WO2015199435A1 - Method for preparing transgenic plant having increased stilbene production and plant prepared thereby - Google Patents

Method for preparing transgenic plant having increased stilbene production and plant prepared thereby Download PDF

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WO2015199435A1
WO2015199435A1 PCT/KR2015/006409 KR2015006409W WO2015199435A1 WO 2015199435 A1 WO2015199435 A1 WO 2015199435A1 KR 2015006409 W KR2015006409 W KR 2015006409W WO 2015199435 A1 WO2015199435 A1 WO 2015199435A1
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plant
gene
stilbene
ibmyb1a
rpsts
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김차영
정유정
안철한
우수경
정형재
전효곤
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한국생명공학연구원
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • 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
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    • C12N15/8257—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 for the production of primary gene products, e.g. pharmaceutical products, interferon
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    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Definitions

  • the present invention relates to a method for producing a transgenic plant with increased stilbene production and to a plant according to the present invention, and more particularly to a gene encoding a stilbene synthase (STS) protein derived from Rheum palmatum .
  • STS stilbene synthase
  • Plants transformed with a recombinant vector containing and a plant transformed with a recombinant vector comprising a gene encoding a MYB1a protein derived from sweet potato ( Ipomoea batatas ) were crossed with a wild type that simultaneously overexpressed the RpSTS gene and IbMYB1a gene.
  • stilbene relates to a method for producing a transgenic plant with increased production, a transgenic plant produced by the method and a seed thereof.
  • Polyphenols with stilbene skeletons are widely found in nature and are antimicrobial, antimalarial, antioxidant, anti-leukemia, antiplatelet aggregation, anticancer, anti-HIV, protein tyrosine kinase inhibitory, anti-inflammatory, anti-mutation, It is known to have various physiological activities such as antifungal and hepatoprotective effects.
  • Resveratrol a type of stilbene, is produced by the enzyme stilbene synthase (STS) in plants, and it is used for the treatment of colon cancer, oral herpes, and ulcers due to its strong antioxidant activity. It has been found to have pharmacological effects for the improvement of human health, such as prevention of blood clots, anti-hyperlipidemia, anti-inflammatory, seizure diseases, and cardiovascular diseases.Resveratrol having such physiological activity is synthesized in more than 72 kinds of plants, In herbaceous plants, they are biosynthesized by phytoalexin activity to protect against external stresses such as wounds, ozone damage, UV, and pest infections. Grapes and peanuts are the best biosynthetic plants for resveratrol.
  • STS stilbene synthase
  • Anthocyanins are red, purple and blue water-soluble flavonoid pigments that are widely contained in plants, fruits, stems and leaves. These anthocyanin pigments are mainly non-toxic in red, and are known to improve eyesight, prevent vascular disease, increase insulin production, anti-inflammatory, and improve memory. Among them, antioxidant effects are commonly known as antioxidants such as vitamins, carotene-based compounds, selenium, and tocophenol. It is reported to be superior to the material. In addition, anthocyanins can be prepared as a by-product of vegetables or fruits, and are used in soft drinks, alcoholic beverages, and other powder products as a substitute for synthetic coloring agents (red No. 2, No. 4 and No. 40), which pose a hazard problem.
  • Metabolic plus control maximizes the production of desired metabolites by expanding or removing specific metabolic pathways, introducing heterologous metabolic pathways that are not present in existing organisms, or by optimizing the flow at the metabolic branching point, based on the introduction of transformation technology. It is to improve the functionality of the cell and use it to produce various chemical and biological materials useful for human activities.
  • Korean Laid-Open Patent Publication No. 2003-0067689 discloses 'production of stilbene in a transgenic plant and a method of producing the same'
  • Korean Laid-open Patent No. 2014-0040372 discloses a plant using ROMT and STS genes. A method of increasing the stilbene content and changing the color of the 'is disclosed, but there is no description of the method for producing a transgenic plant with increased stilbene production of the present invention and the resulting plant.
  • the present invention is derived from the above requirements, the present inventors are transformed with a recombinant vector comprising a gene encoding a stilbene biosynthetic enzyme protein derived from rhubarb rhubarb produced tobacco plants and sweet potato-derived MYB1a protein
  • a transgenic plant transformed with a recombinant vector containing a gene encoding a gene and crosslinked with a tobacco plant containing a high concentration of anthocyanin anthocyanin pigments were increased in leaves and stems, and glycidids and methyl glycosides of resveratrol were increased.
  • the present invention was completed by confirming that the productivity of the derivative form of stilbene material was also greatly improved.
  • the present invention jangyeop rhubarb (Rheum palmatum) derived from stilbene biosynthetic enzyme (STS, stilbene synthase) transformed plants, and sweet potato with a recombinant vector comprising a gene encoding a protein (Ipomoea batatas) derived MYB1a
  • STS stilbene biosynthetic enzyme
  • STS stilbene synthase
  • a recombinant vector comprising a gene encoding a protein (Ipomoea batatas) derived MYB1a
  • a method for producing a transformed plant having increased stilbene production compared to a wild type which simultaneously cross-expresses a RpSTS gene and an IbMYB1a gene by crossing a transformed plant with a recombinant vector including a protein encoding a protein.
  • the present invention also provides a transgenic plant and its seed having increased stilbene production compared to the wild type produced by the method.
  • stilbene compounds having various useful physiological activities can be efficiently increased in various plants, and stilbene compounds are produced in large quantities based on these techniques. It is expected that this will be much easier, and that a method of greatly enhancing other useful metabolite biosynthesis by the hybridization strategy of the two plants of the present invention is also expected to contribute to the development of the high value-added agricultural biomaterial industry.
  • Figure 1 is a diagram showing the biosynthetic pathway of the stilbene compound of the present invention (resveratrol, terrostilbene and pisid).
  • Figure 2 is a result of analyzing the characteristics of stilbene and anthocyanin pigment biosynthetic transgenic plants
  • A is the gene PAL, C4H, 4CL required for the biosynthesis of p- coumaryl -CoA from phenylalanine when IbMYB1a overexpressed in Arabidopsis plants and check the expression pattern of the resulting increase in a
  • B and C are photographs of observing the phenotype of flowers and leaves of tobacco plants overexpressing RpSTS and IbMYB1a.
  • FIG. 3 is a schematic diagram of a plant for overexpression of the stilbene biosynthesis gene and anthocyanin pigment biosynthesis transcription factor of the present invention.
  • STS Cross-plant of pGR-Flag :: RpSTS transgenic plant and pCam-SWPA2-IbMYB1a transgenic plant
  • ROST pGR-HA :: VrROMT-Flag :: RpSTS transgenic plant and pCam-SWPA2-IbMYB1a transgenic plant Mating plant.
  • FIG. 5 is a result of confirming the expression of RpSTS and VrROMT genes in transgenic tobacco plants, Western blot results using a flag antibody.
  • STS crossing of pGR-Flag :: RpSTS transgenic plant and pCam-SPO-IbMYB1a transgenic plant
  • ROST pGR-HA :: VrROMT-Flag :: RpSTS transgenic plant and pCam-SPO-IbMYB1a transgenic plant Mating plant.
  • FIG. 6 is a result of confirming the expression of RpSTS and VrROMT genes in transgenic tobacco plants, Western blot results using a flag antibody and HA antibody.
  • SP pGR-Flag :: RpSTS transgenic plants and pCam-SPO-IbMYB1a transgenic plants
  • RP pGR-HA :: VrROMT-Flag :: RpSTS transgenic plants and pCam-SPO-IbMYB1a transgenic plants
  • RW pGR-HA :: VrROMT-Flag :: RpSTS transgenic plants and pCam-SWPA2-IbMYB1a transgenic plants.
  • Figure 7 is the result of confirming the phenotype in flowers and leaves of transgenic mating tobacco plants.
  • Cross white White petals among flowers bloomed in a hybrid plant of the IbMYB1a gene overexpressing plant and the RpSTS gene overexpressing plant.
  • (c) including the step of selecting the transgenic plant overexpressing RpSTS gene and IbMYB1a genes crossed the (a) step of RpSTS gene transgenic plant and the step (b) of IbMYB1a gene transgenic plants It provides a method for producing a transgenic plant with increased stilbene production compared to the wild type characterized in that the production.
  • the transgenic plant overexpressing the IbMYB1a gene may be prepared by transforming the plant with a recombinant vector comprising a gene encoding MYB1a protein derived from sweet potato ( Ipomoea batatas ). This is not restrictive.
  • the stilbene is a resveratrol glycoside or piceid methyl ether such as trans-piceid or cis-piceid.
  • stilbene in the form of a methyl derivative such as resveratrol methyl ether-O-hexoside, but is not limited thereto.
  • the range of proteins includes proteins having amino acid sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4, and functional equivalents of such proteins, respectively.
  • “Functional equivalent” means at least 70%, preferably at least 80%, more preferably at least 90%, of the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 4 as a result of the addition, substitution, or deletion of the amino acid; More preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with the proteins represented by SEQ ID NO: 2 and SEQ ID NO: 4.
  • the present invention provides a gene for MYB1a derived from STS and sweet potato derived from the rhubarb rhubarb, preferably consisting of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 3, respectively.
  • homologues of the above nucleotide sequences are included within the scope of the present invention.
  • the gene is at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 3, respectively. Base sequences having homology.
  • the "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
  • recombinant refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid.
  • Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form.
  • Recombinant cells can also express genes found in natural cells, but the genes are modified and intracellularly reintroduced by artificial means.
  • the RpSTS or IbMYB1a gene sequence may be inserted into a recombinant expression vector.
  • recombinant expression vector means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.
  • Expression vectors comprising the RpSTS or IbMYB1a gene sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.
  • Preferred examples of recombinant vectors of the invention are Ti-plasmid vectors capable of transferring part of themselves, the so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens.
  • Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have.
  • a particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.
  • viral vectors such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc.
  • CaMV double stranded plant viruses
  • gemini viruses single stranded viruses
  • it may be selected from an incomplete plant viral vector.
  • the use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.
  • the expression vector will preferably comprise one or more selectable markers.
  • the marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance gene, aadA gene, and the like, but are not limited thereto.
  • the promoter may be a CaMV 35S, a peroxidase gene (SWPA2) promoter, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter, but is not limited thereto.
  • the term “promoter” refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription.
  • a "plant promoter” is a promoter capable of initiating transcription in plant cells.
  • a “constitutive promoter” is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.
  • terminators can be used, such as sporamin gene (SPO) terminator, nopalin synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, agro Terminator of the octopine gene of Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), rrnB1 / B2 terminator of E. coli, but is not limited thereto.
