Method for producing transgenic plant with increased saponin content

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

Method for producing a transgenic plant with increased saponin content

[0001] The present invention relates to a method for producing a transgenic plant with increased saponin content.

[0002] Ginseng (Panax ginseng Meyer) is a perennial herb belonging to the genus Panax of the family Araliaceae. The genus name Panax is derived from the Greek word panacea, meaning "all-healing," and "ginseng" refers to the root that resembles a human shape. Known as the king of all medicinal herbs, ginseng root 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 its physiological activities began, and various activities such as antioxidant, anticancer, anti-inflammatory, anti-cardiovascular, and immune system enhancement have been reported.

[0003] The efficacy of ginseng primarily comes from saponins called ginsenosides, which are triterpenoid secondary metabolites produced only in the genus Panax. Ginseng biosynthesizes ginsenosides through the mevalonate (MVA) pathway, which is mediated by 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMGR), which converts acetyl-CoA to mevalonate (MVA). Isopentenyl diphosphate (IPP) and farnesyl diphosphate (FPP) are then produced from MVA, and the two FPPs are joined by squalene synthase (SS) to form squalene. Squalene is epoxidized by squalene epoxidase (SE) to form 2,3-oxidosquelene, a precursor of triterpenes and sterols. 2,3-oxidosqualene is cyclized by dammarenediol synthase (DDS) to form dammarenediol, a precursor of various types of ginsenosides. Next, cytochrome P450 enzymes, protopanaxadiol sythase (hereinafter referred to as PPDS) and protopanaxatriol sythase (hereinafter referred to as PPTS), convert damarendiol into protopanaxadiol (PPD) and convert PPD into protopanaxatriol (PPT), producing PPD- and PPT-type precursors of ginsenosides.

[0004] UGT (UDP-glycosyl transferase) has been identified as a substance that produces various ginsenosides. 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, much remains to be clearly understood 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 fully identified. Therefore, active research is being conducted to identify substances that regulate ginsenoside biosynthesis and to develop ginseng with increased ginsenoside content through such substances.

[0005] Meanwhile, jasmonic acid (JA) is one of the major plant hormones that regulates plant growth and responses to external stimuli. 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 biosynthetic genes, such as SE1 and DDS, in ginseng, thereby increasing JA levels and consequently enhancing ginsenoside accumulation in ginseng.

[0006] The purpose of the present invention is to provide a method for producing a transgenic plant with increased saponin content.

[0007] Another object of the present invention is to provide seeds obtained from the above-mentioned transformed plant.

[0008] Another object of the present invention is to provide a composition for increasing saponin content.

[0009] To achieve the above purpose, the present invention provides a method for producing a transgenic plant with increased 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.

[0010] In addition, the present invention provides seeds obtained from the above-described transformed plant.

[0011] In addition, the present invention provides a composition for increasing saponin content, which comprises 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.

[0012] According to the present invention, it has been confirmed that a nucleotide having a base sequence represented by sequence number 1 or 2 derived from ginseng (Panaxginseng) increases ginsenoside accumulation through regulation of ginsenoside biosynthesis gene expression, and thus can be usefully utilized as a method for producing a transgenic plant with increased saponin content.

[0013] 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. PanaxquinquefoliusbHLH22 (PqbHLH22, UUA80731.1) AaMYC2 (Artemisiaannua, KP119607), AtMYC2 (Arabidopsisthaliana, AT1G32640), CrMYC2 (Catharanthusroseus, AF283507), CsMYC2a (Camelliasinensis, MK336383), CsMYC2b (Camelliasinensis, MK336384), CsMYC2c (Camelliasinensis, MK336385), FhMYC2 (Freesiahybrida, XP_010919958), HbMYC2b (Heveabrasiliensis, GQ859161), OsMYC2 (Oryzasativa, LOC_Os10g42430), PtrMYC2 (Poncirustrifoliata, MT919252), SlMYC2 (Solanumlycopersicum, Solyc08g076930), SmMYC2 (Salviamiltiorrhiza, KJ945636), TaMYC2 (Triticumaestivum, MF679157), TcMYC2 (Taxuschinensis, MG494378), TwMYC2a (Tripterygium wilfordii, MN836714), TwMYC2b (Tripterygium wilfordii, MN836715), WsMYC2 (Withaniasomnifera, MG434696). Figure 2b shows the results of functional domain location analysis of MYC2 TF in the Pg1 and Pg2 genes.Figure 2c shows the predicted protein structures of the Pg1 and Pg2 genes. Figure 2d shows the results of analyzing the sequence of the MYC2 TF, which is identical to the sequence used in the phylogenetic tree analysis, using MEME (Multiple Em for Motif Elicitation).

