KR101374184B1 - A gene encoding patggasa protein from poplar(populus alba × p. tremula var. glandulosa), a recombinant vector containing the patggasa gene, the methods for producing the recombinant vector containing the patggasa gene and the plants showing enhanced drought tolerance using the recombinant vector - Google Patents

Abstract

The present invention provides: a PatgGASA gene which is derived from Populus alba × P. tremula var. glandulosa and has base sequences represented by the sequence number 1; a recombinant vector containing the PatgGASA gene; a method for producing the recombinant vector containing the PatgGASA gene; a plant having an improved drought stress tolerance using the recombinant vector containing the PatgGASA gene; and a method for producing the plant having an improved drought stress tolerance using the recombinant vector containing the PatgGASA gene.

Description

A method for producing a plant having improved resistance to dry stress and a plant having improved resistance to dry stress, a method of producing the recombinant vector, a recombinant vector containing the PATGAspASA gene, the PATGAspASA gene, a recombinant vector using the recombinant vector {A gene encoding PatgGASA protein from poplar (Populus alba × P. tremula var.glandulosa), a recombinant vector containing the PatgGASA gene, the methods for producing the recombinant vector containing the PatgGASA gene and the plants showing enhanced drought tolerance using the recombinant vector}

The present invention is a recombinant vector comprising a hyeonsasi (Populus alba × P. Tremula var . Glandulosa) a recombinant vector, a method for preparing a recombinant vector including the PatgGASA genes including PatgGASA gene, the PatgGASA gene derived from the PatgGASA gene The present invention relates to a method for producing a plant having improved resistance to dry stress and a plant having improved resistance to dry stress using a recombinant vector comprising PatgGASA gene.

In the late 20th century, due to a variety of reasons, global warming causes severe climate change due to global warming, leading to a sudden increase in weather events such as droughts and floods. It is causing great damage to development. In addition, as the global average temperature increases due to global warming, deserts of soils and deserts are gradually increasing around desert areas in the conventional desert areas, and the death of plants with low dry tolerance is increasing. Accordingly, as the desertification of soil due to plant death continues to increase, the problem of gradually deteriorating forests and soils is increasing. Therefore, it is important to study the function of stress response genes for dry stress that affect the productivity of trees.

Gibberellic acid stimulated transcript 1 (GAST1), a gene known to play an important role in growth regulation and biological or abiotic stress, is the gibberellic acid stimulated Arabidopsis (GASA) gene. (Herzog et al, 1995, Plant Mol Biol 27, 743-752).

The GASA family proteins are 15 in Arabidopsis (Alonso-Ramirez et al, 2009, Plant Physiol, 150, 1335-1344), 17 in Poplar (JGI v2.2 http://www.jgi.doe.gov), Five have been reported in petunia (Ben-Nissan et al, 2004, Plant J, 37, 229-238). These GASA family proteins mostly consist of small proteins with 85 to 119 amino acids. The GASA protein also has a signal peptide at the amino terminus and 12 cysteine residues at the carboxy terminus (Herzog et al, 1995, Plant Mol Biol 27, 743-752).

GASA is known to be involved in developmental processes such as cell division or cell extension, organogenesis, stem extension, entourage formation, and flowering regulation (Kotilainen et al, 1999, Plant Cell, 11, 1093-1104; Ben-Nissan et al. , 2004, Plant J, 37, 229-238). And is involved in biological or abiotic stresses such as pathogen defense, antioxidant activity, heat stress response, salt stress response or oxidation stress response (Alonso-Ramirez et al, 2009). , Plant Physiol, 150, 1335-1344; Li et al, 2011, J Plant Physiol, 168, 2153-2160).

In the case of transgenic studies on plant GASA genes, GASA1 overexpressed Arabidopsis accumulates DELLA genes and inhibits root development in response to cold stress (Li et al, 2011, J Plant Physiol, 168, 2153-2160). . GASA4 overexpressed Arabidopsis also improved resistance to salt (150 mM), oxidation (500 uM paraquat) and heat (50 ° C) stress. On the other hand, the Arabidopsis with GASA4 expression inhibition showed no difference in stress resistance compared to the control (Alonso-Ramirez et al, 2009, Plant Physiol, 150, 1335-1344).

