGGTTTTACACCCAAGATCAGCTTAGAGTAGCGTTGACAGCCCGAGTTTCCGAAACAAGATAA

[서열번호 3] (670bp)[Sequence number 3] (670bp)

MTDYRVPTAAMNLWSTTTTDDNTSMMDAFMSADLTSFWPPPTPPPPPPPQSSSTSTSTAAAAVFNQESLQHRLQSLIEGAKESWTYAIFWQWQSASGDIDYLSSQSSLLGWGDGYYKGEDKEKQLKRKPTSAAEQAHRRKVLRELNSLISGSQSFPDDAVDEEVTDTEWFFLVSMTQSFVNGAGLPGQAFFNSSPVWVTRAERLLSSPCERARQAQTFGLQTMVCIPSNNGVVELGSTELIYQSSDLMNKVRILFNFNSIDSGYWPVPSEPNESDPSALWLTDPSPLPNVEIKEIPMNSAKPPQIGFENHSFSTLTENPSTSSVINVQNQHSKQSQQQNGNITRELNFSGFGFDGISSTVRKGNSNSHVCKPESGEKLNFGESKRSCSGNGTLFAGHSQFVGIVEDSKKKRSSSSRGSYHEEGGILSFSSGMILPSSGIVKSSGGGGDSDHSDLEPSVVKEAIVSQVVDPERKPRKRGRKPANGREEPLNHVEAERQRREKLNQKFYALRVVVPNVSKMDKASLLGDAIVFINELKAKLQTSDSEKDELRSQLESLKKELASKESQYSSQMAADKDLKISNDHGNKFINLDIDVKIIGWDAMIRIQCSKKNHPAARLMAALEEMDLEVSHASISVVNDLMIQQATVKMGSRFYTQEQLRVALAAKVSETRMTDYRVPTAAMNLWSTTTTDDNTSMMDAFMSADLTSFWPPPTPPPPPPPQSSSTSTSTAAAAVFNQESLQHRLQSLIEGAKESWTYAIFWQWQSASGDIDYLSSQSSLLGWGDGYYKGEDKEKQLKRKPTSAAEQAHRRKVLRELNSLISGSQSFPDDAVDEEVTDT EWFFLVSMTQSFVNGAGLPGQAFFNSSPVWVTRAERLLSSPCERARQAQTFGLQTMVCIPSNNGVVELGSTELIYQSSDLMNKVRILFNFNSIDSGYWPVPSEPNESDPSALWLTDPSPLPNVEIKEIPMNSAKPPQIGFENHSFSTLTENPSTSSVINVQNQHSKQS QQQNGNITRELNFSGFGFDGISSTVRKGNSNSHVCKPESGEKLNFGESKRSCSGNGTLFAGHSQFVGIVEDSKKKKRSSSSRGSYHEEGGILSFSSGMILPSSGIVKSSGGGGDSDHSDLEPSVVKEAIVSQVVDPERKPRKRGRKPANGREEPLNHVEAERQRREKL NQKFYALRVVVPNVSKMDKASLLGDAIVFINELKAKLQTSDSEKDELRSQLESLKKELASKESQYSSQMAADKDLKISNDHGNKFINLDIDVKIIGWDAMIRIQCSKKNHPAARLMAALEEMDLEVSHASISVVNDLMIQQATVKMGSRFYTQEQLRVALAAKVSETR

[서열번호 4] (652bp)[Sequence number 4] (652bp)

MNLWSTTITTDDNASMLDAFMASDLTSFWPPPPVPQQSSSTSTSAAAVFSQESLQHRLQSLIEGSKESWTYAIFWQSSVADYSSSQTLLGWGDGYYKGEDKEKQLLKRKSTSATEQDHRKKVLRELNSLISGNQASHDDAVDEEVTDTEWFFLVSMTQSFVNGTGLPGQAFFNSSPVWVTGIERLASSHCERARQAQTFGLQTIVCIPSNNGVVELGSTELIFQSSGFMNKVRILFNFNAIESGSWPLPSDPNEPDPSALWLTDPLPVPSVEIKEIPLNSNSKPPQIMFENHSSSTLTENPSTSSVINVHNQHLNQQNGVLHRELNFSGFGFDGISSTNVRNGNLNSHACKPESGEILNFGESKRSSCSGNGNLFSGNSPFGGIVEDNKKKKSPNSRGSHEEGMLSFSSGVILPSSGVVKSSGGGGDSDHSDLEASVVKEVESSRVVDPEKKPRKRGRKPANGREEPLNHVEAERQRREKLNQRFYALRAVVPNVSKMDKASLLGDAISYINELKSKLQESDSEKDELRSQLDSLRKELAKKVSQYPTQATQAAVEQDLKMSNHHGSKLLDLDIDVKIIGWDAMIRFQSTKKNHPAARLMAALKELDLDVHHASVSVVNDLMIQQATVKMVSRFYTQDQLRVALTARVSETRMNLWSTTITTDDNASMLDAFMASDLTSFWPPPPVPQQSSSTSTSAAAVFSQESLQHRLQSLIEGSKESWTYAIFWQSSVADYSSSQTLLGWGDGYYKGEDKEKQLLKRKSTSATEQDHRKKVLRELNSLISGNQASHDDAVDEEVTDTEWFFLVSMTQSFVNG TGLPGQAFFNSSPVWVTGIERLASSHCERARQAQTFGLQTIVCIPSNNGVVELGSTELIFQSSGFMNKVRILFNFNAIESGSWPLPSDPNEPDPSALWLTDPLPVPSVEIKEIPLNSNSKPPQIMFENHSSSTLTENPSTSSVINVHNQHLNQQNGVLHRELN FSGFGFDGISSTNVRNGNLNSHACKPESGEILNFGESKRSSCSGGNNLFSGNSPFGGIVEDNKKKKSPNSRGSHEEGMLSFSSGVILPSSGVVKSSGGGGDSDHSDLEASVVKEVESSRVVDPEKKPRKRGRKPANGREEPLNHVEAERQRREKLNQRFYALR AVVPNVSKMDKASLLGDAISYINELKSKLQESDSEKDELRSQLDSLRKELAKKVSQYPTQATQAAVEQDLKMSNHHGSKLLDLDIDVKIIGWDAMIRFQSTKKNHPAARLMAALKELDLDVHHASVSVVNDLMIQQATVKMVSRFYTQDQLRVALTARVSETR

상기 사포닌은 진세노사이드(ginsenoside)일 수 있다.The above saponin may be ginsenoside.

상기 뉴클레오타이드는 인삼(Panax ginseng)에서 유래한 것일 수 있다.The above nucleotides may be derived from ginseng ( Panax ginseng ).

상기 식물체는 애기장대, 담배, 벼, 밀, 보리, 옥수수, 수수, 귀리 및 콩으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 한정되는 것은 아니다.The above plant may be at least one selected from the group consisting of, but is not limited to, Arabidopsis thaliana, tobacco, rice, wheat, barley, corn, sorghum, oats and soybeans.

또한, 본 발명은 형질전환 식물체로부터 수득한 종자를 제공한다.Additionally, the present invention provides seeds obtained from a transgenic plant.

또한, 본 발명은 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드; 또는 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드를 유효성분으로 포함하는 사포닌(saponin) 함량 증진용 조성물을 제공한다.In addition, the present invention provides a composition for increasing saponin content, comprising as an active ingredient a nucleotide having a base sequence represented by SEQ ID NO: 1; or a nucleotide having a base sequence represented by SEQ ID NO: 2.

이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, in order to help understand the present invention, examples will be given and described in detail. However, the following examples are only intended to illustrate the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to a person having average knowledge in the art.

