KR101260935B1 - A red pepper gene CaBI-1 confers stress-tolerance to plants - Google Patents

Abstract

The present invention relates to the environmental stress resistance induced pepper gene CaBI- 1 ( Capsicum annuum Bax Inhibitor-1), and more particularly to a novel pepper gene CaBI- 1 that induces resistance to high temperature stress, dry stress or high salt stress. CaBI - 1 hot pepper plant, low temperature, high salt road, and over-expression time was applied for drying, immersion or heavy metal stress, CaBI - transgenic plant by introducing a 1 out hayeoteumeuro Obtained ionic, naegeonseong and salt tolerance, for environmental stress It can be usefully used for the development of plants obtained resistance.

CaBI-1 (Capsicum annuum Bax Inhibitor-1), Plant High Temperature Resistance, Plant Dry Resistance, Plant Flame Resistance, Red Pepper, Tobacco, Environmental Stress

Description

A red pepper gene CaBI-1 confers stress-tolerance to plants}

The present invention relates to novel genes that confer resistance to environmental stress.

Plants are constantly exposed to various environmental stresses such as drying, high salinity and low temperature. Thus, it is no exaggeration to say that plant life is a continuation of the response to environmental stress and adaptation processes (Boyer, 1982, Science 218: 443-448).

Plants under environmental stress exhibit a variety of biochemical and physiological responses and adaptation processes. How efficiently a plant can respond to and adapt to environmental stress is a factor in determining how well the plant can withstand environmental stress. Programmed Cell Death (PCD) is considered to be an important mechanism of environmental stress adaptation in the response of organisms to environmental stress (Huckelhoven, 2004, Apoptosis 9: 299-307). PCD has been identified in all eukaryotic cell organisms, and has been studied mainly in metozoan to date, and its function has been confirmed in animal development and response and adaptation to environmental stress (Huckelhoven, 2004). PCD is expected to be widely used in plants, but studies to date have been limited. Some developmental processes, such as the development of tracheary elements and pollen, and the resistance of plants to infection by pathogenic microorganisms. Research has been conducted mainly as a basic mechanism of hypersensitive reaction, a function. PCDs identified in plants are far more limited than those in animals, but changes at the molecular cell level have much in common, suggesting that PCDs in animals and plants have been well preserved through evolution (Lam et al. ., 2001, Nature 411: 848-853; Bolduc et. al ., 2003, Planta 216: 377-386; Huckelhoven, 2004).

As a major regulator of PCD, BCL2 associated x protein (BAX), which has a PCD-inducing function, has been the subject of many studies (Kroemer, 1997, Nat). Med 3: 614-620), while BAX inhibitor-1 (BI-1), a factor that inhibits the development of PCD by inhibiting BAX function, has recently been the subject of much research ( Scorrano et al ., 2003, Biochem Biophys Res Commun 304: 437-444).

BAX was not found from plants, but it was confirmed that BI-1 exists in both animals and plants. Inhibition of apoptosis by BAX in these cells was confirmed by the introduction of plant BI-1 gene from the outside and overexpression in animal cells or yeast. Inducible apoptosis occurred (Sanchez et. al ., 2000, Plant J 21: 393-399; Kawai-Yamada et al ., 2004, Plant Cell 16: 21-32; Lacomme and Cruz, 1999, Proc Natl Acad Sci USA 96: 7956-7961; Huckelhoven et al ., 2003, Proc Natl Acad Sci USA 100: 5550-5560; Eichman et al ., 2004, Mol Plant Microbe Int 17: 484-490; Matsumura et al ., 2003, Plant J 33: 425-434). Therefore, although BAX has not been found in plants, there is a possibility that there is a PCD process associated with BAX in plants.

As described above, the conditions under which the expression of BI-1 is induced are confirmed to be a specific developmental process involving apoptosis and a biological or abiotic environmental stress. For example, induction of the expression of the BI-1 gene by the invasion of pathogenic microorganisms from the Arabidopsis (Sanchez et. al ., 2000; Matsumura et al ., 2003; Eichmann et al ., 2004) and the induction of BI-1 expression in Arabidopsis under high temperature and free radical stress (Watanabe and Lam, 2006, Plant J 45: 884-894).

Crop yield losses due to abiotic environmental stress are enormous, and more than half of the possible yields have been found to be lost to abiotic environmental stress (Bray et al ., 2000, In Biochemistry & Molecular Biology of Plants, BB Buchanan, W Gruissem, and RL Jones ed, American Society of Plant Biologists, Rockville; Wang et al ., 2007, Planta 218: 1-14), but recent rapid changes in the environment have been recorded, and the degree of damage to crops is more concerned (Lane and Jarvis, 2007, EJournal 4: 1-12). As mentioned above, the role of PCD and BI-1 in connection with the adaptation mechanisms of plants to abiotic environmental stresses has been suggested, and this possibility has led to the transient expression of the gene of BI-1 in tobacco leaf slices. As a result, although it is a short time effect, it has been confirmed in part by the reported increase in the resistance of tobacco cells to high and low temperatures and free radicals (Chae et. al ., 2003, Gene 323: 101-113; Watanabe and Lam, 2006). However, it is not producing a transgenic plant using the BI -1 gene, and by using the prepared transgenic plants reported gave show that the BI -1 gene can be given to promote the resistance to environmental stress in plants.

