WO2012108630A2 - Sbta gene derived from synechocystis sp. pcc6803 and use thereof - Google Patents

Sbta gene derived from synechocystis sp. pcc6803 and use thereof Download PDF

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WO2012108630A2
WO2012108630A2 PCT/KR2012/000599 KR2012000599W WO2012108630A2 WO 2012108630 A2 WO2012108630 A2 WO 2012108630A2 KR 2012000599 W KR2012000599 W KR 2012000599W WO 2012108630 A2 WO2012108630 A2 WO 2012108630A2
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plant
sbta
gene
plants
pcc6803
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WO2012108630A9 (en
WO2012108630A3 (en
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유장렬
민성란
정원중
박연일
정화지
김종현
박지현
유재일
이정희
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한국생명공학연구원
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention is of the genus Origin of PCC6803 SbtA Gene and use thereof, and more specifically, Synechocystis sp.) Origin of PCC6803 SbtA (Sodium dependent bicarbonate transporter) gene to increase the flame resistance of plants, the method SbtA Method for producing a transgenic plant with increased salt resistance using a gene, a transgenic plant with increased salt resistance produced by the method and seeds thereof, SbtA Method of increasing plant biomass using genes, said SbtA
  • the present invention relates to a method for producing a transformed plant having an increased biomass using a gene, a transformed plant having an increased biomass produced by the method, and a seed thereof.
  • Plants use solar energy to oxidize water, release oxygen, and reduce carbon dioxide and organics, mainly in the form of sugars.
  • Photosynthesis is the most important chemical reaction that harvests this energy, synthesizes organic matter and uses it as the energy source of life.
  • Most terrestrial plants that do photosynthesis are classified as C3 plants, which directly fix CO 2 in the air through the Calvin Rubisco.
  • Carbon fixation of C3 plants is catalyzed by Rubisco (ribulose bisphosphate carboxylase / oxygenas; Rubisco), and Rubisco has a carboxylase activity and an oxygenase activity, which is a competitive reaction. Fixed efficiency is low.
  • CO 2 immobilization of C4 plants in the tropical environment is anatomically formed by co-operation of foliar cells and endospertic cells, and the first carboxylation of the plants is catalyzed by PEP (phosphoenylpyruvate) carboxylase instead of Rubisco, C4 acids malic and aspartic acid become the first photosynthetic intermediates (Chollet et al., 1996, Annu Rev Plant Physiol Plant Mol Biol 47: 273-298).
  • PEP carboxylase has a high affinity for bicarbonate ions as a substrate and is saturated at CO 2 concentrations similar to atmospheric levels, and does not react with O 2 because it reacts with bicarbonate ions as a substrate.
  • C4 plants can conserve water while fixing CO 2 at high speed even when the pores are partially closed. In addition to the high photosynthesis rate, C4 plants are more productive in subtropical regions due to their higher efficiency of nitrogen and water use.
  • Photosynthetic microalgae have a mechanism for concentrating CO 2 around Rubisco, which compensates for the drawbacks.
  • Micro-algae of the CO 2 concentrator agent CCM CO 2 -concentrating mechanism
  • the transporter acting as a bicarbonate ion pump of the plasma membrane Increase the concentration of inorganic carbon in the cell.
  • the concentrated bicarbonate ions are converted to CO 2 by carbonic anhydrase, and CO 2 enters the Calvin cycle and binds to Rubisco to undergo carboxylation.
  • Rubisco's activity appears to be competitive with each other depending on the concentration of CO 2 and O 2 near the active site. Rubisco's CO 2 affinity is originally low, but it can show high photosynthetic rate by increasing intracellular CO 2 concentration. . Although the amount of the CO 2 concentration in the chloroplast small increase CO 2 fixing efficiency can be improved very high (Price et al, 2008, J Exp Bot 59:. 1441-1461).
  • IctB gene a bicarbonate ion transporter derived from Cyanobacterium Synechococcus PCC 7942, was used for C3 plant Arabidopsis and tobacco.
  • the photosynthetic efficiency and growth rate were increased (Lieman-Hurwitz et al., 2003, Plant Biotechnol J 1: 43-50 ), and when the IctB gene was expressed in rice, photosynthetic efficiency and enzyme activity were increased and CO 2 was increased. Reward points have been lowered and biomass increases have been reported (Yang et al., 2008, Energy from the Sun 20: 1244-1246).
  • bicarbonate transporters in cyanobacteria that are used for various CO 2 enrichment mechanisms.
  • SbtA has high affinity with bicarbonate ions, so it can increase the intracellular CO 2 concentration by efficiently absorbing CO 2 intracellularly even in low CO 2 concentration environment, and as a sodium ion / bicarbonate ion sympoter, SbtA Absorption of bicarbonate ions through is reported to be dependent on sodium ions (Shibata et al., 2002, J Biol Chem 227: 18658-18664).
  • SbtA is also present in Synechocystis PCC6803 as a single-subunit bicarbonate transporter, and is easier to operate than multi-subunit transporters. Transformation is advantageous (Price et al., 2008, J Exp Bot 59: 1441-1461).
  • Cyanobacteria is a primitive form of chloroplast, which contains the outer membrane and chlorophyll a, and is a photosynthetic bacterium that photosynthesizes like plants, and has great genetic and structural similarities with chloroplasts.
  • cyanobacterium cinecosistis PCC6803 is the first photosynthetic prokaryotic genome analysis has been used as a model organism (Tabei et al., 2007, Biochem Biophys Res Commun 355: 1045-1050). Recently, cyanobacteria have been widely used for research on photosynthesis and genetic regulation of photosynthesis due to the characteristics and information by genome sequencing.
  • SbtA sodium dependent bicarbonate transporter gene was isolated from Cinecosystis PCC6803 in consideration of this point, and was introduced to the C3 plant tobacco to develop a plant with increased CO2 utilization efficiency and biomass production.
  • tobacco plants with the SbtA gene were introduced through the chloroplast transformation system, and the enzyme activity, photosynthetic efficiency, carbohydrate content, growth and biomass production were investigated for the T2 generation.
  • Korean Patent Registration No. 10-0895611 A method of increasing the flame resistance of a plant using a PCC6803 derived SyFBP / SBPase gene is disclosed.
  • Korean Patent No. 10-0990333 discloses a method of increasing the flame resistance of a plant using a barley-derived NHX gene.
  • the present invention is derived from the above-described needs, and the present inventors have confirmed that the present invention is confirmed by the increase in resistance to salt stress and the increase in biomass in transgenic tobacco plants to which the SbtA gene derived from the cinecosistis PCC6803 is introduced. It was completed.
  • the present invention (Synechocystis) sp.) Origin of PCC6803 SbtA It provides a method of increasing the flame resistance of plants by using a sodium dependent bicarbonate transporter gene.
  • the present invention is a genus Origin of PCC6803 SbtA Provided is a method for producing a transgenic plant having increased salt resistance using a gene.
  • the present invention also provides a transgenic plant with increased flame resistance produced by the above method and its seeds.
  • the present invention is a genus From PCC6803 SbtA It provides a composition for increasing the salt resistance of a plant, comprising a gene.
  • the present invention is a genus Origin of PCC6803 SbtA Provided are methods for increasing plant biomass using genes.
  • the present invention is a genus Origin of PCC6803 SbtA Provided is a method for producing a transgenic plant having increased biomass using a gene.
  • the present invention also provides a transgenic plant and seed thereof having increased biomass produced by the above method.
  • the present invention is a genus From PCC6803 SbtA Provided is a composition for increasing biomass of a plant, including a gene.
  • SbtA Transforming genes to plant chloroplasts not only increases resistance to salt stress but also increases biomass. Therefore SbtA Genes are expected to be useful for developing transgenic plants that are resistant to salt stress environments.
  • the vector consists of the Rclp promoter, SbtA target gene, two marker genes ( aadA and GFP ), the rrnB1 / B2 terminator and insertion regions ( trnI and trnA ) in the chloroplast genome.
  • Fig. 2 shows the sineticosis introduced into tobacco chloroplasts.
  • SbtA The results of introduction and expression of the gene are shown.
  • A trnA Vector Control and SySbtA Using Probes Southern blot analysis of transgenic plants.
  • B SbtA Northern blot analysis using probes.
  • FIG. 3 shows GFP expression of protoplasts isolated from the leaves of chloroplast transgenic tobacco plants. Expression of GFP was observed by fluorescence microscopy.
  • FIG. 5 shows phenotypes of transgenic tobacco plants and control plants.
  • A Phenotype of tobacco plants grown for 7 weeks.
  • B Phenotype of tobacco plants at initiation of flowering for 14 weeks.
  • C The appearance of the seventh leaf from the top of tobacco plants grown for nine weeks. The plants were grown in Phytotron conditions under a cycle of 16 hours light and 8 hours cancer at 25 ° C. and 60% relative humidity.
  • Figure 6 shows the growth rate, growth weight and dry weight of control plants and SySbtA transgenic tobacco plants.
  • A Ultra long under phytotron conditions after transplantation into soil.
  • B fresh weight.
  • Figure 7 compares the number of leaves and stem diameter of control plants and SySbtA transgenic plants.
  • A Number of leaves.
  • Figure 8 shows the result of measuring the chlorophyll content.
  • A The amounts of chlorophyll a, b and total chlorophyll.
  • Figure 11 shows the results of the measurement of the water-soluble sugar and starch content.
  • A The amount of fructose, glucose and sucrose.
  • B the amount of starch.
  • the present invention (Synechocystis) sp.) Origin of PCC6803 SbtA Plant cells were transformed into a recombinant vector containing a sodium dependent bicarbonate transporter gene. SbtA It provides a method for increasing the flame resistance of a plant comprising overexpressing a gene.
  • the recombinant vector is preferably a chloroplast transformation vector, and more preferably, may be the RclpGAH :: SbtA vector described in FIG. 1, but is not limited thereto.
  • the recombinant vector is transformed into plant cells, preferably chloroplasts of plant cells.
  • the SbtA gene may be preferably composed of the nucleotide sequence of SEQ ID NO: 1.
  • the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include.
  • 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 reintroduced into cells by artificial means.
  • the SbtA gene sequence can 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 SbtA gene sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill 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 currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced which 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, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter.
  • 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 possibility of selection.
  • terminators can be used, for example nopalin synthase (NOS), rice ⁇ -amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens) Terminator of the octopine gene, and the rrnB1 / B2 terminator of E. coli, but are not limited thereto.
  • NOS nopalin synthase
  • rice ⁇ -amylase RAmy1 A terminator phaseoline terminator
  • Agrobacterium tumefaciens Agrobacterium tumefaciens
  • Terminator of the octopine gene and the rrnB1 / B2 terminator of E. coli, but are 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. Therefore, the use of terminators is highly desirable in the context of the present invention.
  • a host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.
  • yeast Saccharomyce cerevisiae
  • insect cells human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) as host cells.
  • human cells e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) as host cells.
  • HepG2, 3T3, RIN and MDCK cell lines and plant cells and the like can be used.
  • the host cell is preferably a plant cell.
  • the method of carrying the vector of the present invention into a host cell is performed by using the CaCl 2 method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method.
  • the vector may be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, or the like. Can be.
  • the present invention (Synechocystis) sp.) Origin of PCC6803 SbtA It provides a method for producing a transgenic plant with increased flame resistance comprising the steps of transforming plant cells with a recombinant vector comprising a sodium dependent bicarbonate transporter gene and regenerating the plant from the transformed plant cells.
  • the SbtA The gene may consist of the nucleotide sequence of SEQ ID NO: 1.
  • the method of the present invention comprises transforming plant cells with a recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumefiaciens .
  • the method also includes the step of regenerating the transgenic plant from said transformed plant cell.
  • the method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.
  • Transformed plant cells should be re-differentiated into whole plants. Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for many different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.).
  • the present invention also provides a transgenic plant with increased flame resistance produced by the above method and its seeds.
  • the plant may be a dicotyledonous plant, but is not limited thereto.
  • the dicotyledonous plant is tobacco.
  • the dicotyledonous plants are Asteraceae (Dolaceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae (Primulaceae), Plumbaginaceae, Persimmonaceae (Ebenaceae) , Styracaceae, Stink bug, Symplocaceae, Ash (Oleaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae, Apocynaceae , Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae).
  • Gesneriaceae Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranun
  • the present invention consists of the nucleotide sequence of SEQ ID NO: 1, Synechocystis genus (Synechocystis sp.) From PCC6803 SbtA It provides a composition for increasing the salt resistance of plants, comprising a sodium dependent bicarbonate transporter gene.