[준비예 1] 실험 준비[Preparation Example 1] Experiment Preparation

1-1. 인삼1-1. Ginseng

인삼(Panax ginseng) 품종 중 하나인 천풍(Chunpoong)’을 실험에 사용하였고, 상기 인삼은 농촌진흥청(the Rural Development Administrations) 인삼밭에서 재배한 종자를 제공받아 사용하였다. 인삼 캘러스(callus)는 2,4-디클로로페녹시아세트산(2,4-dichlorophenoxyacetic acid) 1mg/L 및 6-벤질아미노푸린(6-benzylaminopurine; 이하 D1Ba0.5라 함) 0.5mg/L를 포함하는 Murashige and Skoog 고형 배지(MS, Duchefa Biochie, 네덜란드)에서 배양한 성숙 배아로부터 얻었고, 캘러스 형성 동안 16시간의 명주기, 온도 22℃ 및 상대습도 60% 조건을 유지하였다. 캘러스 형성 후에, 캘러스를 동일한 액상배지로 옮기고, 130RPM 조건으로 배양하여 증식시켰다.One of the ginseng ( Panax ginseng ) varieties, 'Chunpoong', was used in the experiment. The ginseng was provided as seeds grown in a ginseng field of the Rural Development Administrations. Ginseng callus was obtained from mature embryos cultured on Murashige and Skoog solid medium (MS, Duchefa Biochie, Netherlands) containing 1 mg/L of 2,4-dichlorophenoxyacetic acid and 0.5 mg/L of 6-benzylaminopurine (hereinafter referred to as D1Ba0.5), and the conditions of a 16-h light cycle, 22°C temperature, and 60% relative humidity were maintained during callus formation. After callus formation, the callus was transferred to the same liquid medium and cultured under the conditions of 130 RPM for proliferation.

1-2. 담배1-2. Cigarettes

경희대학교 정기홍 교수님에게 분양받은 담배(Nicotiana tabacum) 종자를 파종하고 4℃에서 3일간 휴면 상태를 유지하였다. 그 후, 16시간의 명주기, 22℃ 및 상대습도 60% 조건에서 4주 동안 배양하였다.Tobacco ( Nicotiana) donated by Professor Jeong Gi-hong of Kyunghee University tabacum ) seeds were sown and kept in a dormant state at 4℃ for 3 days. Afterwards, they were cultured for 4 weeks under conditions of a 16-hour light cycle, 22℃, and 60% relative humidity.

[실험예 2] 유전적 및 생물정보학적 분석[Experimental Example 2] Genetic and bioinformatics analysis

유전자 분석; 생물학적 분석; 및 유전자 클로닝을 위해, Pg1(Pg_S1528.8) 및 Pg2(Pg_S1229.1)의 암호화 서열을 SNU ginseng DB(http:/ginsengdb.snu.ac.kr/)에서 얻었다. 다른 식물에서 보고된 18개의 다른 MYC2 전사인자의 암호화 서열은 GenBank(https:/www.ncbi.nlm.nih.gov/genbank/)에서 얻었다. For genetic analysis; biological analysis; and gene cloning, the coding sequences of Pg1 (Pg_S1528.8) and Pg2 (Pg_S1229.1) were obtained from SNU ginseng DB (http:/ginsengdb.snu.ac.kr/). The coding sequences of 18 other MYC2 transcription factors reported in other plants were obtained from GenBank (https:/www.ncbi.nlm.nih.gov/genbank/).

Pg1 및 Pg2의 예측 단백질 구조를 얻기 위해, 상기 Pg1 및 Pg2의 아미노산 서열은 SWISS-MODEL(https:/swissmodel.expasy.org/)에 제출하였고, 예측 단백질 구조에 존재하는 도메인은 PDB(Protein Data Bank, https:/www.rcsb.org/)로 확인하였다.To obtain the predicted protein structures of Pg1 and Pg2, the amino acid sequences of Pg1 and Pg2 were submitted to SWISS-MODEL (https:/swissmodel.expasy.org/), and the domains present in the predicted protein structures were checked with PDB (Protein Data Bank, https:/www.rcsb.org/).

Pg1; Pg2; 및 상기 18개의 MYC2 전사인자의 아미노산 서열은ClustalX 1.83에 정렬시켜, 다른 MYC2 전사인자와 함께 Pg1 및 Pg2 간의 유사성을 확인하였다. 계통발생 트리(Phylogenetic tree) 분석은 MEGA11에 의해 아미노산 서열에 대해 neighbor-joining의 통계적 방법으로 수행하였고, 1,000개의 부트스트랩(bootstrap)을 복제하였다. Pg1 및 Pg2를 포함한 총 20개의 아미노산 서열도 MEME Suite(https:/meme-suite.org/meme/)에 제출하였고, 서열 중 conserved motif의 로고를 얻었다. MEME suite motif 분석 결과은 Tomtom Motif Comparision Tool(https:/meme-suite.org/meme/tools/tomtom) 및 Prosite(https:/prosite.expasy.org/)에 입력하여 conserved motif의 예측 기능을 확인하였다.Pg1; Pg2; and the amino acid sequences of the 18 MYC2 transcription factors were aligned with ClustalX 1.83 to confirm the similarity between Pg1 and Pg2 with other MYC2 transcription factors. Phylogenetic tree analysis was performed by MEGA11 using the statistical method of neighbor-joining for the amino acid sequences, and 1,000 bootstraps were replicated. A total of 20 amino acid sequences including Pg1 and Pg2 were also submitted to MEME Suite (https:/meme-suite.org/meme/), and logos of conserved motifs among the sequences were obtained. The results of MEME suite motif analysis were input into Tomtom Motif Comparison Tool (https:/meme-suite.org/meme/tools/tomtom) and Prosite (https:/prosite.expasy.org/) to confirm the predicted functions of the conserved motifs.

SNU ginseng DB에서 DDS(Pg_Scaffold3318), PPTS(Pg_Scaffold1770) 및 PPDS(Pg_Scaffold4733)의 업스트림(upstream) 1,800bp의 프로모터 영역 서열을 얻었다. PgHMGR1(Pg_Scaffold6083) 및 PgSE1(Pg_Scaffold0129) 서열의 업스트림 1,300bp도 상기와 동일한 방법으로 얻었다. PgHMGR2(Pg_Scaffold1295) 서열의 업스트림 477bp도 다른 서열과 동일하게 분석하였다. 상기 서열들은 Pg2에서 PgHMGR1, PgHMGR2, PgSE1, DDS, PPDS 및 PPTS의 결합 부위를 예측하기 위해, New Place(Plant Cis-active Regulatory DNA element에 대한 데이터베이스, https:/www.dna.affrc.go.jp/PLACE/?action=newplace)에 제출하였다.The promoter region sequences of 1,800 bp upstream of DDS (Pg_Scaffold3318), PPTS (Pg_Scaffold1770), and PPDS (Pg_Scaffold4733) were obtained from SNU ginseng DB. The upstream 1,300 bp of PgHMGR1 (Pg_Scaffold6083) and PgSE1 (Pg_Scaffold0129) sequences were also obtained in the same manner as above. The upstream 477 bp of PgHMGR2 (Pg_Scaffold1295) sequence was also analyzed in the same manner as other sequences. The above sequences were submitted to New Place (Database for Plant Cis-active Regulatory DNA elements, https:/www.dna.affrc.go.jp/PLACE/?action=newplace) to predict the binding sites of PgHMGR1, PgHMGR2, PgSE1, DDS, PPDS and PPTS in Pg2.