Meanwhile, as disclosed in the related art of the present invention, Korean Patent Publication No. 2011-0100417 discloses a composition for improving the dry stress resistance of a plant, a transformed plant into which the composition is introduced, a method for producing the transformed plant, and a method for enhancing dry stress resistance of the plant. And a composition for promoting plant growth or flowering, wherein the nucleotide sequence used in the prior art is a nucleotide sequence encoding a CaSRP1 protein, which is involved in the resistance and growth ability of the plant to dry stress, and the plant is used to transform the plant. The conversion indicates that it can be usefully used as a new functional plant with excellent drought resistance or rapid cultivation.

An object of the present invention is hyeonsasi (Populus alba × P. Tremula var . Glandulosa) a recombinant vector, a method for preparing a recombinant vector including the PatgGASA genes including PatgGASA gene, the PatgGASA gene of origin, a recombinant containing the PatgGASA gene The present invention provides a method for producing a plant having improved resistance to dry stress using a vector and a plant having improved resistance to dry stress using a recombinant vector comprising a PatgGASA gene.

The present invention SEQ ID NO: 1 hyeonsasi having the nucleotide sequence of (Populus alba × P. Tremula var . Glandulosa) a recombinant vector, a method for preparing a recombinant vector including the PatgGASA genes including PatgGASA gene, the PatgGASA gene of origin, the It is possible to provide a method for producing a plant having improved resistance to dry stress using a recombinant vector containing PatgGASA gene and a plant having improved resistance to dry stress using a recombinant vector containing PatgGASA gene.

The present invention can provide a plant having improved productivity of crops and trees even in dry soil by improving the resistance to dry stress by using a recombinant vector comprising PatgGASA gene derived from suspension at the time of having the nucleotide sequence of SEQ ID NO: 1.

Figure 1 (A) is hyeonsasi (Populus alba × P. Tremula var . Glandulosa) the GASA protein (PtGASA), the Arabidopsis GASA proteins PatgGASA protein, black aspen (Populus trichocarpa) isolated from (AtGASA1, AtGASA4, AtGASA5 Multiple sequence alignments), where multiple sequence comparisons were made using the ClustalW program. Signal peptides well conserved at the amino terminus of GASA proteins are boxed and twelve Cysteine (C) residues well conserved at the carboxy terminus are marked with asterisks. Cleavage sites, where the signal peptide is released from the protein, are indicated by arrow heads.
FIG. 1 (B) hyeonsasi (Populus alba × P. Tremula var . Glandulosa) to separate one PatgGASA protein, PtGASA protein, Arabidopsis GASA family proteins of black cottonwood trees in a (AtGASA1, AtGASA2, AtGASA3, AtGASA4 , AtGASA5) and This is a result of phylogenetic analysis of PatgGASA protein comparing the soft relationship between corn GAST1 (gibberellic acid stimulated transcript 1, ZmGAST1). The phylogenetic tree was created using Mega 4.1. The number of branches is the bootstrap value expressed as a percentage. The bootstrap analysis was performed with 1,000 iterations and the values were expressed as a percentage. In protein phylogenetic analysis, PatgGASA showed the highest flexibility with PtGASA of Black Cottonwood.
Figure 2 shows the results of Southern blot analysis using chromosomal DNA at the time of suspension, in Figure 2 B means Bam HI, EI means Eco RI, H means Hin dIII, X means Xba I. PatgGASA gene was found to be present in 1 to 2 copies of the chromosomes at the time of suspension.
Figure 3 (A) to Figure 3 (C) shows the results of the PatgGASA gene expression analysis according to the growth stages and abiotic stress treatment of the growth curve of suspension cultured suspension cells, Figure 3 (A) is suspension culture cells suspended The growth curve according to the culture days (day 0 to day 26) of Figure 3 (B) shows the expression of the PatgGASA gene at the stage of growth (4 days to 24 days) of the suspension growth culture growth curve at suspension, Figure 3 (C) shows no treatment in suspension cultured cells (Con), dried in suspension cultured cells (250 mM mannitol, Man), salt (150 mM sodium chloride, NaCl), low temperature (2 ° C, Cold) , Apsis acid (25 μM, ABA) was treated and shaken for 2 hours and 10 hours, respectively, to show the expression of PatgGASA gene.
4 (A) to 4 (C) are directed to the production of overexpression vectors comprising the gene of SEQ ID NO: 1 encoding PatgGASA protein derived from suspending and to selection of transgenic suspending, FIG. 4 (A) is derived from suspending A gene map of an expression vector prepared by inserting the PatgGASA gene into the pBI121 vector, and FIG. 4 (B) shows the results of genomic DNA PCR and transfected untransfected control (WT) when the PatgGASA gene was introduced. The genomic DNA PCR result of Figure 4 (C) is a result of analyzing the expression of PatgGASA genes in the control (WT) and transgenic suspension (PatgGASA-OX) that is not transformed suspension by qRT-PCR.
5 (A) to 5 (C) show dry stress resistance during transgenic suspension with increased PatgGASA expression, and FIG. 5 (A) shows overexpression during PatgGASA gene (PatgGASA-OX) and transfection after dry stress treatment. The phenotype of unconverted control (WT) suspension was shown, and FIG. 5 (B) shows the amount of chlorophyll fluorescence (PatgGASA-OX) and the untransformed control (WT) suspension of the PatgGASA gene following dry stress treatment. Fv / Fm), and FIG. 5 (C) shows the soil moisture (VWC,%) of PatgGASA gene overexpression (PatgGASA-OX) and untransformed control (WT) suspension following dry stress treatment. It is a change.

