KR20140028655A - Method for producing transgenic plant with inhibited photorespiration and increased resistance to stress using the gene from cyanobacteria and the plant thereof - Google Patents

Abstract

The present invention provides a method for enhancing stress resistance by transforming a plant cell with a plant expression vector and suppressing photorespiration in a plant; a method for producing the transgenic plant with inhibited photorespiration and enhanced stress resistance compared with a wild type by transforming the plant cell with a recombinant vector containing the gene; a transgenic plant produced thereby with inhibited photorespiration and enhanced stress resistance compared with a wild type; and a composition containing seeds of the transgenic plant and the expression vector of the gene for inhibiting photorespiration and enhancing stress resistance in the plant. The plant expression vector contains: a glycolate dehydrogenase 1 (glcD1) coding gene derived from cyanobacteria; or a glycolate carboligase (gcl) coding gene and a tartronic semialdehyde reductase (tsr) coding gene.

Description

Method for producing transgenic plant with inhibited photorespiration and increased resistance to stress using the gene from cyanobacteria and the plant according to the present invention.

The present invention relates to a method for producing a transgenic plant having enhanced photosynthesis inhibition and stress resistance using a cyanobacteria-derived gene, and to a plant according thereto. ) Coding genes; Or inhibiting plant breathing by transforming plant cells with a plant expression vector comprising a glucolate carboligase (gcl) coding gene and a tartronic semialdehyde reductase (tsr) coding gene. And a method for improving stress resistance, transforming with a recombinant vector comprising the gene, thereby inhibiting photorespiration compared to wild type, and producing a transformed plant having enhanced stress resistance, optical respiration compared to wild type produced by the above method. The present invention relates to a composition for enhancing photorespiration and stress resistance of a transformed plant, which is inhibited and has enhanced stress resistance, and a seed thereof and a plant containing an expression vector of the gene.

Photosynthesis is a unique energy conversion process that not only allows plants to sustain their independent nutrition, but also provides the lowest means of living for other organisms on Earth. Ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco), an important enzyme present in the chloroplast, is responsible for CO ₂ fixation (carboxylation) in the photosynthetic cancer reaction. Induces the formation of 3-phosphoglycerate (3PGA). As the name implies, Rubisco also substitutes O _{2 for} carboxylation. Fixation (oxygenation) can also be made, which leads to the formation of one molecule of 3PGA and one 2-phosphoglycolate (2PG). The 2PG produced is toxic to plants because 2PG inhibits distinct steps in the Calvin-Bensin cycle. Thus, decomposition of 2PG is required, which is done by plants using the photorespiratory glycolate pathway, where 2 molecules of 2PG are converted into 1 molecule of 3PGA, CO ₂ and ammonia. C3 plants lose 25% of fixed CO ₂ in this process, consume ATP and NADPH through the photorespiration pathway and catalyze lethal ammonia.

C4 type photosynthetic plants (corn, sugar cane, etc.) have a CO ₂ enrichment mechanism (CCM) to minimize photorespiration, while C3 plants are estimated to lose 6.1% energy through photorespiration. However, research has shown that such phenomena are essential for plant survival. Thus, it has been suggested that such loss can be minimized if it is not completely extinguished by installing new and shorter 2PG metabolic pathways in C3 plants. Cyanobacteria model strain Synechocystis sp . PCC 6803 possesses three different 2PG metabolic pathways: 1) plant-like 2-C cycles, 2) bacterial-like glycerate pathways, and 3) unique dislocations. Distinct decarboxylation pathway. These pathways cause cyanobacteria to re-release CO ₂ near Rubisco, strengthening existing CCM in cyanobacteria and consequently lowering the lower CO ₂ of cyanobacteria-Rubisco even lower than that of C3 plants. Supplement affinity.

The inventors have attempted to modify the components of the cyanobacterial glycerate pathway in potato plants to target the chloroplasts. But transgenic potato "Desiree" plants expressing glcD1 (referred to as synGDH plants) in separate lines and separate gcl And plants expressing the tsr gene together (referred to as synGT plants). The plants showed stable introduction and expression of chimeric genes. At present, we are attempting to re-transform synGT expressing plants with glcD1 to complete the pathway. This is the first report showing stable transformation of components of the cyanobacterial glycerate pathway in crop plants, which can improve potato crop plant function in a constantly changing climate.