  • SPO sporamin gene
  • NOS nopalin synthase
  • rice ⁇ -amylase RAmy1 A terminator phaseoline terminator
  • agro Terminator of the octopine gene of Agrobacterium tumefaciens Agrobacterium tumefaciens
  • rrnB1 / B2 terminator of E. coli but is not limited thereto.
  • terminators With regard to the need for terminators, such regions are generally known to increase the certainty and efficiency of transcription in plant cells
  • yeast Saccharomyce cerevisiae
  • insect cells human cells
  • human cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines
  • plant cells and the like can be used as a host cell.
  • the host cell is preferably a plant cell.
  • the method of transporting the vector of the present invention into the host cell may be performed by injecting the vector into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, or the like. can do.
  • the present invention also provides a transgenic plant and its seed having increased stilbene production compared to the wild type produced by the method.
  • the plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, It may be a dicotyledonous plant such as gat radish, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, green bean, kidney bean, pea, or monocotyledonous plant such as rice, barley, wheat, rye, corn, sugarcane, oats, onions, Preferably it is a dicotyledonous plant, More preferably, it is a tobacco.
  • the gene used to develop the resveratrol production system was used to isolate the resveratrol biosynthesis transcription factor RpSTS (1176bp) from Rheum palmatum.
  • RpSTS resveratrol biosynthesis transcription factor
  • a sample of rhubarb was obtained from the Northern Agricultural Experiment Station, Kangwon-do Agricultural Research and Extension Services, and the total RNA was isolated from the sample. Homology-based primers were used for RT-PCR and overlap extension PCR methods. Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit (Fermantas, Canada).
  • a rhubarb RpSTS transcription factor was amplified by PCR using an Advantage 2 polymerase mix (Clontech, USA). Primer sequences used in this experiment were as follows: RpSTS_F (Nde): 5'-CATATGGCACCGGAGGAGT-3 '(SEQ ID NO: 5), RpSTS_R (Spe): 5'-ACTAGTTCAGGTAATTAGCGGC-3' (SEQ ID NO: 6). PCR amplification was carried out by a final extension reaction at 30 cycles and 10 minutes at 72 ° C.
  • the amplified PCR product was cloned into pT-Blunt vector (solgent, Korea) and analyzed for sequencing. As a result of sequencing, the cloned RpSTS gene was composed of a total of 1176bp.
  • the gene used to develop the telostilbene production system was used to isolate the telostilbene biosynthesis transcription factor gene VrROMT (1074bp) from Vitis riparis. Grape samples were obtained from the Grape Research Institute, Chungcheongbuk-do Agricultural Research and Extension Services. This gene was isolated by RT-PCR and overlap extension PCR using homology-based primers. Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit. Using the synthesized cDNA as a template, the grape VrROMT transcription factor was amplified by PCR using an Advantage 2 polymerase mix.
  • VrROMT_F Nde
  • VrROMT_R Spe
  • PCR amplification of the VrROMT gene was carried out by a final extension reaction for 30 minutes at 72 ° C and 10 minutes at 72 ° C for 2 minutes denaturation at 95 ° C, 15 seconds at 95 ° C, 30 seconds at 55 ° C, and 2 minutes at 72 ° C.
  • the amplified PCR product was cloned into pT-Blunt vector and analyzed for sequencing. Sequence analysis revealed that the cloned VrROMT gene was composed of a total of 1074bp.
  • the gene used to develop the anthocyanin mass production system was used by separating the anthocyanin biosynthesis transcription factor gene IbMYB1a from purple sweet potato ( Shinjami cultivar).
  • Total RNA was isolated from tubers of purple sweet potatoes (Kim CY et al. 2010, Physiologia Plantarum 139: 259-261).
  • Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit.
  • the sweet potato IbMYB1a transcription factor was amplified by PCR using an Advantage 2 polymerase mix.
  • the primer sequences used in this experiment were as follows: IbMYB1a-F: 5'-AGCTAAGAATTTCCGACACCCTTCAATA-3 '(SEQ ID NO: 9), IbMYB1a-R: 5'-GTGAATTTAACGCTTAGCTTAACAGTTCT-3' (SEQ ID NO: 10).
  • PCR amplification was carried out by a final extension reaction at 30 cycles and 10 minutes at 72 ° C. with 2 cycles of denaturation at 95 ° C., 15 seconds at 95 ° C., 30 seconds at 55 ° C., and 2 minutes at 72 ° C.
  • the amplified PCR product was cloned into pGEM-T Easy vector (Promega, USA) and analyzed for sequencing. As a result of sequencing, the cloned IbMYB1a gene was composed of a total of 797bp, and the ORF (open readin frame) was composed of 750 bp.
  • a plant expression vector of pCAMBIA 2300 having kanamycin resistance (Center for Application of Molecular Biology to International Agriculture, Australia) ) potato roots and expression of Spokane's Grameen gene IbMYB1a genes between the SPO promoter and SPO whole IbMYB1a genes between terminator (open reading frame) is inserted into a vector, oxidative stress-induced potato peroxidase are kinase gene SWPA2 promoter and SPO terminator Each vector with an open reading frame was produced.
  • the produced overexpressing carriers were named 3) pCam-SPO-IbMYB1a and 4) pCam-SWPA2-IbMYB1a (FIG. 3).
  • a control wild-type tobacco was added with 1 mg / L of NAA (a-naphthalene acetic acid), 1 mg / L of BA (6-benzyladenine), 10 g / L of sucrose, and 8 g / L of agar.
  • NAA a-naphthalene acetic acid
  • BA a-benzyladenine
  • 10 g / L of sucrose sucrose
  • 8 g / L of agar After 3 days co-culture in MS medium, 1 mg / L NAA, 1 mg / L BA, 300 mg / L carbenicillin, 100 mg / L kanamycin, 10 g / L Cross and 8 g / L agar were incubated in MS medium and then subcultured every three weeks.
  • Regenerated shoots were transferred to MS medium to which 300 mg / L carbenicillin, 100 mg / L kanamycin, 10 g / L sucrose and 8 g / L agar were added to induce rooting. Cultures were incubated under conditions of 26 °C, 16 hours photoperiod / 8 hours dark cycle, rooted individuals were transplanted into pollen after acclimatization and grown in a greenhouse at an average temperature of 26 °C or more.
  • Stilbene biosynthesis STS converts p-coumaryl-CoA to resveratol.
  • the amount of p-coumariyl-CoA biosynthesized in plants is limited, it is not easy to obtain a large amount of stilbene compounds from plants through induction of overexpression of stilbene biosynthesis only.
  • Metabolic control is needed to increase the concentration of p-coumariyl-CoA, an important precursor of the stilbene biosynthetic pathway (FIG. 2A).
  • sikyeoteul overexpressing IbMYB1a in Arabidopsis genes required for biosynthesis of the p- Kumar reel -CoA from phenylalanine PAL, C4H check patterns that increase the expression of 4CL (Fig. 2A) was metabolic flux by inserting the gene into a tobacco plant IbMYB1a By controlling the (Flux) to obtain the precursor required for the biosynthesis of the stilbene.
  • a transformant overexpressing the MYB gene that regulates the stage of the phenylpropanoid synthesis pathway was prepared , and a transgenic plant overexpressing the stilbene biosynthetic gene STS was also produced (FIG. 2B) and crossed. Then was isolated transgenic plant that over-expression with the STS and MYB or ROST and MYB genes in the F1 generation, respectively, and secured (Fig. 2).
  • each part of each transgenic hybrid tobacco plant selected in Example 7 was taken, immersed in liquid nitrogen and frozen, and then powdered using a mortar and pestle.
  • 0.5 g of the plant powder was extracted with 5 ml of 80% methanol, and then centrifuged to obtain a plant extract of the upper layer.
  • the extracted filtrate was dried by injecting air, and the dried product was dissolved in 300 ⁇ l 80% methanol, and 0.2 ⁇ m PTFE filter (hydrophilic, ADVANTEC, Japan) was used for HPLC (High-performance liquid chromatography) analysis.
  • HPLC High-performance liquid chromatography
  • A 0.05% trifluoroacetic acid
  • B 0.05% trifluoroacetic acid
  • LC-MS liquid chromatography-mass spectrometry

Abstract

The present invention relates to: a method for preparing a transgenic plant having increased stilbene production, compared to a wild type overexpressing an RpSTS gene and an IbMYB1a gene at the same time, by crossing a plant, having been transformed with a recombinant vector containing a gene coding for an RpSTS protein, with a plant having been transformed with a recombinant vector containing a gene coding for an IbMYB1a protein; and a transgenic plant prepared by the method. It is expected that mass producing, in various plants, stilbene compounds having various useful physiological activities will become much easier.

Description

스틸벤 생산이 증가된 형질전환 식물체의 제조 방법 및 그에 따른 식물체Method for producing transformed plant with increased stilbene production and plant accordingly
본 발명은 스틸벤 생산이 증가된 형질전환 식물체의 제조 방법 및 그에 따른 식물체에 관한 것으로, 더욱 상세하게는 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환된 식물체 및 고구마 (Ipomoea batatas) 유래 MYB1a 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환된 식물체를 교배하여 RpSTS 유전자 및 IbMYB1a 유전자를 동시에 과발현하는 야생형에 비해 스틸벤 (stilbene) 생산이 증가된 형질전환 식물체의 제조 방법, 상기 방법에 의해 제조된 형질전환 식물체 및 이의 종자에 관한 것이다.The present invention relates to a method for producing a transgenic plant with increased stilbene production and to a plant according to the present invention, and more particularly to a gene encoding a stilbene synthase (STS) protein derived from Rheum palmatum . Plants transformed with a recombinant vector containing and a plant transformed with a recombinant vector comprising a gene encoding a MYB1a protein derived from sweet potato ( Ipomoea batatas ) were crossed with a wild type that simultaneously overexpressed the RpSTS gene and IbMYB1a gene. stilbene) relates to a method for producing a transgenic plant with increased production, a transgenic plant produced by the method and a seed thereof.
스틸벤 (stilbene) 골격을 지닌 폴리페놀류는 천연에서 폭넓게 발견되며 항미생물, 항말라리아, 항산화, 항백혈병, 항혈소판응집, 항암, 항-HIV, 단백질 티로신 카이나제 억제, 항염증, 항돌연변이, 항진균 및 간보호 효과 등 다양한 생리활성을 갖는 것으로 알려져 있다.Polyphenols with stilbene skeletons are widely found in nature and are antimicrobial, antimalarial, antioxidant, anti-leukemia, antiplatelet aggregation, anticancer, anti-HIV, protein tyrosine kinase inhibitory, anti-inflammatory, anti-mutation, It is known to have various physiological activities such as antifungal and hepatoprotective effects.