[실험예 3] 플라스미드 제작 및 형질전환[Experimental Example 3] Plasmid production and transformation

Pg1 및 Pg2의 과발현 인삼 형질전환 라인을 제작하기 위해, 두 유전자의 전체 길이(full-length)를 꽃양배추 모자이크 바이러스(cauliflower mosaic virus) p35S 프로모터 뒤의 pCambia1301 벡터에 복제하였다. 상기 두 유전자 모두 인트론(intron) 부재에 의해, Pg1와 Pg2를 인삼의 게놈 DNA으로부터 증폭시켰고, myc-tag는 Pg2 뒤에 융합시켜 각각 재조합 플라스미드(p35S:Pg1 및 p35S:Pg2-myc)를 제작하였다. To construct ginseng transgenic lines overexpressing Pg1 and Pg2, the full-length of both genes was cloned into the pCambia1301 vector behind the cauliflower mosaic virus p35S promoter. Since both genes lack introns, Pg1 and Pg2 were amplified from ginseng genomic DNA, and the myc-tag was fused behind Pg2 to construct recombinant plasmids (p35S:Pg1 and p35S:Pg2-myc), respectively.

Pg1 및 Pg2의 전체 길이는 p35S를 탑재한 pGreen-GFP에 복제하여 Pg1-GFP 및 Pg2-GFP를 제작하고, subcellular localization 분석에 사용하였다. Pg2는 mCherry tag를 탑재한 pGreen 벡터에 복제하여 Pg2-mCherry를 제작하고, 상기 Pg2-mCherry 및 Pg1-GFP를 co-lacalization 분석에 사용하였다. Pg1 및 Pg2는 PGreen-NV 및 PGreen-CV에 복제하여 Pg1-NV 및 Pg2-CV를 제작하고, BiFC 분석에 사용하였다.Full-length Pg1 and Pg2 were cloned into pGreen-GFP containing p35S to produce Pg1-GFP and Pg2-GFP, which were used for subcellular localization analysis. Pg2 was cloned into pGreen vector containing mCherry tag to produce Pg2-mCherry, and the Pg2-mCherry and Pg1-GFP were used for co-lactalization analysis. Pg1 and Pg2 were cloned into PGreen-NV and PGreen-CV to produce Pg1-NV and Pg2-CV, which were used for BiFC analysis.

promotor-GUS 분석의 경우, Pg1 및 Pg2의 번역(translation) 시작 부위로부터 업스트림 1,281bp 및 1,626bp를 인삼 게놈 DNA으로부터 증폭시켰다. 증폭된 산물은 GUS 유전자를 탑재한 pCambia1301 벡터에 복제하여 pPg1-GUS 및 pPg2-GUS를 제작하였다. For promotor-GUS analysis, 1,281 bp and 1,626 bp upstream from the translation start site of Pg1 and Pg2 were amplified from ginseng genomic DNA. The amplified products were cloned into the pCambia1301 vector carrying the GUS gene to produce pPg1-GUS and pPg2-GUS.

Y2H 분석을 위해, Pg1 및 Pg2의 전체 길이를 pGADT7 및 pGBKT7에 각각 복제하였다. Pg2의 자가-원격 활성(self-transactivation) 특성 확인 후, G-box 서열(CACGTG)을 포함하는 658bp의 업스트림; 및 G-box 서열을 포함하지 않은 659bp 업스트림을 Y1H assay에서 사용된 베이트(bait)를 위해 pHIS2 벡터에 복제하였다. Pg2의 전체 길이는 pGADT7에 삽입하였다. 베이트의 자가-원격 활성을 확인한 후, 링커 서열을 가진 3개의 G-box 서열을 pHIS2에 복제하였다. PgHMGR2 및 PgSE1의 E-box 서열은 Y1H와의 상호작용 assay를 위해 pHIS2에 복제하였다.For Y2H analysis, the full length of Pg1 and Pg2 were cloned into pGADT7 and pGBKT7, respectively. After confirming the self-transactivation property of Pg2, 658 bp upstream including the G-box sequence (CACGTG) and 659 bp upstream not including the G-box sequence were cloned into the pHIS2 vector for the bait used in the Y1H assay. The full length of Pg2 was inserted into pGADT7. After confirming the self-transactivation of the bait, three G-box sequences with a linker sequence were cloned into pHIS2. The E-box sequences of PgHMGR2 and PgSE1 were cloned into pHIS2 for the interaction assay with Y1H.

[실험예 4] 호르몬 처리[Experimental Example 4] Hormone Treatment

인삼 캘러스 내 Pg1 및 Pg2의 JA(jasmonic acid) 반응을 확인하기 위해, 인삼 캘러스를 새로운 D1Ba0.5 액상배지 100ml에 옮겨 3주 동안 계대배양(subcultured)하고, 호르몬(JA)을 처리하였다. 인삼 캘러스를 포함하는 계대배양 플라스크에 JA 100μM을 3일 동안 처리하였다. 대조군에는 JA와 동일한 부피의 에탄올을 처리하였다. 그 후, 캘러스를 수확하자마자 액상질소로 동결하고, -80℃에서 보관하였다.To examine the jasmonic acid (JA) response of Pg1 and Pg2 in ginseng callus, ginseng callus was transferred to 100 ml of fresh D1Ba0.5 liquid medium, subcultured for 3 weeks, and treated with hormone (JA). 100 μM JA was treated to the subculture flask containing ginseng callus for 3 days. The control group was treated with the same volume of ethanol as JA. After harvesting, the callus was frozen in liquid nitrogen and stored at -80°C.

Pg1 및 Pg2의 과발현 라인을 생성한 후, 대조군으로 야생형 인삼 캘러스 형질전환 라인을 새로운 배지에서 3주간 계대배양하고, JA 100μM을 처리하였다. 대조군에는 JA와 동일한 부피의 에탄올을 처리하였다. JA 및 에탄올은 야생형, Pg1(OX #4, OX #10 및 OX #11) 및 Pg2(OX #19, OX #22 및 OX #62)에 각각 3일 동안 처리하고, 수확하자마자 액상질소로 동결하였다.After generating overexpression lines of Pg1 and Pg2, wild-type ginseng callus transformant lines as a control were subcultured in fresh medium for 3 weeks and treated with 100 μM JA. The control group was treated with the same volume of ethanol as JA. JA and ethanol were treated for 3 days to wild type, Pg1 (OX #4, OX #10, and OX #11), and Pg2 (OX #19, OX #22, and OX #62), respectively, and then frozen in liquid nitrogen immediately after harvest.

[실험예 5] Pg1 및 Pg2 과발현 캘러스 생성[Experimental Example 5] Generation of Pg1 and Pg2 overexpressing callus

인삼 캘러스를 D1Ba0.5 액상배지에서 증식시킨 후, 2종의 플라스미드(p35S:Pg1 및 p35S:Pg2-myc)를 근두암종균(Agrobacterium tumefaciens LBA4404)에 형질전환시켰다. 인삼 캘러스는 잘라서 조각 형태로 만들고, 황산마그네슘(MgSO4) 0.05M을 전처리한 후, 근두암종균으로 감염시켰다. 형질전환 후, 감염된 캘러스를 12주 동안 세포탁심(cefotaxime) 및 하이그로마이신(hygromycin)을 포함하는 D1Ba0.5 배지에서 선별하였다. 생존 캘러스를 분석하고, Pg1 및 Pg2 발현을 확인해서, 형질전환 라인을 선별하였다.After ginseng callus was grown in D1Ba0.5 liquid medium, two types of plasmids (p35S:Pg1 and p35S:Pg2-myc) were introduced into the rhizome of Agrobacterium tumefaciens LBA4404) was transformed with ginseng callus. Ginseng callus was cut into pieces, pretreated with 0.05 M magnesium sulfate (MgSO 4 ), and then infected with rhizome. After transformation, the infected callus was selected on D1Ba0.5 medium containing cefotaxime and hygromycin for 12 weeks. Surviving calli were analyzed, and Pg1 and Pg2 expression was confirmed, and transformant lines were selected.