The present invention is a recombinant vector comprising a hyeonsasi (Populus alba × P. Tremula var . Glandulosa) a recombinant vector, a method for preparing a recombinant vector including the PatgGASA genes including PatgGASA gene, the PatgGASA gene derived from the PatgGASA gene By using the recombinant vector containing the plant and PatgGASA gene resistance to dry stress is improved the production method of the plant is improved resistance to dry stress.

The present invention refers to the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1.

The present invention refers to a recombinant vector comprising the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1.

The recombinant vector is a pBI121-PatgGASA vector containing the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1.

The recombinant vector pBI121-PatgGASA including the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1 may increase the expression of the PatgGASA gene.

The present invention shows a method for producing a recombinant vector comprising the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1.

The present invention comprises PCR amplifying a PatgGASA gene having a nucleotide sequence of SEQ ID NO: 1 using a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4; A method of preparing a recombinant vector comprising a PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1 comprising inserting the amplified PCR product into a pBI121 vector comprising a 35S promoter and a kanamycin label is shown.

In the PCR amplification, 50 pg of plasmid DNA of PatgGASA gene having a nucleotide sequence of SEQ ID NO: 1 in a 4 µl Hipi PCR premix (ELPLIS, Korea), a forward primer of SEQ ID NO: 3, and a reverse primer of SEQ ID NO: 4, respectively A total volume of 20 μl containing 1 μM was performed. Amplification was carried out in an automatic summer cycler (Biometra, Germany) for 35 cycles of 30 seconds at 98 ° C., 30 seconds at 58 ° C. and 30 seconds at 72 ° C. The PCR products were separated on 1.0% agarose gel. The isolated PCR product was purified using UltraClean 15 DNA purification Kit (MoBio, Korea) according to the user manual and subcloned into pGEM-T easy (Promega, USA).

In the method for preparing a recombinant vector, the plasmid DNA of PatgGASA introduced into the pGEM-T easy vector (Promega, USA) was completely cleaved with restriction enzymes Xba I and Sac I, followed by electrophoresis and separation on 1.0% agarose gel. The isolated fragment was purified using UltraClean 15 DNA purification Kit (MoBio, Korea) and introduced between the restriction enzyme sites Xba I and SacI of pBI121. The recombinant vector was transduced into the Agrobacterium tumerfaciens cell line LBA4404 by electroporation.

The present invention shows a plant transformed using a recombinant vector comprising the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1.

The recombinant vector including the PatgGASA gene is pBI121-PatgGASA vector.

Plants transformed above represent plants with improved resistance to dry stress.

The transformed plant may be any one selected from poplars at the time of suspension having improved resistance to dry stress.

The present invention shows a method for producing a plant transformed using a recombinant vector comprising the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1.

The plant in the method for producing a transformed plant indicates a plant transformed to improve resistance to dry stress using the pBI121-PatgGASA recombinant vector having increased expression of PatgGASA gene.

Plants in the method of producing a transformed plant in the above may be any one selected from the poplar at the time of suspension, improved resistance to dry stress.

Hereinafter, the present invention will be described in more detail.

The present invention is a PatgGASA gene having a nucleotide sequence of SEQ ID NO: 1 from the present-day suspension, a recombinant vector comprising the gene, a plant with improved resistance to dry stress transformed using a recombinant vector comprising the PatgGASA gene and to dry stress It shows a method of producing plants with improved resistance to.