레스베라트롤 (resveratrol)은 스틸벤의 한 종류로서 식물에서 효소인 스틸벤 합성효소 (stilbene synthase ; STS)에 의해 생성되며, 강한 항산화성으로 결장암, 구강포진, 궤양치료에 이용되고 있으며, 또한 항암, 항혈전, 항고지혈증, 항염증, 발작질환, 심혈관 질병의 예방 등 인간의 건강 증진을 위한 약리 효과가 있음이 밝혀졌는데, 이러한 생리활성을 갖는 레스베라트롤은 72종 이상의 식물체에서 합성되며, 소나무 등 목본류에서는 항상 생산되지만, 초본류에서는 피토알렉신 (phytoalexin) 활성에 의해 상처, 오존에 의한 손상, UV, 병해충 감염 등 외부 스트레스에 의한 자기방어 물질로 생합성되며, 레스베라트롤의 생합성이 가장 우수한 식물은 포도와 땅콩이다. 이러한 레스베라트롤의 함량을 인위적으로 증진시키기 위하여 재배시 포도에 인위적으로 균주를 접종하거나, 수확 후 포도와 땅콩 종자에 초음파 세척 처리나, UV 조사 등을 통하여 레스베라트롤의 함량을 증진시키는 연구들이 시도되고 있다.Resveratrol, a type of stilbene, is produced by the enzyme stilbene synthase (STS) in plants, and it is used for the treatment of colon cancer, oral herpes, and ulcers due to its strong antioxidant activity. It has been found to have pharmacological effects for the improvement of human health, such as prevention of blood clots, anti-hyperlipidemia, anti-inflammatory, seizure diseases, and cardiovascular diseases.Resveratrol having such physiological activity is synthesized in more than 72 kinds of plants, In herbaceous plants, they are biosynthesized by phytoalexin activity to protect against external stresses such as wounds, ozone damage, UV, and pest infections. Grapes and peanuts are the best biosynthetic plants for resveratrol. In order to artificially increase the content of such resveratrol, studies have been attempted to artificially inoculate strains on grapes during cultivation, or to increase the content of resveratrol through ultrasonic cleaning or UV irradiation on grape and peanut seeds after harvesting.
또한, 포도와 땅콩으로부터 분리한 레스베라트롤 합성에 관여하는 유전자를 생명공학기법을 이용하여 레스베라트롤을 생합성하는 작물을 개발하려는 연구가 시도되고 있는데, 포도의 레스베라트롤 합성 유전자를 이용한 형질전환 키위 식물체 잎에서 182㎍/g의 레스베라트롤이 생산되었음을 보고하였다.In addition, a study is being conducted to develop a crop that synthesizes resveratrol using biotechnology for genes involved in the synthesis of resveratrol isolated from grapes and peanuts. It was reported that / g of resveratrol was produced.
안토시아닌 (anthocyanin)은 식물의 꽃, 과실, 줄기, 잎 등에 폭넓게 함유되어 있는 적색, 자색, 청색을 나타내는 수용성 플라보노이드 색소이다. 이러한 안토시아닌 색소는 주로 적색으로 독성이 없고, 시력개선, 혈관질환 예방, 인슐린 생성증대, 항염, 기억력 개선 효과가 알려져 있으며, 그중 항산화 효과는 비타민, 카로틴계 화합물, 셀레늄, 토코페놀 등 일반적으로 알려진 항산화 물질보다 뛰어난 것으로 보고되어 있다. 또한, 안토시아닌은 채소나 과일의 부산물로도 제조가 가능하여 유해성 문제가 대두되는 합성착색료(적색2호, 4호, 40호)의 대체용으로 청량음료, 주류, 기타 분말제품에 이용되고 있다.Anthocyanins are red, purple and blue water-soluble flavonoid pigments that are widely contained in plants, fruits, stems and leaves. These anthocyanin pigments are mainly non-toxic in red, and are known to improve eyesight, prevent vascular disease, increase insulin production, anti-inflammatory, and improve memory. Among them, antioxidant effects are commonly known as antioxidants such as vitamins, carotene-based compounds, selenium, and tocophenol. It is reported to be superior to the material. In addition, anthocyanins can be prepared as a by-product of vegetables or fruits, and are used in soft drinks, alcoholic beverages, and other powder products as a substitute for synthetic coloring agents (red No. 2, No. 4 and No. 40), which pose a hazard problem.
대사 플러스 조절은 형질전환 기술 도입을 바탕으로 특정 대사경로를 확대 혹은 제거하거나 기존의 생물이 갖고 있지 않은 이종 대사경로를 도입 혹은 최적화 대사 분지점에서의 흐름을 조절하여 원하는 대사 산물의 생산을 극대화하거나 세포의 기능성을 향상시켜 이를 인간 활동에 유용한 다양한 화학물질 및 생물학적 물질 생산에 활용하는 것을 말한다.Metabolic plus control maximizes the production of desired metabolites by expanding or removing specific metabolic pathways, introducing heterologous metabolic pathways that are not present in existing organisms, or by optimizing the flow at the metabolic branching point, based on the introduction of transformation technology. It is to improve the functionality of the cell and use it to produce various chemical and biological materials useful for human activities.
한편, 한국공개특허 제2003-0067689호에는 '형질전환 식물에서의 스틸벤의 생산 및 그것의 생산 방법'이 개시되어 있고, 한국공개특허 제2014-0040372호에는 'ROMT 및 STS 유전자를 이용한 식물의 스틸벤 함량을 증가시키고 화색을 변화시키는 방법'이 개시되어 있으나, 본 발명의 스틸벤 생산이 증가된 형질전환 식물체의 제조 방법 및 그에 따른 식물체에 대해서는 기재된 바가 없다.Meanwhile, Korean Laid-Open Patent Publication No. 2003-0067689 discloses 'production of stilbene in a transgenic plant and a method of producing the same', and Korean Laid-open Patent No. 2014-0040372 discloses a plant using ROMT and STS genes. A method of increasing the stilbene content and changing the color of the 'is disclosed, but there is no description of the method for producing a transgenic plant with increased stilbene production of the present invention and the resulting plant.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 장엽대황 유래의 스틸벤 생합성 효소 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환되어 스틸벤을 생산하는 담배 식물체와 고구마 유래 MYB1a 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환되어 안토시아닌을 고농도로 함유하는 담배 식물체를 타가 교배하여 제조한 형질전환 식물체에서, 잎 및 줄기에서 안토시아닌 색소가 증가되고, 레스베라트롤의 배당체인 피시드와 메틸 유도체 형태의 스틸벤 물질의 생산성도 크게 향상된 것을 확인함으로써, 본 발명을 완성하였다.The present invention is derived from the above requirements, the present inventors are transformed with a recombinant vector comprising a gene encoding a stilbene biosynthetic enzyme protein derived from rhubarb rhubarb produced tobacco plants and sweet potato-derived MYB1a protein In a transgenic plant transformed with a recombinant vector containing a gene encoding a gene and crosslinked with a tobacco plant containing a high concentration of anthocyanin, anthocyanin pigments were increased in leaves and stems, and glycidids and methyl glycosides of resveratrol were increased. The present invention was completed by confirming that the productivity of the derivative form of stilbene material was also greatly improved.
상기 과제를 해결하기 위해, 본 발명은 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환된 식물체 및 고구마 (Ipomoea batatas) 유래 MYB1a 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 형질전환된 식물체를 교배하여 RpSTS 유전자 및 IbMYB1a 유전자를 동시에 과발현하는 야생형에 비해 스틸벤 (stilbene) 생산이 증가된 형질전환 식물체의 제조 방법을 제공한다.In order to solve the above problems, the present invention jangyeop rhubarb (Rheum palmatum) derived from stilbene biosynthetic enzyme (STS, stilbene synthase) transformed plants, and sweet potato with a recombinant vector comprising a gene encoding a protein (Ipomoea batatas) derived MYB1a Provided is a method for producing a transformed plant having increased stilbene production compared to a wild type which simultaneously cross-expresses a RpSTS gene and an IbMYB1a gene by crossing a transformed plant with a recombinant vector including a protein encoding a protein.
또한, 본 발명은 상기 방법에 의해 제조된 야생형에 비해 스틸벤 생산이 증가된 형질전환 식물체 및 이의 종자를 제공한다.The present invention also provides a transgenic plant and its seed having increased stilbene production compared to the wild type produced by the method.
본 발명에서 제안된 교배를 통한 대사 플럭스 조절기술을 바탕으로 다양한 유용 생리활성을 가진 스틸벤 화합물을 다양한 식물체에서 효율적으로 그 생산량을 증대시킬 수 있으며, 이러한 기술을 바탕으로 스틸벤 화합물을 대량으로 생산하는 것이 훨씬 용이해질 것으로 기대되며, 또한 본 발명의 두 식물체의 교배 전략으로 다른 유용 대사체 생합성을 크게 증진시킬수 있는 방법은 고부가가치의 농업생물소재 산업의 발달에도 기여할 것으로 기대된다.Based on the metabolic flux control technology proposed in the present invention, stilbene compounds having various useful physiological activities can be efficiently increased in various plants, and stilbene compounds are produced in large quantities based on these techniques. It is expected that this will be much easier, and that a method of greatly enhancing other useful metabolite biosynthesis by the hybridization strategy of the two plants of the present invention is also expected to contribute to the development of the high value-added agricultural biomaterial industry.
도 1은 본 발명의 스틸벤류화합물 (레스베라트롤, 테로스틸벤 및 피시드)의 생합성 경로를 나타낸 그림이다.Figure 1 is a diagram showing the biosynthetic pathway of the stilbene compound of the present invention (resveratrol, terrostilbene and pisid).
도 2는 스틸벤류 및 안토시아닌 색소 생합성 형질전환 식물체의 특성을 분석한 결과로, A는 애기장대 식물체에서 IbMYB1a를 과발현 시켰을 경우 페닐알라닌으로부터 p-쿠마릴-CoA를 생합성하는데 요구되는 유전자 PAL, C4H, 4CL의 발현이 증가되는 패턴을 확인한 결과이며, B와 C는 RpSTS와 IbMYB1a를 과발현하는 담배 식물체의 꽃과 잎의 표현형을 관찰한 사진이다.Figure 2 is a result of analyzing the characteristics of stilbene and anthocyanin pigment biosynthetic transgenic plants, A is the gene PAL, C4H, 4CL required for the biosynthesis of p- coumaryl -CoA from phenylalanine when IbMYB1a overexpressed in Arabidopsis plants and check the expression pattern of the resulting increase in a, B and C are photographs of observing the phenotype of flowers and leaves of tobacco plants overexpressing RpSTS and IbMYB1a.