[실험예 6] 총 RNA 추출, cDNA 합성 및 유전자 발현 분석[Experimental Example 6] Total RNA extraction, cDNA synthesis, and gene expression analysis

발현 분석에 사용한 모든 샘플(인삼 캘러스)은 수확하자마자 동결하였고, 총 RNA는 easy-spin Total RNA Extraction Kit(iNtRON)를 통해 얻었으며, RNA에서 gDNA Eraser(Takara)를 갖춘 PrimeScript RT reagent Kit를 통해 cDNA를 합성하였다.All samples (ginseng calli) used for expression analysis were frozen immediately after harvest, and total RNA was obtained using an easy-spin Total RNA Extraction Kit (iNtRON), and cDNA was synthesized from RNA using a PrimeScript RT reagent Kit with a gDNA Eraser (Takara).

RT-PCR은 Mastercycler Nexus Gradient(Ependorf) 및 T100 Thermal Cycler (Bio-Rad) PCR 기기를 사용하여 수행하였다. RT-PCR 분석에 사용한 증폭 사이클은 35 사이클로 통일하였고, 증폭된 생성물은 1%(w/v) 아가로스 겔(agarose gel)로 로딩하였다.RT-PCR was performed using a Mastercycler Nexus Gradient (Ependorf) and T100 Thermal Cycler (Bio-Rad) PCR instrument. The amplification cycle used for RT-PCR analysis was uniformly 35 cycles, and the amplified products were loaded onto a 1% (w/v) agarose gel.

qRT-PCR은 CFX Connect Real-Time System(Bio-Rad) 및 SYBR Green Mastermix(Takara)를 사용하여 수행하였다. 실험 결과는 2- ΔCt 및 2- ΔΔCt 방법에 의한 내부 대조군 β-Actin의 Ct 정규화를 통해 나타냈다.qRT-PCR was performed using the CFX Connect Real-Time System (Bio-Rad) and SYBR Green Mastermix (Takara). The experimental results were expressed by Ct normalization to the internal control β-Actin by the 2 - ΔCt and 2 - ΔΔCt methods.

[실험예 7] Subcellular localization, co-localization 및 BiFC assay[Experimental Example 7] Subcellular localization, co-localization, and BiFC assay

4주령의 니코티아나 벤타미아나(Nicotiana benthamiana) 식물을 Pg1 및 Pg2의 subcellular localization; co-localization; 및 BiFC assay에 사용하였다. pGreen-Pg1-GFP, pGreen-Pg2-GFP, pGreen-Pg2-mCherry, pGreen-Pg1-NV 및 pGreen-Pg2-CV는 근두암종균(Agrobacterium tumefaciens GV3101)에 세포 감염시키고(transfected), 유전자의 순간적인 발현(transient expression)을 위해, 담배(tabacco) 잎에 침투시켰다(infiltrated). Nicotiana benthamiana, 4 weeks old benthamiana ) plants were used for subcellular localization; co-localization; and BiFC assays of Pg1 and Pg2. pGreen-Pg1-GFP, pGreen-Pg2-GFP, pGreen-Pg2-mCherry, pGreen-Pg1-NV, and pGreen-Pg2-CV were isolated from Agrobacterium tumefaciens GV3101) and infiltrated into tobacco (tabacco) leaves for transient expression of genes.

Pg1 및 Pg2의 subcellular localization assay를 위해, pGreen-Pg1-GFP 및 pGreen-Pg2-GFP를 포함하는 근두암종균을 별도로 담배 잎에 주입하였다. co-localization assay를 위해, pGreen-Pg1-GFP 및 pGreen-Pg2-mCherry를 니코티아나 벤타미아나 잎에 함께 주입하였다. Co-localization assay에서 Pg1 및 Pg2의 발현 위치가 동일해 서로 간의 결합 가능성을 확인하기 위해 BiFC assay를 수행하였고, pGreen-Pg1-NV 및 pGreen-Pg2-CV도 담배 잎에 함께 주입하였다.For the subcellular localization assay of Pg1 and Pg2, the root nematode containing pGreen-Pg1-GFP and pGreen-Pg2-GFP was separately injected into tobacco leaves. For the co-localization assay, pGreen-Pg1-GFP and pGreen-Pg2-mCherry were co-injected into Nicotiana benthamiana leaves. Since the expression sites of Pg1 and Pg2 were identical in the co-localization assay, the BiFC assay was performed to confirm the possibility of their binding, and pGreen-Pg1-NV and pGreen-Pg2-CV were also co-injected into tobacco leaves.

침투(infiltration) 후, 22℃에서 24시간 동안 담배를 dart 상태로 두고, 침투한지 3일 후, 공초점 레이저 주사 현미경(K1-Fluo, Nanoscope systems, Korea)을 이용하여 GFP는 488nm 및 mCherry는 530nm에서 형광 신호를 관찰하였다.After infiltration, the tobacco was kept in a dart state at 22°C for 24 hours, and 3 days after infiltration, fluorescence signals were observed at 488 nm for GFP and 530 nm for mCherry using a confocal laser scanning microscope (K1-Fluo, Nanoscope systems, Korea).

[실험예 8] 진세노사이드 내용물 분석[Experimental Example 8] Analysis of Ginsenoside Contents

Pg1 및 Pg2의 과발현 라인에 대한 진세노사이드(ginsenoside) 분석을 위해, 진세노사이드 추출을 수행하였다. 각 형질전환 라인의 100ml의 D1Ba0.5 액상배지에서 4주 동안 계대배양한 형질전환 캘러스; 및 대조군인 야생형 캘러스를 얻고, 수확하자마자 액상질소로 동결하였다. 동결시킨 샘플은 모두 미세하게 분쇄한 후. 일주일 동안 동결건조하여 건조하였다. 각 샘플 0.2g을 정량하고, 정량한 샘플에 70% 메탄올 2ml를 첨가하였다. 그 후, 50℃에서 30분간 초음파 추출기로 추출하고, 15,000RPM에서 3분 동안 원심분리하여 추출물의 상층액을 얻었다. 메탄올 및 증류수를 이용하여 C18 cartridge column(INOPAK C18, C8, NH2, Sil, Florisil For Solid Phase Extraction)을 전처리하고, SPE(solid-phase extraction) 처리를 위해 각 샘플의 상등액 1ml를 로딩하였다. 진세노사이드는 메탄올로 용출(elution)을 통해 얻었고, HPLC 분석을 위해 0.45μm 멤브레인 필터로 여과하였다.For ginsenoside analysis of the overexpression lines of Pg1 and Pg2, ginsenoside extraction was performed. Transformed calli subcultured for 4 weeks in 100 ml of D1Ba0.5 liquid medium of each transformant line; and wild-type calli as a control were obtained, and frozen in liquid nitrogen immediately after harvest. All frozen samples were finely ground and then freeze-dried for one week. 0.2 g of each sample was quantified, and 2 ml of 70% methanol was added to the quantified sample. Thereafter, the extract was extracted using an ultrasonic extractor at 50°C for 30 minutes, and centrifuged at 15,000 RPM for 3 minutes to obtain the supernatant of the extract. C18 cartridge column (INOPAK C18, C8, NH2 , Sil, Florisil For Solid Phase Extraction) was pretreated with methanol and distilled water, and 1 ml of the supernatant of each sample was loaded for SPE (solid-phase extraction). Ginsenosides were obtained through elution with methanol and filtered with a 0.45 μm membrane filter for HPLC analysis.