[0014] Figures 2a and 2b show the results of analyzing the predicted expression patterns of the Pg1 and Pg2 genes using an RNA-sequencing-based database. Figures 2c and 2d show the results of analyzing the expression of the Pg1 and Pg2 genes in various ginseng tissues.

[0015] Figure 3 shows the results of analyzing the intracellular location, binding potential, and gene transcription activation ability of Pg1 and Pg2.

[0016] Figure 4 shows the results of analyzing the effect of JA (Jasmonic acid) on Pg1 and Pg2 expression.

[0017] Figure 5 shows the results of analyzing the effects of Pg1 and Pg2 on ginsenoside accumulation.

[0018] 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.

[0019] Figure 7 shows the results of analyzing the total ginsenoside content of Pg1 and Pg2 ginseng overexpressors and the appearance of the transformants.

[0020] Hereinafter, the present invention will be described in more detail.

[0021]

[0022] 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.

[0023] 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”.

[0024] 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.

[0025] 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.

[0026] [Sequence number 1] (2013bp)

[0027] ATATAACTAGGGAATTGAATTTCTCTGGATTCGGGTTTGATGGAATAAGTAGTACTGTTAGGAAAGGGAATTCGAATTCACATGTTTGTAAGCCTGAATCCGGAGAAAAACTGAATTTTGGGGAAAGTAAGAGGAGTTGTAGTGGAAATGGGACTTTGTTTGCAGGACATTCACAATTTGTGGGAATTGTTGAGGATAGCAAGAAGAAGAGATCGTCGAGTTCGAGAGGTAGTTATCATGAAGAGGGTGGTATACTTTCCTTCAGTTCTGGTATGATTTTGCCGTCTTCTGGTATCGTAAAATCCAGCGGTGGTGGTGGGGATTCTGACCATTCGGATCTTGAACCCTCTGTAGTTAAAGAGGCTATTGTTAGTCAAGTTGTGGATCCAGAAAGAAAACCACGAAAGCGGGGAAGAAAGCCGGCTAATGGGAGAGAAGAGAGCCATTGAATCATGTTGAAGCAGAACGACAGAGGAGGGAGAAGCTTAACCAGAAGTT CTATGCCCTCCGAGTAGTGGTACCTAATGTATCAAAAATGGACAAGGCTTCACTTCTCGGAGATGCTATTGTTTTCATCAATGAGTTGAAAGCCAAACTCCAGACATCGGATTCAGAAAAGGATGAGTTGCGAAGCCAATTGGAGTCTTTGAAAAAGGAGTTGGCTAGTAAAGAATCGCAATATTCAAGTCAGATGGCAGCTGATAAAGATCTCAAAATATCAAATGACCATGGAAATAAGTTTATTAATTTGGACATAGATGTGAAGATTATTGGTTGGGACGCGATGATCCGAATCCAATGTAGTAAGAAGAACCACCCTGCAGCTAGATTGATGGCGGCTTTGGAAGAGATGGACCTAGAAGTCAGCCACGCGAGTATATCCGTCGTCAATGATTTGATGATTCAACAGGCTACGGTGAAGATGGGTAGTCGATTTTACACCCAAGAACAGCTTAGAGTAGCATTGGCAGCCAAAGTCTCAGAAACAAGATAA

[0028] [Sequence number 2] (1,994 bp)