Accordingly, the present inventors screened a new BI- 1 gene that was subjected to high temperature stress on pepper plants and thus increased expression, and confirmed that the transformed tobacco plants obtained high temperature resistance, dry resistance and flame resistance by introducing the new gene. By this, the present invention was completed.

It is an object of the present invention to provide a novel environmental stress resistance gene derived from red pepper and a transformant into which the gene is introduced.

In order to achieve the above object, the present invention is CaBI-1 ( Capsicum) which is an environmental stress resistance protein having an amino acid sequence of SEQ ID NO: 1 Annuum Bax Inhibitor-1) provides a polypeptide constituting.

The present invention also provides a polynucleotide of the following (a) or (b) encoding the polypeptide:

(a) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; And

(b) the polynucleotide set forth in SEQ ID NO: 2.

In addition, the present invention provides an expression vector comprising the polynucleotide.

The present invention also provides a transformant prepared by introducing the polynucleotide into a host, a plant which is a progeny or clone of the transformant, and seeds, fruit, ear, tuber, tuber, stem, callus or Provide protoplasts.

In addition,

1) preparing an expression vector operably linked to the polynucleotide;

2) introducing the expression vector of step 1) into a host plant cell; And

3) It provides a method for producing an environmental stress resistance inducing gene transformant comprising the step of tissue culture of the transformed plant cells of step 2).

Novel pepper gene CaBI- 1 of the present invention ( Capsicum annuum Bax Inhibitor-1) is useful for the development of transgenic plants that are resistant to environmental stress because plants are overexpressed under high temperature stress and CaBI- 1 transduced transformants acquire temperature resistance, dry resistance and flame resistance. Can be.

Hereinafter, the present invention will be described in detail.

The present invention provides a polypeptide constituting CaBI-1 ( Capsicum annuum Bax inhibitor-1), which is an environmental stress resistant protein having the amino acid sequence set forth in SEQ ID NO: 1.

The polypeptide of the present invention is preferably a polypeptide having at least 70% homology with the polypeptide described in SEQ ID NO: 1, more preferably at least 80% homology, and at least 90% homology. Most preferred, but not limited to, polypeptides having a homology of up to 70% but having at least 90% homology or a biologically equivalent function in peptide fragments up to 15 amino acids with the polypeptide described in SEQ ID NO: 1. It may be included in the polypeptide of the invention.

The present invention also provides a polynucleotide of the following (a) or (b) encoding the polypeptide:

(a) a polynucleotide encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1; And

(b) the polynucleotide set forth in SEQ ID NO: 2.

The environmental stress is any one selected from the group consisting of high temperature, drying, low temperature, high salt, heavy metals and immersion; It is more preferably any one selected from the group consisting of high temperature, drying and high salt, but is not limited thereto.

The polypeptide or polynucleotide of the present invention is preferably derived from a pepper plant.

In addition, the present invention provides an expression vector comprising the polynucleotide.

The gene included in the expression vector of the present invention includes a polynucleotide encoding CaBI-1 described in SEQ ID NO: 1, and preferably includes a polynucleotide described in SEQ ID NO: 2. As the vector used to insert the gene, it is preferable to use any one selected from the group consisting of a pKBS1-1 vector, a pBI102 vector, and a pCAMBIA vector, and any expression vector for plant transformation in the art may be used. It's okay.

The present invention also provides a transformant prepared by introducing the polynucleotide into a host, a plant which is a progeny or clone of the transformant, and seeds, fruit, ear, tuber, tuber, stem, callus or Provide protoplasts.

The host includes microorganisms, animal cells, plant cells, animals or plants, and cultured cells derived therefrom.

Preferably, the plant cell or plant is preferably a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.

In a specific embodiment of the present invention, the inventors of the present invention, in order to obtain genes capable of improving environmental stress resistance in plants, first apply high temperature stress to pepper, and then produce a cDNA library to significantly increase the amount of expression at high temperature stress cDNA clone Were screened using differential screening techniques, plasmid DNA was collected from selected cDNA clones to partially determine sequencing, and then highly homologous to previously reported BIX (BAX inhibitor-1). CDNA clones were isolated and named CaBI-1 (GenBank Accession Number: ACS 36610) (see FIGS. 1 and 2). In addition, the inventors confirmed that CaBI- 1 expression increased even when hot, dry, low temperature, high salinity, heavy metals and submerged stress were applied to the pepper plants (see FIG. 3). Escherichia coli with clone containing CaBI- 1 gene inserted into pBluescript vector coli strain DH5α) was deposited on December 3, 2009 to the Agricultural Genetic Resource Center, 88-20 Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do, Korea (accession number: KACC95101P; deposit date: December 03, 2009).

In addition, the present inventors transformed the CaBI- 1 gene into Agrobacterium, and then infected it with tobacco plants to prepare CaBI- 1 transformed tobacco plants (see FIG. 4). (See Fig. 5) the CaBI -1 transformants confirmed that the conversion CaBI-1 is normally expressed in tobacco then, in the results, CaBI -1 transgenic plants was analyzed and resistance to environmental stress, high temperature, drying (see FIG. 6) and It was confirmed that resistance to high salts (see FIG. 7) occurred.