도 3은 본 발명의 스틸벤류 생합성 유전자 및 안토시아닌 색소 생합성 전사인자의 식물체 내 과발현용 운반체의 모식도이다.3 is a schematic diagram of a plant for overexpression of the stilbene biosynthesis gene and anthocyanin pigment biosynthesis transcription factor of the present invention.
도 4는 형질전환 담배 식물체에서 RpSTS와 VrROMT 유전자의 발현 여부를 확인한 결과로, flag 항체를 이용한 웨스턴 블랏 결과이다. STS: pGR-Flag::RpSTS 형질전환 식물체와 pCam-SWPA2-IbMYB1a 형질전환 식물체의 교배 식물체, ROST: pGR-HA::VrROMT-Flag::RpSTS 형질전환 식물체와 pCam-SWPA2-IbMYB1a 형질전환 식물체의 교배 식물체.4 is a result of confirming the expression of RpSTS and VrROMT genes in transgenic tobacco plants, Western blot results using a flag antibody. STS: Cross-plant of pGR-Flag :: RpSTS transgenic plant and pCam-SWPA2-IbMYB1a transgenic plant, ROST: pGR-HA :: VrROMT-Flag :: RpSTS transgenic plant and pCam-SWPA2-IbMYB1a transgenic plant Mating plant.
도 5는 형질전환 담배 식물체에서 RpSTS와 VrROMT 유전자의 발현 여부를 확인한 결과로, flag 항체를 이용한 웨스턴 블랏 결과이다. STS: pGR-Flag::RpSTS 형질전환 식물체와 pCam-SPO-IbMYB1a 형질전환 식물체의 교배 식물체, ROST: pGR-HA::VrROMT-Flag::RpSTS 형질전환 식물체와 pCam-SPO-IbMYB1a 형질전환 식물체의 교배 식물체.5 is a result of confirming the expression of RpSTS and VrROMT genes in transgenic tobacco plants, Western blot results using a flag antibody. STS: crossing of pGR-Flag :: RpSTS transgenic plant and pCam-SPO-IbMYB1a transgenic plant, ROST: pGR-HA :: VrROMT-Flag :: RpSTS transgenic plant and pCam-SPO-IbMYB1a transgenic plant Mating plant.
도 6은 형질전환 담배 식물체에서 RpSTS와 VrROMT 유전자의 발현 여부를 확인한 결과로, flag 항체 및 HA 항체를 이용한 웨스턴 블랏 결과이다. SP: pGR-Flag::RpSTS 형질전환 식물체와 pCam-SPO-IbMYB1a 형질전환 식물체의 교배 식물체, RP: pGR-HA::VrROMT-Flag::RpSTS 형질전환 식물체와 pCam-SPO-IbMYB1a 형질전환 식물체의 교배 식물체, RW: pGR-HA::VrROMT-Flag::RpSTS 형질전환 식물체와 pCam-SWPA2-IbMYB1a 형질전환 식물체의 교배 식물체.6 is a result of confirming the expression of RpSTS and VrROMT genes in transgenic tobacco plants, Western blot results using a flag antibody and HA antibody. SP: pGR-Flag :: RpSTS transgenic plants and pCam-SPO-IbMYB1a transgenic plants, RP: pGR-HA :: VrROMT-Flag :: RpSTS transgenic plants and pCam-SPO-IbMYB1a transgenic plants Crossbreeding plants, RW: pGR-HA :: VrROMT-Flag :: RpSTS transgenic plants and pCam-SWPA2-IbMYB1a transgenic plants.
도 7은 형질전환 교배 담배 식물체의 꽃과 잎에서의 표현형을 확인한 결과이다.Figure 7 is the result of confirming the phenotype in flowers and leaves of transgenic mating tobacco plants.
도 8은 형질전환 교배 담배 식물체의 추출물에서 HPLC (high performance liquid chromatography)를 이용한 스틸벤 화합물 생합성 효율을 조사한 결과이다. Control(EV): 야생형 담배 식물체, IbMYB1a-OX: IbMYB1a 유전자 과발현 식물체, RpSTS-OX: RpSTS 유전자 과발현 식물체, SP425#3(cross): IbMYB1a 유전자 과발현 식물체와 RpSTS 유전자 과발현 식물체의 교배 식물체.8 is a result of investigating the efficiency of stilbene compound biosynthesis using HPLC (high performance liquid chromatography) in extracts of transgenic hybrid tobacco plants. Control (EV): wild type tobacco plant, IbMYB1a-OX: IbMYB1a gene overexpressing plant, RpSTS-OX: RpSTS gene overexpressing plant, SP425 # 3 (cross): IbMYB1a gene overexpressing plant and RpSTS gene overexpressing plant.
도 9는 형질전환 교배 담배 식물체 추출물에서 스틸벤 화합물을 LC-MS (liquid chromatography-mass spectrometry)로 분석한 결과이다. Control(EV): 야생형 담배 식물체, IbMYB1a-OX: IbMYB1a 유전자 과발현 식물체, RpSTS-OX: RpSTS 유전자 과발현 식물체, Cross red: IbMYB1a 유전자 과발현 식물체와 RpSTS 유전자 과발현 식물체의 교배 식물체에서 핀 꽃 중 붉은색 꽃잎, Cross white: IbMYB1a 유전자 과발현 식물체와 RpSTS 유전자 과발현 식물체의 교배 식물체에서 핀 꽃 중 흰색 꽃잎.9 is a result of analyzing the stilbene compound by LC-MS (liquid chromatography-mass spectrometry) in the transgenic hybrid tobacco plant extract. Control (EV): wild-type tobacco plants, IbMYB1a-OX: IbMYB1a gene overexpressing plants, RpSTS-OX: RpSTS gene overexpressing plants, Cross red: IbMYB1a gene overexpressing plants and RpSTS gene overexpressing plants, red petals among the flowering flowers Cross white: White petals among flowers bloomed in a hybrid plant of the IbMYB1a gene overexpressing plant and the RpSTS gene overexpressing plant.
본 발명의 목적을 달성하기 위하여, 본 발명은In order to achieve the object of the present invention, the present invention
(a) 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질을 코딩하는 RpSTS 유전자를 과발현하는 형질전환 식물체를 제조하는 단계;(a) preparing a transgenic plant that overexpresses the RpSTS gene encoding a stilbene synthase (STS) protein derived from Rheum palmatum ;
(b) 고구마 (Ipomoea batatas) 유래 MYB1a 단백질을 코딩하는 IbMYB1a 유전자를 과발현하는 형질전환 식물체를 제조하는 단계; 및(b) preparing a transgenic plant that overexpresses the IbMYB1a gene encoding MYB1a protein from sweet potato ( Ipomoea batatas ); And
(c) 상기 (a) 단계의 RpSTS 유전자 과발현 형질전환 식물체와 상기 (b) 단계의 IbMYB1a 유전자 과발현 형질전환 식물체를 교배하여 RpSTS 유전자 및 IbMYB1a 유전자를 동시에 과발현하는 형질전환 식물체를 선발하는 단계를 포함하여 제조하는 것을 특징으로 하는 야생형에 비해 스틸벤 (stilbene) 생산이 증가된 형질전환 식물체의 제조 방법을 제공한다.(c) including the step of selecting the transgenic plant overexpressing RpSTS gene and IbMYB1a genes crossed the (a) step of RpSTS gene transgenic plant and the step (b) of IbMYB1a gene transgenic plants It provides a method for producing a transgenic plant with increased stilbene production compared to the wild type characterized in that the production.
본 발명의 일 구현 예에 따른 제조 방법에 있어서, 상기 RpSTS 유전자를 과발현하는 형질전환 식물체는 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물체를 형질전환시켜 제조할 수 있으나, 이에 제한되지 않는다.In the production method according to an embodiment of the invention, transgenic plants overexpressing the RpSTS gene jangyeop rhubarb (Rheum palmatum) derived from stilbene biosynthetic enzyme (STS, stilbene synthase) a recombinant vector comprising a gene encoding a protein It can be prepared by transforming plants, but is not limited thereto.
본 발명의 일 구현 예에 따른 제조 방법에 있어서, 상기 IbMYB1a 유전자를 과발현하는 형질전환 식물체는 고구마 (Ipomoea batatas) 유래 MYB1a 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물체를 형질전환시켜 제조할 수 있으나, 이에 제한되지 않는다.In the production method according to an embodiment of the present invention, the transgenic plant overexpressing the IbMYB1a gene may be prepared by transforming the plant with a recombinant vector comprising a gene encoding MYB1a protein derived from sweet potato ( Ipomoea batatas ). This is not restrictive.
본 발명의 일 구현 예에 따른 제조 방법에 있어서, 상기 스틸벤은 트랜스-피시드(trans-piceid) 또는 시스-피시드(cis-piceid) 등의 레스베라트롤 배당체 또는 피시드 메틸 에테르(piceid methyl ether) 또는 레스베라트롤 메틸 에테르-O-헥소사이드(resveratrol methyl ether-O-hexoside) 등의 메틸 유도체 형태의 스틸벤일 수 있으나, 이에 제한되지 않는다.In the production method according to an embodiment of the present invention, the stilbene is a resveratrol glycoside or piceid methyl ether such as trans-piceid or cis-piceid. Or stilbene in the form of a methyl derivative such as resveratrol methyl ether-O-hexoside, but is not limited thereto.
본 발명에 따른 장엽대황 유래의 STS 및 고구마 유래의 MYB1a 단백질의 범위는 각각 서열번호 2 및 서열번호 4로 표시되는 아미노산 서열을 갖는 단백질 및 상기 단백질의 기능적 동등물을 포함한다. "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 2 및 서열번호 4로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더 바람직하게는 90% 이상, 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 2 및 서열번호 4로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. MYB1a from STS and sweet potato derived from Rhubarb rhubarb according to the present invention The range of proteins includes proteins having amino acid sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4, and functional equivalents of such proteins, respectively. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, of the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 4 as a result of the addition, substitution, or deletion of the amino acid; More preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with the proteins represented by SEQ ID NO: 2 and SEQ ID NO: 4.