On the other hand, Korean Patent No. 0769755 discloses a PCK type C4 circuit, but as described in the present invention, a method for producing a transgenic plant having enhanced photosynthesis inhibition and stress resistance using a cyanobacteria-derived gene and a plant according thereto are described. It has not been disclosed.

The present invention is derived from the above requirements, and in the present invention, a plant (named synGDH) and glycolic acid carbo-linking enzyme (incorporated with cyglate dehydrogenase1, glcD1) encoding genes are introduced. A plant (named synGT) that expresses glucolate carboligase (gcl) and tartronic semialdehyde reductase (tsr) coding genes simultaneously was constructed, and the introduction and expression of the genes in these plants were determined by PCR and RT-PCR. As a result, the glyoxylic acid content in synGDH plants increased by 19%, and in the two types of transgenic plants synGDH and synGT, the biomass was increased by 25% and 19%, respectively, compared to the non-transformants. The weight of potato tubers was increased 38% and 26%, respectively, compared to the non-transformants. In addition, synGDH plants maintained 8-10% higher photosynthetic efficiency (Fv / Fm) than nontransformers and synGT plants under high temperature and stress conditions, and the chlorophyll content (6%) decreased in synGDH plants under the same conditions. Was lower than nontransformers (11.6%) and synGT plants (9%). In addition, the dry weight of synGDH plants after stress treatment at high temperature and hardening was 16.5% and 14.5% higher than nontransformers and synGT plants, respectively. Through these results, the present invention was completed by confirming the utility of the genes in plants.

In order to solve the above problems, the present invention is a glycolic acid dehydrogenase (glycolate dehydrogenase 1, glcD1) coding gene derived from cyanobacteria; Or transforming a plant cell with a plant expression vector comprising a glycolic acid carboigase (gcl) coding gene and a tartronic semialdehyde reductase (tsr) coding gene. It provides a method of suppressing photorespiration and enhancing stress resistance.

The present invention also provides a glycolic acid dehydrogenase (glcD1) coding gene derived from cyanobacteria; Or compared to the wild type comprising the step of transforming with a recombinant vector comprising a glycolic acid carbo ligase (gcl) coding gene and tartron acid semialdehyde reductase (tsr) coding gene; It provides a method for producing a transformed plant.

In addition, the present invention provides a transgenic plant and its seeds that are suppressed in photorespiration and enhanced stress resistance compared to the wild type produced by the method.

The present invention also provides a glycolic acid dehydrogenase (glcD1) coding gene derived from cyanobacteria; Or it provides a composition for inhibiting photorespiration and stress resistance of a plant containing a recombinant expression vector comprising a glycolic acid carbo-linking enzyme (gcl) coding gene and tartron acid semialdehyde reductase (tsr) coding gene.

Glycolic acid dehydrogenase (glcD1) coding gene derived from cyanobacteria of the present invention; Or synGDH or synGT potato plants prepared by transformation of plant expression vectors, including; glycolic acid carbo-linking enzyme (gcl) coding gene and tartron acid semialdehyde reductase (tsr) coding gene. Significantly increased compared to wild type, in particular, synGDH potato transformant increased the photosynthetic efficiency, chlorophyll content reduction ratio width at high temperature and intense stress conditions compared to synGT and wild type, the method of producing a transgenic plant according to the present invention It can be usefully used to increase the amount of photosynthesis or biomass.