[실험예 9] RNA 서열 분석[Experimental Example 9] RNA Sequence Analysis

Pg1 OX #4 및 Pg2 OX #19의 총 RNA를 얻고, 대조군으로 야생형 캘러스의 총 RNA를 준비하였다. 3번의 반복 수행을 통해 3개의 RNA 라인을 추출하고, 상기 라인의 서열을 마크로젠(https:/dna.macrogen.com/main.do#)을 통해 판독하였다. 야생형 대조군과 비교해서, Pg1 OX #4 및 Pg2 OX #19의 유전자 발현 변화를 확인하고, log2fold 값으로 발현 수준이 낮은 유전자를 정규화하여 DEGs를 확인하였다.Total RNA of Pg1 OX #4 and Pg2 OX #19 was obtained, and total RNA of wild-type callus was prepared as a control. Three RNA lines were extracted through three repetitions, and the sequences of the lines were read through Macrogen (https:/dna.macrogen.com/main.do#). Compared to the wild-type control, the gene expression changes of Pg1 OX #4 and Pg2 OX #19 were confirmed, and genes with low expression levels were normalized by log2fold values to identify DEGs.

[실시예 1] Pg1 및 Pg2 특성 분석[Example 1] Analysis of Pg1 and Pg2 characteristics

PgLOX6가 JA(Jasmonic acid) 및 진세노사이드 생합성을 증가시키고, MYC2 TF 후보가 PgLOX6 과발현 라인에서 유전자 발현 수준을 증가시키는 것을 확인하였다. 이미 공지된 인삼 게놈 서열 중에서 2개의 MYC2를 확인하였다. Pg_S1528.8 및 Pg_S1229.1에 해당하는 게놈 및 암호화 서열을 얻었고, 서열 블라스트(sequence blast)를 통해 이 상기 2종의 JA 반응인자가 MYC2에 해당하는 것을 확인하였다. 따라서 상기 2종의 JA 반응인자를 각각 Pg1(Pg_S1528.8) 및 Pg2(Pg_S1229.1)로 명명하였다. Pg1 및 Pg2는 모두 인트론이 나타나지 않았고, 암호화 서열은 각각 2,013bp 및 1,959bp의 뉴클레오타이드이며, 80%의 동일성을 나타냈다. Pg1 및 Pg2의 아미노산 서열을 비교하여, 상기 서열들이 79%의 유사성을 나타내는 것을 확인하였다(도 1a). We confirmed that PgLOX6 increases JA (Jasmonic acid) and ginsenoside biosynthesis, and that the MYC2 TF candidate increases gene expression levels in PgLOX6 overexpression lines. Two MYC2s were identified among the previously known ginseng genome sequences. The genomic and coding sequences corresponding to Pg_S1528.8 and Pg_S1229.1 were obtained, and the two JA response factors were confirmed to correspond to MYC2 through sequence blast. Therefore, the two JA response factors were named Pg1 (Pg_S1528.8) and Pg2 (Pg_S1229.1), respectively. Both Pg1 and Pg2 did not show introns, and the coding sequences were 2,013 bp and 1,959 bp of nucleotides, respectively, and showed 80% identity. By comparing the amino acid sequences of Pg1 and Pg2, it was confirmed that the sequences showed 79% similarity (Fig. 1a).

Panax 종 내에서 2개의 PgMYC의 분류학적 관계를 확인하기 위해, Pg1 및 Pg2를 NCBI BLAST를 통해 분석한 결과, 가장 높은 유사한 서열은 화기삼(Panax quinquefolius)의 bHLH 유전자로 나타났고, 구체적으로 상기 bHLH 유전자는 bPg1와 97%, Pg2와 78%의 유사성을 나타냈으나, Araliaceae 과(family)와는 유사하지 않았다. Pg1 및 Pg2와 다른 MYC2 TF((transcription factor)와의 유사성을 확인하기 위해, 2차 대사산물의 생합성을 조절하는 것으로 보고된 MYC2 TF의 모든 암호화 서열을 얻고, 상기 서열을 이용하여 아미노산 서열 정렬(amino acid sequence alignment) 및 계통발생 트리(phylogenetic tree) 분석을 수행하였다(도 1a). To confirm the taxonomic relationship of the two PgMYCs within Panax species, Pg1 and Pg2 were analyzed by NCBI BLAST. The most similar sequence was found to be the bHLH gene of Panax quinquefolius . Specifically, the bHLH gene showed 97% similarity to bPg1 and 78% similarity to Pg2, but was not similar to the Araliaceae family. To confirm the similarity of Pg1 and Pg2 to other MYC2 TFs (transcription factors), all coding sequences of MYC2 TFs reported to regulate the biosynthesis of secondary metabolites were obtained, and amino acid sequence alignment and phylogenetic tree analysis were performed using these sequences (Fig. 1a).

아미노산 서열 분석 결과, Pg1 및 Pg2는 차나무(Camellia sinensis) 내 인돌(indole) 생합성을 조절하는 것으로 보고된 CsMYC2a; 및 뇌공등(Tripterygium wilfordii) 내 디테르펜(diterpene) 생합성을 조절하는 것으로 알려진 TwMYC2s(TwMYC2a 및 TwMYC2b)와 밀접한 관련성을 나타냈다(도 1a). 또한, CsMYC2a는 Pg1 및 Pg2와 67%의 유사성을 나타냈고, TwMYC2a는 Pg1 및 Pg2와 각각 63% 및 67%의 유사성을 나타냈으며, TwMYC2b는 Pg1 및 Pg2와 각각 62%의 유사성을 나타냈다다(도 1a). 또한, Pg1 및 Pg2는 WsMYC2 및 SlMYC2와도 약 60%의 유사성을 나타냈다. Amino acid sequence analysis results showed that Pg1 and Pg2 are closely related to CsMYC2a, which has been reported to regulate indole biosynthesis in Camellia sinensis ; and TwMYC2s (TwMYC2a and TwMYC2b), which are known to regulate diterpene biosynthesis in Tripterygium wilfordii (Fig. 1a). In addition, CsMYC2a showed 67% similarity to Pg1 and Pg2, TwMYC2a showed 63% and 67% similarity to Pg1 and Pg2, respectively, and TwMYC2b showed 62% similarity to Pg1 and Pg2, respectively (Fig. 1a). Additionally, Pg1 and Pg2 showed approximately 60% similarity to WsMYC2 and SlMYC2.

또한, 계통발생 트리(phylogenetic tree) 분석 결과, Pg1 및 Pg2가 MYC2 TF 과(family)에 속해있어 트리트리펜(triterpenes)과 같은 2차 대사산물 생합성에 관련된 유전자 발현을 조절하는 기능이 있음을 확인하였다. 상기 결과로부터, Pg1 및 Pg2는 Araliaceae 과(family) 내에서 트리테르펜 생합성 조절 기능이 있음을 확인하였다. 또한, 아미노산 서열 분석 결과, Pg1 및 Pg2는 이전에 보고되었던 18개의 다른 MYC2 TF와 유사하게 분류학적으로 MYC2 TF 과(family)에 속해있음을 확인하였다(도 1b).In addition, phylogenetic tree analysis results showed that Pg1 and Pg2 belong to the MYC2 TF family, which indicates that they have the function of regulating the expression of genes involved in the biosynthesis of secondary metabolites such as triterpenes. From the above results, it was confirmed that Pg1 and Pg2 have the function of regulating triterpene biosynthesis within the Araliaceae family. In addition, amino acid sequence analysis results confirmed that Pg1 and Pg2 belong to the MYC2 TF family taxonomically, similar to 18 other MYC2 TFs previously reported (Fig. 1b).