  • the composition consists of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient SbtA It includes a gene, which can increase the flame resistance of the plant by transforming the gene into the chloroplast of the plant.
  • the present invention (Synechocystis) sp.) Origin of PCC6803 SbtA Plant cells were transformed into a recombinant vector containing a sodium dependent bicarbonate transporter gene.
  • SbtA It provides a method of increasing the biomass of a plant comprising overexpressing a gene.
  • the recombinant vector is preferably a chloroplast transformation vector, and more preferably, may be the RclpGAH :: SbtA vector described in FIG. 1, but is not limited thereto.
  • the recombinant vector is transformed into plant cells, preferably chloroplasts of plant cells.
  • the SbtA The gene may consist of the nucleotide sequence of SEQ ID NO: 1.
  • the present invention (Synechocystis) sp.) Origin of PCC6803 SbtA It provides a method for producing a transformed plant with an increased biomass comprising the step of transforming the plant cell with a recombinant vector comprising a sodium dependent bicarbonate transporter gene and regenerating the plant from the transformed plant cell. .
  • the SbtA The gene may consist of the nucleotide sequence of SEQ ID NO: 1.
  • the present invention also provides a transgenic plant and seed thereof having increased biomass produced by the above method.
  • the plant may be a dicotyledonous plant, but is not limited thereto.
  • the dicotyledonous plants are as described above.
  • the dicotyledonous plant is tobacco.
  • the present invention consists of the nucleotide sequence of SEQ ID NO: 1, Synechocystis genus (Synechocystis sp.) From PCC6803 SbtA It provides a composition for increasing the biomass of a plant, including a sodium dependent bicarbonate transporter gene.
  • the composition consists of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient SbtA It includes a gene, and by converting the gene into the chloroplast of the plant it is possible to increase the biomass of the plant.
  • Cigarettes in this experiment were Nicotiana tabacum L. cv. Samsun varieties were used, surface sterilization in 10% (v / v) sodium hypoglolite (5% effective chlorine content) solution for 15 minutes, washed three times with sterile water, and washed with MS medium containing 3% sucrose. Leaves incubated for about 5 weeks in a culture chamber maintained at 25 ° C., 16 hours light condition and 30 ⁇ mol / m 2 / s were used as chloroplast transforming materials.
  • the amplified fragment was digested with EcoRV and then subcloned with the RclpGAH vector and Rclp- SbtA Was cut with XhoI / SpeI and blunted and then introduced into TIA (PvuII), a tobacco chromatin transformation vector (FIG. 1).
  • Plant material preparations for particle balm-borders were prepared by sterile filter paper (diameter 70 mm) in MS medium to which 1 mg / L benzyladenin (BA) and 0.1 mg / L naphthaleneacetic acid (NAA) were added one day prior to the balm-border.
  • Advantec Toyo, Tokyo Japan 3 cm tobacco leaves were placed on the leaves, with the back of the leaves facing up.
  • DNA was coated on gold particles (0.6 ⁇ m) and subjected to a balm body at 9 cm height using a particle delivery system (PDS 1000 / He, Bio-Rad, USA) and a 1,100 psi (7,580 MPa) rupture disk. It was.
  • the tobacco leaves were placed in dim light for 48 hours and then cut into 5 ⁇ 5 mm, 1 mg / L benzyladenin (BA) and 0.1 mg / L naphthaleneacetic acid (NAA), 500 mg / L It was dentured in MS medium (spectral selection medium) containing spectinomycin. After about 4-6 weeks, the leaves of shoots generated from the transformed cells were chopped and placed on the same medium, and then re-differentiated through three repeated selection (rounding) processes. The roots were transferred, purified, and grown under conditions of 25 ° C. and 16 hours of phytotron.
  • BA 1 mg / L benzyladenin
  • NAA 0.1 mg / L naphthaleneacetic acid
  • Total genomic DNA was isolated from tobacco leaves using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), 4 ⁇ g of genomic DNA was cut with BamHI and BglII and electrophoresed on 0.8% agarose gel, followed by Zeta-probe GT Blotting. Membrane (Bio-Rad, Hercules, Calif.) was transferred using 10 ⁇ SSC. The insertion of the transgene was confirmed by labeling the trnA probe (P1, 150 bp) PCR-amplified on the trAI-containing BamHI-BglII DNA fragment in the chromatin genome with a radioisotope [ ⁇ - 32 P] dCTP.
  • Prehybridization and hybridization were performed overnight at 65 ° C. using 0.25 M sodium phosphate buffer (pH 7.2) containing 7% (w / v) SDS, Wash Buffer I (20 mM sodium phosphate buffer, pH 7.2). ) And 5% SDS) and Wash Buffer II (20 mM sodium phosphate buffer (pH 7.2) and 1% SDS) at 65 ° C. for 15 minutes each, and then the membrane is image plate (Fuji Film) After 3 hours exposure to the band was confirmed.
  • One SbtA gene segment (P2 probe) was labeled with [ ⁇ - 32 P] dCTP and hybridization and washing were the same as the Southern blotting method.
  • Protoplasts were isolated to observe GFP fluorescence. Take the leaves of tobacco grown in soil for 2 weeks and remove the epidermis, and then in enzyme solution (1-1.5% Cellulase R10, 0.2-0.4% Mecerozyme R10, 0.4 M mannitol, 20 mM MES, pH 5.7 and 0.1% BSA). Put and treated for 3-4 hours in the dark. The separated protoplasts were observed by fluorescence microscopy.
  • T2 seeds were healed in MS medium containing NaCl 0 mM, 25 mM and 100 mM, respectively, and then cultured at 25 ° C. for 16 hours under light conditions, and 9 days after germination The root length of was investigated.
  • chlorophyll a and b The content of chlorophyll a and b was measured by taking 100 mg of 10-week-old tobacco leaves, frozen with liquid nitrogen, crushed, and adding 1 mL of 80% acetone and stirring for 20 minutes in the dark to obtain a supernatant. Chlorophyll was completely isolated by repeating the above procedure three times, and the measurement was performed at a wavelength of 645 nm and 663 nm using a spectrophotometer (UV-2450, SHIMADZU Inc., Japan) (Jeong et al., 2002, Plant Physiol 129: 112-121).
  • Starch was measured for glucose by hydrolysis of the remaining pellets with amylase and amyloglucosidase. Sugar and starch were measured for absorbance of NADPH at 340 nm using a UV-Spectorphotometer (UV-2450, SHIMADZU Inc., Japan).
  • Chloroplast transformation vectors include trn I and trnA, insertion sequences for homologous recombination, and spectinomycin resistance selection markers along with the SbtA gene, a target gene found in PCC 6803 of the genus Scincosistis, with RclpGAH as the backbone. Phosphorus aadA gene and GFP reporter gene were used, and the clp promoter found in rice and the rrnB1 / B2 terminator of Escherichia coli were used (FIG. 1).
  • T1 seeds received from T0 plants were germinated and selected in spectinomycin medium, and Southern and Northern blot analysis revealed that the genes introduced in T1 plants were expressed and homozygous (homoplasmic).
  • the generation was once again advanced and the experiment was conducted on the T2 generation.
  • Southern blot analysis using trnA probe on T2 plants revealed bands of approximately 4.5 kb in all three independent chloroplast transformants, approximately 3.3 kb in the vector control group, and in wild-type. A band of 0.88 kb was detected (FIG. 2).
  • SySbtA In order to determine the salt resistance of the transgenic plants, the response to the salt was examined. T2 seeds were seeded in medium supplemented with NaCl 0 mM, 25 mM and 100 mM, respectively, and root development was observed after 9 days. In the medium without NaCl, that is, under normal conditions, the SySbtA plants showed faster root growth in the roots, whereas the vector control group showed slower root growth and developed lateral roots (FIG. 4A). Root growth in the state without the addition of NaCl, which is a general condition, is different from each other. Thus, the root growth when the length of the root is 100 is expressed as a relative value.
  • SySbtA T2 plants were grown in phytotron environment, and the height, leaves, stem diameter, fresh weight and dry weight of the plants were measured. SySbtA grew rapidly after transfer to soil compared to the control and began to bloom from week 13 (FIG. 5). As a result of comparing the height of the flowering period, SySbtA plants showed a high growth rate of about 50% (FIG. 6A). As a result of comparing the fresh weight and dry weight at the flowering period, the fresh weight was 36-50% higher in SySbtA, especially the dry weight was higher by 50% and more than double in the first line (Figs. 6B and 6C). . Conifers also showed 14-24% more SySbtA plants (FIG. 7A), and the diameter of the stems was also increased by 6-9% (FIG. 7B).
  • the CO 2 compensation point is the CO 2 exchange the zero point, i.e. the minimum CO 2 environment because CO 2 compensation point lower the plant even in a low concentration of CO 2 environment for there plants can survive being adapted to use the CO 2 efficiently It means that you have increased your ability to do so.
  • SySbtA line 1 and line 4 were 12-16% lower than the vector control (Fig. 10). This result is equivalent to the Ci-curve (Ci-curve) and the SySbtA plant was found to increase the CO 2 utilization efficiency.

Abstract

The present invention relates to: a method for enhancing salt tolerance of plants using a SbtA (Sodium dependent bicarbonate transporter) gene derived from Synechocystis sp. PCC6803; a method for preparing transgenic plants with enhanced salt tolerance using the SbtA gene; transgenic plants with enhanced salt tolerance prepared by the same method and seeds thereof; a method for increasing biomass of plants using the SbtA gene; a method for preparing the transgenic plants with increased biomass using the SbtA gene; and transgenic plants with increased biomass using the SbtA gene and seeds thereof.

Description

시네코시스티스 속 ＰＣＣ6803 유래 ＳｂｔＡ 유전자 및 이의 용도SC PatA Gene Derived from the CPC6803 Genus
본 발명은 시네코시스티스 속 PCC6803 유래 SbtA 유전자 및 이의 용도에 관한 것으로서, 더욱 상세하게는 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 이용하여 식물의 내염성을 증가시키는 방법, 상기 SbtA 유전자를 이용한 내염성이 증가된 형질전환 식물체의 제조 방법, 상기 방법에 의해 제조된 내염성이 증가된 형질전환 식물체 및 이의 종자, 상기 SbtA 유전자를 이용하여 식물의 바이오매스를 증가시키는 방법, 상기 SbtA 유전자를 이용한 바이오매스가 증가된 형질전환 식물체의 제조 방법, 상기 방법에 의해 제조된 바이오매스가 증가된 형질전환 식물체 및 이의 종자에 관한 것이다.The present invention is of the genus Origin of PCC6803SbtA Gene and use thereof, and more specifically, Synechocystis sp.) Origin of PCC6803SbtA(Sodium dependent bicarbonate transporter) gene to increase the flame resistance of plants, the methodSbtAMethod for producing a transgenic plant with increased salt resistance using a gene, a transgenic plant with increased salt resistance produced by the method and seeds thereof,SbtAMethod of increasing plant biomass using genes, saidSbtAThe present invention relates to a method for producing a transformed plant having an increased biomass using a gene, a transformed plant having an increased biomass produced by the method, and a seed thereof.
식물은 태양에너지를 이용하여 물을 산화시키며 산소를 방출하고 이산화탄소와 유기물을 주로 당의 형태로 환원시킨다. 광합성은 이러한 에너지를 수확하고 유기물질을 합성하여 생명의 에너지원으로 이용하는 가장 중요한 화학 반응이라고 할 수 있다. 광합성을 하는 대부분의 육상식물은 캘빈회로의 루비스코를 통해 공기중의 CO2를 바로 고정하는 C3 식물로써 구분된다. C3 식물의 탄소고정은 루비스코 (ribulose bisphosphate carboxylase/oxygenas; Rubisco)에 의하여 촉매되며, 루비스코는 카르복실라제(carboxylase) 활성과 옥시게나제(oxygenase) 활성을 동시에 지니고 있어 경쟁적으로 반응하기 때문에 탄소 고정 효율이 낮다. 또한 루비스코의 산화반응에 의한 광호흡은 탄소고정 능력을 30~50%까지 저하시키며 가뭄, 고조도 및 고온 등의 스트레스 조건에서는 탄소 고정율이 지속적으로 감소된다(Sharkey, 1988, Physiologia Plantarum 73:147-152).Plants use solar energy to oxidize water, release oxygen, and reduce carbon dioxide and organics, mainly in the form of sugars. Photosynthesis is the most important chemical reaction that harvests this energy, synthesizes organic matter and uses it as the energy source of life. Most terrestrial plants that do photosynthesis are classified as C3 plants, which directly fix CO 2 in the air through the Calvin Rubisco. Carbon fixation of C3 plants is catalyzed by Rubisco (ribulose bisphosphate carboxylase / oxygenas; Rubisco), and Rubisco has a carboxylase activity and an oxygenase activity, which is a competitive reaction. Fixed efficiency is low. In addition, photorespiration by Rubisco's oxidation decreases the carbon fixation capacity by 30-50%, and the carbon fixation rate is continuously decreased under stress conditions such as drought, high illumination and high temperature (Sharkey, 1988, Physiologia Plantarum 73: 147). -152).