[0029] ATTTGGGTTTGATGGAATTAGTAGTACTAATGTTAGGAATGGAAATTTGAATTCACACGCATGTAAGCCTGAATCCGAGGAAATACTGAATTTTGGGGAAAGTAAGAGGAGTTCTTGTAGTGGAAATGGGAATTTGTTTTCGGGAAATTCACCATTTGGTGGAATTGTTGAAGACAATAAAAAGAAGAAATCTCCGAATTCCAGGGGTAGTCATGAGGAGGGTATGCTATCGTTTTCTTCCGGTGTGATTTTACCGTCTTCCGGTGTGGTAAAATCGAGTGGTGGTGGTGGGGATTCGGATCACTCGGATCTTGAAGCCTCGGTTGTTAAAGAGGTTGAGAGTAGTCGAGTTGTAGACCCGGGAAAAGAAACCACGAAAAAGGGGGAGAAAGCCGGCAAATGGGAGGGAAGAGCCATTGAATCATGTTGAGGCGGAGCGACAGAGGAGGGAGAAGCTCAACCAGAGGTTCTACGCTCTTCGCGCAGT GGTACCTAATGTATCGAAAATGGACAAGGCCTCACTTCTTGGGGATGCTATTTCTTACATTAATGAGCTTAAATCCAAGCTCCAGGAATCGGATTCAGAGAAAGATGAGTTGAGAAGCCAATTGGATTCATTGAGAAAGGAATTAGCTAAAAAAGTATCACAATATCCAACTCAAGCAACTCAAGCAGCAGTCGAGCAAGATCTCAAAATGTCAAACCACCATGGGAGCAAGTTGCTTGATTT GGATATAGATGTGAAAATCATTGGTTGGGACGCCATGATCCGATTCCAATCCACTAAGAAAAACCACCCTGCAGCAAGATTGATGGCGGCTTTGAAAGAGCTGGACCTAGATGTTCATCACGCAAGCGTATCTGTTGTCAATGATTTGATGATTCAACAAGCTACGGTGAAGATGGTCAGTCGGTTTTACACCCAAGATCAGCTTAGAGTAGCGTTGACAGCCCGAGTTTCCGAAACAAGATAA

[0030] [서열번호 3] (670bp)

[0031] MTDYRVPTAAMNLWSTTTTDDNTSMMDAFMSADLTSFWPPPTPPPPPPPQSSSTSTSTAAAAVFNQESLQHRLQSLIEGAKESWTYAIFWQWQSASGDIDYLSSQSSLLGWGDGYYKGEDKEKQLKRKPTSAAEQAHRRKVLRELNSLISGSQSFPDDAVDEEVTDTEWFFLVSMTQSFVNGAGLPGQAFFNSSPVWVTRAERLLSSPCERARQAQTFGLQTMVCIPSNNGVVELGSTELIYQSSDLMNKVRILFNFNSIDSGYWPVPSEPNESDPSALWLTDPSPLPNVEIKEIPMNSAKPPQIGFENHSFSTLTENPSTSSVINVQNQHSKQSQQQNGNITRELNFSGFGFDGISSTVRKGNSNSHVCKPESGEKLNFGESKRSCSGNGTLFAGHSQFVGIVEDSKKKRSSSSRGSYHEEGGILSFSSGMILPSSGIVKSSGGGGDSDHSDLEPSVVKEAIVSQVVDPERKPRKRGRKPANGREEPLNHVEAERQRREKLNQKFYALRVVVPNVSKMDKASLLGDAIVFINELKAKLQTSDSEKDELRSQLESLKKELASKESQYSSQMAADKDLKISNDHGNKFINLDIDVKIIGWDAMIRIQCSKKNHPAARLMAALEEMDLEVSHASISVVNDLMIQQATVKMGSRFYTQEQLRVALAAKVSETR

[0032] [서열번호 4] (652bp)

[0033] MNLWSTTITTDDNASMLDAFMASDLTSFWPPPPVPQQSSSTSTSAAAVFSQESLQHRLQSLIEGSKESWTYAIFWQSSVADYSSSQTLLGWGDGYYKGEDKEKQLLKRKSTSATEQDHRKKVLRELNSLISGNQASHDDAVDEEVTDTEWFFLVSMTQSFVNG TGLPGQAFFNSSPVWVTGIERLASSHCERARQAQTFGLQTIVCIPSNNGVVELGSTELIFQSSGFMNKVRILFNFNAIESGSWPLPSDPNEPDPSALWLTDPLPVPSVEIKEIPLNSNSKPPQIMFENHSSSTLTENPSTSSVINVHNQHLNQQNGVLHRELN FSGFGFDGISSTNVRNGNLNSHACKPESGEILNFGESKRSSCSGGNNLFSGNSPFGGIVEDNKKKKSPNSRGSHEEGMLSFSSGVILPSSGVVKSSGGGGDSDHSDLEASVVKEVESSRVVDPEKKPRKRGRKPANGREEPLNHVEAERQRREKLNQRFYALR AVVPNVSKMDKASLLGDAISYINELKSKLQESDSEKDELRSQLDSLRKELAKKVSQYPTQATQAAVEQDLKMSNHHGSKLLDLDIDVKIIGWDAMIRFQSTKKNHPAARLMAALKELDLDVHHASVSVVNDLMIQQATVKMVSRFYTQDQLRVALTARVSETR

[0034] The above saponin may be ginsenoside.