In addition,

1) preparing an expression vector operably linked to the polynucleotide;

2) introducing the expression vector of step 1) into a host plant cell; And

3) It provides a method for producing an environmental stress resistance inducing gene transformant comprising the step of tissue culture of the transformed plant cells of step 2).

The plant cell of step 2) is preferably derived from a monocotyledonous plant or a dicotyledonous plant, but is not limited thereto.

The monocotyledonous plants include Alismataceae, Hydrocharitaceae, Juncaginaceae, Schuchzeriaceae, Pomomotontonaceae, Najadaceae, Zosteraceae, Liliaceae, Haemodoraceae, Agavaceae, Amaryllidaceae, Dioscoreaceae, Pontederiaceae, Iridaceae, Burmanniaceae, Juncaceae, Commelinaceae ), Eriocaulaceae, Flower Family (Grapeaceae, Gramineae, Poaceae), Araceae, Lemnaceae, Spaganiaceae, Typhaceae, Cycloaceae, Cyperaceae, Pachoaceae ( Musaceae), Zingiberaceae, Cannaaceae, Orchidaceae (Orchidaceae) is preferably, but not limited to.

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, Rubisaceae, 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, Myraceae, Juglandaceae, Salicaceae, Birchaceae, Beechaceae, Fagaceae, Ulmaceae, Mulberry, Nettle Urticaceae, Santalaceae, Mistletoe, Lothanthaceae, Polygonaceae, Apoaceae, Phytolaccaceae, Nyctaginaceae, Aizoaceae, Portulacaceae ), Caryophyllaceae, Chenopodiaceae, Amaranthaceae, Cactaceae, Magnoliaceae, Illiciaceae, Lauraceae, Cecidiphyllaceae, Minaria (Ranuncul aceae, Berberidaceae, Lardizabalaceae, Bird breeze (Amandiaceae, Menispermaceae), Nymphaeaceae, Ceratophyllaceae, Cabombaceae, Saururaceae , Piperaceae, Chloranthaceae, Aristolochiaceae, Actinidiaceae, Camellia, Theaceae, Guttaiferae, Droseraceae, Poppyaceae ), 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 Callitrichaceae, Rutaceae, Simaroubaceae, Meliaceae, Polygalaceae, Anacardiaceae, Mapleaceae, Aceraceae, Sapindaceae, Mapleaceae (Hippocastanaceae), Sabiaceae, Balsam (Azalea, Balsaminaceae), Aquifoliaceae, Nova (Celastraceae), Staphyleaceae, Buxaceae, Empetraceae , Rhamnaceae, Vitaceae, Elaeocarpaceae, Tiliaceae, Malvaceae, Sterculiaceae, Adenaceae, Thymelaeaceae, Ellaeagnaceae, Flacourtiaceae, Violaceae, Passifloraceae, Tamaricaceae, Elatinaceae, Begoniaceae, Cucurbitaceae, Liliumaceae, Lythraceae Pomegranate Punicaceae, Onnagraceae, Haloragaceae, Bataceae (Alangiaceae), Dogwood (Hortisaceae, Cornaceae), Arboraceae (Araliaceae), Umbelaceae (Umbelliferae (Apiaceae) )), But is not limited thereto.

In addition, the scope of the present invention also includes a plant that is a progeny or clone of the stress-resistant transformed plant and seeds, fruit, ear, tuber, tuber, main, callus or protoplast of the plant.

The step of introducing the expression vector of step 2) into the host plant cell may be introduced into the plant by a known plant transformation method. That is, a well-known method known to those skilled in the art, such as Agrobacterium mediated method, gene gun, method using PEG, and electroporation, can be used. Preferably, the dicotyledonous plants are agrobacterium-mediated method (RB Horsch, et al ., A simple and general method for transferring genes into plants, Science 227 (1985), pp. 1229-1231), Generation of DREB Transgenic Plants by Gene Gun (P. Christou, Particle Bombardment, Methods Cell Biol 50 (1995), pp. 375-382) or Agrobacterium Mediated (Oh et al . Plant Physiol 138: 341-351, 2005; Ito et al . Plant Cell Physiol 47: 141-153, 2006), but is not limited thereto.

After transformation, plant cells should be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known for many different species (Handbook of Plant Cell Culture, Vol. 1-5, 1983-1989 Momillan, N.Y.). Thus, once transformation is realized, it is within the knowledge of the art to regenerate mature plants from transformed plant cells.

The environmental stress of step 3) is preferably any one selected from the group consisting of high temperature, drying, low temperature, high salt, heavy metals and immersion, and any one selected from the group consisting of high temperature, drying and high salt, but is not limited thereto. .

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 contents of the present invention are not limited by the following examples.