Pg1 및 Pg2의 예측 단백질 구조를 예측하고, Pg1 및 Pg2 모두에서 JID, bHLH 및 BIF/ACT 유사 도메인을 표시하였다(도 1c). 그 결과, Pg1 및 Pg2의 예측 구조는 모두 NtMYC2a와 유사하였다. Pg1 및 Pg2는 bHLH를 나타내는 2중 나선형 가닥을 포함하고, α-helix 및 β 가닥(strand)의 두 가닥에 의해 매개되는 BIF/ACT 유사 도메인과 유사한 구조를 보였다. 또한, PgMYC 내 3개의 conserved motif 및 다른 식물의 MYC를 MEME 도구를 통해 분석하여(도 1d), DNA 결합 도메인이 가장 높은 conservation인 것을 확인하였다(도 1d). 결과적으로, Pg1 및 Pg2는 MYC2의 특징인 conserved 도메인 및 특이 구조를 가지는 것을 확인하였다. 따라서, 진세노사이드 생합성 경로를 가진 인삼에서 Pg1 및 Pg2를 특성 확인을 위한 추가 실험을 수행하였다.The predicted protein structures of Pg1 and Pg2 were predicted, and JID, bHLH, and BIF/ACT-like domains were displayed in both Pg1 and Pg2 (Fig. 1c). As a result, the predicted structures of Pg1 and Pg2 were both similar to NtMYC2a. Pg1 and Pg2 showed a structure similar to BIF/ACT-like domain mediated by two strands of α-helix and β strand, including double helix strand representing bHLH. In addition, three conserved motifs in PgMYC and MYC of other plants were analyzed using MEME tool (Fig. 1d), and it was confirmed that DNA binding domain was the most highly conserved (Fig. 1d). As a result, it was confirmed that Pg1 and Pg2 have conserved domains and unique structures that are characteristic of MYC2. Therefore, additional experiments were performed to characterize Pg1 and Pg2 in ginseng with ginsenoside biosynthetic pathway.

[실시예 2] 인삼 조직 내 Pg1 및 Pg2의 발현 분석[Example 2] Expression analysis of Pg1 and Pg2 in ginseng tissue

Pg1 및 Pg2가 생장 동안 전사(transcription)되는지 확인하기 위해, 인삼의 다양한 조직에서 유전자 발현 패턴을 분석하였다. Pg1 및 Pg2의 유전자 패턴은 인삼 내 다양한 조직에서 나타난다고 예측되었다(도 2a 및 도 2b). RNA-seq 데이터베이스를 통한 히트맵(heatmaps) 분석 결과, 종자, 잎, 줄기, 뿌리 및 꽃에서 Pg1(Pg_S1528.8 및 Pg_S4049.3) 및 Pg2(Pg_S1229.1 및 Pg_S7955.1)의 상동체의 유전자 발현을 확인하였고, Pg1 및 Pg2의 유전자 발현 수준은 다른 인삼 뿌리 대비 4년생 인삼 뿌리에서 낮을 것으로 예측되었다. 각 상동체의 거의 동일한 CDS 서열 때문에(98% 유사성), 각 상동체의 유전자 발현 수준은 구분이 어려웠다. 따라서, qRT-PCR을 통해 Pg1 및 Pg2의 두 상동체의 유전자 발현을 분석하였다. 그 결과, Pg1 및 Pg2의 발현은 도 2a 및 도 2b에서 예측한 유전자 발현 패턴과 유사하게 나타났다. Pg1 및 Pg2의 발현 모두 4년생 인삼 뿌리에서 가장 낮게 나타났다. Pg1의 발현은 인삼 열매에서 가장 높게 나타났고(도 2c), Pg2의 발현은 2년생 인삼 뿌리에서 가장 높게 나타났다(도 2d). 인삼 조직에서는 전반적으로 Pg2 대비 Pg1의 발현이 더 높게 나타났다. 상기 결과로부터, 인삼에서 Pg1 및 Pg2의 유전자 발현이 나타나는 것을 확인하였다.To determine whether Pg1 and Pg2 are transcribed during growth, gene expression patterns in various tissues of ginseng were analyzed. Gene patterns of Pg1 and Pg2 were predicted to appear in various tissues of ginseng (Figs. 2a and 2b). Heatmap analysis using the RNA-seq database confirmed gene expression of homologs of Pg1 (Pg_S1528.8 and Pg_S4049.3) and Pg2 (Pg_S1229.1 and Pg_S7955.1) in seeds, leaves, stems, roots, and flowers, and the gene expression levels of Pg1 and Pg2 were predicted to be lower in 4-year-old ginseng roots than in other ginseng roots. Because of the nearly identical CDS sequences of each homolog (98% similarity), it was difficult to distinguish the gene expression levels of each homolog. Therefore, gene expression of the two homologs of Pg1 and Pg2 was analyzed using qRT-PCR. As a result, the expression of Pg1 and Pg2 was similar to the gene expression pattern predicted in Fig. 2a and Fig. 2b. The expression of both Pg1 and Pg2 was lowest in 4-year-old ginseng roots. The expression of Pg1 was highest in ginseng fruit (Fig. 2c), and the expression of Pg2 was highest in 2-year-old ginseng roots (Fig. 2d). In ginseng tissues, the expression of Pg1 was generally higher than that of Pg2. From the above results, it was confirmed that the gene expression of Pg1 and Pg2 occurred in ginseng.

[실시예 3] Subcellular localization, co-localization 및 BiFC assay[Example 3] Subcellular localization, co-localization, and BiFC assay

핵 내 DNA와 결합하는 역할 때문에 식물 세포의 핵에 위치하는 TF(transcription factor)로서 니코티아나 벤타미아나 잎 내 순간적인 발현을 통해 Pg1 및 Pg2의 subcellular localization을 분석하였다. C-말단에 GFP(green fluorescence protein)가 융합된 단백질인 Pg1-GFP 및 Pg2-GFP을 각각 근두암종균에서 매개되는 담배 잎에 침투시켰다. GFP의 형광 신호는 대조군 대비 담배 세포의 핵에서만 나타났고(도 3a), 이를 통해 Pg1 및 Pg2는 TF로써 핵 내 기능을 수행하는 것을 확인하였다. Pg1 및 Pg2의 subcellular localization을 비교하기 위해, co-localization assay를 수행하여 Pg1-GFP 및 Pg2-mCherry의 발현을 분석하였다. 그 결과, GFP 및 mCherry의 융합 방출을 통해 핵 내에서 노란색 형광 신호가 나타났고, Pg1 및 Pg2의 localization과 동일하였다(도 3b). The subcellular localization of Pg1 and Pg2, transcription factors (TFs) that are localized in the nucleus of plant cells due to their role in binding to nuclear DNA, was analyzed through transient expression in Nicotiana benthamiana leaves. Pg1-GFP and Pg2-GFP, proteins whose C-terminus is fused with green fluorescence protein (GFP), were respectively introduced into tobacco leaves mediated by the root cyst fungus. The fluorescence signal of GFP appeared only in the nucleus of tobacco cells compared to the control group (Fig. 3a), confirming that Pg1 and Pg2 perform their functions as TFs in the nucleus. To compare the subcellular localization of Pg1 and Pg2, the expression of Pg1-GFP and Pg2-mCherry was analyzed by performing a co-localization assay. As a result, a yellow fluorescence signal appeared in the nucleus through the fusion release of GFP and mCherry, which was identical to the localization of Pg1 and Pg2 (Fig. 3b).

동일한 세포 위치에서 Pg1 및 Pg2의 co-localization가 관찰되었기 때문에, Pg1 및 Pg2 사이의 물리적 상호작용을 확인하기 위해 BiFC(bimolecular-fluorescence complementation) assay를 수행하였다. Pg1-N-fragment of venus(NV) 및 Pg2-C-fragment of venus(CV)의 조합은 담배 세포의 핵 내에서 양성(positive) 신호를 나타냈고(도 3c), 이를 통해 Pg1 및 Pg2는 핵 내에서 서로 상호작용하는 것을 확인하였다.Since co-localization of Pg1 and Pg2 was observed at the same cellular location, a bimolecular-fluorescence complementation (BiFC) assay was performed to confirm the physical interaction between Pg1 and Pg2. The combination of Pg1-N-fragment of venus (NV) and Pg2-C-fragment of venus (CV) showed positive signals in the nucleus of tobacco cells (Fig. 3c), confirming that Pg1 and Pg2 interact with each other in the nucleus.