또한, 본 발명은 상기 장엽대황 유래의 STS 및 고구마 유래의 MYB1a 유전자를 제공하는데, 바람직하게는 서열번호 1의 염기서열 및 서열번호 3의 염기서열로 각각 이루어질 수 있다. 또한, 상기 염기 서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 염기서열 또는 서열번호 3의 염기 서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In addition, the present invention provides a gene for MYB1a derived from STS and sweet potato derived from the rhubarb rhubarb, preferably consisting of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence of SEQ ID NO: 3, respectively. In addition, homologues of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene is at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 3, respectively. Base sequences having homology. The "% sequence homology" for a polynucleotide is identified by comparing two optimally arranged sequences with a comparison region, wherein part of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) for the optimal alignment of the two sequences. It may include the addition or deletion (ie, gap) compared to).
용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로서 인위적인 수단에 의해 세포내 재도입된 것이다.The term “recombinant” refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. Recombinant cells can express genes or gene fragments that are not found in their natural form in either the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes are modified and intracellularly reintroduced by artificial means.
본 발명에서, 상기 RpSTS 또는 IbMYB1a 유전자 서열은 재조합 발현 벡터 내로 삽입될 수 있다. 용어 "재조합 발현 벡터"는 세균 플라스미드, 파아지, 효모 플라스미드, 식물 세포 바이러스, 포유동물 세포 바이러스, 또는 다른 벡터를 의미한다. 대체로, 임의의 플라스미드 및 벡터는 숙주 내에서 복제 및 안정화할 수 있다면 사용될 수 있다. 상기 발현 벡터의 중요한 특성은 복제 원점, 프로모터, 마커 유전자 및 번역 조절 요소(translation control element)를 가지는 것이다.In the present invention, the RpSTS or IbMYB1a gene sequence may be inserted into a recombinant expression vector. The term "recombinant expression vector" means a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector. In principle, any plasmid and vector can be used as long as it can replicate and stabilize in the host. An important feature of the expression vector is that it has an origin of replication, a promoter, a marker gene and a translation control element.
RpSTS 또는 IbMYB1a 유전자 서열 및 적당한 전사/번역 조절 신호를 포함하는 발현 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관내 재조합 DNA 기술, DNA 합성 기술 및 생체 내 재조합 기술 등을 포함한다. 상기 DNA 서열은 mRNA 합성을 이끌기 위해 발현 벡터 내의 적당한 프로모터에 효과적으로 연결될 수 있다. 또한 발현 벡터는 번역 개시 부위로서 리보좀 결합 부위 및 전사 터미네이터를 포함할 수 있다.Expression vectors comprising the RpSTS or IbMYB1a gene sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence can be effectively linked to a suitable promoter in the expression vector to drive mRNA synthesis. Expression vectors may also include ribosomal binding sites and transcription terminators as translation initiation sites.
본 발명의 재조합 벡터의 바람직한 예는 아그로박테리움 투머파시엔스와 같은 적당한 숙주에 존재할 때 그 자체의 일부, 소위 T-영역을 식물 세포로 전이시킬 수 있는 Ti-플라스미드 벡터이다. 다른 유형의 Ti-플라스미드 벡터(EP 0 116 718 B1호 참조)는 현재 식물 세포, 또는 잡종 DNA를 식물의 게놈 내에 적당하게 삽입시키는 새로운 식물이 생산될 수 있는 원형질체로 잡종 DNA 서열을 전이시키는데 이용되고 있다. Ti-플라스미드 벡터의 특히 바람직한 형태는 EP 0 120 516 B1호 및 미국 특허 제4,940,838호에 청구된 바와 같은 소위 바이너리(binary) 벡터이다. 본 발명에 따른 DNA를 식물 숙주에 도입시키는데 이용될 수 있는 다른 적합한 벡터는 이중 가닥 식물 바이러스(예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래될 수 있는 것과 같은 바이러스 벡터, 예를 들면 비완전성 식물 바이러스 벡터로부터 선택될 수 있다. 그러한 벡터의 사용은 특히 식물 숙주를 적당하게 형질전환하는 것이 어려울 때 유리할 수 있다.Preferred examples of recombinant vectors of the invention are Ti-plasmid vectors capable of transferring part of themselves, the so-called T-region, to plant cells when present in a suitable host such as Agrobacterium tumerfaciens. Another type of Ti-plasmid vector (see EP 0 116 718 B1) is used to transfer hybrid DNA sequences to protoplasts from which current plant cells or new plants can be produced that properly insert hybrid DNA into the plant's genome. have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors, such as those which can be derived from double stranded plant viruses (eg CaMV) and single stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous especially when it is difficult to properly transform a plant host.
발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 것이다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 그 예로는 글리포세이트(glyphosate) 또는 포스피노트리신(phosphinothricin)과 같은 제초제 저항성 유전자, 카나마이신(kanamycin), G418, 블레오마이신(Bleomycin), 하이그로마이신(hygromycin), 클로람페니콜(chloramphenicol)과 같은 항생제 내성 유전자, aadA 유전자 등이 있으나, 이에 한정되는 것은 아니다.The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinothricin, kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. Resistance gene, aadA gene, and the like, but are not limited thereto.
본 발명의 재조합 벡터에서, 프로모터는 CaMV 35S, 퍼록시다아제 유전자 (SWPA2) 프로모터, 액틴, 유비퀴틴, pEMU, MAS, 히스톤 프로모터, Clp 프로모터일 수 있으나, 이에 제한되지 않는다. "프로모터"란 용어는 구조 유전자로부터의 DNA 업스트림의 영역을 의미하며 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다. "식물 프로모터"는 식물 세포에서 전사를 개시할 수 있는 프로모터이다. "구성적(constitutive) 프로모터"는 대부분의 환경 조건 및 발달 상태 또는 세포 분화하에서 활성이 있는 프로모터이다. 형질전환체의 선택이 각종 단계에서 각종 조직에 의해서 이루어질 수 있기 때문에 구성적 프로모터가 본 발명에서 바람직할 수 있다. 따라서, 구성적 프로모터는 선택 가능성을 제한하지 않는다.In the recombinant vector of the present invention, the promoter may be a CaMV 35S, a peroxidase gene (SWPA2) promoter, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter, but is not limited thereto. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constitutive promoters may be preferred in the present invention because selection of the transformants may be made by various tissues at various stages. Thus, the constitutive promoter does not limit the selection possibilities.
본 발명의 재조합 벡터에서, 통상의 터미네이터를 사용할 수 있으며, 그 예로는 스포라민 유전자 (SPO) 터미네이터, 노팔린 신타아제 (NOS), 벼 α-아밀라아제 RAmy1 A 터미네이터, 파세올린 (phaseoline) 터미네이터, 아그로박테리움 투메파시엔스 (Agrobacterium tumefaciens)의 옥토파인 (Octopine) 유전자의 터미네이터, 대장균의 rrnB1/B2 터미네이터 등이 있으나, 이에 한정되는 것은 아니다. 터미네이터의 필요성에 관하여, 그러한 영역이 식물 세포에서의 전사의 확실성 및 효율을 증가시키는 것으로 일반적으로 알려져 있다. 그러므로, 터미네이터의 사용은 본 발명의 내용에서 매우 바람직하다.In the recombinant vectors of the present invention, conventional terminators can be used, such as sporamin gene (SPO) terminator, nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, agro Terminator of the octopine gene of Agrobacterium tumefaciens ( Agrobacterium tumefaciens ), rrnB1 / B2 terminator of E. coli, but is not limited thereto. With regard to the need for terminators, such regions are generally known to increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly desirable in the context of the present invention.
본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주세포로서, 효모 (Saccharomyce cerevisiae), 곤충세포, 사람세포 (예컨대, CHO 세포주(Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 및 식물세포 등이 이용될 수 있다. 숙주세포는 바람직하게는 식물세포이다.When transforming the vector of the present invention to eukaryotic cells, as a host cell, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2) , 3T3, RIN and MDCK cell lines) and plant cells and the like can be used. The host cell is preferably a plant cell.
본 발명의 벡터를 숙주세포 내로 운반하는 방법은 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법, 및 유전자 밤바드먼트 등에 의해 벡터를 숙주세포 내로 주입할 수 있다.The method of transporting the vector of the present invention into the host cell may be performed by injecting the vector into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, or the like. can do.
또한, 본 발명은 상기 방법에 의해 제조된 야생형에 비해 스틸벤 생산이 증가된 형질전환 식물체 및 이의 종자를 제공한다.The present invention also provides a transgenic plant and its seed having increased stilbene production compared to the wild type produced by the method.
본 발명의 일 구현 예에서, 상기 식물체는 애기장대, 감자, 가지, 담배, 고추, 토마토, 우엉, 쑥갓, 상추, 도라지, 시금치, 근대, 고구마, 샐러리, 당근, 미나리, 파슬리, 배추, 양배추, 갓무, 수박, 참외, 오이, 호박, 박, 딸기, 대두, 녹두, 강낭콩, 완두 등의 쌍자엽 식물 또는 벼, 보리, 밀, 호밀, 옥수수, 사탕수수, 귀리, 양파 등의 단자엽 식물일 수 있으며, 바람직하게는 쌍자엽 식물이며, 더욱 바람직하게는 담배이다.In one embodiment of the invention, the plant is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, beetroot, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, It may be a dicotyledonous plant such as gat radish, watermelon, melon, cucumber, pumpkin, gourd, strawberry, soybean, green bean, kidney bean, pea, or monocotyledonous plant such as rice, barley, wheat, rye, corn, sugarcane, oats, onions, Preferably it is a dicotyledonous plant, More preferably, it is a tobacco.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.