The range of PatgGASA protein according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 2 isolated from suspension and functional equivalents of the proteins.

The above-mentioned "functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% or more of the amino acid sequence shown in SEQ ID NO: 2 as a result of addition, substitution or deletion Preferably 95% or more, of a protein having substantially the same physiological activity as the protein represented by SEQ ID NO: 2. "Substantially homogenous bioactivity" means an activity promoting dry stress tolerance of a plant.

The present invention provides a gene encoding PatgGASA protein derived from the present suspension. The gene of the present invention may be DNA or RNA encoding PatgGASA protein. DNA includes cDNA, genomic DNA, or artificial synthetic DNA. The DNA may be single stranded or double stranded. The DNA may be a coding strand or a non-coding strand.

Preferably, the gene of the present invention may include the nucleotide sequence shown in SEQ ID NO: 1.

The present invention provides a recombinant vector comprising the PatgGASA gene derived from the present invention according to the present invention.

In the present invention, the recombinant vector containing the PatgGASA gene is preferably a plant expression vector, more preferably the recombinant vector described in Fig. 4A, but is not limited thereto. 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.

The term "recombinant" as used herein 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.

The term "vector" is used herein to refer to a DNA fragment (s), a nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism.

A preferred example of the plant expression vector of the present invention is a Ti-plasmid vector capable of transferring a so-called T-region to plant cells when present in a suitable host such as Agrobacterium tuberculosis. Other types of Ti-plasmid vectors are currently being used to transfer hybrid DNA sequences to plant cells, or to protoplasts where new plants can be produced that appropriately insert hybrid DNA into the plant's genome.

Other suitable vectors that can be used to introduce the DNA according to the present invention into a plant host include viral vectors such as those derived from double-stranded plant viruses (e.g., CamV) and single-stranded virus, germinavirus, 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.

PatgGASA gene derived from the present invention of the present invention, a recombinant vector comprising the PatgGASA gene, using the recombinant vector was carried out under various conditions for the production method of plants with improved dry stress resistance and plants with improved resistance to dry stress, Plants transformed with the PatgGASA gene derived from suspending showed high chlorophyll fluorescence compared to untransfected control (WT) suspending under dry stress. In addition, the resilience when watering again was superior to the control. Therefore, in order to achieve the object of the present invention under the above-mentioned conditions, PatgGASA gene derived from the suspension, recombinant vector containing the PatgGASA gene, plants with improved dry stress resistance using the recombinant vector and resistance to dry stress It is desirable to provide an improved method for producing plants.

Hereinafter, the content of the present invention will be described in detail through experimental examples. However, these are for the purpose of illustrating the present invention in more detail, and the scope of the present invention is not limited thereto.

<Experimental Example>

[1] materials and methods

(1) plant materials, culture conditions and stress treatment

Suspension (Pulus alba × P. tremula var. Glandulosa ) was used as the plant material (Choi et al, 2001, J Plant Biotech, 3, 83-88). 100 ml of liquid medium was dispensed into a 250 ml Erlenmeyer flask, and 0.4 g of suspension cultured cells were added and suspended in 130 rpm, 22 ± 1 ° C, and light conditions of 20 μmol m ² s ^-1 (Lee et al, 2005). , Plant Sci, 169, 1118-1124).

Liquid medium added 2,4-D 1.0mg / L, NAA 0.1mg / L, 6-BAP 0.01mg / L and 3% sucrose to MS medium (Murashige and Skoog 1962, Physiol Plant, 15, 473-497) After the medium was titrated to pH 5.8 by 1M NaOH treatment and autoclaved at 121 ° C. for 15 minutes, it was used.

In order to investigate the cell growth curve of suspension cultured cells, cultured cells were harvested at intervals of 2 days for 26 days after passage (Lee et al, 2007, Physiol Plant, 131, 599-613).