반면 열대환경에서 나타나는 C4 식물의 CO2 고정은 해부학적으로 엽육세포와 유관속초 세포가 협동하여 이루어지며, 상기 식물의 최초의 카르복시화는 루비스코 대신 PEP(phosphoenylpyruvate) 카르복시화 효소에 의하여 촉매되며, C4 산인 말산 및 아스파르트산이 최초의 광합성 중간 산물이 된다(Chollet et al., 1996, Annu Rev Plant Physiol Plant Mol Biol 47:273-298). PEP 카르복시화 효소는 기질인 중탄산 이온에 대한 친화도가 높아 대기 수준과 비슷한 CO2 농도에서도 포화되며, 기질로 중탄산 이온과 반응하기 때문에 O2와는 반응하지 않는다. 이처럼 PEP 카르복시화 효소의 활성이 높기 때문에 C4 식물은 기공을 부분적으로 닫아도 CO2를 빠른 속도로 고정하면서도 물을 보존할 수 있다. 이렇듯 C4 식물은 높은 광합성율 외에도 질소와 물 이용 효율이 높아 아열대 지역에서 생산성이 높다.On the other hand, CO 2 immobilization of C4 plants in the tropical environment is anatomically formed by co-operation of foliar cells and endospertic cells, and the first carboxylation of the plants is catalyzed by PEP (phosphoenylpyruvate) carboxylase instead of Rubisco, C4 acids malic and aspartic acid become the first photosynthetic intermediates (Chollet et al., 1996, Annu Rev Plant Physiol Plant Mol Biol 47: 273-298). PEP carboxylase has a high affinity for bicarbonate ions as a substrate and is saturated at CO 2 concentrations similar to atmospheric levels, and does not react with O 2 because it reacts with bicarbonate ions as a substrate. Because of the high activity of PEP carboxylase, C4 plants can conserve water while fixing CO 2 at high speed even when the pores are partially closed. In addition to the high photosynthesis rate, C4 plants are more productive in subtropical regions due to their higher efficiency of nitrogen and water use.
최근에는 이러한 C4 식물과 광합성 조류에서 나타나는 CO2 농축 메커니즘을 C3 식물에 도입하기 위한 연구가 진행되고 있다. C4 식물의 광합성 관련 주요 효소들의 유전자를 조작함으로써 C3 식물의 광합성 효율을 높이는 연구가 진행되고 있다(Miyao and Fukayama, 2003, Arch Biochem Biophys 414:197-203). 벼에 옥수수의 PEPC (phosphoenylpyruvate carboxylase), PPDK (pyruvate orthophosphate dikinase) 및 NADP-ME (NADP-malic enzyme) 유전자를 각각 도입한 결과, 높은 효소활성도를 보였으며(Fukayama et al., 2001, Plant Physiol 127:1136-1146), PEPC와 NADP-ME를 함께 발현시킨 담배의 경우 광호흡율이 감소되었다(Hausler et al., 2002, J Exp Bot 53:591-607).Recently, studies have been conducted to introduce CO 2 enrichment mechanisms in C4 plants and photosynthetic algae into C3 plants. Research on enhancing photosynthetic efficiency of C3 plants by engineering genes of key enzymes related to photosynthesis of C4 plants (Miyao and Fukayama, 2003, Arch Biochem Biophys 414: 197-203). The introduction of the corn phosphoenylpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK) and NADP-ME (NADP-malic enzyme) genes into rice showed high enzymatic activity (Fukayama et al., 2001, Plant Physiol 127). : 1136-1146), the photorespiratory rate was decreased in cigarettes expressing PEPC and NADP-ME (Hausler et al., 2002, J Exp Bot 53: 591-607).
한편 광합성 미세 조류는 루비스코 주변에서 CO2를 농축하는 메커니즘을 가지고 있어 결점을 보완한다. 미세 조류의 CO2 농축기작인 CCM (CO2-concentrating mechanism)은 광합성 미세 조류가 낮은 CO2 환경에서 무기 탄소를 농축할 수 있는 능력을 발달시키는데, 원형질막의 중탄산 이온펌프로 작용하는 트랜스포터(transporter)를 사용하여 세포내 무기 탄소의 농도를 높여준다. 농축된 중탄산 이온은 탄산탈수소효소(carbonic anhydrase)에 의하여 CO2로 전환되며, CO2는 캘빈회로로 들어가 루비스코와 결합하여 카르복시화가 진행된다. 루비스코의 활성은 활성부위 근처에서의 CO2와 O2의 농도에 따라 서로 경쟁적으로 나타나는데, 루비스코의 CO2 친화도는 원래 낮지만 세포내 CO2 농도를 높여 줌으로써 높은 광합성 속도를 나타낼 수 있다. 엽록체 내에 CO2 농도가 적은 양이 증가하더라도 CO2 고정효율은 매우 높게 향상될 수 있다(Price et al., 2008, J Exp Bot 59:1441-1461).Photosynthetic microalgae, on the other hand, have a mechanism for concentrating CO 2 around Rubisco, which compensates for the drawbacks. Micro-algae of the CO 2 concentrator agent CCM (CO 2 -concentrating mechanism) is sikineunde photosynthetic micro algae developed the ability to concentrate the inorganic carbon in the low CO 2 environment, the transporter acting as a bicarbonate ion pump of the plasma membrane (transporter) Increase the concentration of inorganic carbon in the cell. The concentrated bicarbonate ions are converted to CO 2 by carbonic anhydrase, and CO 2 enters the Calvin cycle and binds to Rubisco to undergo carboxylation. Rubisco's activity appears to be competitive with each other depending on the concentration of CO 2 and O 2 near the active site. Rubisco's CO 2 affinity is originally low, but it can show high photosynthetic rate by increasing intracellular CO 2 concentration. . Although the amount of the CO 2 concentration in the chloroplast small increase CO 2 fixing efficiency can be improved very high (Price et al, 2008, J Exp Bot 59:. 1441-1461).
이러한 미세 조류의 CO2 농축기작인 CCM 메커니즘을 C3식물에 도입하기 위한 연구로 시아노박테리움(Cyanobacterium) 시네코코커스(Synechococcus) PCC 7942 유래 중탄산 이온 트랜스포터인 IctB 유전자를 C3 식물인 애기장대와 담배에 도입시킨 결과, 광합성 효율과 생장율이 증가되었으며(Lieman-Hurwitz et al., 2003, Plant Biotechnol J 1:43-50), IctB 유전자를 벼에서 발현 시켰을 때 광합성 효율과 효소 활성이 증가되고 CO2 보상점이 낮아졌으며 바이오매스가 증가된 결과가 보고되었다(Yang et al., 2008, Energy from the Sun 20:1244-1246). 상기 연구결과는 CO2 농축 메커니즘에 관련된 유전자를 C3 식물에 발현시킴으로써 CO2 고정효율을 증가시키고, 물과 질소 이용 효율을 높이는 등 C3식물의 결점을 보완할 수 있는 가능성을 제시하고 있다. C4 효소를 C3 식물에서 발현시킴과 동시에 미세 조류의 CO2 농축 메커니즘을 적용한다면 CO2 고정을 극대화함은 물론, 단세포 C4-주기(single-cell C4-cycle) 시스템을 C3 식물에서 실현시킬 수 있는 원동력이 될 것이다.In order to introduce the CCM mechanism, which is a CO 2 enrichment mechanism of microalgae, to C3 plants, IctB gene, a bicarbonate ion transporter derived from Cyanobacterium Synechococcus PCC 7942, was used for C3 plant Arabidopsis and tobacco. As a result, the photosynthetic efficiency and growth rate were increased (Lieman-Hurwitz et al., 2003, Plant Biotechnol J 1: 43-50 ), and when the IctB gene was expressed in rice, photosynthetic efficiency and enzyme activity were increased and CO 2 was increased. Reward points have been lowered and biomass increases have been reported (Yang et al., 2008, Energy from the Sun 20: 1244-1246). The results suggest that the genes involved in the CO 2 enrichment mechanism can be expressed in C3 plants, thereby increasing the CO 2 fixation efficiency and improving the water and nitrogen utilization efficiency. By expressing the C4 enzyme in C3 plants and applying the CO 2 enrichment mechanism of microalgae, it is possible to maximize CO 2 fixation and to realize a single-cell C4-cycle system in C3 plants. It will be the driving force.
한편 시아노박테리아에는 여러 가지 CO2 농축기작에 사용되는 중탄산 이온 트랜스포터(bicarbonate transporter)가 존재한다. 그 중 SbtA는 중탄산 이온과 높은 친화력을 가지고 있어 낮은 CO2 농도의 환경에서도 CO2를 세포내로 효율적으로 흡수시킴으로써 세포내 CO2 농도를 높일 수 있으며, 나트륨 이온/중탄산 이온 심포터(sympoter)로써 SbtA를 통한 중탄산 이온의 흡수가 나트륨 이온에 의존적인 것으로 보고되었다(Shibata et al., 2002, J Biol Chem 227:18658-18664). 또한 SbtA는 시네코시스티스(Synechocystis) PCC6803에 단일-서브유닛 중탄산 이온 트랜스포터(Single-subunit bicarbonate transporter)로 존재하며, 다중-서브유닛 트랜스포터(multi-subunit transporter)에 비하여 조작이 용이하므로 고등식물로의 형질전환이 유리하다(Price et al., 2008, J Exp Bot 59:1441-1461). On the other hand, there are bicarbonate transporters in cyanobacteria that are used for various CO 2 enrichment mechanisms. Among them, SbtA has high affinity with bicarbonate ions, so it can increase the intracellular CO 2 concentration by efficiently absorbing CO 2 intracellularly even in low CO 2 concentration environment, and as a sodium ion / bicarbonate ion sympoter, SbtA Absorption of bicarbonate ions through is reported to be dependent on sodium ions (Shibata et al., 2002, J Biol Chem 227: 18658-18664). SbtA is also present in Synechocystis PCC6803 as a single-subunit bicarbonate transporter, and is easier to operate than multi-subunit transporters. Transformation is advantageous (Price et al., 2008, J Exp Bot 59: 1441-1461).
시아노박테리아는 엽록체의 원시적인 형태로 외막 및 엽록소 a를 함유하며, 식물처럼 광합성을 하는 광합성세균으로 엽록체와 유전적, 구조적 유사성이 크다. 특히 시아노박테리움 시네코시스티스 PCC6803은 광합성 원핵생물로는 최초로 게놈 분석이 완성되어 모델생물로서 이용되고 있다(Tabei et al., 2007, Biochem Biophys Res Commun 355:1045-1050). 상기 특성과 게놈 시퀀싱에 의한 정보로 인해 최근에는 시아노박테리아가 광합성 및 광합성의 유전적 조절 등의 연구에 많이 이용되고 있다. 본 발명에서는 이러한 점을 고려하여 시네코시스티스 PCC6803으로부터 SbtA (Sodium dependent bicarbonate transporter) 유전자를 분리하였으며, C3 식물인 담배에 도입함으로써 CO2 이용 효율과 바이오매스 생산이 증가된 식물체를 개발하고자 하였다. 이를 위하여 엽록체 형질전환 시스템을 통하여 SbtA 유전자가 도입된 담배 식물체를 제작하고, 확보된 T2 세대를 대상으로 효소활성과 광합성 효율, 탄수화물 함량, 생장량 및 바이오매스 생산 등을 조사하였다.Cyanobacteria is a primitive form of chloroplast, which contains the outer membrane and chlorophyll a, and is a photosynthetic bacterium that photosynthesizes like plants, and has great genetic and structural similarities with chloroplasts. In particular, cyanobacterium cinecosistis PCC6803 is the first photosynthetic prokaryotic genome analysis has been used as a model organism (Tabei et al., 2007, Biochem Biophys Res Commun 355: 1045-1050). Recently, cyanobacteria have been widely used for research on photosynthesis and genetic regulation of photosynthesis due to the characteristics and information by genome sequencing. In the present invention, SbtA (Sodium dependent bicarbonate transporter) gene was isolated from Cinecosystis PCC6803 in consideration of this point, and was introduced to the C3 plant tobacco to develop a plant with increased CO2 utilization efficiency and biomass production. To this end, tobacco plants with the SbtA gene were introduced through the chloroplast transformation system, and the enzyme activity, photosynthetic efficiency, carbohydrate content, growth and biomass production were investigated for the T2 generation.