< Example  1> Related to environmental stress tolerance from pepper Bax inhibitor -1 gene ( CaBI -One )of Cloning

<1-1> high temperature stress specific pepper cDNA  library( library Manufacturing and environmental stress tolerance CaBI -One of Cloning

Capsicum of young developmental stage (leaves 6-7) annuum cv. Bu Gang) was subjected to high temperature stress by applying a thermal shock of 42 ° C. for 30 minutes, and extracted mRNA from it (Cho and Hong, J. Plant Biol . 47: 149-159, 2004; Sambrook, J. et al ., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989) to synthesize cDNA (Sambrook, J. et. al ., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989), was inserted into UNI-XR vector (Stratagene, USA) to prepare a high temperature stress specific cDNA library. Differential screening was performed on the prepared cDNA library as described below. The labeled mRNA is extracted from the pepper and grown in normal conditions, that is not the high-temperature stress conditions, mRNA for the cDNA (experimental cDNA) labeled with a ³² P prepared as a template derived from a stress applied to the ³² P prepared as the template cDNA (Control cDNA) was performed as a probe. Escherichia coli cultured with pepper cDNA library inserted in UniZAP-XR vector on 23 cm × 23 cm LB bottom agar plate coli ) was plated on KW251. After about 1 × 10 ⁵ plaques had formed on the plate, the plate was blotted onto a Hybond-N nylon membrane for primary screening. Then, a typical DNA-DNA hybridization reaction was performed using the experimental group cDNA as a probe. In addition, a high-bond-N nylon membrane blotted from the same plate was subjected to DNA-DNA hybridization using a control cDNA probe (Sambrook, J. et. al ., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989).

As a result, gene clones contained in the pBluescript II SK (-) plasmid vector were removed from the plaques that showed positive response in the experimental cDNA probe but negative in the control cDNA probe by using the Exassist helper phage (M13). Isolated (see Stratagene's manual), and the sequences were determined using an automated DNA base sequencer (ABI 3730; Applied Bio-systems, USA). The nucleotide sequence determined above was compared with a previously reported BAX inhibitor gene by searching for homology in a public database (//www.ncbi.nlm.nih.gov) operated by the US NCBI. Lycopersicon esculentum Bax inhibitor (AY380778.1) mRNA and 91%, Nicotiana tabacum Bax inhibitor 1 (GenAF390556.1) mRNA and 85% high phase It has been shown to be homosexual and is known as “Capsicum Backs Inhibitor 1 ( Capsicum annuum Bax Inhibitor-1) "( CaBI- 1; Fig. 1, GenBank Accession Number: ACS36610.1). Escherichia coli with a clone in which the CaBI- 1 gene is inserted into the pBluescript vector. coli strain DH5α) was deposited on December 3, 2009 to the Agricultural Genetic Resource Center, 88-20 Seodun-dong, Gwonseon-gu, Suwon-si, Gyeonggi-do, Korea (accession number: KACC95101P; deposit date: December 03, 2009)

<1-2> CaBI -One Sequence Sequence Determination

A sequencing kit using a exonuclease III was used to create a one-way deletion series, and the sequencing of these deletion series was performed using a sequencing kit for kilo-sequencing (Takara, Japan). Sanger's dyeoxy chain termination, Sambrook J., Fritsch EF, and Maniatis T., Molecular The entire sequence of CaBI- 1 was determined using an automated DNA sequencing (ABI 3730; Applied Bio-systems, USA) using Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989). The nucleotide sequence determined according to the general gene codon was translated into an amino acid sequence (FIG. 2). CaBI- 1 had an open reading frame (ORF) consisting of 744 bases set forth in SEQ ID NO: 2 and translated into 248 amino acid sequences set forth in SEQ ID NO: 1.

<1-3> CaBI -One  For gene clones RNA Blot  analysis

Genes induced by expression of environmental stresses isolated from plants are commonly known to be induced by various environmental stresses. In Example <1-1>, BAX inhibitor cDNA clone CaBI-1 of red pepper isolated in Example <1-1> The expression patterns in red pepper under stress of high temperature, dryness, low temperature, high salinity, heavy metals and submersion respectively were verified by RNA blot analysis.

The young pepper plants 5 weeks after sowing 6-8 egg leaves were treated with high, dry, low temperature, high salt, heavy metals and immersion stress under the following conditions. First, the high temperature stress treatment was performed at 42 ° C. for 1 hour, 2 hours, 3 hours, and 4 hours in an incubator having a relative humidity of 90% or more. The dry stress treatment was carefully taken out of the roots of the pepper plants, washed with water, and then placed on a petri dish with dry filter paper, and placed in a plant culture room maintained at 25 ° C. and 16 hours photoperiod for up to 12 hours. Cold stress treatment was placed pepper seedlings up to 24 hours in 4 ℃ low temperature room maintained 16 hours photoperiod. In the high salt stress treatment, the pepper plants were carefully extracted to prevent root damage and washed with water, and the roots were submerged in 250 mM NaCl solution for 4 hours. The heavy metal stress treatment was performed by carefully extracting the red pepper plants with water so as not to damage the roots and washing the roots with water in a 100 mM CaCl ₂ , MgCl ₂ , MnCl ₂ , CoCl ₂ or LiCl solution at 25 ° C. for 16 hours in a plant culture room. Immerse by time. Immersion stress treatment was placed for 72 hours so that the whole pepper seedlings were submerged in the plant culture room maintained for 16 hours photoperiod.

