KR101315068B1 - SbtA gene from Synechocystis sp. PCC6803 and uses thereof - Google Patents

Abstract

The present invention is of the genus Synechocystis sp.) Method for increasing the flame resistance of plants using a PCC6803 derived SbtA (Sodium dependent bicarbonate transporter) gene, the SbtA Method for producing a transgenic plant with increased salt resistance using a gene, the transgenic plant with increased salt resistance produced by the method and its seeds, the SbtA The present invention relates to a method of increasing the biomass of a plant using a gene, a method of producing a transformed plant having an increased biomass using the SbtA gene, a transformed plant having an increased biomass produced by the method, and a seed thereof. .

Description

SC PatA gene derived from PCC6803 genus, and its use {SbtA gene from Synechocystis sp. PCC6803 and uses approximately}

The present invention is of the genus SbtA gene derived from PCC6803 and its use, and more specifically, Synechocystis sp.) Method for increasing the flame resistance of a plant using a PCC6803-derived SbtA (Sodium dependent bicarbonate transporter) gene, a method for producing a transgenic plant with increased flame resistance using the SbtA gene, a transgenic plant with increased salt resistance prepared by the method And a seed thereof, a method of increasing the biomass of a plant using the SbtA gene, the 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. 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).

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.

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).

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. . Even small increases in the concentration of CO _{2 in the} chloroplasts can lead to very high CO ₂ fixation efficiencies (Price et al., 2008, J Exp Bot 59: 1441-1461).

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 plants, 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.

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 manipulate 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. 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.

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.

In order to solve the above problems, the present invention (Synechocystis) sp.) SbtA from PCC6803 It provides a method of increasing the flame resistance of plants by using a sodium dependent bicarbonate transporter gene.

In addition, the present invention is a genus Provided is a method for producing a transgenic plant having increased salt resistance using the SbtA gene derived from PCC6803 .

The present invention also provides a transgenic plant with increased flame resistance produced by the above method and its seeds.

In addition, the present invention is a genus Provided is a composition for increasing the salt resistance of a plant, comprising the SbtA gene derived from PCC6803 .

In addition, the present invention is a genus Provided is a method for increasing plant biomass using SbtA gene derived from PCC6803 .

In addition, the present invention is a genus Provided is a method for producing a transgenic plant having increased biomass using the SbtA gene derived from PCC6803 .

The present invention also provides a transgenic plant and seed thereof having increased biomass produced by the above method.

In addition, the present invention is a genus Provided is a composition for increasing biomass of a plant, including the SbtA gene derived from PCC6803 .

The genus Cinecosis of the present invention Transforming the SBTA gene from PCC6803 to the chloroplasts of plants not only increases resistance to salt stress but also increases biomass. Therefore, the SbtA gene is expected to be useful for developing transgenic plants that are resistant to salt stress environments.

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.
Figure 2 shows the results of the introduction and expression of the Synecocistis SbtA gene introduced into tobacco chloroplasts. (A) Vector control and SySbtA with trnA probe Southern blot analysis of transgenic plants. (B) Northern blot analysis using SbtA probe.
Figure 3 shows GFP expression of protoplasts isolated from the leaves of chloroplast transgenic tobacco plants. Expression of GFP was observed by fluorescence microscopy.
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.
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. (C) dry weight. The results are expressed as mean ± standard deviation (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).
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).
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).
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).
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).

In order to achieve the object of the present invention, the present invention (Synechocystis) sp.) SbtA from PCC6803 It provides a method for increasing the flame resistance of a plant comprising the step of transforming a recombinant vector comprising a sodium dependent bicarbonate transporter gene to plant cells overexpressing the SbtA 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. 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. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

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, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

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, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, any plasmid and vector can be used if it can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, 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 technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector to drive mRNA synthesis. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

A preferred example of the recombinant vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to a plant cell when present in a suitable host, such as Agrobacterium tumefaciens. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences to plant cells or protoplasts in which new plants capable of properly inserting hybrid DNA into the plant's genome can be produced have. A particularly preferred form of the Ti-plasmid vector is a so-called binary vector as claimed in EP 0 120 516 B1 and U.S. Patent No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into the plant host include viral vectors such as those that can be derived from double-stranded plant viruses (e. G., CaMV) and single- For example, from non -complete plant virus vectors. The use of such vectors may be particularly advantageous when it is difficult to transform the plant host properly.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. 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.

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 the region of DNA upstream from the 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. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline 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 the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The 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.

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.

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. When the host cell is a eukaryotic cell, the vector is injected into the host cell by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, and gene bombardment .

In addition, the present invention (Synechocystis) sp.) A method for producing a transgenic plant having increased flame resistance comprising transforming a plant cell with a recombinant vector comprising a PCC6803-derived SbtA (Sodium dependent bicarbonate transporter) gene and regenerating the plant from the transformed plant cell. to provide. Preferably, the SbtA gene may be composed of the nucleotide sequence of SEQ ID NO: 1.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of 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. Preferably, the plant may be a dicotyledonous plant, but is not limited thereto. Preferably, 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, 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 (Bamsamaceae), Aquifoliaceae, Nova (Celastraceae), Staphyleaceae, Buxaceae, Empyaceae, Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Eraeagnaceae (Flacourtiaceae), Violaceae, Passifloraceae, Tamaricanaceae, 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.

In addition, the present invention consists of the nucleotide sequence of SEQ ID NO: 1, Synechocystis genus (Synechocystis sp.) Provided is a composition for increasing the flame resistance of plants, including SbtA (Sodium dependent bicarbonate transporter) gene derived from PCC6803 . The composition comprises the SbtA gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient, by increasing the salt resistance of the plant by transforming the gene into the chloroplast of the plant.