1 is glcD1 alone and gcl And tsr Transgenic potato plants expressing genes in chloroplasts together. (A) Schematic representation of T-DNA of pSGT used for potato transformation. 35S pro is the cauliflower mosaic virus promoter, TEV is the transcriptional enhancer, chlTP is the transport peptide from the sequence of the large subunit of Rubisco (tomato), gcl is glyoxylate carbolinkase cDNA, 35S Ter is A termination sequence from cauliflower mosaic virus, tsr is a tartronic semialdehyde reductase cDNA. (B) T-DNA region of pSGD used for potato transformation. All abbreviations are the same as A except that glcD1 is glycolate dehydrogenase 1 cDNA. (C) Representative view of shoot regeneration from stem and leaf explants.
2 shows RT-PCR and total glyoxylate content of non-transformed (NT) and transformed (synGT and synGDH) plants. (A) RT-PCR analysis of gcl, tsr and glcD1 and NPTII gene expression in the leaves of non-transgenic and transgenic plants. First-strand cDNA synthesis and PCR analysis from total RNA was performed according to the manufacturer's instructions. Actin was used as internal control. The reaction product (8 μl) was analyzed via gel electrophoresis. NT is a non-transgenic potato plant, synGT is a transgenic potato plant expressing the gcl and tsr genes, and synGD is a transgenic potato plant expressing the glcD1 gene. (B) Analysis of total glyoxylate content from leaves of NT and transgenic plants by spectrometer. Data is expressed as mean ± standard deviation (SD) of three repeats.
3 shows a comparison of phenotypic and biomass related parameters of NT and transgenic plants. (A) Pictures of 4 week old NT and transformed synGT and synGDH plants. (B) DW is dry weight, TW is tuber weight and BM is total biomass content of NT and transgenic plants after 6 weeks of growth in greenhouse. Data is expressed as mean ± standard deviation (SD) of five repeats.
4 shows the effect of high temperature (38 ° C.) and high light (350 μE) on 6-week non-transformation (NT) and transgenic (synGDH, synGT) plants grown in soil. (A) Effects of high temperature and light on light-inhibition measured by means of Fv / Fm values. (B) Relative decrease in chlorophyll content after stress treatment. (C) Partial dry weight of NT, synGDH and synGT plants after stress under greenhouse conditions. Data is expressed as mean ± standard deviation (SD) of three repeats.

In order to achieve the object of the present invention, the present invention is a glycolic acid dehydrogenase (glycolate dehydrogenase 1, glcD1) coding gene derived from cyanobacteria; Or transforming a plant cell with a plant expression vector comprising a glycolic acid carboigase (gcl) coding gene and a tartronic semialdehyde reductase (tsr) coding gene. It provides a method of suppressing photorespiration and enhancing stress resistance.

In the method according to an embodiment of the present invention, the glcD1 , gcl and tsr genes may be preferably composed of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene is at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, respectively Base sequences having sequence homology. "% 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 glcD1 Alternatively, the gcl and tsr gene sequences 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.

glcD1 Or expression vectors comprising gcl and tsr gene sequences and appropriate transcriptional / translational control signals can be constructed by methods well known to those skilled in the art. Such methods include in vitro recombinant DNA 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 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. 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 method of carrying the vector of the present invention into the host cell may be carried out by microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment or the like. Can be injected.

In the method according to an embodiment of the present invention, the stress may be a high temperature or a strong light, preferably a high temperature of 30 to 40 ° C. or a hard light of 300 to 400 μE, and more preferably a high temperature of 38 ° C. or a hardening of 350 μE. May be, but is not limited thereto.

In addition, the present invention is a glycolic acid dehydrogenase (glycolate dehydrogenase 1, glcD1) coding gene consisting of the nucleotide sequence of SEQ ID NO: 1 from cyanobacteria; Or a glycolic acid carboligase (gcl) coding gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a tartronic semialdehyde reductase (tsr) coding gene consisting of the nucleotide sequence of SEQ ID NO: 3; Transforming the plant cell with the recombinant vector; And

Provided is a method for producing a transgenic plant in which photorespiration is suppressed and stress resistance is enhanced as compared to the wild type comprising regenerating a plant from the transformed plant cell.

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.).

In the method according to an embodiment of the present invention, the stress may be a high temperature or a strong light, preferably a high temperature of 30 to 40 ° C. or a hard light of 300 to 400 μE, and more preferably a high temperature of 38 ° C. or a hardening of 350 μE. May be, but is not limited thereto.

In addition, the present invention provides a transgenic plant and its seeds that are suppressed in photorespiration and enhanced stress resistance compared to the wild type produced by the method.

In a transformed plant according to an embodiment of the present invention, the plant may be a C3 plant, such as grains, peanuts, tobacco, spinach, potatoes, sugar beet, soybeans, preferably may be a potato plant, but is not limited thereto. .

In a transformed plant according to an embodiment of the present invention, the potato plant may be an increase in the weight of potato tubers compared to wild type. In particular, synGDH plants showed an increase in 38 and 16% tuber weight, respectively, compared to NT and synGT plants.