To examine the response to abiotic stress, dry (250 mM mannitol, Man) and salt (150 mM sodium chloride, NaCl) were added to suspension cultured cells at five days of age. The abscisic acid, a plant hormone associated with abiotic stress, was treated to a final concentration of 25 μM. In the low temperature treatment, the Erlenmeyer flask containing suspension culture cells was placed in an ice box containing ice and shaken at 130 rpm, and the temperature of the medium was 2 ° C. Incubation time according to the treatment was carried out for 2 hours and 10 hours, and the suspension cultured cells were recovered. The control was used to recover the same suspension cultured cells used for stress treatment after 2 hours and 10 hours, respectively, without any treatment. All suspension cultured cells were removed from the medium using a vacuum pump, frozen in liquid nitrogen and stored at -70 ° C.

Plants were grown for 8 weeks in the photoperiod of 24 ± 1 ° C., 50% relative humidity, and 16 h light / 8 h cancer to investigate the dry stress resistance during transgenic suspension. One day before the drying treatment, water was supplied until the maximum soil moisture content (about 48%) was reached, followed by no water for 11 days. Water was observed and the condition of the plants was observed until the soil moisture content was restored to the maximum at 11 days of drying. Soil moisture content was measured in three repetitions using a moisture probe meter (ICT international Pty Ltd, Australia). Resistance to transgenic susceptibility to dry stress was confirmed by measuring chlorophyll fluorescence. Chlorophyll fluorescence was measured using a Poket PEA Chlorophyll Fluorimeter (Hansatech, England).

(2) Gene isolation and sequence analysis

Expressed sequence tag (EST) analysis from suspension cultured cells (Lee et al, 2005, Plant Sci, 169, 1118-1124) during suspension showed that cDNA clones showing sequence homology with GASA genes reported in plants were selected.

After determining the nucleotide sequence of the selected cDNA clone, the expected amino acid sequence and molecular weight were examined using Vector NTI advance 10.0 (Invitrogen, USA). Analysis of the signal peptide was carried out using SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/).

Protein homology analysis was performed using the ClustalW algorithm with the Uniprot program (http://www.uniprot.org/align/). Phylogenetic tree was written by NJ method using MEGA4.1 (beta).

(3) Genomic DNA and total RNA isolation

Genomic DNA was isolated from a fully developed suspension leaf using MagaExtractor plant genome (Toyobo Co, Japan) according to the user manual. Total RNA was poured into suspension cultured cells or leaves and poured into nitrogen, followed by induction, and isolated according to the user's manual using TRI Reagent (Molecular Research Center, USA).

(4) Southern and northern blot analysis

10 μg of genomic DNA was completely digested with restriction enzymes Bam HI, Eco RI, Hi nd III and Xba I, and then recovered by ethanol precipitation. Restricted enzyme digested genomic DNA was electrophoresed on 1% agarose gel and transferred to Hybond-XL nylon membrane (Amersham-Pharmacia Biotech Inc, UK) by capillary transfer method.

10 μg of total RNA was electrophoresed with a 1.2% formaldehyde agarose gel and then transferred to a Hybond-XL nylon membrane using a capillary transfer method.

For the radiolabeling of cDNA to be used as a probe (Probe) was reacted according to the user manual using a Multiprime labeling kit (Amersham Co, USA). Membrane 10ml 1 × PerfectHYB plus hybridization buffer (Sigma, USA) and pretreated at 68 ° C for 1 hour. Suspended GASA (Patg GASA) cDNA labeled with α - ³² P was boiled for 5 minutes to denature and then cooled on ice. The denatured probe was added to the hybridization reaction solution and hybridization was performed at 68 ° C for 12 hours. After the reaction, the membrane was washed with 0.2 × SSC-0.1% SDS solution at 65 ° C. for 15 minutes three times. Membranes were attached to the X-ray film, exposed at -70 ° C for 3 days, and then developed.

(5) Transformation vector production and transformation at present time

PCR was amplified using a forward primer (5′-GAGTCTAGAGGATCCAGCACCATG-3 ′; SEQ ID NO: 3) and a reverse primer (5′-GGTAGCTAGAGCTCAAGGTCAAGGG-3 ′; SEQ ID NO: 4) for the overexpression of the PatgGASA gene.