RNA extraction from pepper plants was performed by Cho and Hong, J. Plant. Biol . 47: 149-159, 2004). Pepper plants were frozen in liquid nitrogen and then ground with a mortar to prepare powder. Lysis buffer (lysis buffer: 25 mM Tris-Cl pH 8.0, 50 mM LiCl, 35 mM EDTA, 35 mM EGTA, 0.5% SDS) was added to the powder and mixed by vigorous stirring. Since the same amount of phenol (phenol) was added and then vigorously stirred. Thereafter, an additional 1/4 volume of chloroform was added and mixed with vigorous stirring, and centrifuged for 15 minutes at 6,000 g at 20 ° C. After centrifugation, the supernatant was taken and an equal amount of 4 M LiCl solution was added and centrifuged at 15,000 rpm for 15 minutes at 4 ° C. to obtain RNA as a precipitate.

RNA obtained above was separated according to size by formaldehyde-agarose gel electrophoresis and blotted with a high-bond-N membrane (Sambrook, J. et. al ., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989). RNA blot membranes were hybridized with CaBI - 1 labeled with α- ³² P-dCTP. Hybridization methods are described by Sambrook's method (Sambrook, J. et. al ., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989). The membrane was prehybridized in solution [0.5 M sodium phosphate, pH 7.2, 7% (w / v) SDS and 1 mM EDTA, pH 7.0), which contained CaBI - 1 labeled with ³² P-dCTP. Add and hybridize at 65 ° C. for 16-22 hours. Hybridized membranes were washed at 65 ° C. with 1 × SSPE, 0.1% SDS solution and photosensitive to X-ray films (Sambrook J., Fritsch EF, and Maniatis T., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989).

As a result, it was confirmed that the expression is increased, although the degree of difference under various environmental stresses analyzed (FIG. 3).

< Example  2> CaBI -One  Environmental Stress Tolerance in Transgenic Plants Transgenic with Genes

In order to prepare a transgenic plant transformed with the CaBI- 1 gene of the present invention, the present inventors used tobacco, an eggplant plant such as red pepper, as a host plant. Nicotiana Tabacum WI 38 for tobacco transformation tabacum cv. The young leaf sections of Wisconsin 38) were used.

<2-1> CaBI -One  Preparation of Expression Vector for Plant Transformation Comprising Genes

Primer pairs designed along the bases of both outer regions of the ORF of CaBI- 1 in the pBluescript II SK (-) / CaBI- 1 vector to insert and express the protein recording site of the CaBI- 1 gene into the chromosome of the plant. Sense primer (SEQ ID NO: 3): 5'-ATATGGATCCATGGAGGGTTTCACGGTCGT-3 'and antisense primer (SEQ ID NO: 4) 5'-ATATGGATCCCTAGTTTCTCCTCTTCTTCTTC-3'; the underlined portion is Bam HI outside the ORF to introduce the ORF into the plant transformation vector. PCR was performed using a restriction enzyme) to isolate the ORF from CaBI - 1 , which was then transformed into a plant transformation vector, pBKS1-1 (Suh et. al ., Mol . Cells 4: 211-129, 1994) at the Bam HI restriction site following the CaMV35S promoter. That is, PCR amplified DNA was subjected to Bam HI treatment and electrophoresed on agarose gel, and the DNA fragments corresponding to the protein recording sites were purely separated from agarose gel using GeneClean II kit (Bio 101, USA). The isolated DNA fragment was transferred to plant transformation vector pBKS1-1 (Suh MC et al. , Molecules and Expression vector pBKS1-1 / CaBI - 1 for plant transformation of CaBI- 1 gene by attaching to Bam HI site of Cells , 4, 211-219, 1994) using T4 DNA conjugated enzyme for 8 hours at 19 ° C. (FIG. 4A) (Sambrook, J. Fritsch, EF and Maniatis, T., Molecular Cloning : A laboratory manual, Cold Spring Harbor Laboratory Press, 2nd Ed., 1989).

That in the expression vector of the protein portion of the recording CaBI -1 gene pBKS1-1 have correctly inserted in between CaMV35S promoter and the terminator regions of the furnace sold synthase (nopaline synthase), pBKS1-1 / CaBI - 1 DNA samples The 3 'terminal portion of the CaMV35S promoter was identified using a DNA sequence kit (Sequenase version 2.0 DNA sequencing kit) using a didioxy chain termination method such as Shanger using primers. 2 μl of sequenase buffer (200 mM TrisHCl, pH 7.5, 100 mM MgCl ₂ , 250 mM NaCl) and 0.5 pmol primer were added to the purified DNA, followed by reaction at 65 ° C. for 2 minutes, and then left at room temperature for 30 minutes. It was. Here, 1 μl of 0.1 M DTT, 2 μl of 1/5 diluted labeling mix, 0.5 μl of [α- ³⁵ S] dATP, 2 μl of 1/8 diluted secinase in order After adding and mixing, the mixture was reacted at room temperature for 2 to 3 minutes. 2.5 μl of ddGTP, ddATP, ddTTP, and ddCTP were respectively dispensed into four tubes at 37 ° C., 3.5 μl of the labeled mixture was added thereto, centrifuged, and the reaction mixtures were mixed. . 4 μl of the reaction solution was added to each tube to terminate the reaction, and then heated at 75 to 80 ° C. for 2 minutes, and then loaded to the electrophoretic gel for 2 to 3 μl, followed by electrophoresis, to the X-ray film. The nucleotide sequence was analyzed.