실시예 1. 레스베라트롤 생합성 전사인자 Example 1 Resveratrol Biosynthetic Transcription Factors RpSTSRpSTS 유전자의 클로닝 Cloning of genes
레스베라트롤 생산시스템을 개발하기 위해 사용된 유전자는 장엽대황 (Rheum palmatum)으로부터 레스베라트롤 생합성 관련 전사인자 유전자 RpSTS(1176bp)를 분리하여 이용하였다. 장엽대황 시료는 농업진흥청 강원도 농업기술원 북부농업시험장에서 확보하였으며, 이 시료로부터 총 RNA를 분리하였다. Homology-based 프라이머를 이용하여 RT-PCR 및 overlap extension PCR 방법으로 분리하였다. 분리된 총 RNA는 First-Strand cDNA Synthesis Kit (Fermantas, 캐나다)를 이용하여 설명서대로 cDNA를 합성하였다. 합성된 cDNA를 주형으로 하여 Adavantage 2 polymerase mix (Clontech, 미국)를 이용하여 장엽대황 RpSTS 전사인자를 PCR을 통하여 증폭하였다. 본 실험에 사용한 프라이머 염기서열은 다음과 같다 : RpSTS_F(Nde): 5'-CATATGGCACCGGAGGAGT-3' (서열번호 5), RpSTS_R(Spe): 5'-ACTAGTTCAGGTAATTAGCGGC-3' (서열번호 6). PCR 증폭은 95℃에서 2분간 변성, 95℃에서 15초, 55℃에서 30초, 72℃에서 2분을 1 사이클로 하여 30 사이클 및 72℃에서 10분간 최종 연장반응에 의해 수행되었다. 증폭된 PCR 산물은 pT-Blunt 벡터 (solgent, 한국)에 클로닝한 후 염기서열을 분석하였다. 염기서열분석 결과 클로닝된 RpSTS 유전자는 총 1176bp로 구성되었다.The gene used to develop the resveratrol production system was used to isolate the resveratrol biosynthesis transcription factor RpSTS (1176bp) from Rheum palmatum. A sample of rhubarb was obtained from the Northern Agricultural Experiment Station, Kangwon-do Agricultural Research and Extension Services, and the total RNA was isolated from the sample. Homology-based primers were used for RT-PCR and overlap extension PCR methods. Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit (Fermantas, Canada). Using the synthesized cDNA as a template, a rhubarb RpSTS transcription factor was amplified by PCR using an Adavantage 2 polymerase mix (Clontech, USA). Primer sequences used in this experiment were as follows: RpSTS_F (Nde): 5'-CATATGGCACCGGAGGAGT-3 '(SEQ ID NO: 5), RpSTS_R (Spe): 5'-ACTAGTTCAGGTAATTAGCGGC-3' (SEQ ID NO: 6). PCR amplification was carried out by a final extension reaction at 30 cycles and 10 minutes at 72 ° C. with 2 cycles of denaturation at 95 ° C., 15 seconds at 95 ° C., 30 seconds at 55 ° C., and 2 minutes at 72 ° C. The amplified PCR product was cloned into pT-Blunt vector (solgent, Korea) and analyzed for sequencing. As a result of sequencing, the cloned RpSTS gene was composed of a total of 1176bp.
실시예 2. 테로스틸벤 생합성 전사인자 Example 2. Terostilbene Biosynthetic Transcription Factors VrROMTVrROMT 유전자의 클로닝 Cloning of genes
테로스틸벤 생산시스템을 개발하기 위해 사용된 유전자는 포도 (Vitis riparis)로부터 테로스틸벤 생합성 관련 전사인자 유전자 VrROMT(1074bp)를 분리하여 이용하였다. 포도 시료는 충청북도농업기술원 포도연구소에서 확보하였다. 이 유전자를 homology-based 프라이머를 이용하여 RT-PCR 및 overlap extension PCR 방법으로 분리하였다. 분리된 총 RNA는 First-Strand cDNA Synthesis Kit를 이용하여 설명서대로 cDNA를 합성하였다. 합성된 cDNA를 주형으로 하여 Adavantage 2 polymerase mix를 이용하여 포도 VrROMT 전사인자를 PCR을 통하여 증폭하였으며, 본 실험에 사용한 프라이머 염기서열은 다음과 같다 : VrROMT_F(Nde): 5'-CATATGGATTTGGCAAACG-3' (서열번호 7), VrROMT_R(Spe): 5'-ACTAGTTCAAGGATAAACCTCAA-3' (서열번호 8). VrROMT 유전자의 PCR 증폭은 95℃에서 2분간 변성, 95℃에서 15초, 55℃에서 30초, 72℃에서 2분을 1 사이클로 하여 30 사이클 및 72℃에서 10분간 최종 연장반응에 의해 수행되었다. 증폭된 PCR 산물은 pT-Blunt 벡터에 클로닝한 후 염기서열을 분석하였다. 염기서열분석 결과 클로닝된 VrROMT 유전자는 총 1074bp로 구성되었다. The gene used to develop the telostilbene production system was used to isolate the telostilbene biosynthesis transcription factor gene VrROMT (1074bp) from Vitis riparis. Grape samples were obtained from the Grape Research Institute, Chungcheongbuk-do Agricultural Research and Extension Services. This gene was isolated by RT-PCR and overlap extension PCR using homology-based primers. Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit. Using the synthesized cDNA as a template, the grape VrROMT transcription factor was amplified by PCR using an Adavantage 2 polymerase mix. The primer sequences used in this experiment were as follows: VrROMT_F (Nde): 5'-CATATGGATTTGGCAAACG-3 '( SEQ ID NO: 7), VrROMT_R (Spe): 5'-ACTAGTTCAAGGATAAACCTCAA-3 '(SEQ ID NO: 8). PCR amplification of the VrROMT gene was carried out by a final extension reaction for 30 minutes at 72 ° C and 10 minutes at 72 ° C for 2 minutes denaturation at 95 ° C, 15 seconds at 95 ° C, 30 seconds at 55 ° C, and 2 minutes at 72 ° C. The amplified PCR product was cloned into pT-Blunt vector and analyzed for sequencing. Sequence analysis revealed that the cloned VrROMT gene was composed of a total of 1074bp.
실시예 3. 안토시아닌 색소 생합성 전사인자 Example 3. Anthocyanin Pigment Biosynthesis Transcription Factors IbMYB1aIbMYB1a 유전자의 클로닝 Cloning of genes
안토시아닌 대량 생산시스템을 개발하기 위해 사용된 유전자는 자색고구마 (신자미 품종)로부터 안토시아닌 생합성 관련 전사인자 유전자 IbMYB1a를 분리하여 이용하였다. 자색고구마의 덩이뿌리에서 총 RNA를 분리하였다 (Kim CY et al. 2010, Physiologia Plantarum 139: 259-261). 분리된 총 RNA는 First-Strand cDNA Synthesis Kit를 이용하여 설명서대로 cDNA를 합성하였다. 합성된 cDNA를 주형으로 하여 Adavantage 2 polymerase mix를 이용하여 고구마 IbMYB1a 전사인자를 PCR을 통하여 증폭하였다. 본 실험에 사용한 프라이머 염기서열은 다음과 같다 : IbMYB1a-F: 5'-AGCTAAGAATTTCCGACACCCTTCAATA-3' (서열번호 9), IbMYB1a-R: 5'-GTGAATTTAACGCTTAGCTTAACAGTTCT-3' (서열번호 10). PCR 증폭은 95℃에서 2분간 변성, 95℃에서 15초, 55℃에서 30초, 72℃에서 2분을 1 사이클로 하여 30 사이클 및 72℃에서 10분간 최종 연장반응에 의해 수행되었다. 증폭된 PCR 산물은 pGEM-T Easy 벡터 (Promega, 미국)에 클로닝한 후 염기서열을 분석하였다. 염기서열분석 결과 클로닝된 IbMYB1a 유전자는 총 797bp로 구성되어 있고, ORF(open readin frame)는 750 bp로 구성되었다. The gene used to develop the anthocyanin mass production system was used by separating the anthocyanin biosynthesis transcription factor gene IbMYB1a from purple sweet potato ( Shinjami cultivar). Total RNA was isolated from tubers of purple sweet potatoes (Kim CY et al. 2010, Physiologia Plantarum 139: 259-261). Total RNA isolated was synthesized cDNA as described using the First-Strand cDNA Synthesis Kit. Using the synthesized cDNA as a template, the sweet potato IbMYB1a transcription factor was amplified by PCR using an Adavantage 2 polymerase mix. The primer sequences used in this experiment were as follows: IbMYB1a-F: 5'-AGCTAAGAATTTCCGACACCCTTCAATA-3 '(SEQ ID NO: 9), IbMYB1a-R: 5'-GTGAATTTAACGCTTAGCTTAACAGTTCT-3' (SEQ ID NO: 10). PCR amplification was carried out by a final extension reaction at 30 cycles and 10 minutes at 72 ° C. with 2 cycles of denaturation at 95 ° C., 15 seconds at 95 ° C., 30 seconds at 55 ° C., and 2 minutes at 72 ° C. The amplified PCR product was cloned into pGEM-T Easy vector (Promega, USA) and analyzed for sequencing. As a result of sequencing, the cloned IbMYB1a gene was composed of a total of 797bp, and the ORF (open readin frame) was composed of 750 bp.
실시예 4. 스틸벤류 생합성 유전자 식물체 과발현용 운반체 제작Example 4. Preparation of stilbene biosynthetic gene plant overexpression
상기 실시예 1, 2에서 클로닝된 RpSTS, VrROMT 유전자를 식물체에서 과발현 시켰을 때 레스베라트롤과 테로스틸벤의 생합성을 증대시킬 수 있는지를 알아보기 위하여, 카나마이신 저항성을 지니는 pGR0229의 식물발현 벡터 (Center for Application of Molecular Biology to International Agriculture, 호주)의 duplicated 35S 프로모터와 35S 터미네이터 사이에 RpSTS가 삽입된 벡터, duplicated 35S 프로모터와 35S 터미네이터 사이에 RpSTS 및 VrROMT가 삽입된 벡터를 각각 제작하였고, 각각을 1) pGR-Flag::RpSTS(pGR-STS), 2) pGR-HA::VrROMT-Flag::RpSTS(pGR-ROST)로 명명하였다(도 3).In order to investigate whether the RpSTS and VrROMT genes cloned in Examples 1 and 2 can be increased in plants, the biosynthesis of resveratrol and theoestylbene can be increased, the plant expression vector of pGR0229 having kanamycin resistance (Center for Application of Molecular Biology to International Agriculture, Australia) a duplicated 35S promoter and the 35S the RpSTS is inserted between the terminator vector, were respectively produced duplicated 35S promoter and the 35S vector RpSTS and VrROMT is inserted between the terminator, 1) pGR-Flag respectively :: RpSTS (pGR-STS), 2) pGR-HA :: VrROMT-Flag :: RpSTS (pGR-ROST) (Fig. 3).