The PCR reaction was performed using Hipi PCR premix (ELPLIS, Korea) according to the user's manual, 50 pg of plasmid DNA of PatgGASA gene having a nucleotide sequence of SEQ ID NO. 1, a forward primer of SEQ ID NO. Reverse primers were performed with a total volume of 20 μl each containing 10 μM. PCR amplification was performed in an automatic summer cycler (Biometra, Germany) for 35 cycles of 30 seconds at 98 ° C., 30 seconds at 58 ° C. and 30 seconds at 72 ° C. PCR products were separated on 1.0% agarose gel. The separated PCR products were purified using UltraClean 15 DNA purification kit (MoBio, Korea) according to the user's manual and subcloned into pGEM-T easy vector (Promega, USA). Plasmid DNA with PatgGASA insertion was verified by complete digestion with restriction enzymes Bam HI and Sac I, followed by electrophoresis and separation on 1.0% agarose gel. The isolated fragment was purified using an UltraClean 15 DNA purification Kit (MoBio, Korea), and inserted into the Bam HI / Sac I position of the pBI121 vector to prepare a PatgGASA overexpression vector, pBI121-PatgGASA.

Standard test method for transfection of the current phase after introducing PatgGASA overexpression vector into Agrobacterium tumefaciens LBA4404 (Noh et al, 1994, Res Rep For Gen Res Inst Kor, 30, 114-120; Kim et al, 2011, Plant Biotecchnol J, 9, 334-347).

Callus was induced from transverse interstitial tissues, and the callus selected from kanamycin was transferred to stem-induced medium to induce differentiation, and then to root-induced medium. The callus induction medium was prepared by adding 4.4 g of MS medium, 1 mg / L of 2,4-D, 0.1 mg / L of NAA, 0.01 mg / L of BA and 3% of sucrose and titrating to pH 5.8. Stem induction medium was titrated to pH 5.5 with 2.46 g of WPM, 1 mg / L Zeatin, 0.1 mg / L BAP, 0.01 mg / L NAA, and 3% sucrose. Root induction medium was prepared by adding 4.4 g of MS medium, 0.2 mg / L of IBA and 3% of sucrose, and titrating to pH 5.8. 50 mg / L of kanamycin and 250 mg / L of Cytaxime were added to all the media.

The transgenic plants isolated genomic DNA of the redifferentiated plant from the medium containing kanamycin and PCR analysis to confirm the introduction of the gene. PCR was amplified using a forward primer (5'-TGCTTCACAAACCAAGGCAAGTAAT-3 '; SEQ ID NO: 5) and a reverse primer (5'-CTTGTTGCCATAAGTACCAGGAGG-3'; SEQ ID NO: 6) to verify the introduction of the gene. The PCR reaction was performed by adding 10 μM of each of the forward primer of SEQ ID NO: 5 having a partial sequence of the 35S promoter and the reverse primer of SEQ ID NO: 6 having a partial sequence of the PatgGASA gene to Ex-Taq DNA polymerase (Takara, Japan), and PatgGASA. A total volume of 50 µl containing 100 pg of genomic DNA in transfected or transgenic untransfected genomic DNA (WT) was performed. PCR amplification was performed in an automatic thermocycler (Biometra, Germany) for 35 cycles of 5 seconds at 98 ° C., 30 seconds at 98 ° C., 30 seconds at 55 ° C., and 30 seconds at 72 ° C. for 40 seconds. PCR products were electrophoresed on 1.0% agarose gel and visualized in UV light.

Expression of the transgene at transfections was confirmed by Quantitative Real-Time PCR (qRT-PCR). At this time, a forward primer (5'-TAGCCTCAGTCGCAATTCTCCC-3 '; SEQ ID NO: 7) and a reverse primer (5'- AGGCATGTCGCACAGCATTT-3'; SEQ ID NO: 8) specific for the PatgGASA gene were used. For qRT-PCR, first strand cDNA was synthesized according to the user's manual using PrimeScript RT reagent Kit with gDNA Eraser (Takara, Japan) with 1 μg of total RNA of transgenic and non-transgenic WT Respectively. The qRT-PCR reaction was carried out using a total volume of 20 μl containing 2 μl of 10 μl of the first strand cDNA reaction product, 10 μM of the gene-specific primer and 10 μl of SYBR Premix EX Taq II (Takara, Japan). qRT-PCR amplification was performed in a multi-color Real-time PCR System CFX96 (Bio-Rad, USA) for 40 cycles of 95 ° C for 3 minutes, 95 ° C for 10 seconds, 60 ° C for 10 seconds and 72 ° C for 30 seconds . The PCR products were electrophoresed on 1.0% agarose gel and visualized in UV light. A forward primer (5'-CGGCTTGGAGGCGATAAAACTGAT-3 '; SEQ ID NO: 9) and a reverse primer (5'-CGCGGCCGAGGTACTATGATGGAC-3'; SEQ ID NO: 10) of Rubisco Small Subunit were used as internal control genes.