In addition, the present invention is a glycolic acid dehydrogenase (glycolate dehydrogenase 1, glcD1) coding gene consisting of the nucleotide sequence of SEQ ID NO: 1 from cyanobacteria; Or a glycolic acid carboligase (gcl) coding gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a tartronic semialdehyde reductase (tsr) coding gene consisting of the nucleotide sequence of SEQ ID NO: 3; It provides a composition for inhibiting photorespiration and enhancing stress resistance of a plant containing a recombinant expression vector. The composition is a glycolic acid dehydrogenase (glycolate dehydrogenase 1, glcD1) coding gene consisting of the nucleotide sequence of SEQ ID NO: 1 from cyanobacteria as an active ingredient; Or a glycolic acid carboligase (gcl) coding gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a tartronic semialdehyde reductase (tsr) coding gene consisting of the nucleotide sequence of SEQ ID NO: 3; Comprising a recombinant expression vector, and by transforming the recombinant expression vector to the plant is to suppress photorespiration of the plant, it is possible to enhance the stress resistance.

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

Plasmid Preparation

The coding sequences of glcd1 (sll0404), gcl (slr2088) and tsr (slr0229) were downloaded from the publicly available http://genome.kazusa.or.jp/cyanobase . Primers corresponding to each gene were designed to have a Bam HI restriction site (Table 1). The gene is Synechocystis sp.) strain PCC 6803 (hereinafter, PCC 6803) was amplified using genomic DNA.

After sequencing, coding regions were ligated to shuttle vectors using Bam HI. The shuttle vector has cauliflower mosaic virus 35S dual promoter (35S), tobacco eth virus 5′-UTR translation enhancer (TEV) and chloroplast transport peptide (chlTP) from a small subunit of tomato rubis and 35S terminator. syngcl And Cassettes for gene expression of syntsr were introduced into the Hin dIII and Sal I sites in the same T-DNA of pCAMBIA2300, respectively, and were designated pSGT. The resulting cassette of glcd1 gene expression was introduced into the pCAMBIA2300 vector using the Pst I site and named pSGD. The plant expression vector of frozen-thawed by Agrobacterium tumefaciens methods Solarium Tome Pacifico Enschede (Agrobacterium tumefaciens ) strain GV3101.

Plant material, transformation and regeneration

Infertile Potato Plants ( Solanum tuberosum L .; cv. Desiree) was grown for the transformation experiments of the present invention. The plants were sterilely propagated in petri-plates, and transformation and regeneration were carried out by Ahmad et al. 2008 Plant Cell Rep 27: 687-698, except for the alone modification of kanamycin (100 mgL ⁻¹ ) plant selection marker. It was performed as described in.

PCR  And RT - PCR  analysis

Genomic DNA was extracted using the method defined in the prior art (Kim and Hamada 2005 Biotechnol Lett 27: 1841-1845). PCR reactions were performed using genomic DNA purified from PCR premixes (Bioneer, Korea) using two astringent gene specific primers. The amplification reaction consisted of 5 minutes (1 cycle) at 94 ° C. followed by 27 cycles (30 seconds at 94 ° C., 30 seconds at 60 ° C. and 1 minute at 72 ° C.) and finally an extension cycle of 7 minutes at 72 ° C. PCR products were separated on 1% agarose gels, stained with ethidium bromide and visualized under UV.

For RT-PCR analysis, total RNA was extracted with RNeasy Plant Mini Kit according to manufacturer's instructions (QIAGEN, Germany) and treated with DNase I without RNase to remove any contaminating genomic DNA. RNA (2 μg) was used for reverse transcription according to manufacturer's instructions (Fermentas, USA). PCR was performed using 1.00 μl 3-fold dilution first-stranded cDNA. Primers and chimeric genes specific for actin (internal standard) are listed in Table 1. PCR conditions for the actin, glcD1, gcl and tsr genes are as described above, except for the elongation time, which was 30 seconds per cycle.

gun Glyoxylate  Analysis of the content

Total glyoxylate content was determined as described in Rojano-Deldgado et al. (Rojano-Deldgado et al. 2010 Talanta 82: 1757-1762). Briefly, 50 mg of leaf tissue was submerged in 1 mL sterile distilled water and extracted by ultrasound for 10 minutes. The extracted liquid was divided into two 1.5 mL tubes containing 450 μl of extract each, and aliquots A and B were defined. Then 50 μl of freshly prepared 1% phenylhydrazine solution with 100 mM hydrochloric acid (HCl) was added to each tube and incubated at 60 ° C. for 10 minutes. After the sample was cooled to 20 ° C., 250 μl of concentrated HCl was added to each tube. Thereafter, 100 μl of 1.6% potassium ferricyanide (dissolved in water) was added to Aliquot A, while the same amount of sterile distilled water was added to Aliquot B. The absorbance difference at 520 nm, corresponding to the glyoxylate content, was measured after 12 minutes of incubation at room temperature.