실시예 5. 안토시아닌 색소 생합성 전사인자의 식물체 과발현용 운반체 제작Example 5 Preparation of Plant Overexpression Carrier of Anthocyanin Pigment Biosynthesis Transcription Factor
상기 실시예 3에서 클로닝된 IbMYB1a 유전자를 식물체에서 과발현 시켰을 때 안토시아닌 색소 합성을 증대시킬 수 있는지를 알아보기 위해서, 카나마이신 저항성을 지니는 pCAMBIA 2300의 식물발현 벡터 (Center for Application of Molecular Biology to International Agriculture, 호주)에 고구마 뿌리 고발현 스포라민 유전자 SPO 프로모터와 SPO 터미네이터 사이에 IbMYB1a 유전자의 전체 (open reading frame)가 삽입된 벡터, 산화 스트레스 유도성 고구마 퍼록시다아제 유전자 SWPA2 프로모터와 SPO 터미네이터 사이에 IbMYB1a 유전자의 전체 (open reading frame)가 삽입된 벡터를 각각 제작하였다. 상기 제작된 과발현 운반체는 3) pCam-SPO-IbMYB1a, 4) pCam-SWPA2-IbMYB1a로 각각 명명하였다(도 3).In order to determine whether anthocyanin pigment synthesis can be enhanced when the IbMYB1a cloned in Example 3 is overexpressed in a plant, a plant expression vector of pCAMBIA 2300 having kanamycin resistance (Center for Application of Molecular Biology to International Agriculture, Australia) ) potato roots and expression of Spokane's Grameen gene IbMYB1a genes between the SPO promoter and SPO whole IbMYB1a genes between terminator (open reading frame) is inserted into a vector, oxidative stress-induced potato peroxidase are kinase gene SWPA2 promoter and SPO terminator Each vector with an open reading frame was produced. The produced overexpressing carriers were named 3) pCam-SPO-IbMYB1a and 4) pCam-SWPA2-IbMYB1a (FIG. 3).
실시예 6. 스틸벤류 및 안토시아닌 색소 생합성 담배 형질전환체의 제조Example 6 Preparation of Stilbenes and Anthocyanin Pigment Biosynthetic Tobacco Transformants
상기 실시예 4, 5에서 제작된 각각의 1) pGR-Flag::RpSTS(pGR-STS), 2) pGR-HA::VrROMT-Flag::RpSTS(pGR-ROST), 3) pCam-SPO-IbMYB1a 및 4) pCam-SWPA2-IbMYB1a 식물발현벡터를 아그로박테리움 투메파시엔스 LBA4404 (H, R. and Willmitzer L. 1988, Nucleic Acids Res 16:9877)에 동결-해동 (freeze-thaw) 방법을 이용하여 형질전환하였다. 형질전환된 아그로박테리움과 담배 (Nicotiana tabaccum cv SR-1) 잎 절편을 공동 배양하여 담배를 형질전환하였다. 형질전환을 위해 대조군인 야생형 담배를 1mg/L의 NAA (a-naphthalene acetic acid), 1mg/L의 BA (6-benzyladenine), 10g/L의 수크로스 및 8g/L의 한천 (agar)이 첨가된 MS 배지에서 3일간 공동배양한 후, 형질전환된 재분화 식물체를 얻기 위하여 1mg/L의 NAA, 1mg/L의 BA, 300mg/L의 카르베니실린, 100mg/L의 카나마이신, 10g/L의 수크로스 및 8g/L 한천이 첨가된 MS 배지에서 배양한 다음, 3주마다 계대배양 하였다. 재분화된 신초는 300mg/L의 카르베니실린, 100mg/L의 카나마이신, 10g/L의 수크로스 및 8g /L의 한천이 첨가된 MS 배지로 옮겨 발근을 유도하였다. 배양은 26℃, 16시간 광주기/8시간 암주기의 조건에서 배양하였고, 발근된 개체는 순화 후 화분으로 이식하여 온실에서 평균 기온 26℃ 이상의 조건에서 생육시켰다. 결과 IbMYB1a 유전자를 과발현시킨 형질전환식물체의 잎에서 안토시아닌 색소의 축적을 확인할 수 있었으며 SWPA2-IbMYB1a 형질전환 식물체보다 SPO-IbMYB1a 형질전환 식물체에서 안토시아닌 색소의 축적이 더 높은 것을 확인 하였다(도 2C).1) pGR-Flag :: RpSTS (pGR-STS), 2) pGR-HA :: VrROMT-Flag :: RpSTS (pGR-ROST), 3) pCam-SPO- IbMYB1a and 4) pCam-SWPA2-IbMYB1a plant expression vector was applied to Agrobacterium tumefaciens LBA4404 (H, R. and Willmitzer L. 1988, Nucleic Acids Res 16: 9877) using a freeze-thaw method. By transformation. Tobacco was transformed by co-culturing the transformed Agrobacterium and tobacco ( Nicotiana tabaccum cv SR-1) leaf sections. For transformation, a control wild-type tobacco was added with 1 mg / L of NAA (a-naphthalene acetic acid), 1 mg / L of BA (6-benzyladenine), 10 g / L of sucrose, and 8 g / L of agar. After 3 days co-culture in MS medium, 1 mg / L NAA, 1 mg / L BA, 300 mg / L carbenicillin, 100 mg / L kanamycin, 10 g / L Cross and 8 g / L agar were incubated in MS medium and then subcultured every three weeks. Regenerated shoots were transferred to MS medium to which 300 mg / L carbenicillin, 100 mg / L kanamycin, 10 g / L sucrose and 8 g / L agar were added to induce rooting. Cultures were incubated under conditions of 26 ℃, 16 hours photoperiod / 8 hours dark cycle, rooted individuals were transplanted into pollen after acclimatization and grown in a greenhouse at an average temperature of 26 ℃ or more. Results The accumulation of anthocyanin pigments in the leaves of the transgenic plants overexpressing the IbMYB1a gene was confirmed, and the accumulation of anthocyanin pigments in the SPO-IbMYB1a transgenic plants was higher than in the SWPA2-IbMYB1a transgenic plants (Fig. 2C).
실시예 7. 교배를 통한 스틸벤류 고함유 담배 형질전환체의 제조Example 7 Preparation of Stilbene-Containing Tobacco Transformants by Crossing
스틸벤 생합성효소 STS의 발현은 p-쿠마릴-CoA를 레스베라톨로 전환시킨다. 그러나 식물체내에 생합성되는 p-쿠마릴-CoA의 양은 한정적이기 때문에 스틸벤 생합성효소만의 과발현 유도를 통해 많은 양의 스틸벤 화합물을 식물체에서 얻기란 쉽지 않다. 스틸벤 생합성 경로의 중요 전구물질인 p-쿠마릴-CoA의 농도를 높일 수 있는 대사공학적 조절이 필요하다(도 2A).Expression of stilbene biosynthesis STS converts p-coumaryl-CoA to resveratol. However, since the amount of p-coumariyl-CoA biosynthesized in plants is limited, it is not easy to obtain a large amount of stilbene compounds from plants through induction of overexpression of stilbene biosynthesis only. Metabolic control is needed to increase the concentration of p-coumariyl-CoA, an important precursor of the stilbene biosynthetic pathway (FIG. 2A).
애기장대에서 IbMYB1a를 과발현 시켰을 경우 페닐알라닌으로부터 p-쿠마릴-CoA를 생합성하는데 요구되는 유전자 PAL, C4H, 4CL의 발현이 증가되는 패턴을 확인(도 2A)하였고 IbMYB1a 유전자를 담배식물체에 삽입하여 대사 플럭스 (Flux)를 조절함으로써 스틸벤 화합물이 생합성 되는데 필요한 전구체를 확보하고자 하였다. 그리하여 상시 실시예 6에서 페닐프로파노이드 합성 경로의 단계를 조절하는 MYB 유전자를 과발현하는 형질전환체를 제작하고, 스틸벤 생합성 유전자 STS를 과발현하는 형질전환 식물체 또한 제작(도 2B)하여 교배 시켰다. 그 후 F1 세대에서 STS와 MYB 또는 ROST와 MYB 유전자를 함께 과발현하는 형질전환 식물체를 각각 분리하고, 확보하였다(도 2).If sikyeoteul overexpressing IbMYB1a in Arabidopsis genes required for biosynthesis of the p- Kumar reel -CoA from phenylalanine PAL, C4H, check patterns that increase the expression of 4CL (Fig. 2A) was metabolic flux by inserting the gene into a tobacco plant IbMYB1a By controlling the (Flux) to obtain the precursor required for the biosynthesis of the stilbene. Thus, in Example 6, a transformant overexpressing the MYB gene that regulates the stage of the phenylpropanoid synthesis pathway was prepared , and a transgenic plant overexpressing the stilbene biosynthetic gene STS was also produced (FIG. 2B) and crossed. Then was isolated transgenic plant that over-expression with the STS and MYB or ROST and MYB genes in the F1 generation, respectively, and secured (Fig. 2).
실시예 8. 형질전환 담배의 레스베라트롤 생합성 단백질의 발현 분석Example 8 Analysis of Expression of Resveratrol Biosynthetic Protein in Transgenic Tobacco
상기 실시예 7에서 선발된 각각의 형질전환 담배에서 RpSTS와 VrROMT 유전자가 안정적으로 번역되는지 확인하기 위하여 각각을 Flag-tag와 HA-tag으로 웨스턴 블럿 분석을 수행하였다. 카나마이신이 포함된 배지에서 선발된 형질전환 담배 식물체를 대상으로 분석한 결과, 레스베라트롤과 테로스틸벤의 전사인자인 RpSTS와 VrROMT 유전자가 안정적으로 발현되어 번역되는 것을 확인하였다(도 4, 5 및 6).In order to confirm that the RpSTS and VrROMT genes are stably translated in each transgenic tobacco selected in Example 7, Western blot analysis was performed using Flag-tag and HA-tag. Analysis of the transgenic tobacco plants selected from the medium containing kanamycin confirmed that the RpSTS and VrROMT genes, which are transcription factors of resveratrol and terrostilbene, were stably expressed and translated (FIGS. 4, 5, and 6). .
실시예 9. 형질전환 담배의 표현형 관찰Example 9 Phenotypic Observation of Transgenic Tobacco
상기 실시예 7에서 선발된 각각의 형질전환된 담배 식물체는 비형질전환 대조구 및 RpSTS와 IbMYB1a 유전자가 각각 발현된 형질전환 대조구에 비교하여 유식물체에서 외형적인 표현형의 차이가 나타났다. RpSTS와 VrROMT 유전자가 도입된 형질전환 담배 식물체의 경우 꽃잎에서는 안토시아닌 색소의 감소를 확인할 수 있었고 잎에서는 색소의 변화가 보이지 않았다(도 7). IbMYB1a 유전자가 도입된 형질전환 담배 식물체의 경우 꽃잎과 잎 등 식물체 전반에 걸쳐 안토시아닌 색소의 증가를 관찰하였다(도 7). 교배담배 식물체의 경우 꽃잎에서 안토시아닌 색소가 STS 발현 형질전환 식물체에 비해 증가하고 비형질전환 대조구에 비해 감소되는 표현형과 비형질전환 대조구에 비해 증가하고 MYB 발현 형질전환 식물체에 비해 감소한 표현형의 두 종류 꽃이 확인되었다(도 7). 그리고 교배담배 식물체의 잎에서는 비형질전환 식물체와 MYB 발현 형질전환 식물체 잎에서 나타나는 표현형 중간정도의 안토시아닌 색소 축적을 확인하였다(도 7).In each of the transformed tobacco plants selected in Example 7, the phenotypic differences in the seedlings were compared with those of the non-transformed control and the transformed control expressing the RpSTS and IbMYB1a genes, respectively. In the transgenic tobacco plants in which the RpSTS and VrROMT genes were introduced, the anthocyanin pigment was decreased in the petals and the pigment was not seen in the leaves (FIG. 7). In the transgenic tobacco plants to which the IbMYB1a gene was introduced, an anthocyanin pigment was observed to increase throughout the plants, including petals and leaves (FIG. 7). In the case of mating tobacco plants, two kinds of flowers, the phenotype of anthocyanin pigments in petals increased compared to STS expressing transgenic plants, decreased compared to non transgenic control plants, and increased compared to non transgenic control plants and reduced phenotypes compared to MYB expressing transgenic plants. This was confirmed (FIG. 7). In the leaves of the mating tobacco plants, the anthocyanin pigment accumulation of the phenotype was observed in non-transgenic plants and MYB- expressing transgenic plants (Fig. 7).