As a result, the target ORF is inserted in the sense or antisense direction between the CaMV35S promoter and the terminator of the nopaline synthase included in the expression vector. Confirmation (FIG. 4A).

<2-2> pBKS1 -One/ CaBI -One  Transformation of Expression Vectors into Plants

The pBKS1-1 / CaBI- 1 expression vector obtained in Example <2-1> was used as Agrobacterium tumefaciens ( Agrobacterium). tumefaciens ) was introduced into LBA4404. DNA was added to a transforming competent cell prepared by treatment with 20 mM CaCl _2, and then reacted with ice for 30 minutes and left in liquid nitrogen for 1 minute. The solution was dissolved in a 37 ° C. water bath for 5 minutes, dissolved, and then incubated with 1 ml YEP medium for 5 hours at 28 ° C. at 200 rpm, followed by centrifugation at 12,000 rpm to precipitate Agrobacterium. Precipitated Agrobacterium was resuspended in 0.1 ml YEP medium and plated in LB solid medium containing kanamycin and incubated at 28 ° C. (An G., Methods in Enzymology , 153, 292-305, 1987). Strains that formed colonies in kanamycin medium were selected as CaBI- 1 transgenic strains.

The CaBI- 1 transformed strain obtained above was infected with tobacco organisms in the following manner to prepare CaBI- 1 transformed tobacco. That is, Agrobacterium tumefaciens transformed with pBKS1-1 / CaBI- 1 expression vector After incubating the leaf sections of tobacco in suspension for 48 hours in the dark (Suh MC, Hong CB, et al ., Molecules and Cells , 4, 211-219. 1994), MS stem induction medium (Murashige T. and Skoog F., Physiol ) containing BAP 2.0 mg / l, NAA 0.1 mg / l, kanamycin 100 mg / l and cefotaxim 100 mg / l . Plant., in-flight and cultured for 4 to 6 weeks at 15, 473-497, 1962). When the stem is induced by the in-flight culture, MS root induction medium containing 200 mg / L of kanamycin again (Murashige T. and Skoog F., Physiolia) Plantarum ., 15, 473-497, 1962). Root-derived plants were transferred to the soil, and the growth and flowering and seed development were analyzed by cultivation in a greenhouse maintained at 25 ± 2 ℃ under natural light and blocked by outside and double glazing. When the seeds matured, they were harvested, dried at room temperature for 2 weeks, and then stored at 4 ° C.

<2-3> pBKS1 -One/ CaBI -One  Environmental Stress Tolerance Analysis of Expressing Transgenic Plants

The CaBI- 1 transgenic tobacco obtained in Example <2-2> was subjected to RNA blot analysis to confirm that CaBI- 1 introduced was properly expressed. The method used for RNA blot analysis was done in the same manner as described in Example <1-2> above.

As a result, it was confirmed that most of the plants produced were transgenic plants expressing CaBI- 1 . That is, in the transgenic plants introduced in the sense direction, strong RNA blot results were confirmed in 11 lines of the 12 lines analyzed, and in the antisense case, strong RNA blot results were detected in 8 lines of the analyzed 9 lines. . On the other hand, RNA blots were negative in both sense and antisense from plants in which only wild type and vector were introduced (FIGS. 4B and 4C).

The CaBI- 1 transgenic tobacco was cultivated in a greenhouse to harvest seeds by inducing self pollination. The harvested seed was sown to obtain a plant and a bioanalytical experiment was conducted to determine whether the environmental stress resistance was increased.

Bioassays to confirm the increase in resistance to environmental stress were performed as follows. For high temperature and dry resistance analysis, the seeds harvested in common are germinated and grown in MS-agar medium containing 200 mg / ml of kanamycin, and then only green seedlings that turn green are transplanted into pots containing culture soil and used in culture. It was.

1) High Temperature Tolerance Analysis: A 8-10 leaf CaBI- 1 transgenic tobacco was heated at 48 ° C. for 2 hours in an incubator maintained at 90% or higher humidity, and then transferred to a 25 ° C. culture chamber for 1 week. The leaves were collected and frozen in liquid nitrogen and stored in -70 ℃ ultra low temperature freezer. Protein and chlorophyll were extracted from this and quantified to verify the high temperature resistance of the plant.

Extraction of the protein from the plant was according to a method developed by the Columbia Proteomic Center in Missouri, USA. In summary. The leaves frozen in liquid nitrogen and stored in cryogenic freezers were ground in a liquid nitrogen and ground with a mortar and pestle. 0.3 g of powder was placed in a 1.5 ml microtube and 0.5 ml of 4 × protein extract buffer (0.175 M Tris-Cl pH 8.8, 5% SDS, 15% glycerol and 0.3M dithiothreitol) was added. The mixture was mixed well by vortex and left on ice for 15 minutes. Then, the microcentrifuge was centrifuged at 4 ° C. for 15 minutes at 15000 rpm, and the supernatant was placed in a new 1.5 ml microtube and centrifuged at 4 ° C. for 15 minutes at 15000 rpm. Supernatants were taken and quantified using the Bio-Rad (US) protein analysis system based on the Bradford method.