Morphological features of transgenic plants

Transformed and non-transformed (NT) plants were prepared by in vitro rooting development. After 10 days of subculture, shoots produced enough roots to support transplantation into soil pollen. The plants were grown in greenhouse conditions without any fertilizer supplementation. After 6 weeks of growth, the plants were harvested and their ground weight and tuber fresh weight determined. The dry weight of the ground portion was measured after drying the plants at 60 ° C. for 72 hours.

Growth analysis at high temperature and high light conditions

The plants (6 weeks old) were each applied to high temperature 38/32 ° C. in 16/8 hr light and dark conditions for 3 days inside a growth chamber at 60% relative humidity. The light breathing conditions were also introduced by setting the light conditions to slightly higher values (350 μE). Plants were watered at regular intervals to avoid drought conditions caused by high temperatures. At the beginning of treatment, the fourth fully unfolded leaf from the top was marked to record chlorophyll fluorescence (Fv / Fm) and chlorophyll content during the entire assay and served as a point for scoring biomass accumulation. Fv / Fm and chlorophyll content were measured at 0 hours to indicate the start of treatment, which was then measured after 2, 24 and 72 hours of high temperature treatment. Fv / Fm was recorded via a handheld chlorophyll fluorimeter (Handy PEA, Hansatech, UK) after 30 min dark adaptation from the indicated leaves. Chlorophyll content was measured using a portable non-destructive chlorophyll meter (SPAD-502 plus, Konica Minolta, Japan).

After three days of stress treatment, the plants were transferred to the greenhouse under normal growth conditions and allowed to grow for another two weeks. Thereafter, the stems were cut out from the leaves, and the dry weight (specified as partial dry weight) was measured after drying the sample as described above. The tubers were also harvested, recorded and compared for their fresh weight.

Example  1. Development of Transgenic Potato Plants

gcl And have cloned the cDNA corresponding to the vector tsr pSGD for Meanwhile, glcD cloning in plant expression vectors pSGT (Fig. 1A) (Figure 1B). Chloroplast transport peptide (chlTP) from a small subunit of tomato rubis was ligated upstream of the coding region to target the protein to the chloroplasts of the transgenic plant. The resulting plasmid was transformed with Agrobacterium tumefaciens GV3101 strain for potato transformation. Putative redifferentiated potato plants were selected on MS medium (redifferentiation medium with appropriate hormones) containing kanamycin as plant selection marker. Putative putative transformation lines (FIG. 1C) re-differentiated from stem or leaf explants on selection medium were transferred to MS medium without kanamycin to ensure rapid rooting of shoots. After gaining potent force through growth on non-selective medium, shoots were transferred to MS medium containing kanamycin to finally screen for transgenic lines. In addition, polymerase chain reaction (PCR) analysis was performed using genomic DNA of shoots with strong rooting compared to NT plants on selection medium (data not shown). PCR screened positive strains of glcD1 expressing plants were defined as synGDH (syn and GD correspond to Synechocystis PCC 6803 and glycolate dehydrogenase), while gcl And The tsr expression lineage was defined as synGT, where GT corresponds to gcl and tsr . Genomic DNA PCR analysis of selected strains of synGT and synGDH showed stable transformation of the chimeric gene into the potato genome (data not shown).

Example  2. Analysis of Foreign Gene Expression in Transgenic Plants

To assess the stable introduction and transcription of foreign genes in the transgenic line, RT-PCR was performed from the total RNA (no DNA) of the greenhouse growing transgenic plants. The amplification profile of each foreign gene obtained from the diluted cDNA (FIG. 2A) showed stable introduction and transcription of the foreign gene. Introduction and expression of the NPTII gene allows synGT and synGDH plants to grow in selection media. foreign genes gcl and in synGT tsr was expressed at remarkable levels, but tsr was expressed at a higher rate than gcl (FIG. 2). Similarly, the glcD1 gene also showed satisfactory expression in synGDH plants.