한국등록특허 제10-0895611호에는 시네코시스티스 PCC6803 유래 SyFBP/SBPase 유전자를 이용하여 식물체의 내염성을 증가시키는 방법이 개시되어 있으며, 한국등록특허 제10-0990333호에는 보리 유래 NHX 유전자를 이용하여 식물체의 내염성을 증가시키는 방법이 개시되어 있다. Korean Patent Registration No. 10-0895611 A method of increasing the flame resistance of a plant using a PCC6803 derived SyFBP / SBPase gene is disclosed. Korean Patent No. 10-0990333 discloses a method of increasing the flame resistance of a plant using a barley-derived NHX gene.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 시네코시스티스 PCC6803 유래 SbtA 유전자를 도입시킨 형질전환 담배 식물체에서 염 스트레스에 대한 내성이 증가하고, 바이오매스가 증가한 것을 확인함으로써 본 발명을 완성하게 되었다.The present invention is derived from the above-described needs, and the present inventors have confirmed that the present invention is confirmed by the increase in resistance to salt stress and the increase in biomass in transgenic tobacco plants to which the SbtA gene derived from the cinecosistis PCC6803 is introduced. It was completed.
상기 과제를 해결하기 위해, 본 발명은 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 이용하여 식물의 내염성을 증가시키는 방법을 제공한다. In order to solve the above problems, the present invention (Synechocystis) sp.) Origin of PCC6803SbtAIt provides a method of increasing the flame resistance of plants by using a sodium dependent bicarbonate transporter gene.
또한, 본 발명은 시네코시스티스 속 PCC6803 유래 SbtA 유전자를 이용한 내염성이 증가된 형질전환 식물체의 제조 방법을 제공한다.In addition, the present invention is a genus Origin of PCC6803SbtAProvided is a method for producing a transgenic plant having increased salt resistance using a gene.
또한, 본 발명은 상기 방법에 의해 제조된 내염성이 증가된 형질전환 식물체 및 이의 종자를 제공한다. The present invention also provides a transgenic plant with increased flame resistance produced by the above method and its seeds.
또한, 본 발명은 시네코시스티스 속 PCC6803 유래의 SbtA 유전자를 포함하는, 식물체의 내염성 증가용 조성물을 제공한다.In addition, the present invention is a genus From PCC6803SbtA It provides a composition for increasing the salt resistance of a plant, comprising a gene.
또한, 본 발명은 시네코시스티스 속 PCC6803 유래 SbtA 유전자를 이용하여 식물의 바이오매스를 증가시키는 방법을 제공한다. In addition, the present invention is a genus Origin of PCC6803SbtAProvided are methods for increasing plant biomass using genes.
또한, 본 발명은 시네코시스티스 속 PCC6803 유래 SbtA 유전자를 이용한 바이오매스가 증가된 형질전환 식물체의 제조 방법을 제공한다. In addition, the present invention is a genus Origin of PCC6803SbtAProvided is a method for producing a transgenic plant having increased biomass using a gene.
또한, 본 발명은 상기 방법에 의해 제조된 바이오매스가 증가된 형질전환 식물체 및 이의 종자를 제공한다.The present invention also provides a transgenic plant and seed thereof having increased biomass produced by the above method.
또한, 본 발명은 시네코시스티스 속 PCC6803 유래의 SbtA 유전자를 포함하는, 식물체의 바이오매스 증가용 조성물을 제공한다.In addition, the present invention is a genus From PCC6803SbtA Provided is a composition for increasing biomass of a plant, including a gene.
본 발명의 시네코시스티스 속 PCC6803 유래 SbtA 유전자를 식물체의 엽록체에 형질전환시키면 염 스트레스에 대한 내성이 증가할 뿐만 아니라 바이오매스 또한 증가한다. 따라서 SbtA 유전자를 이용하면 염 스트레스 환경에 대해 저항성이 강한 형질전환 식물체를 개발하는데 유용할 것으로 기대된다.The genus Cinecosis of the present invention Origin of PCC6803SbtA Transforming genes to plant chloroplasts not only increases resistance to salt stress but also increases biomass. thereforeSbtA Genes are expected to be useful for developing transgenic plants that are resistant to salt stress environments.
도 1은 시네코시스티스 SbtA 유전자의 엽록체 형질전환 벡터를 나타낸다. 상기 벡터는 Rclp 프로모터, SbtA 표적 유전자, 2개의 마커 유전자(aadA 및 GFP), rrnB1/B2 터미네이터 및 엽록체 게놈 내의 삽입 영역(trnI 및 trnA)으로 이루어진다.1 shows the chloroplast transforming vector of the cinecosistis SbtA gene. The vector consists of the Rclp promoter, SbtA target gene, two marker genes ( aadA and GFP ), the rrnB1 / B2 terminator and insertion regions ( trnI and trnA ) in the chloroplast genome.
도 2는 담배 엽록체에 도입된 시네코시스티스 SbtA 유전자의 도입과 발현 결과를 나타낸다. (A) trnA 프로브를 이용한 벡터 대조군 및 SySbtA 형질전환 식물의 서던 블롯 분석. (B) SbtA 프로브를 이용한 노던 블롯 분석.Fig. 2 shows the sineticosis introduced into tobacco chloroplasts.SbtA The results of introduction and expression of the gene are shown. (A)trnA Vector Control and SySbtA Using Probes Southern blot analysis of transgenic plants. (B)SbtA Northern blot analysis using probes.
도 3은 엽록체 형질전환 담배 식물체의 잎으로부터 분리된 원형질체의 GFP 발현을 나타낸 것이다. GFP의 발현은 형광현미경으로 관찰하였다.Figure 3 shows GFP expression of protoplasts isolated from the leaves of chloroplast transgenic tobacco plants. Expression of GFP was observed by fluorescence microscopy.
도 4는 대조군 식물 및 SySbtA 형질전환 식물의 생장 및 뿌리 발달에 대한 염의 효과를 나타낸 것이다. (A) 0 mM, 25 mM 및 100 mM의 NaCl을 포함하는 배지에서 9일 동안 대조군 및 형질전환 식물을 직접 발아시킨 결과. (B) 뿌리 발달에 대한 다양한 염 농도의 효과.4 shows the effect of salt on growth and root development of control plants and SySbtA transgenic plants. (A) Direct germination of control and transgenic plants for 9 days in medium containing 0 mM, 25 mM and 100 mM NaCl. (B) Effect of varying salt concentrations on root development.
도 5는 형질전환 담배 식물 및 대조군 식물의 표현형을 나타낸 것이다. (A) 7주 동안 키운 담배 식물의 표현형. (B) 14주 동안 키운 개화 개시기의 담배 식물의 표현형. (C) 9주 동안 키운 담배 식물의 꼭대기로부터 7번째 잎의 모양. 식물은 25℃ 및 60% 상대습도로 16시간 광 및 8시간 암의 주기하의 피토트론(Phytotron) 조건에서 생장되었다. 5 shows phenotypes of transgenic tobacco plants and control plants. (A) Phenotype of tobacco plants grown for 7 weeks. (B) Phenotype of tobacco plants at initiation of flowering for 14 weeks. (C) The appearance of the seventh leaf from the top of tobacco plants grown for nine weeks. The plants were grown in Phytotron conditions under a cycle of 16 hours light and 8 hours cancer at 25 ° C. and 60% relative humidity.
도 6은 대조군 식물 및 SySbtA 형질전환 담배 식물의 생장율, 생중량 및 건중량을 나타낸 것이다. (A) 토양에 이식 후 피토트론 조건하의 초장. (B) 생중량. (C) 건조중량. 결과는 평균±표준편차로 나타내었다(n=10). Figure 6 shows the growth rate, growth weight and dry weight of control plants and SySbtA transgenic tobacco plants. (A) Ultra long under phytotron conditions after transplantation into soil. (B) fresh weight. (C) dry weight. The results are expressed as mean ± standard deviation (n = 10).
도 7은 대조군 식물 및 SySbtA 형질전환 식물체의 잎 수와 줄기 직경을 비교한 것이다. (A) 잎의 수. (B) 줄기 직경. 결과는 평균±표준편차로 나타내었다(n=10).Figure 7 compares the number of leaves and stem diameter of control plants and SySbtA transgenic plants. (A) Number of leaves. (B) stem diameter. The results are expressed as mean ± standard deviation (n = 10).
도 8은 엽록소 함량을 측정한 결과를 나타낸 것이다. (A) 엽록소 a, b 및 총 엽록소의 양. (B) 엽록소 a/b 비. 담배 식물은 10주 동안 키웠다. 결과는 평균±표준편차로 나타내었다(n=3).Figure 8 shows the result of measuring the chlorophyll content. (A) The amounts of chlorophyll a, b and total chlorophyll. (B) Chlorophyll a / b ratio. Tobacco plants were grown for 10 weeks. The results are expressed as mean ± standard deviation (n = 3).
도 9는 대조군 식물 및 SySbtA 형질전환 식물에서 세포내 CO2 농도에 따른 광합성율을 나타낸 것이다. 담배 식물은 9주 동안 키웠다. 700 μmol/m2/s의 광도에서 증가하는 세포내 CO2 농도(Ci)에 대한 CO2 동화율의 반응(n=7). 9 shows photosynthesis rate according to intracellular CO 2 concentration in control plants and SySbtA transgenic plants. Tobacco plants were grown for 9 weeks. Response of CO 2 assimilation to increasing intracellular CO 2 concentration (Ci) at a light intensity of 700 μmol / m 2 / s (n = 7).
도 10은 벡터 대조군 및 형질전환 담배 식물에서의 CO2 보상점을 나타낸 것이다. 보상점은 0~400 mL/L 범위의 CO2 농도에서 CO2 교환율을 측정하여 추정되었다. 담배 식물은 9주 동안 키웠다. 결과는 평균±표준편차로 나타내었다(n=4).10 shows CO 2 reward points in vector control and transgenic tobacco plants. The compensation point was estimated by measuring the CO 2 exchange rate at a CO 2 concentration in the range of 0 to 400 mL / L. Tobacco plants were grown for 9 weeks. The results are expressed as mean ± standard deviation (n = 4).
도 11은 수용성 당 및 전분 함량의 측정 결과를 나타낸 것이다. (A) 프룩토오스, 글루코오스 및 수크로오스의 양. (B) 전분의 양. 당 및 전분은 식물 꼭대기로부터 5번째에 해당하는 잎 추출물에서 측정되었다. 담배 식물은 10주 동안 키웠다. 결과는 평균±표준편차로 나타내었다(n=4).Figure 11 shows the results of the measurement of the water-soluble sugar and starch content. (A) The amount of fructose, glucose and sucrose. (B) the amount of starch. Sugar and starch were measured in the fifth leaf extract from the plant top. Tobacco plants were grown for 10 weeks. The results are expressed as mean ± standard deviation (n = 4).
본 발명의 목적을 달성하기 위하여, 본 발명은 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터를 식물세포에 형질전환시켜 SbtA 유전자를 과발현하는 단계를 포함하는 식물의 내염성을 증가시키는 방법을 제공한다. 상기 재조합 벡터는 바람직하게는 엽록체 형질전환 벡터이며, 더욱 바람직하게는 도 1에 기재된 RclpGAH::SbtA 벡터일 수 있으나, 이에 제한되지 않는다. 상기 재조합 벡터를 식물세포, 바람직하게는 식물세포의 엽록체에 형질전환시킨다.In order to achieve the object of the present invention, the present invention (Synechocystis) sp.) Origin of PCC6803SbtAPlant cells were transformed into a recombinant vector containing a sodium dependent bicarbonate transporter gene.SbtAIt provides a method for increasing the flame resistance of a plant comprising overexpressing a gene. The recombinant vector is preferably a chloroplast transformation vector, and more preferably, may be the RclpGAH :: SbtA vector described in FIG. 1, but is not limited thereto. The recombinant vector is transformed into plant cells, preferably chloroplasts of plant cells.
상기 SbtA 유전자는 바람직하게는 서열번호 1의 염기서열로 이루어질 수 있다. 또한, 상기 염기 서열의 변이체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 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.The SbtA gene may be preferably composed of the nucleotide sequence of SEQ ID NO: 1. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. 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 세포 내 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 reintroduced into cells by artificial means.
본 발명에서, 상기 SbtA 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 SbtA gene sequence can 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.