Chlorophyll extraction from plants was performed as follows. 0.2 g of the powder was placed in a conical tube, 80% acetone was added, vortexed, left in the dark for 48 hours at room temperature, and only the supernatant was taken. The absorbance of the supernatant was measured at 645 nm and 663 nm using a spectrophotometer. Chlorophyll was quantified by substituting the measured absorbance into Equation 1 below.

Chlorophyll a + Chlorophyll b = 0.0202 A ₆₄₅ + 0.00802 A ₆₆₃ (Mochizuki et al ., 2001, Proc Natl Acad Sci USA 98: 2053-2058).

2) Dry Tolerance Analysis: CaBI- 1 transgenic tobacco with 4-5 leaves was dried in the culture room after stopping watering for 35 days. After water was added thereto, dry resistance was measured by measuring the biomass and the relative moisture after 2 days. The biomass was determined by measuring the weight of the ground. Relative water content was determined by submerging only the ground portion and submerged in water for two hours and the measured ground portion and drying the cut ground portion at 54 ° C. for 48 hours, and then substituting the measured weight in Equation 2 below.

Relative water content = (biomass-dry weight) / (weight after immersion-dry weight) x 100 (Bray et al., 2000).

3) High Salinity Tolerance Assay: CaBI- 1 transgenic tobacco germinated in MS-agar medium containing kanamycin and incubated for 15 days was transferred to MS-agar medium containing NaCl 0, 50, 100 and 200 mM, respectively. And the whole was upright and incubated in the culture room for 10 days, the length of the root was measured to quantify high salt tolerance.

As a result, as shown in FIG. 5, in the case of the control group (transformant in which only wild type and vector were introduced), leaves were sulphated and growth stopped, but transformed in CaBI- 1 was introduced. The yellowing of the leaves and the decrease in growth were apparently small.

For quantification, the plants were cultured in the culture room for 7 days after the high temperature treatment and the amount of chlorophyll and protein were measured to compare the degree of damage caused by the high temperature stress of the control and the transformed CaBI- 1 .

As a result, as shown in FIG. 5, the control group had a chlorophyll content of 1.46-1.64 mg / g of biomass, whereas the CaBI- 1 introduced transformant had a chlorophyll content of 2.09-2.36 mg / g of biomass. It was. The protein content also showed a similar trend, whereas the control group had a protein content of 3.28-3.68 mg / g of biomass, whereas the CaBI-1-induced transformant had a protein content of 4.11-5.79 mg / g of biomass ( 5).

In addition, as shown in FIG. 6, the transformant with CaBI- 1 introduced showed better resistance to dry stress, which stopped watering for 35 days. Comparison of biomass measured after 2 days of resumption of water cycle showed that the transformants with CaBI- 1 introduced were biomass 1.7 to 2.4 times larger than controls. In addition, in the relative moisture content, the transformants introduced with CaBI- 1 showed higher values by 4.58-17.91% compared to the control (FIG. 6).

In addition, as shown in FIG. 7, the results of the resistance analysis for high salinity showed that the transformants into which CaBI- 1 was introduced were superior to the control. In the medium containing 50 mM NaCl, the control group observed an average decrease in root length of 17%, whereas the transformants introduced with CaBI- 1 showed an average root growth of 9%. In the medium containing NaCl 100 mM, the growth of root was increased in the control group, but in the transformants with CaBI- 1 , no decrease in root growth was observed. In medium containing 200 mM NaCl, serious damage was observed in both control and CaBI- 1 introduced transformants except the S-2 line. In addition, in the case of biomass under high salinity stress conditions, it was observed that similar to the control and biomass reduction except for the S-2 and S-7 lines among the transformant lines to which CaBI- 1 was introduced. If CaBI -1 NaCl 50 mM in both the introduction of the transgenic and the control is contained in the culture medium has an average 25% decrease in biomass, if contained 100 mM has been observed that reduction of the average biomass 45%, S-2 All plants except line were observed to die when 200 mM NaCl was contained (FIG. 7).

Is a gene map of / CaBI vector -1 - Figure 1 is a plasmid vector comprising a CaBI -1 Ⅱ pBluescript SK ().

Figure 2 is the base sequence and amino acid sequence of the environmental stress resistance inducing gene of the present invention.

Figure 3 shows the result of confirming the CaBI- 1 gene expression by RNA blot after applying hot, dry, low temperature, high salinity, heavy metals, immersion stress to pepper plants.

4A is a genetic map of the plant expression vector pBKS1-1 / CaBI- 1 , used to express CaBI- 1 gene in plants.

4B and C are RNA blot analysis results confirming that the plant expression vector pBKS1-1 / CaBI- 1 of the present invention is introduced to express CaBI-1 in the sense or antisense direction in the transformed tobacco plants.

FIG. 5 shows the results of biological experiments comparing the tobacco plants transformed with the pBKS1-1 / CaBI- 1 vector of the present invention and the tobacco plants not introduced with the vector after high temperature stress.

FIG. 6 shows the results of biological experiments comparing dry tobacco to tobacco plants transformed with the pBKS1-1 / CaBI- 1 vector of the present invention and transformed tobacco plants.

FIG. 7 shows the results of bio experiments comparing the tobacco plants transformed with the pBKS1-1 / CaBI- 1 vector of the present invention and the tobacco plants not transformed with the vector after introduction.