Example  3. Gun Glyoxylate  Determination of content

Total glyoxylate content from NT and transgenic lines grown in greenhouses was determined. The analysis showed that synGDH plants accumulate higher glyoxylate content compared to NT and synGT plants. Total glyoxylates determined from the leaf samples accumulated 2.65 μg / g FW (raw weight) for NT plants, while synGT plants accumulated 2.72 μg / g FW. However, synGDH plants accumulated 3.14 μg / g FW glyoxylate content (FIG. 2B). Compared to NT and synGT plants, synGDH plants accumulated 18 and 19% higher glyoxylate content, respectively.

Example  4. Improved Transgenic Plants Biomass  accumulation

Transgenic and NT plants of equal size and health were grown in pots with the same amount of soil under greenhouse conditions. Plants were irrigated at regular intervals for trophic growth and tuber biomass assessment of NT and transgenic plants to avoid any kind of stress (hypoxia or drought). Representative drawings taken after 5 weeks of growth (FIG. 3A) show that synGDH plants are taller than NT and synGT, and that synGT plants are better in function than NT plants.

Comparing biomass of trophic growth, it was found that synGDH plants accumulated 11% more dry weight above the ground than synGT and NT plants (FIG. 3B). Interestingly, synGDH plants showed an increase in 38 and 16% tuber weight, respectively, compared to NT and synGT plants. The total increase in biomass accumulation was 25 and 15% higher in synGDH plants compared to NT and synGT plants (FIG. 3B).

Example  5. Effect of high temperature and high light on plant growth

To evaluate plants under photorespiratory conditions, soil-grown 6 week old plants were tested at high temperature (38 ° C.) and high light conditions (35 μE). Fv / Fm and chlorophyll content were determined from the indicated leaves of all plants before and after the assay run. On average, synGDH plants maintained a 10% higher Fv / Fm reading compared to NT, and 8% from synGT plants after 72 hours of stress analysis (FIG. 4A). On the other hand, in NT the relative chlorophyll content decrease was 11.6%, whereas the synGT and synGDH plants showed 9 and 6% reductions, respectively (FIG. 4B). After 72 hours of the stress assay, the plants were grown for two more weeks to assess for biomass accumulation. For this purpose, plants were cut from the indicated leaves and dried in an oven to determine dry weight content. synGDH plants accumulated 16.5 and 14.5% more nutrient growth biomass than NT and synGT plants, respectively (FIG. 4C).