SbtA 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 SbtA gene sequence and appropriate transcriptional / translational control signals can be constructed by methods well known to those of skill 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 currently used to transfer hybrid DNA sequences to protoplasts from which plant cells or new plants can be produced which 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, 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, but is not limited to, CaMV 35S, actin, ubiquitin, pEMU, MAS, histone promoter, Clp promoter. 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 possibility of selection.
본 발명의 재조합 벡터에서, 통상의 터미네이터를 사용할 수 있으며, 그 예로는 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, for example nopalin synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens ( Agrobacterium tumefaciens) Terminator of the octopine gene, and the rrnB1 / B2 terminator of E. coli, but are 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 원핵세포에 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주세포는 당업계에 공지된 어떠한 숙주세포도 이용할 수 있으며, 예컨대, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스, 바실러스 츄린겐시스와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움, 세라티아 마르세슨스 및 다양한 슈도모나스 종과 같은 장내균과 균주 등이 있다. A host cell capable of continuously cloning and expressing the vector of the present invention in a prokaryotic cell while being stable can be used in any host cell known in the art, for example, E. coli JM109, E. coli BL21, E. coli RR1. , Bacillus genus strains, such as E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas Enterobacteria such as species and strains.
또한, 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.In addition, when transforming the vector of the present invention into eukaryotic cells, yeast ( Saccharomyce cerevisiae ), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293) as host cells. , 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, 숙주 세포가 원핵 세포인 경우, CaCl2 방법, 하나한 방법 (Hanahan, D., J. Mol. Biol., 166:557-580(1983)) 및 전기천공 방법 등에 의해 실시될 수 있다. 또한, 숙주세포가 진핵세포인 경우에는, 미세주입법, 칼슘포스페이트 침전법, 전기천공법, 리포좀-매개 형질감염법, DEAE-덱스트란 처리법, 및 유전자 밤바드먼트 등에 의해 벡터를 숙주세포 내로 주입할 수 있다.The method of carrying the vector of the present invention into a host cell is performed by using the CaCl 2 method or one method (Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) when the host cell is a prokaryotic cell. And the electroporation method. In addition, when the host cell is a eukaryotic cell, the vector may be injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, or the like. Can be.
또한, 본 발명은 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계 및 상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 내염성이 증가된 형질전환 식물체의 제조 방법을 제공한다. 바람직하게는, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어질 수 있다. In addition, the present invention (Synechocystis) sp.) Origin of PCC6803SbtAIt provides a method for producing a transgenic plant with increased flame resistance comprising the steps of transforming plant cells with a recombinant vector comprising a sodium dependent bicarbonate transporter gene and regenerating the plant from the transformed plant cells. Preferably, theSbtA The gene may consist of the nucleotide sequence of SEQ ID NO: 1.
본 발명의 방법은 본 발명에 따른 재조합 벡터로 식물 세포를 형질전환하는 단계를 포함하는데, 상기 형질전환은 예를 들면, 아그로박테리움 튜머파시엔스(Agrobacterium tumefiaciens)에 의해 매개될 수 있다. 또한, 본 발명의 방법은 상기 형질전환된 식물 세포로부터 형질전환 식물을 재분화하는 단계를 포함한다. 형질전환 식물 세포로부터 형질전환 식물을 재분화하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다.The method of the present invention comprises transforming plant cells with a recombinant vector according to the present invention, which transformation can be mediated by, for example, Agrobacterium tumefiaciens . The method also includes the step of regenerating the transgenic plant from said transformed plant cell. The method for regenerating the transformed plant from the transformed plant cell may use any method known in the art.
형질전환된 식물세포는 전식물로 재분화되어야 한다. 캘러스 또는 원형질체 배양으로부터 성숙한 식물의 재분화를 위한 기술은 수많은 여러 가지 종에 대해서 당업계에 주지되어 있다(Handbook of Plant Cell Culture, 1-5권, 1983-1989 Momillan, N.Y.).Transformed plant cells should be re-differentiated into whole plants. Techniques for regeneration of mature plants from callus or protoplast cultures are well known in the art for many different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.).
또한, 본 발명은 상기 방법에 의해 제조된 내염성이 증가된 형질전환 식물체 및 이의 종자를 제공한다. 바람직하게는, 상기 식물체는 쌍자엽 식물일 수 있으나, 이에 제한되지는 않는다. 바람직하게는, 상기 쌍자엽 식물은 담배이다.The present invention also provides a transgenic plant with increased flame resistance produced by the above method and its seeds. Preferably, the plant may be a dicotyledonous plant, but is not limited thereto. Preferably, the dicotyledonous plant is tobacco.
상기 쌍자엽 식물은 암매과(돌매화나무과, Diapensiaceae), 매화오리나무과(Clethraceae), 노루발과(Pyrolaceae), 진달래과(Ericaceae), 자금우과(Myrsinaceae), 앵초과(Primulaceae), 갯질경이과 (Plumbaginaceae), 감나무과(Ebenaceae), 때죽나무과(Styracaceae), 노린재나무과, 회목과(Symplocaceae), 물푸레나무과(목서과, Oleaceae), 마전과(Loganiaceae), 용담과(Gentianaceae), 조름나물과(Menyanthaceae), 협죽도과(마삭나무과, Apocynaceae), 박주가리과(Asclepiadaceae), 꼭두서니과(Rubiaceae), 꽃고비과(Polemoniaceae), 메꽃과(Convolvulaceae), 지치과(Boraginaceae), 마편초과(Verbenaceae), 꿀풀과(Labiatae), 가지과(Solanaceae), 현삼과(Scrophulariaceae), 능소화과(Bignoniaceae), 쥐꼬리망초과(Acanthaceae), 참깨과(Pedaliaceae), 열당과 (Orobanchaceae). 제스네리아과(Gesneriaceae), 통발과(Lentibulariaceae), 파리풀과(Phrymaceae), 질경이과(Plantaginaceae), 인동과(Caprifoliaceae), (연복초과 Adoxaceae), 마타리과(Valerianaceae), 산토끼꽃과(Dipsacaceae), 초롱꽃과 (Campanulaceae), 국화과(Compositae), 소귀나무과(Myricaceae), 가래나무과 (Juglandaceae), 버드나무과(Salicaceae), 자작나무과(Betulaceae), 너도 밤나무과(참나무과, Fagaceae), 느릅나무과(Ulmaceae), 뽕나무과(Moraceae), 쐐기풀과 (Urticaceae), 단향과(Santalaceae), 겨우살이과(Loranthaceae), 마디풀과(여뀌과, Polygonaceae), 자리공과(상륙과, Phytolaccaceae), 분꽃과(Nyctaginaceae), 석류풀과(Aizoaceae), 쇠비름과(Portulacaceae), 석죽과(Caryophyllaceae), 명아주과 (Chenopodiaceae), 비름과(Amaranthaceae), 선인장과(Cactaceae), 목련과(Magnoliaceae), 붓순나무과(Illiciaceae), 녹나무과(Lauraceae), 계수나무과 (Cercidiphyllaceae), 미나리아재비과(Ranunculaceae), 매자나무과(Berberidaceae), 으름덩굴과(Lardizabalaceae), 새모래덩굴과(방기과, Menispermaceae), 수련과(Nymphaeaceae), 붕어마름과(Ceratophyllaceae), 어항마름과(Cabombaceae), 삼백초과(Saururaceae), 후추과(Piperaceae), 홀아비꽃대과(Chloranthaceae), 쥐방울덩굴과(Aristolochiaceae), 다래나무과(Actinidiaceae), 차나무과(동백나무과, Theaceae), 물레나물과(Guttiferae), 끈끈이주걱과(Droseraceae), 양귀비과(Papaveraceae), 풍접초과(Capparidaceae), 십자화과(겨자과, Cruciferae), 플라타너스과(버즘나무과, Platanaceae), 조록나무과(금루매과, Hamamelidaceae), 꿩의비름과(돌나물과, Crassulaceae), 범의귀과(Saxifragaceae), 두충과(Eucommiaceae), 돈나무과(Pittosporaceae), 장미과(Rosaceae), 콩과(Leguminosae), 괭이밥과(Oxalidaceae), 쥐손이풀과(Geraniaceae), 한련과(Tropaeolaceae), 남가새과(Zygophyllaceae), 아마과(Linaceae), 대극과(Euphorbiaceae), 별이끼과(Callitrichaceae), 운향과(Rutaceae), 소태나무과(Simaroubaceae), 멀구슬나무과(Meliaceae), 원지과(Polygalaceae), 옻나무과(Anacardiaceae), 단풍나무과(단풍과, Aceraceae), 무환자나무과(Sapindaceae), 칠엽수과(Hippocastanaceae), 나도밤나무과(Sabiaceae), 봉선화과(물봉선과, Balsaminaceae), 감탕나무과(Aquifoliaceae), 노박덩굴과(화살나무과, Celastraceae), 고추나무과(Staphyleaceae), 회양목과 (Buxaceae), 시로미과(Empetraceae), 갈매나무과(Rhamnaceae), 포도과(Vitaceae), 담팔수과(Elaeocarpaceae), 피나무과(Tiliaceae), 아욱과(Malvaceae), 벽오동과 (Sterculiaceae), 팥꽃나무과(서향나무과, Thymelaeaceae), 보리수나무과 (Elaeagnaceae), 이나무과(Flacourtiaceae), 제비꽃과(Violaceae), 시계꽃과 (Passifloraceae), 위성류과(Tamaricaceae), 물별과(Elatinaceae), 베고니아과 (Begoniaceae), 박과(Cucurbitaceae), 부처꽃과(배롱나무과, Lythraceae), 석류나무과(Punicaceae), 바늘꽃과(Onagraceae), 개미탑과(Haloragaceae), 박쥐나무과 (Alangiaceae), 층층나무과(산수유나무과, Cornaceae), 두릅나무과(오갈피나무과, Araliaceae) 또는 산형과(미나리과)(Umbelliferae(Apiaceae))일 수 있으나, 이에 제한되지는 않는다. The dicotyledonous plants are Asteraceae (Dolaceae, Diapensiaceae), Asteraceae (Clethraceae), Pyrolaceae, Ericaceae, Myrsinaceae, Primaceae (Primulaceae), Plumbaginaceae, Persimmonaceae (Ebenaceae) , Styracaceae, Stink bug, Symplocaceae, Ash (Oleaceae), Loganiaceae, Gentianaceae, Menyanthaceae, Oleaceae, Apocynaceae , Asclepiadaceae, Rubiaceae, Polemoniaceae, Convolvulaceae, Boraginaceae, Verbenaceae, Labiatae, Solanaceae, Scrophulariaceae , Bignoniaceae, Acanthaceae, Sesame (Pedaliaceae), Fructose (Orobanchaceae). Gesneriaceae, Lentibulariaceae, Phrymaceae, Plantaginaceae, Caprifoliaceae, (Perox Adoxaceae), Valerianaceae, Dipsacaceae, Campanaceae ( Campanulaceae, Compositae, Myricaceae, Sapaceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Elmaceae, Moraceae , Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Landaceae, Phytolaccaceae, Nyctaginaceae, Pomegranate, Azizaceae (Portulacaceae), Caryophyllaceae, Chinopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cassia family, Cecidiphyllaceae, Ranunculus eae), Berberidaceae, Lardizabalaceae, Bird breeze (Mentaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Papaveraceae ), Capparidaceae, Cruciferaceae (Mustaceae, Cruciferae), Planeaceae (Plataceae, Platanaceae), Verruaceae, Hamamelidaceae, Pheasant (Snaphaceae, Crassulaceae), Panaxaceae (Saxifragaceae) Eucommiaceae, Pittosporaceae, Rosaceae, Leguminosae, Oxalidaceae, Geraniaceae, Tropaeolaceae, Zygophyllaceae, Linaceae Euphorbiaceae), star moss (Callitrichaceae), Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae Hippocastanaceae, Sabiaceae, Balsam (Balsaminaceae), Aquifoliaceae, Nova (Celastraceae), Staphyleaceae, Buxaceae, Empyaceae, Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Eraeagnaceae (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Buddha (Lythraceae), Pomegranate Punicac eae), Onagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hornaceae, Cornaceae), Arboraceae (Agaraceae) or Umbelliferae (Apiaceae) It may be, but is not limited thereto.
또한, 본 발명은 서열번호 1의 염기서열로 이루어진, 시네코시스티스 속(Synechocystis sp.) PCC6803 유래의 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는, 식물체의 내염성 증가용 조성물을 제공한다. 상기 조성물은 유효성분으로 서열번호 1의 염기서열로 이루어진 SbtA 유전자를 포함하며, 상기 유전자를 식물체의 엽록체에 형질전환시킴으로써 식물체의 내염성을 증가시킬 수 있는 것이다.In addition, the present invention consists of the nucleotide sequence of SEQ ID NO: 1, Synechocystis genus (Synechocystis sp.) From PCC6803SbtA It provides a composition for increasing the salt resistance of plants, comprising a sodium dependent bicarbonate transporter gene. The composition consists of the nucleotide sequence of SEQ ID NO: 1 as an active ingredientSbtA It includes a gene, which can increase the flame resistance of the plant by transforming the gene into the chloroplast of the plant.