[2] results

Results of the above [1] experimental materials and methods are shown in Figs. 1 (A) to 5 (C).

FIG. 1 (A) is a multiple sequence alignment of the hyeonsasi (Populus alba × P. Tremula var . Glandulosa) to separate one PatgGASA protein, PtGASA protein, Arabidopsis GASA family proteins of black cottonwood trees in a (AtGASA1, AtGASA4, AtGASA5) As shown, multiple sequence comparisons were performed using the ClustalW program. Amino acid sequencing results in a signal peptide at the amino terminus and 12 cysteine residues at the carboxy terminus. Signal peptides well conserved at the amino terminus are indicated by boxes, and twelve Cysteine (C) residues well conserved at the carboxy terminus are indicated by asterisks. The cleavage site, where the signal peptide is released from the protein, is indicated by the arrow head.

FIG. 1 (B) hyeonsasi (Populus alba × P. Tremula var . Glandulosa) to separate one PatgGASA protein and GASA family of black cottonwood PtGASA of trees protein, Arabidopsis proteins from (AtGASA1, AtGASA2, AtGASA3, AtGASA4 , AtGASA5) and This is a result of phylogenetic analysis of PatgGASA protein comparing the soft relationship between corn GAST1 (gibberellic acid stimulated transcript 1, ZmGAST1). The phylogenetic tree was created using Mega 4.1. The number of branches is the bootstrap value expressed as a percentage. The bootstrap analysis was performed with 1,000 iterations and the values were expressed as a percentage. In protein phylogenetic analysis, PatgGASA was 98% identical to the GASA of black cottonwood and AtGASA5 among the GASA family proteins of Arabidopsis was 68% identical. Hence, PatgGASA at present was the most flexible to GASA of cottonwood trees.

FIG. 2 shows Southern blot analysis results using chromosomal DNA at the time of suspension, and Southern blot analysis using chromosomal DNA at the time of presence showed that PatgGASA gene was present in 1 to 2 copies of the chromosomes at the time of suspension. Meanwhile, in FIG. 2, B means Bam HI, EI means Eco RI, H means Hin dIII, and X means Xba I.

Figure 3 (A) to Figure 3 (C) is a result of analysis of the expression of the PatgGASA gene according to various conditions in suspension cultured cells, Figure 3 (A) is a culture day (0 days to 24 days) of suspension culture cells during suspension Figure 3 (B) shows the expression of the PatgGASA gene at the stage of growth (4 days to 24 days) of the suspension growth culture growth curve of suspension culture cells (4), Figure 4 (C) shows no treatment to suspension culture cells Treated (Con), suspended suspension cultured cells (250 mM mannitol, Man), salt (150 mM sodium chloride, NaCl), low temperature (2 ℃, Cold), abscisic acid (25 μM, ABA) respectively After 2 and 10 hours shaking culture, PatgGASA gene expression is shown. 3 (A) to 3 (B) show that the PatgGASA gene at the time of suspension is expressed differently according to the growth stage of suspension cultured cells. Higher expression was followed by a significant decrease in expression. Figure 3 (C) is a result of examining the expression of PatgGASA gene for abiotic stress, PatgGASA gene is no expression difference compared to the control at 2 hours of drying and salt stress treatment, but the expression was not at 10 hours of drying and salt stress treatment Decreased.

4 (A) to 4 (C) are directed to the production of overexpression vector of PatgGASA gene and selection at the time of transformation, and FIG. 4 (A) is a gene map of the expression vector prepared by inserting PatgGASA gene into pBI121 vector. 4 (B) shows the results of genomic PCR of the transgenic suspension with the PatgGASA gene and the result of the genomic PCR of the control (WT) without the untransfected suspension, and FIG. The expression of PatgGASA gene for WT) and transgenic suspension (PatgGASA-OX) is analyzed by qRT-PCR.

An overexpression vector of PatgGASA gene was constructed and transformed at the time of manifestation. The introduction of PatgGASA gene was confirmed by genomic DNA PCR. As a result, the PatgGASA gene amplification product was not observed in the non-transformed control group (WT), whereas the amplification product appeared in the transformed suspension (PatgGASA-OX27 or 28) to confirm that the transformation was performed normally. As a result of confirming the expression level of PatgGASA gene by qRT-PCR in transgenic suspension, the expression of PatgGASA gene was increased by 200-250 times in transgenic suspension compared to the control.