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for producing transgenic plant with inhibited          photorespiration and increased resistance to stress using the          gene from cyanobacteria and the plant <130> PN12188 <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 1479 <212> DNA <213> Synechocystis sp. PCC 6803 <400> 1 atggccattt tctcccccgt caacgccgtt accgatatta ttccccagct cgaaaaaatt 60 gttggccagg atggagtaat taaacgcaaa gacgagctat tcacctacga atgcgacggt 120 ttaacgggtt atcgacaacg gccggccctg gtggttttgc cccgcacaac ggaacaggta 180 gccacaatag tgaaactttg tcacgatcgc caaattcctt ggattgccag gggggctggc 240 acagggttat cggggggagc cttgccgggg gccgatagcc tattgattgt caccactcgc 300 atgcggcaaa ttttggcagt agattacgac aaccagacca ttgttgtcca gccgggggtg 360 gtgaataact gggttaccca aaccgttagt ggggctggct tttactatgc ccctgatcct 420 tccagtcaga ttgtctgctc cattggcggt aatattgcgg aaaattccgg tggagttcat 480 tgtttgaaat atggcaccac caccaaccat gtgctgggct tgaaactggt tattcccgat 540 ggctccattg tggaagtagg ggggcaagtc cccgaaacgc cgggctacga tttaaccggt 600 ttatttgttg gttccgaagg aaccctaggc atcgccacag aaatcaccct aaaaattctc 660 aaaaccccag aatctatctg tgtcgtattg gcggattttc tttctctcga agccaccgcc 720 caatccgtgg ccgatatcat tgcggcgggc atcgtcccag cgggcatgga aattatggac 780 aatttcagca tcaatgcggt ggaagacgtg gtggccacca attgttaccc cagggatgcg 840 gcggccattt tgttagtgga actggacggt ctgcccatcg aagtggaatt aaaccaagcc 900 aaagtagaag aaatttgccg caacaatgga gcccgcaaca cggcgatcgc ctacgaccaa 960 gaaacccgcc taaaaatgtg gaaaggaaga aaagcggcct ttgcggcggc gggtaaacta 1020 agccccagtt actttgtcca agatggtgtg gtaccccgga ctcaattggt acagatttta 1080 agcgacatta atgatttaag taagaaatat ggctttgcca ttgccaatgt tttccatgcc 1140 ggagacggta atttacatcc cctaattttg tatgatcaaa aagtaccagg agcctgggaa 1200 aaagtggaag aattgggggg agaaatcctt aaacgctgtg tggaattggg gggaagttta 1260 tccggagaac acggcattgg cattgataaa aattgcttta tgcccaatat gttcaacgaa 1320 gtagatttag aaacaatgca atgggtcaga caatgtttta atcctgataa cttagctaat 1380 cctggtaagc tttttcctac cccccgcagt tgtggagaag tggccaatgc ccaacggctt 1440 aacctaggcc aggacaagaa aatggaggaa atttattga 1479 <210> 2 <211> 1785 <212> DNA <213> Synechocystis sp. PCC 6803 <400> 2 atggatagcc tgaaacgcca tggggtcaaa cacatttttg gctatcccgg cggggcaatt 60 ttgcccatct atgatgaact gtaccgcttt gaagcggcgg gggaaattga gcatattttg 120 gtgcgccatg aacaaggagc ttcccatgcg gcggatgggt atgccagagc cacaggtaaa 180 gtgggagttt gtttcggtac atctggacca ggggcgacta acttggtgac cggcattgcc 240 aatgcccatt tggactcggt gcccatggtg gtgattactg gacaggtggg ccgtgccatg 300 attggtagcg atgctttcca ggaaattgac atttttggca tcaccttacc gatcgttaag 360 cactcctatg tggtacgtag tgcggcggat atggctcgca ttgttactga ggctttccat 420 cttgctagca ccggtcgtcc cgggccggtt ttgatcgata ttcccaagga tgtgggctta 480 gaagaatgtg agtacattcc cctcgacccc ggtgacgtta atctaccggg ttatcgcccc 540 acggttaaag gtaatccccg acaaattaat gcggcattgc aattgttgga gcaggccaga 600 aatcccttgc tctacgtagg gggaggggcg atcgccgcca atgcccatgc ccaggtgcag 660 gaatttgcgg aaaggttcca gttgccggta acaaccaccc tgatgggaat tggggctttt 720 gacgaaaacc atcccctttc ggtgggtatg ttgggtatgc atggcaccgc ctatgccaac 780 tttgccgtca gcgaatgtga tttgttgatt gcggtggggg cccgtttcga cgaccgggta 840 actggcaaac tagacgaatt tgctagccgc gccaaagtaa ttcacattga catcgacccg 900 gcggaggtgg gaaaaaacag ggctcccgat gtgcccattg tgggggatgt acgccatgtt 960 ttagaacagc ttttgcagcg ggcccgggaa ttggattacc ccacccatcc ccataccacc 1020 caggcatggt taaatcgcat tgatcattgg cggaccgatt accccctcca ggtgccccac 1080 tatgaggata ctattgcccc ccaggaggta gtacacgaaa ttggtcgcca ggcccccgat 1140 gcctactaca ccaccgatgt gggacaacac caaatgtggg cggcccagtt tttgaacaat 1200 ggcccccgcc gatggatttc cagtgctggc ttgggtacga tgggctttgg tttacctgcc 1260 gccatgggag ccaaagtggg agtgggggac gaagcggtca tttgcatcag tggagatgcc 1320 agcttccaaa tgaatcttca ggaactggga accctagccc agtacgacat ccaggttaaa 1380 actattattc tcaataacgg ttggcagggg atggtgcgtc agtggcaaca aactttctac 1440 gaagaacgtt attctgcttc taacatgtcc cagggcatgc cagacattaa tctcctctgt 1500 gaagcctatg gcatcaaggg tattactgtg cgcaagcggg aagatttggc cccggcgatc 1560 gccgaaatgc tagcccacaa tggtcctgtg gtgatggatg tggtggtcaa aaaagatgaa 1620 aactgttacc ctatgattgc ccccggcatg agtaatgccc aaatgctagg tttaccggaa 1680 gtgccggtac gggacaatgg tccccggatg gtggagtgca accattgcca aacccaaaat 1740 ttcatcaccc atcgtttctg ttctggttgt ggagccaaac tctaa 1785 <210> 3 <211> 873 <212> DNA <213> Synechocystis sp. PCC 6803 <400> 3 atgaacgttt ccaaaattgc agttttcggt ttaggagtta tgggcagtcc tatggctcaa 60 aacctagtta aaaatggtta tcaaacagtt ggttataacc gcaccctaga gcgtccttct 120 gtccaggaag cagcaaaggc aggggttaaa gttgttacta gcattgcggt ggcggcggca 180 aatgcagata ttattctcac ctgtgttgga gatgaaaaag atgttcaaca gcttatttta 240 ggttcgggag ggattgctga atacgctaaa ccccaagcct taataattga ttgcagtacc 300 attggtaaaa ctgcggctta tgaactagct acaaatttaa aactccaggg tttacgcttt 360 ttggatgcgc cggtaacagg aggtgacgtt ggcgcaatca acggtacgtt aacgattatg 420 gtggggggtg atatttcgga ttttgaagaa gctttacccg tcctcaaatc cataggagaa 480 aaaatagtcc actgtggtcc cagtggcagt ggtcaagctg ttaagctttg taatcaggta 540 ctttgtggca tccatgcgat cgccgctgcg gaagctatac aactttctga gcagttgggg 600 attgccccag agttagtaat tgatacttgt ggcagtgggg cggcgggttc ctgggccttg 660 actaatttag caccgaaaat gtcggaggct gattttgccc caggctttat ggtcaaacat 720 cttcttaagg atctgcgcct cgtccgggaa gccgctgaaa atggcccatt accaggggtt 780 acgttggcag aaagcttgtt tacatcggtt caattattgg ggggagaaga ccagggttcc 840 caagccatta tccgtgccta tcgcgaagct tag 873 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggatccatgg ccattttctc ccccgt 26 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggatcctcaa taaatttcct ccattttct 29 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggatccatgg atagcctgaa acgc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggatccttag agtttggctc caca 24 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggatccatga acgtttccaa aattgca 27 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ggatccctaa gcttcgcgat aggc 24 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gatcgcctac gaccaagaaa 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 attggccact tctccacaac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 gccaatggtt gcgaagtagt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggctggttgg agcatgttat 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cagggtttac gctttttgga 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ccccaataat tgaaccgatg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 tggactctgg tgatggtgtc 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cctccaatcc aaacactgta 20