In addition, the present invention (Synechocystis) sp.) Provided is a method for increasing the biomass of a plant, comprising the step of overexpressing the SbtA gene by transforming a recombinant vector comprising a PCC6803 derived SbtA (Sodium dependent bicarbonate transporter) gene into plant cells. 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, the SbtA gene may be composed of the nucleotide sequence of SEQ ID NO: 1.

In addition, the present invention (Synechocystis) sp.) Method for producing a transformed plant with increased biomass comprising transforming plant cells with a recombinant vector comprising a PCC6803 derived SbtA (Sodium dependent bicarbonate transporter) gene and regenerating the plant from the transformed plant cells To provide. Preferably, the SbtA gene may be composed 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. 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.

In addition, the present invention consists of the nucleotide sequence of SEQ ID NO: 1, Synechocystis genus (Synechocystis sp.) Provided is a composition for increasing biomass of a plant, including a SbtA (Sodium dependent bicarbonate transporter) gene derived from PCC6803 . The composition comprises the SbtA gene consisting of the nucleotide sequence of SEQ ID NO: 1 as an active ingredient, by increasing the biomass of the plant by transforming the gene into the chloroplast of the plant.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Materials and methods

Plant material

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.

Cynecosistis ( Synechocystis ) SbtA  Gene Cloning and Chromosome Transformation Vector Construction

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 subcloned with the RclpGAH vector, and the Rclp- SbtA was digested with XhoI / SpeI and blunted and then introduced into the tobacco pigment transforming vector TIA (PvuII) (FIG. 1). .

Chloroplast transformation and selection of transformants

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 the day before the balm-border was carried out; 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 at 25 ° C. and 16 hours of phytotron conditions.

Southern and Northern Blot  analysis

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.

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.

Observe GFP Expression

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.

Flameproof Assay and Morphological Observation

In order to see the response to the salts of SySbtA chloroplast-transformed tobacco, T2 seeds were soaked 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.

Investigation of plant growth conditions and growth

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 ℃.

Chlorophyll content determination

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).

Determination of Water Soluble Sugar and Starch Content

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 After the treatment, 0.5 mL of 80% ethanol was added, 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).

CO ₂ Exchange and CO ₂ Compensation Point Measurement

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%. CO ₂ The 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 at which the value became 0 was used as the CO ₂ compensation point.

Example 1 Identification of Chloroplast Transformation Vectors and Transgenic Plants

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).

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).

Example 2: GFP Expression of Tobacco Transgenic Plants

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).

Example 3: Salt Tolerance Assay and Phenotypic Analysis of SySbtA Plants

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 of the roots, whereas the vector control group showed slower root growth and developed lateral roots (FIG. 4A). Root growth in the absence of NaCl, which is a general condition, is different from each other. Thus, the root growth of the root at 100 at this time is expressed as a relative value. As a result, root growth decreased with increasing NaCl concentration in the vector control group, and root growth increased by 11 ~ 38% in NaCl 25 mM in SySbtA plants, while root length decreased in 100 mM NaCl. It was found that the resistance was increased compared to the control (Fig. 4B).

Example 4: SySbtA Physiological Characteristics and Growth of Plants

The seeded SySbtA T2 plants were grown in a phytotron environment, and the plant height, leaves, stem diameter, fresh weight and dry weight 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).

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).

Example 5 Photosynthetic Efficiency and CO of SySbtA Transgenic Plants ₂  Reward points

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).

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 corresponds to the Ci-curve and the SySbtA plant is CO ₂ It was found that the utilization efficiency was increased.

Example  6: SySbtA  Changes in Water-soluble Sugars and Starch Contents of Transgenic Plants

Increased CO ₂ in SySbtA Plants To determine whether the assimilation affects the biosynthesis of carbohydrates, the content of water-soluble sugars and starch in tobacco leaves collected at noon was analyzed for carbohydrate content 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.

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Claims (18)

Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) A method of increasing the flame resistance of eggplant plants, comprising overexpressing the SbtA gene by transforming a recombinant vector comprising a PCC6803-derived SbtA (Sodium dependent bicarbonate transporter) gene into Solanaceae plant cells. delete Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) Transforming Solanaceae plant cells with a recombinant vector comprising a PCC6803-derived SbtA (Sodium dependent bicarbonate transporter) gene; And
A method for producing a transformed eggplant plant with increased flame resistance, comprising the step of regenerating the eggplant plant from the transformed eggplant plant cells. delete Transgenic eggplant plants of increased salt resistance prepared by the method of claim 3. delete delete Seeds of the eggplant plant according to claim 5. Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) A composition for increasing the salt resistance of Solanaceae plants, including SbtA (Sodium dependent bicarbonate transporter) gene derived from PCC6803 . Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) A method of increasing the biomass of an eggplant plant comprising the step of overexpressing the SbtA gene by transforming a recombinant vector comprising a PCC6803-derived SbtA (Sodium dependent bicarbonate transporter) gene into Solanaceae plant cells. delete Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) Transforming Solanaceae plant cells with a recombinant vector comprising a PCC6803-derived SbtA (Sodium dependent bicarbonate transporter) gene; And
A method for producing a transformed eggplant plant with increased biomass comprising the step of regenerating the eggplant plant from the transformed eggplant plant cells. delete Transgenic eggplant plants with increased biomass produced by the method of claim 12. delete delete Seeds of the branch family according to claim 14. Synechocystis, consisting of the nucleotide sequence of SEQ ID NO: 1 sp.) Composition for increasing the biomass of Solanaceae plants, including SbtA (Sodium dependent bicarbonate transporter) gene derived from PCC6803 .

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