5 (A) to FIG. 5 (C) show dry stress resistance at the time of transgenic suspension with increased expression of PatgGASA gene, wherein plants grown in a greenhouse for 8 weeks were exposed to dry conditions for 11 days and then watered again. The degree of recovery was checked.

FIG. 5 (A) shows control (WT) suspension without PatgGASA gene overexpression (PatgGASA-OX27 or 28). FIG. 5 (B) shows PatgGASA gene overexpression (PatgGASA-OX27) following dry stress treatment. Or 28) and untransformed control (WT) chlorophyll fluorescence (Fm / Fv) change in suspension, Figure 5 (C) is PatgGASA gene overexpression suspension (PatgGASA-OX27 or 28) following dry stress treatment And soil moisture content (VWC,%) at the time of untransformed control (WT) suspension.

As a result of confirming the dry stress resistance, both the overexpressed transgenic suspension (PatgGASA-OX27 or 28) and the untransfected control (WT) suspension gradually decreased the soil moisture content to reach 0 after 11 days of treatment. Untreated control (WT) suspensions failed to recover even after watering, but PatgGASA overexpressed transgenic suspensions (PatgGASA-OX27 or 28) maintained normal growth and showed strong resistance to dry stress. In the results of chlorophyll fluorescence, untransformed control (WT) suspension showed 0 fluorescence after 11 days of treatment due to plant death, but PatgGASA overexpression (PatgGASA-OX27 or 28) was 0.13 to 0.51. By maintaining the amount of light (Fv / Fm) it was confirmed that PatgGASA over-expression was resistant to dry stress.

Although described with reference to the preferred experimental example of the present invention as described above, those skilled in the art without departing from the spirit and scope of the present invention described in the claims below It will be understood that various modifications and variations can be made to the invention.

The transformed plants provided by the present invention can provide improved plants and improved productivity of crops and trees even in dry soils, thereby contributing to the development of technology in the forest-related field, and thus have industrial applicability.

Attach an electronic file to a sequence list

Claims (13)

PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1. The method of claim 1,
Wherein the nucleotide sequence of SEQ ID NO: 1 is derived from a prefecture. Recombinant vector comprising a PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1. The method of claim 3,
The recombinant vector is a recombinant vector characterized by increased expression of the PatgGASA gene. PCR amplifying the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1 using a forward primer of SEQ ID NO: 3 and a reverse primer of SEQ ID NO: 4;
A method of preparing a recombinant vector comprising a PatgGASA gene having a nucleotide sequence of SEQ ID NO: 1 comprising inserting the amplified PCR product into a pBI121 vector comprising a 35S promoter and a kanamycin label. 6. The method of claim 5,
PCR amplification was performed in the Hipi PCR premix (ELPLIS, Korea) with 50 pg of plasmid DNA of PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1, the forward primer of SEQ ID NO: 3, and the reverse primer of SEQ ID NO: 4, respectively. A total volume of 20 μl containing the PatgGASA introduced in pGEM-T easy, which was carried out in an automatic thermocycler (Biometra, Germany) for 35 cycles of 30 seconds at 98 ° C., 30 seconds at 58 ° C. and 30 seconds at 72 ° C. A method for producing a recombinant vector using a method in which plasmid DNA is completely cleaved with restriction enzymes Xba I and Sac I, electrophoresed and separated from a 1.0% agarose gel, and introduced between the restriction enzyme sites Xba I and SacI of pBI121. A plant transformed with a recombinant vector comprising a PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1. 8. The method of claim 7,
The recombinant vector is a plant characterized in that the pBI121-PatgGASA overexpression vector. 8. The method of claim 7,
A plant transformed to improve resistance to dry stress using a recombinant vector having increased expression of PatgGASA gene. 8. The method of claim 7,
The plant is a plant that is characterized in that any one selected from the time of the poplar, poplar Method for producing a plant transformed using a recombinant vector containing the PatgGASA gene having the nucleotide sequence of SEQ ID NO: 1. 12. The method of claim 11,
A method for producing a plant transformed to improve resistance to dry stress using a recombinant vector having increased PatgGASA gene expression. 12. The method of claim 11,
Plant is a method of producing a plant, characterized in that any one selected from the poplar, at the time of

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