Claims (10)

A glycolic acid dehydrogenase 1 (glcD1) coding gene consisting of the nucleotide sequence of SEQ ID NO: 1 derived from cyanobacteria; Or a glycolic acid carboligase (gcl) coding gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a tartronic semialdehyde reductase (tsr) coding gene consisting of the nucleotide sequence of SEQ ID NO: 3; A method for inhibiting photorespiration of plants and enhancing stress resistance, comprising the step of transforming plant cells with a plant expression vector. The method of claim 1, wherein the stress is high temperature or intense light. A glycolic acid dehydrogenase 1 (glcD1) coding gene consisting of the nucleotide sequence of SEQ ID NO: 1 derived from cyanobacteria; Or a glycolic acid carboligase (gcl) coding gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a tartronic semialdehyde reductase (tsr) coding gene consisting of the nucleotide sequence of SEQ ID NO: 3; Transforming the plant cell with the recombinant vector; And
Compared to the wild type comprising the step of regenerating the plant from the transformed plant cells, the photorespiration is suppressed, the production method of the transformed plant enhanced stress resistance. 4. The method of claim 3 wherein the stress is high temperature or intense light. Transgenic plants in which photorespiration is suppressed and stress resistance is enhanced compared to the wild type produced by the method of claim 3. The transgenic plant of claim 5, wherein the plant is a C3 plant. The transgenic plant of claim 6, wherein the plant is a potato. 8. The transgenic plant of claim 7, wherein the potato plant has an increased weight of potato tubers compared to the wild type. Seed of the transgenic plant according to claim 5. A glycolic acid dehydrogenase 1 (glcD1) coding gene consisting of the nucleotide sequence of SEQ ID NO: 1 derived from cyanobacteria; Or a glycolic acid carboligase (gcl) coding gene consisting of the nucleotide sequence of SEQ ID NO: 2 and a tartronic semialdehyde reductase (tsr) coding gene consisting of the nucleotide sequence of SEQ ID NO: 3; A composition for inhibiting photorespiration and enhancing stress resistance of a plant containing a recombinant expression vector.

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