또한, 본 발명은 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터를 식물세포에 형질전환시켜 SbtA 유전자를 과발현하는 단계를 포함하는 식물의 바이오매스를 증가시키는 방법을 제공한다. 상기 재조합 벡터는 바람직하게는 엽록체 형질전환 벡터이며, 더욱 바람직하게는 도 1에 기재된 RclpGAH::SbtA 벡터일 수 있으나, 이에 제한되지 않는다. 상기 재조합 벡터를 식물세포, 바람직하게는 식물세포의 엽록체에 형질전환시킨다. 바람직하게는, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어질 수 있다.In addition, the present invention (Synechocystis) sp.) Origin of PCC6803SbtAPlant cells were transformed into a recombinant vector containing a sodium dependent bicarbonate transporter gene.SbtAIt provides a method of increasing the biomass of a plant comprising overexpressing a gene. The recombinant vector is preferably a chloroplast transformation vector, and more preferably, may be the RclpGAH :: SbtA vector described in FIG. 1, but is not limited thereto. The recombinant vector is transformed into plant cells, preferably chloroplasts of plant cells. Preferably, theSbtA The gene may consist of the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명은 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계 및 상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 바이오매스가 증가된 형질전환 식물체의 제조 방법을 제공한다. 바람직하게는, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어질 수 있다. In addition, the present invention (Synechocystis) sp.) Origin of PCC6803SbtAIt provides a method for producing a transformed plant with an increased biomass comprising the step of transforming the plant cell with a recombinant vector comprising a sodium dependent bicarbonate transporter gene and regenerating the plant from the transformed plant cell. . Preferably, theSbtA The gene may consist of the nucleotide sequence of SEQ ID NO: 1.
또한, 본 발명은 상기 방법에 의해 제조된 바이오매스가 증가된 형질전환 식물체 및 이의 종자를 제공한다. 바람직하게는, 상기 식물체는 쌍자엽 식물일 수 있으나, 이에 제한되지는 않는다. 상기 쌍자엽 식물은 전술한 바와 같다. 바람직하게는, 상기 쌍자엽 식물은 담배이다. The present invention also provides a transgenic plant and seed thereof having increased biomass produced by the above method. Preferably, the plant may be a dicotyledonous plant, but is not limited thereto. The dicotyledonous plants are as described above. Preferably, the dicotyledonous plant is tobacco.
또한, 본 발명은 서열번호 1의 염기서열로 이루어진, 시네코시스티스 속(Synechocystis sp.) PCC6803 유래의 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는, 식물체의 바이오매스 증가용 조성물을 제공한다. 상기 조성물은 유효성분으로 서열번호 1의 염기서열로 이루어진 SbtA 유전자를 포함하며, 상기 유전자를 식물체의 엽록체에 형질전환시킴으로써 식물체의 바이오매스를 증가시킬 수 있는 것이다.In addition, the present invention consists of the nucleotide sequence of SEQ ID NO: 1, Synechocystis genus (Synechocystis sp.) From PCC6803SbtA It provides a composition for increasing the biomass of a plant, including a sodium dependent bicarbonate transporter gene. The composition consists of the nucleotide sequence of SEQ ID NO: 1 as an active ingredientSbtA It includes a gene, and by converting the gene into the chloroplast of the plant it is possible to increase the biomass of the plant.
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.
재료 및 방법Materials and methods
식물 재료Plant material
본 실험에서 담배는 Nicotiana tabacum L. cv. Samsun 품종을 사용하였으며, 10%(v/v) 소듐 하이포글로라이트(유효 염소 함유량 5%) 용액에 15분간 표면 살균 후 멸균수로 3회 세척하고 3% 수크로오스가 첨가된 MS 배지에 치상하여 25℃, 16시간 광조건, 30 μmol/m2/s로 유지되는 배양실에서 약 5주 동안 배양된 잎을 엽록체 형질전환 재료로 사용하였다.Cigarettes in this experiment were Nicotiana tabacum L. cv. Samsun varieties were used, surface sterilization in 10% (v / v) sodium hypoglolite (5% effective chlorine content) solution for 15 minutes, washed three times with sterile water, and washed with MS medium containing 3% sucrose. Leaves incubated for about 5 weeks in a culture chamber maintained at 25 ° C., 16 hours light condition and 30 μmol / m 2 / s were used as chloroplast transforming materials.
시네코시스티스(Synechocystis) SbtA 유전자의 클로닝 및 색소체 형질전환용 벡터 제작Cloning of Synechocystis SbtA Gene and Construction of Chromosome Transformation Vector
시네코시스티스 PCC6803의 게놈 DNA를 주형으로 하여 SbtA F (5'- ATG GAT TTT TTG TCC AAT TTC TTG -3'; 서열번호 2) 및 SbtA R (5'- TTA ACC TGC ACC AAG GGT CTG GGC -3'; 서열번호 3) 프라이머를 사용하여 PCR 증폭(1.1 kb)하였으며, 염기서열을 시퀀싱하여 확인하였다. 증폭한 단편을 EcoRV으로 절단한 후 RclpGAH 벡터로 서브클로닝하고, Rclp-SbtA를 XhoI/SpeI으로 절단하여 블런팅(blunting)한 후 담배 색소체 형질전환 벡터인 TIA (PvuII)로 도입시켰다(도 1).The genomic DNA of Cynecosistis PCC6803 as a template SbtA PCR amplification using F (5'- ATG GAT TTT TTG TCC AAT TTC TTG-3 '; SEQ ID NO: 2) and SbtA R (5'- TTA ACC TGC ACC AAG GGT CTG GGC -3'; SEQ ID NO: 3) primers (1.1 kb) and confirmed by sequencing the sequence. The amplified fragment was digested with EcoRV and then subcloned with the RclpGAH vector and Rclp-SbtAWas cut with XhoI / SpeI and blunted and then introduced into TIA (PvuII), a tobacco chromatin transformation vector (FIG. 1).
엽록체 형질전환과 형질전환체의 선발Chloroplast transformation and selection of transformants
입자 밤바드먼트를 위한 식물 재료 준비는 밤바드먼트 수행 하루 전에 1 mg/L 벤질아데닌(BA) 및 0.1 mg/L 나프탈렌아세트산(NAA)이 첨가된 MS 배지에 멸균된 필터 페이퍼(직경 70 mm ; Advantec Toyo, Tokyo Japan)에 올려놓고, 그 위에 3 cm 크기의 담배 잎을 취하여 잎 뒷면이 위를 향하게 하여 올려놓았다. DNA는 금 입자(gold particle, 0.6 ㎛)에 코팅하여 particle delivery system (PDS 1000/He, Bio-Rad, USA)과 1,100 psi (7,580 MPa) rupture disk를 사용하여 9 cm 높이에서 밤바드먼트를 시행하였다. 밤바드먼트 후 담배 잎을 48시간 동안 딤 라이트(dim light) 조건에 둔 다음 5×5 mm로 잘라서 1 mg/L 벤질아데닌(BA) 및 0.1 mg/L 나프탈렌아세트산(NAA), 500 mg/L 스펙티노마이신(spectinomycin)이 포함된 MS 배지(신초 선발 배지)에 치상하였다. 약 4-6주 후 형질전환된 세포로부터 발생된 신초의 잎을 잘게 잘라 동일 배지에 치상 한 후 3번의 반복 선발과정(라운딩 과정)을 거쳐 재분화된 식물체는, 호르몬이 첨가되지 않은 MS 선발 배지에 옮겨 뿌리를 발생시킨 후 순화과정을 거쳐 피토트론(Phytotron)의 25℃, 16시간 일장 조건하에서 생육시켰다.Plant material preparations for particle balm-borders were prepared by sterile filter paper (diameter 70 mm) in MS medium to which 1 mg / L benzyladenin (BA) and 0.1 mg / L naphthaleneacetic acid (NAA) were added one day prior to the balm-border. Advantec Toyo, Tokyo Japan), 3 cm tobacco leaves were placed on the leaves, with the back of the leaves facing up. DNA was coated on gold particles (0.6 μm) and subjected to a balm body at 9 cm height using a particle delivery system (PDS 1000 / He, Bio-Rad, USA) and a 1,100 psi (7,580 MPa) rupture disk. It was. After balm, the tobacco leaves were placed in dim light for 48 hours and then cut into 5 × 5 mm, 1 mg / L benzyladenin (BA) and 0.1 mg / L naphthaleneacetic acid (NAA), 500 mg / L It was dentured in MS medium (spectral selection medium) containing spectinomycin. After about 4-6 weeks, the leaves of shoots generated from the transformed cells were chopped and placed on the same medium, and then re-differentiated through three repeated selection (rounding) processes. The roots were transferred, purified, and grown under conditions of 25 ° C. and 16 hours of phytotron.
서던 및 노던 블롯 분석Southern and Northern Blot Analysis
담배 잎에서 DNeasy Plant Mini Kit (Qiagen, Hilden, Germany)를 이용하여 총 게놈 DNA를 분리하였으며, 게놈 DNA 4 ㎍을 BamHI 및 BglII로 잘라 0.8 % 아가로스 겔에서 전기영동한 후, Zeta-프로브 GT Blotting Membrane (Bio-Rad, Hercules, CA)에 10×SSC를 이용하여 옮겼다. 색소체 게놈안의 trnI을 포함하는 BamHI-BglII DNA 절편에 PCR 증폭한 trnA 프로브(P1, 150 bp)를 방사선 동위원소 [α-32P]dCTP로 라벨링 하여 도입 유전자의 삽입을 확인하였다. 전혼성화(prehybridization) 및 혼성화는 7% (w/v) SDS가 들어가 있는 0.25 M 인산 나트륨 완충액(pH 7.2)을 이용하여 65℃에서 밤새 수행하였으며, 세척 완충액 I(20 mM 인산 나트륨 완충액(pH 7.2) 및 5% SDS)과 세척 완충액 II(20 mM 인산 나트륨 완충액(pH 7.2) 및 1% SDS)로 65℃에서 각각 15분씩 씻어낸 다음, 막(membrane)을 이미지 플레이트(Image plate, Fuji Film)에 3시간 노출시킨 뒤 밴드를 확인하였다.Total genomic DNA was isolated from tobacco leaves using DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), 4 μg of genomic DNA was cut with BamHI and BglII and electrophoresed on 0.8% agarose gel, followed by Zeta-probe GT Blotting. Membrane (Bio-Rad, Hercules, Calif.) Was transferred using 10 × SSC. The insertion of the transgene was confirmed by labeling the trnA probe (P1, 150 bp) PCR-amplified on the trAI-containing BamHI-BglII DNA fragment in the chromatin genome with a radioisotope [α- 32 P] dCTP. Prehybridization and hybridization were performed overnight at 65 ° C. using 0.25 M sodium phosphate buffer (pH 7.2) containing 7% (w / v) SDS, Wash Buffer I (20 mM sodium phosphate buffer, pH 7.2). ) And 5% SDS) and Wash Buffer II (20 mM sodium phosphate buffer (pH 7.2) and 1% SDS) at 65 ° C. for 15 minutes each, and then the membrane is image plate (Fuji Film) After 3 hours exposure to the band was confirmed.
총 RNA는 Trizol Reagent (Invitrogen, Carlsbad, CA)를 이용하여 형질전환된 잎으로부터 추출하였다. 총 RNA(2 ㎍)를 5.1% (v/v) 포름알데히드가 들어간 1% 아가로스 겔에 전기영동 후, Zeta-probe GT blotting Membrane(Bio-Rad, Hercules, CA)에 RNA를 옮기고 PCR로 증폭한 SbtA 유전자 절편(P2 프로브)에 [α-32P]dCTP로 라벨링 하였으며, 혼성화 및 세척 과정은 서던 블롯팅 방법과 동일하였다.Total RNA was extracted from the transformed leaves using Trizol Reagent (Invitrogen, Carlsbad, Calif.). Total RNA (2 μg) was electrophoresed on a 1% agarose gel containing 5.1% (v / v) formaldehyde, followed by transfer of RNA to Zeta-probe GT blotting Membrane (Bio-Rad, Hercules, CA) and amplification by PCR. One SbtA gene segment (P2 probe) was labeled with [α- 32 P] dCTP and hybridization and washing were the same as the Southern blotting method.