[실시예 4] Pg1 및 Pg2; 및 진세노사이드 생합성의 관련성 분석[Example 4] Analysis of the relationship between Pg1 and Pg2 and ginsenoside biosynthesis

진세노사이드 생합성에 있어서 Pg1 및 Pg2의 관계를 추가로 확인하기 위해, 아그로박테리움(agobacterium) 매개 형질전환을 통해 Pg1 및 Pg2 각각의 과발현 라인을 생성하였다. 상기 Pg1 및 Pg2의 형질전환 라인은 약 20%의 효율로 얻었다. 전이유전자(transgene)의 발현 효과 떄문에, 형질전환 라인의 발현 수준은 각 라인에 따라 다양하게 나타났다. Pg1의 과발현 라인은 공통적으로 발현 수준이 야생형 대비 약 2배 증가하였다. 그러나 야생형 대비 발현이 유의하게 증가하지 않은 라인도 있었다. Pg2의 과발현 라인은 발현 수준이 야생형 대비 약 3배 증가하였고, 야생형에 비해 발현이 더 낮은 라인도 있었다. 성장 조건과 발현 수준을 모두 고려하여, Pg1(OX #4, OX #10 및 OX #11) 및 Pg2(OX #19, OX #22 및 OX #62)를 추가 분석 대상으로 선정하였다(도 6). Pg1 및 Pg2의 과발현이 진세노사이드 축적에 영향을 미치는지 확인하기 위해, HPLC(high performance liquid chromatography)를 수행하여 진세노사이드를 추출하여 분석하였다. 진세노사이드 분석을 위해, Pg1 및 Pg2의 과발현 라인 중에서 발현 수준이 가장 높은 3개 라인을 각각 선정하였다[Pg1(OX #4, OX #10 및 OX #11) 및 Pg2(OX #19, OX #22 및 OX #62)(도 6)]. 모든 형질전환 라인은 증식하였고(도 7), 진세노사이드 함량 분석을 위해, 야생성 캘러스를 형질전환 라인과 동일한 절차로 대조군으로 준비하였다. 그 결과, 대조군 대비 과발현 라인에서 진세노사이드 축적이 유의하게 증가하였다(도 5a). 구체적으로, 과발현 라인은 500mg/L 이상의 진세노사이드를 함유하였고, 대조군은 약 200mg/L의 진세노사이드를 함유하였다(도 7). 진세노사이드 Re, Rg1, Rf, Rg1, Rg2, Rh1, Rc, Rb2, Rb3 및 Rd를 포함하는 각 진세노사이드의 함량은 과발현 라인에서 증가하였으나, 일부 라인에서는 Rb2, Rb3, Rd 및 Rh1의 증가가 나타나지 않았다(도 5a).To further confirm the relationship between Pg1 and Pg2 in ginsenoside biosynthesis, overexpression lines of Pg1 and Pg2, respectively, were generated through Agrobacterium-mediated transformation. The Pg1 and Pg2 transformant lines were obtained with an efficiency of approximately 20%. Due to the expression effect of the transgene, the expression levels of the transformant lines varied depending on each line. The Pg1 overexpression lines commonly showed an expression level that was approximately 2-fold higher than that of the wild type. However, there were also lines in which the expression did not significantly increase compared to the wild type. The Pg2 overexpression lines showed an expression level that was approximately 3-fold higher than that of the wild type, and there were also lines in which the expression was lower than that of the wild type. Considering both the growth conditions and the expression levels, Pg1 (OX #4, OX #10, and OX #11) and Pg2 (OX #19, OX #22, and OX #62) were selected as additional analysis subjects (Fig. 6). To determine whether overexpression of Pg1 and Pg2 affects ginsenoside accumulation, ginsenosides were extracted and analyzed by high performance liquid chromatography (HPLC). For ginsenoside analysis, three lines with the highest expression levels among the overexpression lines of Pg1 and Pg2 were selected [Pg1 (OX #4, OX #10, and OX #11) and Pg2 (OX #19, OX #22, and OX #62) (Fig. 6)]. All transgenic lines were proliferated (Fig. 7), and for ginsenoside content analysis, wild callus was prepared as a control using the same procedure as the transgenic lines. As a result, ginsenoside accumulation was significantly increased in the overexpression lines compared to the control group (Fig. 5a). Specifically, the overexpression lines contained more than 500 mg/L of ginsenoside, and the control group contained about 200 mg/L of ginsenoside (Fig. 7). The content of each ginsenoside, including ginsenosides Re, Rg1, Rf, Rg1, Rg2, Rh1, Rc, Rb2, Rb3, and Rd, increased in the overexpression lines, but in some lines, no increase in Rb2, Rb3, Rd, and Rh1 was observed (Fig. 5a).

진세노사이드 생합성 유전자(Pg1 및 Pg2)가 향상된 진세노사이드에 기여하는지 확인하기 위해, QRT-PCR을 수행하여 HMGR1, HMGR2, SE1 및 SE2의 발현을 분석하였다(도 5b~5e). 그 결과, HMR1의 발현은 Pg1(OX #4, OX #11) 및 Pg2(OX #19 및 OX #22)에 의해 유의하게 증가하였다(도 5b). HMGR2 발현도 대조군(야생형 캘러스) 대비 6개의 형질전환 라인(Pg1 OX #4, #10, #11, Pg2 OX #19, #22 및 #62)(도 5c)에 의해 증가하였다. 또한, Pg1 및 Pg2의 6개 과발현 라인에서도 SE1 및 SE2의 발현 수준이 모두 증가하였다(도 5d~5e). 또한, Pg1 OX #4는 PPTS 및 PPDS의 발현이 증가하였다(도 5f). 상기 결과로부터, Pg1 및 Pg2의 과발현이 인삼 캘러스에서 진세노사이드 생합성을 유도하는 것을 확인하였다.To determine whether ginsenoside biosynthetic genes (Pg1 and Pg2) contribute to the enhanced ginsenosides, QRT-PCR was performed to analyze the expression of HMGR1, HMGR2, SE1, and SE2 (Fig. 5b-5e). As a result, the expression of HMR1 was significantly increased by Pg1 (OX #4, OX #11) and Pg2 (OX #19 and OX #22) (Fig. 5b). HMGR2 expression was also increased by six transgenic lines (Pg1 OX #4, #10, #11; Pg2 OX #19, #22, and #62) (Fig. 5c) compared to the control (wild-type callus). In addition, the expression levels of SE1 and SE2 were both increased in six overexpression lines of Pg1 and Pg2 (Fig. 5d-5e). In addition, Pg1 OX #4 showed increased expression of PPTS and PPDS (Fig. 5f). From the results above, it was confirmed that overexpression of Pg1 and Pg2 induced ginsenoside biosynthesis in ginseng callus.