[0035] The above nucleotides may be derived from ginseng (Panaxginseng).

[0036] The above plant may be one or more selected from the group consisting of, but is not limited to, Arabidopsis thaliana, tobacco, rice, wheat, barley, corn, sorghum, oats, and soybeans.

[0037]

[0038] In addition, the present invention provides seeds obtained from a transgenic plant.

[0039]

[0040] In addition, the present invention provides a composition for increasing saponin content, which comprises 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.

[0041] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0042]

[0043] [Example 1] Experimental Preparation

[0044] 1-1. Ginseng

[0045] One of the ginseng (Panaxginseng) varieties, 'Chunpoong', was used in the experiment, and the ginseng seeds were provided by the Rural Development Administrations from the ginseng field. 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 at 130 RPM for proliferation.

[0046]

[0047] 1-2. Cigarettes

[0048] Tobacco (Nicotianatabacum) seeds, provided by Professor Jeong Gi-hong of Kyung Hee University, were sown and left dormant at 4°C for 3 days. They were then cultured for 4 weeks under conditions of a 16-h light cycle, 22°C, and 60% relative humidity.

[0049]

[0050] [Experimental Example 2] Genetic and Bioinformatic Analysis

[0051] For genetic analysis, biological analysis, and gene cloning, the coding sequences of Pg1 (Pg_S1528.8) and Pg2 (Pg_S1229.1) were obtained from the 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 / ).

[0052] 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 identified with PDB (Protein Data Bank, https: / www.rcsb.org / ).

[0053] The amino acid sequences of Pg1; Pg2; and 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 using MEGA11 with the statistical method of neighbor-joining for amino acid sequences, and 1,000 bootstrap replicates were performed. 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.

[0054] The 1,800-bp promoter region sequences upstream of DDS (Pg_Scaffold3318), PPTS (Pg_Scaffold1770), and PPDS (Pg_Scaffold4733) were obtained from the SNU ginseng DB. The 1,300-bp upstream of PgHMGR1 (Pg_Scaffold6083) and PgSE1 (Pg_Scaffold0129) sequences were also obtained using the same method as above. The 477-bp upstream of PgHMGR2 (Pg_Scaffold1295) sequence was also analyzed in the same manner as the 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.

[0055]

[0056] [Experimental Example 3] Plasmid Construction and Transformation

[0057] To generate 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 a myc-tag was fused to the back of Pg2 to generate recombinant plasmids (p35S:Pg1 and p35S:Pg2-myc), respectively.

[0058] 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 the pGreen vector containing the 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.

[0059] For the promotor-GUS analysis, 1,281 bp and 1,626 bp upstream from the translation start sites 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.

[0060] For Y2H analysis, the full-length Pg1 and Pg2 were cloned into pGADT7 and pGBKT7, respectively. After confirming the self-transactivation property of Pg2, the 658-bp upstream region containing the G-box sequence (CACGTG) and the 659-bp upstream region without the G-box sequence were cloned into the pHIS2 vector for use as bait in the Y1H assay. The full-length 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 interaction assays with Y1H.

[0061]

[0062] [Experimental Example 4] Hormone Treatment

[0063] To examine the JA (jasmonic acid) response of Pg1 and Pg2 in ginseng callus, ginseng callus was subcultured in 100 ml of fresh D1Ba0.5 liquid medium for 3 weeks and treated with hormone (JA). 100 μM JA was treated in 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.

[0064] After generating overexpression lines of Pg1 and Pg2, wild-type ginseng callus transformant lines as a control were subcultured on fresh medium for 3 weeks and treated with 100 μM JA. The control line was treated with the same volume of ethanol as JA. JA and ethanol were treated for 3 days to the wild type, Pg1 (OX #4, OX #10, and OX #11), and Pg2 (OX #19, OX #22, and OX #62), respectively, and harvested and frozen in liquid nitrogen immediately.

[0065]

[0066] [Experimental Example 5] Generation of Pg1 and Pg2 overexpressing callus

[0067] After ginseng calli were grown in D1Ba0.5 liquid medium, two types of plasmids (p35S:Pg1 and p35S:Pg2-myc) were transformed into Agrobacterium tumefaciens LBA4404. Ginseng calli were cut into pieces, pretreated with 0.05 M magnesium sulfate (MgSO4), and then infected with Agrobacterium tumefaciens. After transformation, the infected calli were selected on D1Ba0.5 medium containing cefotaxime and hygromycin for 12 weeks. Surviving calli were analyzed, and the expression of Pg1 and Pg2 was confirmed, and the transformant lines were selected.