GFP 발현 관찰Observe GFP Expression
GFP 형광을 관찰하기 위하여 원형질체를 분리하였다. 2주간 흙에서 키운 담배의 잎을 채취하여 표피를 벗겨낸 뒤, 효소 용액(1~1.5% Cellulase R10, 0.2~0.4% Mecerozyme R10, 0.4 M 만니톨, 20 mM MES, pH 5.7 및 0.1% BSA)에 넣고 암 상태로 3~4시간 처리하였다. 분리한 원형질체는 형광현미경을 통해 관찰하였다.Protoplasts were isolated to observe GFP fluorescence. Take the leaves of tobacco grown in soil for 2 weeks and remove the epidermis, and then in enzyme solution (1-1.5% Cellulase R10, 0.2-0.4% Mecerozyme R10, 0.4 M mannitol, 20 mM MES, pH 5.7 and 0.1% BSA). Put and treated for 3-4 hours in the dark. The separated protoplasts were observed by fluorescence microscopy.
내염성 검정 및 형태적 관찰Flameproof Assay and Morphological Observation
SySbtA 엽록체 형질전환 담배의 염에 대한 반응을 보기 위하여 T2 종자를 NaCl 0 mM, 25 mM 및 100 mM이 첨가된 MS 배지에 각각 치상한 후, 25℃, 16 시간 광조건으로 배양하여 발아 9일 후 식물체의 뿌리 길이를 조사하였다.In order to see the response to the salts of SySbtA chloroplast-transformed tobacco, T2 seeds were healed in MS medium containing NaCl 0 mM, 25 mM and 100 mM, respectively, and then cultured at 25 ° C. for 16 hours under light conditions, and 9 days after germination The root length of was investigated.
식물 생육조건과 생장량 조사Investigation of plant growth conditions and growth
모든 분석법은 형질전환 T2 식물로 수행하였으며, 대조군 식물체와 형질전환체는 MS 기본배지에 스펙티노마이신 500 mg/L가 들어있는 배지에서 25℃, 16시간 광조건으로 발아시키고 5주 후 흙으로 옮겼으며, 피토트론 환경(25℃, 16시간 일장)에서 생육하였다. 생장률 분석을 위해 10개의 독립 개체를 일주일 간격으로 초장을 측정하였고, 개화기 때의 식물체를 수확하여 지상부의 생중량 및 건중량을 측정하였으며, 줄기의 직경과 잎의 개수를 조사하였다. 건중량은 70℃에서 10일간 건조시킨 후 측정하였다.All assays were performed with transgenic T2 plants. Control plants and transformants were germinated at 25 ° C. for 16 hours in medium containing 500 mg / L of spectinomycin in MS medium and transferred to soil after 5 weeks. And grown in phytotron environment (25 ° C., 16 h length). For the growth rate analysis, the height of 10 independent individuals was measured at weekly intervals, the plants were harvested during flowering period, and the fresh and dry weights of the ground were measured, and the diameters of stems and the number of leaves were examined. Dry weight was measured after drying for 10 days at 70 ℃.
엽록소 함량 측정Chlorophyll content determination
엽록소 a 및 b의 함량 측정은 10주 된 담배 잎 100 mg을 채취하여 액체 질소로 얼린 뒤 분쇄하고, 80% 아세톤 1 mL을 첨가하여 암 상태로 20분간 교반하여 상등액을 취하였다. 상기 과정을 3번 반복하여 엽록소를 완전히 분리하였으며, 측정은 spectrophotometer (UV-2450, SHIMADZU Inc., Japan)를 이용하여 645 nm 및 663 nm의 파장에서 실시하였다(Jeong et al., 2002, Plant Physiol 129:112-121). The content of chlorophyll a and b was measured by taking 100 mg of 10-week-old tobacco leaves, frozen with liquid nitrogen, crushed, and adding 1 mL of 80% acetone and stirring for 20 minutes in the dark to obtain a supernatant. Chlorophyll was completely isolated by repeating the above procedure three times, and the measurement was performed at a wavelength of 645 nm and 663 nm using a spectrophotometer (UV-2450, SHIMADZU Inc., Japan) (Jeong et al., 2002, Plant Physiol 129: 112-121).
수용성 당 및 전분 함량 측정Determination of Water Soluble Sugar and Starch Content
수용성 당 및 전분의 함량을 측정하기 위하여 효소 분석법을 이용하였다 (Stitt et al., 1989, Method Enzymol 174:518-522). 6시간 광적응 된 식물의 잎 0.1 g (FW)을 채취하여 액체 질소로 동결시켜 마쇄한 뒤, 0.5 mL의 킬링(Killing) 용액(2 : 8 = 포름산 : 100% 에탄올)을 넣고 80℃에서 20분간 처리하고, 80% 에탄올 0.5 mL을 첨가한 뒤 다시 80℃에서 20분간 처리하여 추출하였다. 증발기를 이용하여 완전히 건조시킨 뒤, 펠렛을 500 ㎕의 증류수에 녹여 풀어준 다음 원심 분리하여 상등액을 취하여 수용성 당을 측정하였다. 전분의 측정은 남아 있는 펠렛을 아밀라제(amylase)와 아밀로글루코시다제(amyloglucosidase)로 가수분해시켜 글루코오스를 측정하였다. 당 및 전분은 UV-Spectorphotometer (UV-2450, SHIMADZU Inc., Japan)를 이용하여 340 nm에서 NADPH의 흡광도를 측정하였다.Enzyme assays were used to determine the content of water soluble sugars and starch (Stitt et al., 1989, Method Enzymol 174: 518-522). Take 0.1 g (FW) of the leaves of the plants which have been adapted for 6 hours, freeze them with liquid nitrogen, crush them, and then add 0.5 mL of Killing solution (2: 8 = formic acid: 100% ethanol) at 20 ° C. After the treatment, 0.5 mL of 80% ethanol was added thereto, followed by extraction at 80 ° C. for 20 minutes. After drying completely using an evaporator, the pellet was dissolved in 500 µl of distilled water, released, and centrifuged to obtain a supernatant to measure water-soluble sugars. Starch was measured for glucose by hydrolysis of the remaining pellets with amylase and amyloglucosidase. Sugar and starch were measured for absorbance of NADPH at 340 nm using a UV-Spectorphotometer (UV-2450, SHIMADZU Inc., Japan).
CO2 교환 및 CO2 보상점 측정CO 2 exchange and CO 2 compensation point measurement
식물의 잎에서의 기체 교환과 CO2 보상점의 측정은 fluorescence chamber head (Li-6400-40 leaf chamber fluorometer; Li-Cor Inc., USA)가 장착된 LI-6400 (Li-Cor Inc., USA)을 이용하였다. 비슷한 시기의 식물체의 성숙한 잎을 선정하였고 IRGA (infra red gas analyzer) leaf chamber의 온도는 25℃, 상대습도는 50±2%로 하였다. CO2 농도는 400 μmol CO2/mol, 광도는 700 μmol/m2/s로 하였다. 광원은 적색 및 푸른색 LED 광원이며, 이산화탄소는 Li-6400 CO2 혼합기체를 사용하였다. CO2 보상점 조사는 CO2 농도를 400 μmol CO2/mol에서 0 μmol CO2/mol로 변화시키면서 CO2 교환값을 측정하여 값이 0이 되는 지점을 CO2 보상점으로 하였다. Gas exchange and CO 2 compensation points in the leaves of plants were measured using LI-6400 (Li-Cor Inc., USA) equipped with a fluorescence chamber head (Li-6400-40 leaf chamber fluorometer; Li-Cor Inc., USA). ) Was used. Mature leaves of plants of similar period were selected and IRGA (infra red gas analyzer) leaf chamber temperature was 25 ℃ and relative humidity was 50 ± 2%. The CO 2 concentration was 400 μmol CO 2 / mol and the brightness was 700 μmol / m 2 / s. The light source was a red and blue LED light source, and carbon dioxide was mixed with a Li-6400 CO 2 gas. In the CO 2 compensation point investigation, the CO 2 exchange value was measured while changing the CO 2 concentration from 400 μmol CO 2 / mol to 0 μmol CO 2 / mol, and the point where the value became 0 was used as the CO 2 compensation point.
실시예 1: 엽록체 형질전환 벡터와 형질전환 식물체 확인Example 1 Identification of Chloroplast Transformation Vectors and Transgenic Plants
엽록체 형질전환 벡터에는 동형 재조합을 위한 삽입 염기서열 부위인 trnI와 trnA가 있으며, RclpGAH를 백본(backbone)으로 하여 시네코시스티스 속 PCC 6803에서 찾아낸 표적 유전자인 SbtA 유전자와 함께 스펙티노마이신 저항성 선발 마커인 aadA 유전자 및 GFP 리포터 유전자를 사용하였으며, 벼에서 찾아낸 clp 프로모터 및 대장균의 rrnB1/B2 터미네이터를 사용하였다(도 1).Chloroplast transformation vectors include trn I and trnA, insertion sequences for homologous recombination, and spectinomycin resistance selection markers along with the SbtA gene, a target gene found in PCC 6803 of the genus Scincosistis, with RclpGAH as the backbone. Phosphorus aadA gene and GFP reporter gene were used, and the clp promoter found in rice and the rrnB1 / B2 terminator of Escherichia coli were used (FIG. 1).
형질전환된 3개의 T0 독립 라인을 얻었으며, T0 식물체에서 받은 T1 종자를 스펙티노마이신 배지에 발아시켜 선발하였고, 서던 및 노던 블롯 분석 결과 T1 식물체에서도 도입된 유전자가 발현됨을 확인하였으며, 동형(homoplasmic) 라인을 얻기 위하여 다시 한 번 세대 진전시켜 T2 세대를 대상으로 이후 실험을 진행하였다. T2 식물체를 대상으로 trnA probe를 이용하여 서던 블롯 분석을 수행한 결과 3개의 독립적인 엽록체 형질전환체 모두 약 4.5 kb 크기의 밴드가 검출되었고, 벡터 대조군에서는 약 3.3 kb 크기의 밴드가 검출되었으며 야생형에서는 0.88 kb의 밴드가 검출되었다(도 2). 상기 결과는 형질전환 식물체의 엽록체 게놈에 목적 유전자인 SbtA 유전자가 동형(homoplasmy) 상태로 도입되고 다음 세대로 유전되었음을 의미한다(도 2A). 또한 노던 블롯 분석을 위하여 SbtA 프로브 (P2)를 사용하였으며, 벡터 대조구를 제외한 모든 라인에서 SbtA 유전자가 발현하는 것을 확인하였다(도 2B).Three transformed T0 independent lines were obtained, T1 seeds received from T0 plants were germinated and selected in spectinomycin medium, and Southern and Northern blot analysis revealed that the genes introduced in T1 plants were expressed and homozygous (homoplasmic). In order to obtain the line, the generation was once again advanced and the experiment was conducted on the T2 generation. Southern blot analysis using trnA probe on T2 plants revealed bands of approximately 4.5 kb in all three independent chloroplast transformants, approximately 3.3 kb in the vector control group, and in wild-type. A band of 0.88 kb was detected (FIG. 2). The results indicate that the SbtA gene, the target gene, was introduced into the homoplasmy state and inherited to the next generation in the chloroplast genome of the transgenic plant (FIG. 2A). In addition, SbtA probe (P2) was used for Northern blot analysis, and it was confirmed that the SbtA gene was expressed in all lines except the vector control (FIG. 2B).
실시예 2: 담배 형질전환 식물체의 GFP 발현Example 2: GFP Expression of Tobacco Transgenic Plants
도입된 GFP 유전자의 발현 여부를 확인하기 위하여 야생형, 벡터 대조군 및 SySbtA 4번 라인을 대상으로 식물체 잎으로부터 원형질체를 분리하여 세포수준에서 관찰한 결과, 표지 유전자인 GFP가 식물 엽록체 내에서 발현하는 것을 UV 형광으로 확인하였다(도 3).In order to confirm the expression of the introduced GFP gene, protoplasts were isolated from plant leaves in wild-type, vector control, and SySbtA line 4, and observed at the cellular level, indicating that GFP, a marker gene, was expressed in plant chloroplasts. It was confirmed by fluorescence (Fig. 3).
