WO2015199435A1 - Procédé de préparation de plante transgénique ayant une meilleure production de stilbène et plante ainsi préparée - Google Patents

Procédé de préparation de plante transgénique ayant une meilleure production de stilbène et plante ainsi préparée 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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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C12N9/10—Transferases (2.)
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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

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Abstract

La présente invention concerne : un procédé de préparation d'une plante transgénique ayant une meilleure production de stilbène, par rapport à un type sauvage surexprimant en même temps un gène RpSTS et un gène IbMYB1a , par croisement d'une plante, ayant été transformée par un vecteur recombiné contenant un gène codant pour une protéine RpSTS, avec une plante ayant été transformée par un vecteur recombiné contenant un gène codant pour une protéine IbMYB1a ; et une plante transgénique préparée au moyen du procédé. On s'attend à ce que la production en masse, dans diverses plantes, de composés de stilbène présentant diverses activités physiologiques utiles soit bien plus facile.
PCT/KR2015/006409 2014-06-26 2015-06-24 Procédé de préparation de plante transgénique ayant une meilleure production de stilbène et plante ainsi préparée Ceased WO2015199435A1 (fr)

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KR20140040372A (ko) * 2012-09-26 2014-04-03 한국생명공학연구원 Ｒｏｍｔ 및 ｓｔｓ 유전자를 이용한 식물의 스틸벤 함량을 증가시키고 화색을 변화시키는 방법

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