[0068]

[0069] [Experimental Example 6] Total RNA extraction, cDNA synthesis, and gene expression analysis

[0070] 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).

[0071] RT-PCR was performed using a Mastercycler Nexus Gradient (Ependorf) and a 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.

[0072] qRT-PCR was performed using the CFX Connect Real-Time System (Bio-Rad) and SYBR Green Mastermix (Takara). The experimental results are 2 - ΔCt and 2 - ΔΔCt It was expressed through Ct normalization of internal control β-Actin by method.

[0073]

[0074] [Experimental Example 7] Subcellular localization, co-localization, and BiFC assays

[0075] Four-week-old Nicotiana 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 transfected into Agrobacterium tumefaciens GV3101 and infiltrated into tobacco (tabacco) leaves for transient expression of the genes.

[0076] For the subcellular localization assay of Pg1 and Pg2, the root cap fungus 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, a BiFC assay was performed to confirm the possibility of their binding. pGreen-Pg1-NV and pGreen-Pg2-CV were also co-injected into tobacco leaves.

[0077] After infiltration, the tobacco was left 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).

[0078]

[0079] [Experimental Example 8] Analysis of ginsenoside contents

[0080] For ginsenoside analysis of Pg1 and Pg2 overexpression lines, ginsenoside extraction was performed. Transformed calli from each transgenic line were subcultured for 4 weeks in 100 ml of D1Ba0.5 liquid medium; and wild-type calli as a control were obtained and frozen in liquid nitrogen immediately after harvest. All frozen samples were finely ground and lyophilized for one week. 0.2 g of each sample was quantified, and 2 ml of 70% methanol was added to the quantified sample. The extract was then extracted using an ultrasonic extractor at 50°C for 30 minutes, and the supernatant was obtained by centrifugation at 15,000 RPM for 3 minutes. A 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 from each sample was loaded for SPE (solid-phase extraction). Ginsenosides were obtained through elution with methanol and filtered through a 0.45 μm membrane filter for HPLC analysis.

[0081]

[0082] [Experimental Example 9] RNA Sequence Analysis

[0083] Total RNA was obtained from Pg1 OX #4 and Pg2 OX #19, and total RNA from wild-type calli was prepared as a control. Three RNA lines were extracted through three replicates, and the sequences of the lines were analyzed through Macrogen (https: / dna.macrogen.com / main.do#). Compared to the wild-type control, changes in gene expression of Pg1 OX #4 and Pg2 OX #19 were confirmed, and genes with low expression levels were normalized by log2-fold values ​​to identify DEGs.

[0084]

[0085] [Example 1] Characteristic analysis of Pg1 and Pg2

[0086] 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 sequence blast confirmed that these two JA response factors correspond to MYC2. 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 their 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).

[0087] To determine the taxonomic relationship of the two PgMYCs within the Panax species, Pg1 and Pg2 were analyzed using 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 determine 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).

[0088] Amino acid sequence analysis revealed that Pg1 and Pg2 are closely related to CsMYC2a, which has been reported to regulate indole biosynthesis in Camelliasinensis; 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). In addition, Pg1 and Pg2 also showed approximately 60% similarity to WsMYC2 and SlMYC2.

[0089] In addition, phylogenetic tree analysis results confirmed that Pg1 and Pg2 belong to the MYC2 TF family and thus have the function of regulating gene expression related to 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).

[0090] The predicted protein structures of Pg1 and Pg2 were predicted, and JID, bHLH, and BIF / ACT-like domains were indicated in both Pg1 and Pg2 (Fig. 1c). As a result, the predicted structures of Pg1 and Pg2 were similar to NtMYC2a. Pg1 and Pg2 contained a double-stranded helix representing bHLH, and showed a structure similar to a BIF / ACT-like domain mediated by two strands of an α-helix and a β-strand. In addition, three conserved motifs in PgMYC and MYCs from other plants were analyzed using the MEME tool (Fig. 1d), and the DNA-binding domain was confirmed to be the most highly conserved (Fig. 1d). Consequently, it was confirmed that Pg1 and Pg2 have the conserved domains and unique structures characteristic of MYC2. Therefore, additional experiments were performed to characterize Pg1 and Pg2 in ginseng, which has a ginsenoside biosynthetic pathway.