실시예 3: SySbtA 식물체의 내염성 검정 및 표현형 분석Example 3: Salt Tolerance Assay and Phenotypic Analysis of SySbtA Plants
SySbtA 형질전환 식물체의 내염성을 알아보기 위하여, 염에 대한 반응을 조사하였다. T2 종자를 각각 NaCl 0 mM, 25 mM 및 100 mM이 첨가된 배지에 치상하여 9일 후 뿌리 발달을 관찰하였다. NaCl을 첨가하지 않은 배지 즉, 일반 조건에서 SySbtA 식물체는 원뿌리의 뿌리생장이 빨랐으며, 반면 벡터 대조군은 뿌리생장이 느리고 측뿌리가 발달한 것을 볼 수 있었다(도 4A). 일반 조건인 NaCl이 첨가되지 않은 상태에서의 뿌리의 형태가 서로 다르기 때문에, 이를 보정하기 위해서 이때의 뿌리의 길이를 100으로 하여 NaCl을 첨가하였을 때의 뿌리생장을 상대적인 수치로 표현하였다. 그 결과 벡터 대조군에서는 NaCl의 농도가 증가할수록 뿌리생장이 감소하였고, SySbtA 식물체에서는 NaCl 25 mM일 때 오히려 뿌리생장이 11~38% 증가하였으며, NaCl 100 mM일 때에는 뿌리길이가 감소하였지만 SySbtA식물체가 벡터 대조구에 비하여 내성이 증가된 것을 알 수 있었다(도 4B).SySbtA In order to determine the salt resistance of the transgenic plants, the response to the salt was examined. T2 seeds were seeded in medium supplemented with NaCl 0 mM, 25 mM and 100 mM, respectively, and root development was observed after 9 days. In the medium without NaCl, that is, under normal conditions, the SySbtA plants showed faster root growth in the roots, whereas the vector control group showed slower root growth and developed lateral roots (FIG. 4A). Root growth in the state without the addition of NaCl, which is a general condition, is different from each other. Thus, the root growth when the length of the root is 100 is expressed as a relative value. As a result, the root growth decreased with increasing NaCl concentration in the vector control group, and the root growth was increased by 11 ~ 38% in NaCl 25 mM in the SySbtA plant. It was found that the resistance was increased compared to the control (Fig. 4B).
실시예 4: SySbtA 식물체의 생리적 특징과 생장량Example 4: SySbtA Physiological Characteristics and Growth of Plants
파종된 SySbtA T2 식물체들은 피토트론 환경에서 키웠으며, 식물체의 초장, 엽수, 줄기의 직경, 생중량 및 건중량을 측정하였다. SySbtA는 대조군에 비해 흙으로 옮긴 후부터 빠르게 성장하였으며 13주부터 개화가 시작되었다(도 5). 개화기 때의 초장을 비교해 본 결과 SySbtA 식물체가 50% 정도 성장률이 높게 조사되었다(도 6A). 개화기 때의 생중량과 건중량을 비교해 본 결과, 생중량은 SySbtA가 36~50% 높았고, 특히 건중량은 50% 이상 높아졌으며 1번 라인의 경우 두 배 이상 높아진 것을 볼 수 있었다(도 6B 및 6C). 엽수 또한 SySbtA 식물체가 14~24% 많은 것으로 조사되었고(도 7A), 줄기의 직경도 6~9% 증가한 것을 볼 수 있었다(도 7B).The seeded SySbtA T2 plants were grown in phytotron environment, and the height, leaves, stem diameter, fresh weight and dry weight of the plants were measured. SySbtA grew rapidly after transfer to soil compared to the control and began to bloom from week 13 (FIG. 5). As a result of comparing the height of the flowering period, SySbtA plants showed a high growth rate of about 50% (FIG. 6A). As a result of comparing the fresh weight and dry weight at the flowering period, the fresh weight was 36-50% higher in SySbtA, especially the dry weight was higher by 50% and more than double in the first line (Figs. 6B and 6C). . Conifers also showed 14-24% more SySbtA plants (FIG. 7A), and the diameter of the stems was also increased by 6-9% (FIG. 7B).
엽록소 함량에 있어서 SySbtA 식물체는 벡터 대조군에 비하여 총 엽록소 함량이 45-84% 증가하였으며, 엽록소 a/b 비율에는 별다른 변화를 보이지 않았다(도 8).In the chlorophyll content, SySbtA plants showed a 45-84% increase in total chlorophyll content compared to the vector control, and showed no change in the chlorophyll a / b ratio (FIG. 8).
실시예 5: SySbtA 형질전환 식물체의 광합성 효율과 CO2 보상점Example 5 Photosynthetic Efficiency and CO 2 Reward Points of SySbtA Transgenic Plants
담배의 엽록체에 직접적으로 SbtA를 도입시켰을 때 식물의 생장과 바이오매스가 증가되었으므로 광합성 효율에는 어떠한 변화가 있는지를 확인하기 위해 Phytotron에서 9주 생육된 T2 형질전환 식물체의 손상되지 않은 성숙된 잎을 사용하여 CO2 동화율을 측정하였다. 엽육세포 내의 CO2　농도(Ci)에 따른 CO2 동화율(A/Ci response curve)을 측정하기 위하여 광도를 700 μmol/m2/s로 고정하고 CO2 농도를 0-2100 μmol CO2/mol까지 변화시키면서 측정하였다. 측정 결과 벡터 대조군에 비하여 SySbtA 식물체가 광합성 효율이 높게 나타났다(도 9).The introduction of SbtA directly into the chloroplasts of tobacco increased plant growth and biomass, so intact mature leaves of T2 transgenic plants grown 9 weeks in Phytotron were used to determine what changes in photosynthetic efficiency. CO 2 assimilation rate was measured. To measure the CO 2 assimilation rate (A / Ci response curve) according to the CO 2 concentration (Ci) in the leaf cells, the intensity was fixed at 700 μmol / m 2 / s and the CO 2 concentration was 0-2100 μmol CO 2 / mol Measured while changing to. As a result, SySbtA plants showed higher photosynthetic efficiency than the vector control (FIG. 9).
한편 CO2 보상점은 CO2 교환이 0이 되는 시점, 즉 식물이 살아갈 수 있는데 필요한 최소한의 CO2 환경이므로 CO2 보상점이 낮을수록 저농도의 CO2 환경에서도 식물이 CO2를 효율적으로 이용하여 적응할 수 있는 능력이 커진 것을 의미한다. CO2 보상점을 조사한 결과 SySbtA 1번 라인 및 4번 라인이 벡터 대조군에 비하여 각각 12-16% 낮았다(도 10). 이 결과는 상기 Ci-곡선(Ci-curve)과 상응하는 결과이며 SySbtA 식물체가 CO2 이용 효율이 증가된 것을 알 수 있었다. The CO 2 compensation point is the CO 2 exchange the zero point, i.e. the minimum CO 2 environment because CO 2 compensation point lower the plant even in a low concentration of CO 2 environment for there plants can survive being adapted to use the CO 2 efficiently It means that you have increased your ability to do so. As a result of examining the CO 2 reward point, SySbtA line 1 and line 4 were 12-16% lower than the vector control (Fig. 10). This result is equivalent to the Ci-curve (Ci-curve) and the SySbtA plant was found to increase the CO 2 utilization efficiency.
실시예 6: SySbtA 형질전환 식물체의 수용성 당 및 전분 함량의 변화Example 6: Changes in Water-soluble Sugars and Starch Contents of SySbtA Transgenic Plants
SySbtA 식물체의 증가된 CO2 동화율이 탄수화물의 생합성에 영향을 주는지 알아보기 위해 SySbtA 식물체의 탄수화물 함량 분석을 위해 정오에 채취한 담배 잎에서의 수용성 당 및 전분의 함량을 측정하였다. 그 결과 수용성 당의 경우 프룩토오스는 벡터 대조군과 SySbtA 형질전환 식물체가 유사한 수준이었으나 글루코오스는 소폭 증가하였으며, 수크로오스의 경우에는 함량이 124-189%로 대폭 증가하였다(도 11). 상기 수용성 당 함량의 증가는 CO2 동화작용이 증가됨으로써 탄소대사 산물인 글루코오스 및 수크로오스 합성을 증가시켰을 것으로 생각된다.To determine whether the increased CO 2 assimilation of SySbtA plants affects the carbohydrate biosynthesis, the water content of water-soluble sugars and starch in tobacco leaves collected at noon was analyzed for carbohydrate content analysis of SySbtA plants. As a result, fructose in water-soluble sugars was similar in vector control and SySbtA transgenic plants, but glucose was slightly increased, and in case of sucrose, the content was greatly increased to 124-189% (FIG. 11). The increase in the water-soluble sugar content is thought to increase the synthesis of glucose and sucrose, carbon metabolites by increasing CO 2 assimilation.

Claims (18)

  1. 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터를 식물세포에 형질전환시켜 SbtA 유전자를 과발현하는 단계를 포함하는 식물의 내염성을 증가시키는 방법.Synechocystis sp.) Origin of PCC6803SbtAPlant cells were transformed into a recombinant vector containing a sodium dependent bicarbonate transporter gene.SbtAA method of increasing the flame resistance of a plant comprising overexpressing a gene.
  2. 제1항에 있어서, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어진 것을 특징으로 하는 방법.According to claim 1, wherein the SbtA gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
  3. 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계; 및Synechocystis sp.) Origin of PCC6803SbtATransforming plant cells with a recombinant vector comprising a sodium dependent bicarbonate transporter gene; And
    상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 내염성이 증가된 형질전환 식물체의 제조 방법.A method for producing a transgenic plant having increased flame resistance, comprising the step of regenerating a plant from the transformed plant cell.
  4. 제3항에 있어서, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어진 것을 특징으로 하는 방법.The method of claim 3, wherein the SbtA gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
  5. 제3항의 방법에 의해 제조된 내염성이 증가된 형질전환 식물체. Transgenic plants with increased salt resistance prepared by the method of claim 3.
  6. 제5항에 있어서, 상기 식물체는 쌍자엽 식물인 것을 특징으로 하는 형질전환 식물체. The transgenic plant of claim 5, wherein the plant is a dicotyledonous plant.
  7. 제6항에 있어서, 상기 쌍자엽 식물은 담배인 것을 특징으로 하는 형질전환 식물체.The transgenic plant of claim 6, wherein the dicotyledonous plant is tobacco.
  8. 제5항에 따른 식물체의 종자. Seeds of plants according to claim 5.
  9. 서열번호 1의 염기서열로 이루어진, 시네코시스티스 속(Synechocystis sp.) PCC6803 유래의 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는, 식물체의 내염성 증가용 조성물.Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) From PCC6803SbtA (Sodium dependent bicarbonate transporter) gene, comprising a composition for increasing the flame resistance of plants.
  10. 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터를 식물세포에 형질전환시켜 SbtA 유전자를 과발현하는 단계를 포함하는 식물의 바이오매스를 증가시키는 방법.Synechocystis sp.) Origin of PCC6803SbtAPlant cells were transformed into a recombinant vector containing a sodium dependent bicarbonate transporter gene.SbtAA method of increasing plant biomass comprising overexpressing a gene.
  11. 제10항에 있어서, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어진 것을 특징으로 하는 방법.The method of claim 10, wherein the SbtA gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
  12. 시네코시스티스 속(Synechocystis sp.) PCC6803 유래 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는 재조합 벡터로 식물세포를 형질전환하는 단계; 및Synechocystis sp.) Origin of PCC6803SbtATransforming plant cells with a recombinant vector comprising a sodium dependent bicarbonate transporter gene; And
    상기 형질전환된 식물세포로부터 식물을 재분화하는 단계를 포함하는 바이오매스가 증가된 형질전환 식물체의 제조 방법.A method for producing a transformed plant with increased biomass, comprising: regenerating a plant from the transformed plant cells.
  13. 제12항에 있어서, 상기 SbtA 유전자는 서열번호 1의 염기서열로 이루어진 것을 특징으로 하는 방법.The method of claim 12, wherein the SbtA gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 1.
  14. 제12항의 방법에 의해 제조된 바이오매스가 증가된 형질전환 식물체. Transgenic plant with increased biomass produced by the method of claim 12.
  15. 제14항에 있어서, 상기 식물체는 쌍자엽 식물인 것을 특징으로 하는 형질전환 식물체. The transgenic plant of claim 14, wherein the plant is a dicotyledonous plant.
  16. 제15항에 있어서, 상기 쌍자엽 식물은 담배인 것을 특징으로 하는 형질전환 식물체. The transgenic plant of claim 15, wherein the dicotyledonous plant is tobacco.
  17. 제14항에 따른 식물체의 종자.Seeds of plants according to claim 14.
  18. 서열번호 1의 염기서열로 이루어진, 시네코시스티스 속(Synechocystis sp.) PCC6803 유래의 SbtA (Sodium dependent bicarbonate transporter) 유전자를 포함하는, 식물체의 바이오매스 증가용 조성물.Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) From PCC6803SbtA (Sodium dependent bicarbonate transporter) gene, comprising a composition for increasing the biomass of the plant.
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