<110> SNU R & D Foundation <120> A red pepper gene CaBI-1 confers stress-tolerance to plants <130> 9p-11-68 <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 248 <212> PRT <213> Capsicum annuum <400> 1 Met Glu Gly Phe Thr Ser Phe Phe Glu Ser Gln Ser Ala Ser Arg Ser   1 5 10 15 Arg Trp Asn Tyr Asp Ala Leu Lys Asn Phe His Gln Ile Ser Pro Arg              20 25 30 Val Gln Thr His Leu Lys Gln Val Tyr Leu Thr Leu Cys Cys Ala Leu          35 40 45 Val Ala Ser Ala Ala Gly Ala Tyr Leu His Ile Leu Trp Asn Ile Gly      50 55 60 Gly Phe Leu Thr Thr Leu Ala Cys Ile Gly Ser Met Val Trp Leu Leu  65 70 75 80 Ala Thr Pro Pro Tyr Gln Glu Gln Lys Arg Val Ala Leu Leu Met Ala                  85 90 95 Ala Ala Leu Phe Glu Gly Ala Ser Ile Gly Pro Leu Ile Glu Leu Gly             100 105 110 Ile Asn Phe Asp Pro Ser Ile Val Leu Gly Ala Phe Val Gly Cys Gly         115 120 125 Val Val Phe Gly Cys Phe Ser Ala Ala Ala Met Leu Ala Arg Arg Arg     130 135 140 Glu Tyr Leu Tyr Leu Gly Gly Leu Leu Ser Ser Gly Val Ser Leu Leu 145 150 155 160 Met Trp Leu His Phe Ala Ser Ser Ile Phe Gly Gly Ala Met Ala Leu                 165 170 175 Phe Lys Phe Glu Val Tyr Phe Gly Phe Leu Val Phe Val Gly Tyr Ile             180 185 190 Val Phe Asp Thr Gln Glu Ile Ile Glu Lys Ala His Leu Gly Asp Met         195 200 205 Asp Tyr Val Lys His Ala Leu Thr Leu Phe Thr Asp Phe Val Ala Val     210 215 220 Phe Val Arg Ile Leu Ile Ile Met Leu Lys Asn Ala Phe Glu Lys Glu 225 230 235 240 Glu Lys Lys Lys Lys Arg Arg Asn                 245 <210> 2 <211> 747 <212> RNA <213> Capsicum annuum <400> 2 auggaggguu ucacgucguu cuucgaaucg caaucggcuu cucgcagucg cuggaauuau 60 gaugcucuca aaaacuucca ucagaucucu ccucguguuc aaacucaucu caaacagguc 120 uaccucacac uaugcugugc uuuagucgca ucagcugcug gggcuuaccu ucacauucuu 180 uggaacaucg guggcuuccu cacaacacug gcuugcauug gaagcauggu guggcuucug 240 gcaacuccuc cuuaucaaga gcaaaaaagg guggcacuuc ugauggcagc ugcacucuuu 300 gaaggcgcuu caauuggucc ucugauugaa cugggcauca acuucgaccc aagcauugug 360 cuuggugcuu uuguagguug uggugugguu uuugguugcu ucucagcugc ugccauguug 420 gcaaggcgca gggaguacuu guaccuugga ggccuucuuu caucuggugu cucccuccuc 480 augugguugc acuuugcauc cuccauuuuu gguggugcca uggcccuuuu caaguuugag 540 guguauuuug guuucuuggu guuugugggc uacauaguuu uugacaccca agaaaucauu 600 gagaaggcuc acuuggguga uauggauuac gucaagcaug cacucacccu cuucacagau 660 uuuguugcag ucuuugugcg gauuuugauc aucauguuga agaaugcauu ugagaaggaa 720 gagaagaaga agaagaggag aaacuag 747 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> CaBI-1 sense primer <400> 3 atatggatcc atggagggtt tcacggtcgt 30 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> CaBI-1 antisense primer <400> 4 atatggatcc ctagtttctc ctcttcttct tc 32  

Claims (13)

delete delete delete delete A transformant exhibiting environmental stress resistance selected from the group consisting of high temperature, dryness and high salt prepared by transducing a polynucleotide of SEQ ID NO: 2 encoding the amino acid sequence of SEQ ID NO: 1 into a host. A plant which is a progeny or clone of the transformant of claim 5. Seed, fruit, ear, tuber, tuber, strain, callus or protoplast of claim 5. The transformant of claim 5, wherein the host is a microorganism, an animal cell, a plant cell, an animal or a plant, and cultured cells derived therefrom. The plant of claim 6, wherein the plant is a monocotyledonous plant or a dicotyledonous plant. 1) preparing an expression vector operably linked to the polynucleotide of claim 5; 2) introducing the expression vector of step 1) into a host plant cell; And 3) A method for producing an environmental stress resistance induced gene transformant selected from the group consisting of high temperature, dryness and high salt comprising the step of tissue culture of the transformed plant cells of step 2). The method according to claim 10, wherein the step of introducing the expression vector of step 2) into the host plant cell is performed by an Agrobacterium mediated method, a gene gun, a method using PEG, and electroporation. A method for producing an environmental stress resistance inducing gene transformant. The method of claim 10, wherein the plant cells of step 2) are monocotyledonous or dicotyledonous plants. delete

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