[0091]

[0092] [Example 2] Expression analysis of Pg1 and Pg2 in ginseng tissue

[0093] To determine whether Pg1 and Pg2 are transcribed during growth, gene expression patterns in various ginseng tissues were analyzed. Gene patterns of Pg1 and Pg2 were predicted to appear in various ginseng tissues (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.

[0094]

[0095] [Example 3] Subcellular localization, co-localization, and BiFC assays

[0096] We analyzed the subcellular localization of Pg1 and Pg2 through transient expression in Nicotiana benthamiana leaves, which are transcription factors (TFs) that localize to the nucleus of plant cells due to their role in binding to nuclear DNA. Pg1-GFP and Pg2-GFP, proteins fused to green fluorescent protein (GFP) at their C-terminus, were respectively introduced into tobacco leaves mediated by the fungus Rhizobium rhizome. The fluorescence signal of GFP appeared only in the nucleus of tobacco cells compared to the control (Fig. 3a), confirming that Pg1 and Pg2 function as TFs in the nucleus. To compare the subcellular localization of Pg1 and Pg2, we performed a co-localization assay to analyze the expression of Pg1-GFP and Pg2-mCherry. As a result, the fusion release of GFP and mCherry resulted in a yellow fluorescence signal in the nucleus, which was identical to the localization of Pg1 and Pg2 (Fig. 3b).

[0097] Because 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) elicited positive signals within the nucleus of tobacco cells (Fig. 3c), confirming that Pg1 and Pg2 interact with each other within the nucleus.

[0098]

[0099] [Example 4] Analysis of the Relationship between Pg1 and Pg2 and Ginsenoside Biosynthesis

[0100] To further confirm the relationship between Pg1 and Pg2 in ginsenoside biosynthesis, overexpression lines of Pg1 and Pg2 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 the 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 where the expression level did not increase significantly 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 where the expression level 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 for further analysis (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, the 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-type calli were prepared as controls 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, while the control group contained approximately 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 line, but in some lines, no increase in Rb2, Rb3, Rd, and Rh1 was observed (Fig. 5a).

[0101] To determine whether ginsenoside biosynthetic genes (Pg1 and Pg2) contribute to enhanced ginsenosides, we performed QRT-PCR to analyze the expression of HMGR1, HMGR2, SE1, and SE2 (Figs. 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 in 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 the six overexpression lines of Pg1 and Pg2 (Figs. 5d-5e). Additionally, Pg1 OX #4 increased the expression of PPTS and PPDS (Fig. 5f). From the above results, it was confirmed that overexpression of Pg1 and Pg2 induced ginsenoside biosynthesis in ginseng callus.

[0102]

[0103] [Example 5] Analysis of the mechanism for increased ginsenoside accumulation through overexpression of Pg1 and Pg2.

[0104] To confirm the mechanism for increased ginsenoside accumulation through overexpression of Pg1 and Pg2 confirmed in 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 differentially expressed genes (DEGs). 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, HMGR2 expression was decreased in Pg1 OX #4, whereas it was increased in Pg2 OX #19. This result was also confirmed by qRT-PCR analysis (Fig. 5c), and from the results, it was confirmed that Pg2 significantly affects HMGR2 expression.

[0105] 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 identical to the qRT-PCR analysis results (Fig. 5d). From the above 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 above results, it was confirmed that Pg1 and Pg2 regulate the expression of ginsenoside biosynthetic genes, including HMGR2, PPTS, SE1, and Pg_S0245.36.

[0106]

[0107] [Example 6] Analysis of JA reactivity of Pg1 and Pg2

[0108] Since MYC2 is known as a JA-responsive TF, the expression of Pg1 and Pg2 was analyzed after JA treatment. When 200 μM JA was treated in ginseng callus, the expression of Pg1 and Pg2 increased (Fig. 4a). In the wild type, Pg1 expression increased 2-fold compared to the control group (Fig. 4), and Pg2 expression increased approximately 3-fold compared to the control group. Pg1 and Pg2 expression was also increased by JA in the overexpression lines of Pg1 and Pg2, respectively. Specifically, the expression of Pg1 and Pg2 increased approximately 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.

[0109]

[0110] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for producing a transformed 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.

2. 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.

3. 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.

4. A manufacturing method according to claim 1, characterized in that the saponin is ginsenoside.

5. A manufacturing method according to claim 1, characterized in that the nucleotide is derived from ginseng (Panaxginseng).

6. 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.

7. Seeds obtained from the transformed plant of paragraph 1.

8. 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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