실시예 10. 형질전환 교배 담배에서의 스틸벤 화합물 함량 분석Example 10 Analysis of Stilbene Compound Content in Transgenic Cross Tobacco
상기 실시예 7에서 선발된 각각의 형질전환 교배 담배 식물체의 각 부위를 채취하여 액체질소에 담궈 동결시킨 후 막자사발을 이용하여 분말화 하였다. 상기 식물체 분말 0.5g을 5ml의 80% 메탄올로 추출한 다음 원심분리 함으로써 상층부의 식물체 추출물을 얻었다. 추출한 여액은 공기를 주입하여 건조시키고 건조물을 300㎕ 80% 메탄올에 녹이고 0.2㎛ PTFE 필터(hydrophilic, ADVANTEC, 일본)하여 HPLC (High-performance liquid chromatography) 분석에 이용하였다. HPLC는 agilent technology 1200 시리즈를 사용하여 분석하였다. 펌프 시스템은 quaternary pump를 사용하였으며 컬럼은 agilent사의 ZORBAX SB-18 (5mm, 4.6 X 150mm)을 사용하였으며 이동상은 물 (A, 0.05% trifluoroacetic acid)과 아세토니트릴(B, 0.05% trifluoroacetic acid)을 이용하여 기울기 용리를 이용하여 분석하였다. 상기 형질전환체의 잎과 꽃잎에서 HPLC를 이용한 스틸벤 화합물 생합성 효율을 조사한 결과, RpSTS 유전자만 단독으로 과발현된 형질전환체에 비해 RpSTS와 IbMYB1a 유전자의 과발현 또는 RpSTS, VrROMT와 IbMYB1a 유전자가 과발현된 식물체 라인의 잎에서 각각 스틸벤 화합물의 생합성이 증가하는 양상을 나타내었다(도 8). Each part of each transgenic hybrid tobacco plant selected in Example 7 was taken, immersed in liquid nitrogen and frozen, and then powdered using a mortar and pestle. 0.5 g of the plant powder was extracted with 5 ml of 80% methanol, and then centrifuged to obtain a plant extract of the upper layer. The extracted filtrate was dried by injecting air, and the dried product was dissolved in 300 µl 80% methanol, and 0.2 µm PTFE filter (hydrophilic, ADVANTEC, Japan) was used for HPLC (High-performance liquid chromatography) analysis. HPLC was analyzed using agilent technology 1200 series. The pump system used quaternary pump, and the column was agilent ZORBAX SB-18 (5mm, 4.6 X 150mm) and the mobile phase was water (A, 0.05% trifluoroacetic acid) and acetonitrile (B, 0.05% trifluoroacetic acid). Was analyzed using gradient elution. Examining the leaf and a stilbene compound biosynthesis efficiency by HPLC in the petals of the transformants result, RpSTS gene only overexpression RpSTS and IbMYB1a gene compared to the transformants alone overexpression or RpSTS, VrROMT and IbMYB1a gene-overexpressed plant The biosynthesis of stilbene compounds was increased in the leaves of the lines, respectively (FIG. 8).
실시예 11. 형질전환 교배 담배에서의 스틸벤 화합물 정성 분석Example 11 Qualitative Assay of Stilbene in Transgenic Cross Tobacco
상기 실시예 10에서 준비된 각각의 형질전환 교배 담배 식물체 추출물을 이용하여 LC-MS (liquid chromatography-mass spectrometry) 분석에 이용하였다. LC-MS는 agilent technology 1200 시리즈를 사용하여 분석하였다. 펌프 시스템은 binary pump를 사용하였으며 컬럼은 agilent사의 ZORBAX SB-18 (5mm, 4.6 X 150mm)을 사용하였으며 이동상은 물 (A, 0.05% trifluoroacetic acid)과 아세토니트릴(B, 0.05% trifluoroacetic acid)을 이용하여 기울기 용리를 이용하여 분석하였다. MS 분석결과 HPLC 상에서 보이는 5개의 피크는 순서대로 trans-piceid, cis-piceid, piceid methyl ether 및 resveratrol methyl ether-O-hexoside 인 것으로 확인되었다(도 9 및 표 1).Each transgenic hybrid tobacco plant extract prepared in Example 10 was used for liquid chromatography-mass spectrometry (LC-MS) analysis. LC-MS was analyzed using agilent technology 1200 series. The pump system used binary pump, and the column was agilent's ZORBAX SB-18 (5mm, 4.6 X 150mm) and the mobile phase used water (A, 0.05% trifluoroacetic acid) and acetonitrile (B, 0.05% trifluoroacetic acid). Was analyzed using gradient elution. MS analysis showed that the five peaks shown on the HPLC were trans-piceid, cis-piceid, piceid methyl ether and resveratrol methyl ether-O-hexoside in order (FIG. 9 and Table 1).
[규칙 제91조에 의한 정정 24.09.2015]　
표 1
Figure WO-DOC-TABLE-1
 [Revision 24.09.2015 under Rule 91]
Table 1
Figure WO-DOC-TABLE-1

Claims (10)

  1. (a) 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질을 코딩하는 RpSTS 유전자를 과발현하는 형질전환 식물체를 제조하는 단계;(a) preparing a transgenic plant that overexpresses the RpSTS gene encoding a stilbene synthase (STS) protein derived from Rheum palmatum ;
    (b) 고구마 (Ipomoea batatas) 유래 MYB1a 단백질을 코딩하는 IbMYB1a 유전자를 과발현하는 형질전환 식물체를 제조하는 단계; 및(b) preparing a transgenic plant that overexpresses the IbMYB1a gene encoding MYB1a protein from sweet potato ( Ipomoea batatas ); And
    (c) 상기 (a) 단계의 RpSTS 유전자 과발현 형질전환 식물체와 상기 (b) 단계의 IbMYB1a 유전자 과발현 형질전환 식물체를 교배하여 RpSTS 유전자 및 IbMYB1a 유전자를 동시에 과발현하는 형질전환 식물체를 선발하는 단계를 포함하여 제조하는 것을 특징으로 하는 야생형에 비해 스틸벤 (stilbene) 생산이 증가된 형질전환 식물체의 제조 방법.(c) including the step of selecting the transgenic plant overexpressing RpSTS gene and IbMYB1a genes crossed the (a) step of RpSTS gene transgenic plant and the step (b) of IbMYB1a gene transgenic plants A method for producing a transformed plant, wherein stilbene production is increased compared to wild type, characterized in that the production.
  2. 제1항에 있어서, 상기 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질은 서열번호 2의 아미노산 서열로 이루어진 것을 특징으로 하는 형질전환 식물체의 제조 방법.The method of claim 1, wherein the stilbene synthase (STS) protein derived from Rheum palmatum is composed of the amino acid sequence of SEQ ID NO: 2.
  3. 제1항에 있어서, 상기 고구마 (Ipomoea batatas) 유래 MYB1a 단백질은 서열번호 4의 아미노산 서열로 이루어진 것을 특징으로 하는 형질전환 식물체의 제조 방법.The method of claim 1, wherein the sweet potato ( Ipomoea batatas ) -derived MYB1a protein is a method for producing a transgenic plant, characterized in that consisting of the amino acid sequence of SEQ ID NO: 4.
  4. 제1항에 있어서, 상기 RpSTS 유전자를 과발현하는 형질전환 식물체는 장엽대황 (Rheum palmatum) 유래 스틸벤 생합성 효소 (STS, stilbene synthase) 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물체를 형질전환시켜 제조하는 것을 특징으로 하는 형질전환 식물체의 제조 방법.The transgenic plant overexpressing the RpSTS gene is prepared by transforming the plant with a recombinant vector comprising a gene encoding a stilbene synthase (STS) protein derived from Rheum palmatum . Method for producing a transgenic plant, characterized in that.
  5. 제1항에 있어서, 상기 IbMYB1a 유전자를 과발현하는 형질전환 식물체는 고구마 (Ipomoea batatas) 유래 MYB1a 단백질을 코딩하는 유전자를 포함하는 재조합 벡터로 식물체를 형질전환시켜 제조하는 것을 특징으로 하는 형질전환 식물체의 제조 방법.The method of claim 1, wherein the transgenic plant overexpressing the IbMYB1a gene is prepared by transforming the plant with a recombinant vector comprising a gene encoding a MYB1a protein derived from sweet potato ( Ipomoea batatas ) Way.
  6. 제1항에 있어서, 상기 스틸벤은 레스베라트롤 배당체 또는 메틸 유도체 형태의 스틸벤인 것을 특징으로 하는 형질전환 식물체의 제조 방법.The method of claim 1, wherein the stilbene is a stilbene in the form of resveratrol glycoside or methyl derivative.
  7. 제1항 내지 제6항 중 어느 한 항의 방법에 의해 제조된 야생형에 비해 스틸벤 (stilbene) 생산이 증가된 형질전환 식물체.A transgenic plant with increased stilbene production compared to the wild type produced by the method of any one of claims 1 to 6.
  8. 제7항에 있어서, 상기 식물체는 쌍자엽 식물인 것을 특징으로 하는 형질전환 식물체.8. The transgenic plant of claim 7, wherein the plant is a dicotyledonous plant.
  9. 제8항에 있어서, 상기 쌍자엽 식물은 담배인 것을 특징으로 하는 형질전환 식물체.The transgenic plant of claim 8, wherein the dicotyledonous plant is tobacco.
  10. 제7항에 따른 식물체의 종자.Seeds of plants according to claim 7.
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