[실시예 5] Pg1 및 Pg2의 과발현을 통한 진세노사이드 축적 증가 기전 분석[Example 5] Analysis of the mechanism of increased ginsenoside accumulation through overexpression of Pg1 and Pg2

상기 실시예 4에서 확인한 Pg1 및 Pg2의 과발현을 통한 진세노사이드 축적 증가에 대한 기전을 확인하기 위해, RNA-seq 분석을 수행하였다. 그 결과, HMGR2 (Pg_S1295.30), PPTS(Pg_S1770.12), SE1(Pg_S0129.28) 및 Pg_S0245.36을 포함하는 진세노사이드 생합성 유전자의 존재가 DEGs(differentially expressed genes) 내에서 확인되었다. 과발현 라인에서, HMGR2가 Pg1 OX #4 및 Pg2 OX #19에서 모두 확인되었다. DEG 내 Pg_S0245.36은 compound K 및 진세노사이드 Rd의 생합성에 관여하는 UGT71 계열로 분류될 수 있는 candidate UGT로 확인되었다. RNA-seq 분석에서 HMGR2의 발현은 Pg1 OX #4에서 감소한 반면, 반대로 Pg2 OX #19에서는 증가하였다. 상기 결과는 qRT-PCR 분석에서도 확인된 내용이고(도 5c), 상기 결과로부터, Pg2이 HMGR2 발현에 유의한 영향을 주는 것을 확인하였다.To confirm the mechanism for increased ginsenoside accumulation through overexpression of Pg1 and Pg2 confirmed in the above Example 4, RNA-seq analysis was performed. As a result, the presence of ginsenoside biosynthetic genes including HMGR2 (Pg_S1295.30), PPTS (Pg_S1770.12), SE1 (Pg_S0129.28), and Pg_S0245.36 was confirmed in DEGs (differentially expressed genes). In the overexpression lines, HMGR2 was confirmed in both Pg1 OX #4 and Pg2 OX #19. Pg_S0245.36 in DEGs was identified as a candidate UGT that can be classified into the UGT71 family involved in the biosynthesis of compound K and ginsenoside Rd. In RNA-seq analysis, the expression of HMGR2 was decreased in Pg1 OX #4, whereas it was increased in Pg2 OX #19. The results were also confirmed by qRT-PCR analysis (Fig. 5c), and from the results, it was confirmed that Pg2 significantly affects HMGR2 expression.

SE1의 발현 증가도 Pg1 및 Pg2의 과발현 라인에서 확인되었다. 구체적으로, Pg1 OX #4 및 Pg2 OX #19에서 SE1 발현이 유의하게 증가하였고, 상기 SE1 발현 패턴은 qRT-PCR 분석 결과와 동일하게 나타났다(도 5d). 상기 결과로부터, 또한, Pg1 OX #4 및 Pg2 OX #19에서 PPTS 및 Pg_S0245.36 발현 패턴도 확인하였다. PPTS 발현은 Pg1 OX #4에서 증가한 반면, Pg2 OX #19에서는 감소하였다. 반면, Pg_S0245.36 발현은 Pg1 OX #4에서 감소한 반면, Pg2 OX #19에서는 증가하였다. 상기 결과로부터, Pg1 및 Pg2는 HMGR2, PPTS, SE1 및 Pg_S0245.36을 포함하는 진세노사이드 생합성 유전자 발현을 조절하는 것을 확인하였다.Increased expression of SE1 was also confirmed in the overexpression lines of Pg1 and Pg2. Specifically, SE1 expression was significantly increased in Pg1 OX #4 and Pg2 OX #19, and the SE1 expression pattern was the same as the qRT-PCR analysis result (Fig. 5d). From the results, the expression patterns of PPTS and Pg_S0245.36 were also confirmed in Pg1 OX #4 and Pg2 OX #19. PPTS expression increased in Pg1 OX #4, whereas it decreased in Pg2 OX #19. On the other hand, Pg_S0245.36 expression decreased in Pg1 OX #4, whereas it increased in Pg2 OX #19. From the results, it was confirmed that Pg1 and Pg2 regulate the expression of ginsenoside biosynthetic genes including HMGR2, PPTS, SE1, and Pg_S0245.36.

[실시예 6] Pg1 및 Pg2의 JA 반응성 분석[Example 6] Analysis of JA reactivity of Pg1 and Pg2

MYC2는 JA 반응 TF로 알려져 있으므로, JA 처리 후, Pg1 및 Pg2의 발현을 분석한 결과, 인삼 캘러스에 JA 200μM 처리 시, Pg1 및 Pg2 발현이 증가하였다(도 4a). 야생형에서, Pg1 발현은 대조군 대비 2배 증가하였고(도 4), Pg2 발현은 대조군 대비 약 3배 증가하였다. Pg1 및 Pg2 각각의 과발현 라인에서도 JA에 의해 Pg1 및 Pg2 발현이 증가하였다. 구체적으로, Pg1 및 Pg2의 발현이 대조군 대비 과발현 라인에서 약 3~4배 증가하였다(도 4). 상기 결과로부터, JA가 Pg1 및 Pg2 발현을 촉진하는 것을 확인하였다.Since MYC2 is known as a JA response TF, the expression of Pg1 and Pg2 was analyzed after JA treatment. When 200 μM JA was treated on ginseng callus, Pg1 and Pg2 expression increased (Fig. 4a). In the wild type, Pg1 expression increased 2-fold compared to the control group (Fig. 4), and Pg2 expression increased about 3-fold compared to the control group. Pg1 and Pg2 expression was also increased by JA in each of the overexpression lines of Pg1 and Pg2. Specifically, the expression of Pg1 and Pg2 increased about 3-4 times in the overexpression lines compared to the control group (Fig. 4). From the above results, it was confirmed that JA promotes the expression of Pg1 and Pg2.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific description is merely a preferred embodiment and that the scope of the present invention is not limited thereby. In other words, the actual scope of the present invention is defined by the appended claims and their equivalents.

Claims (8)

서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드; 또는 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드를 포함하는 재조합 벡터로 식물체를 형질전환하는 단계를 포함하는, 사포닌 함량이 증진된 형질전환 식물체 제조방법.A method for producing a transgenic plant with enhanced saponin content, comprising the step of transforming a plant with a recombinant vector comprising a nucleotide having a base sequence represented by sequence number 1; or a nucleotide having a base sequence represented by sequence number 2. 제1항에 있어서, 상기 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드는 서열번호 3으로 표시되는 아미노산 서열을 가지는 펩타이드를 암호화하는 것을 특징으로 하는 제조방법.A manufacturing method according to claim 1, characterized in that the nucleotide having the base sequence represented by sequence number 1 encodes a peptide having the amino acid sequence represented by sequence number 3. 제1항에 있어서, 상기 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드는 서열번호 4로 표시되는 아미노산 서열을 가지는 펩타이드를 암호화하는 것을 특징으로 하는 제조방법.A manufacturing method according to claim 1, characterized in that the nucleotide having the base sequence represented by sequence number 2 encodes a peptide having the amino acid sequence represented by sequence number 4. 제1항에 있어서, 상기 사포닌은 진세노사이드(ginsenoside)인 것을 특징으로 하는 제조방법.A manufacturing method according to claim 1, characterized in that the saponin is ginsenoside. 제1항에 있어서, 상기 뉴클레오타이드는 인삼(Panax ginseng)에서 유래한 것을 특징으로 하는 제조방법.A manufacturing method according to claim 1, characterized in that the nucleotide is derived from ginseng ( Panax ginseng ). 제1항에 있어서, 상기 식물체는 애기장대, 담배, 벼, 밀, 보리, 옥수수, 수수, 귀리 및 콩으로 이루어진 군에서 선택된 하나 이상인 것을 특징으로 하는 제조방법.A manufacturing method according to claim 1, characterized in that the plant is at least one selected from the group consisting of Arabidopsis thaliana, tobacco, rice, wheat, barley, corn, sorghum, oats, and soybeans. 제1항의 형질전환 식물체로부터 수득한 종자.Seeds obtained from the transformed plant of claim 1. 서열번호 1로 표시되는 염기서열을 가지는 뉴클레오타이드; 또는 서열번호 2로 표시되는 염기서열을 가지는 뉴클레오타이드를 유효성분으로 포함하는 사포닌(saponin) 함량 증진용 조성물.A composition for increasing saponin content, comprising as an active ingredient a nucleotide having a base sequence represented by sequence number 1; or a nucleotide having a base sequence represented by sequence number 2.
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