CN101061227B

CN101061227B - Recombinant expression vector for production of plants having multiple stress tolerances, and method for preparing multiple stress-tolerant plants using the same

Info

Publication number: CN101061227B
Application number: CN2005800391729A
Authority: CN
Inventors: 郭尚洙; 权锡胤; 李幸顺; 汤莉; 林纯; 李炳显
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2004-11-17
Filing date: 2005-01-28
Publication date: 2012-02-29
Anticipated expiration: 2025-01-28
Also published as: WO2006054815A1; CN101061227A

Abstract

The present invention relates to a recombinant expression vector for production of multiple stress tolerant plants, which is prepared by attaching multiple stress tolerant genes to oxidative stress inducible promoter, multiple stress tolerant plants transformed with the expression vector and a preparation method for the plants, more precisely, a recombinant expression vector for production of multiple stress tolerant plants that is prepared by combining oxidative stress inducible peroxidase promoter (SWPA2) originated from sweetpotato with multiple stress tolerant genes (SOD (superoxide dismutase) and APX (ascobate peroxidase)) to express the multiple stress tolerant genes in chloroplasts, multiple stress tolerant plants transformed with the expression vector above and a preparation method for the plants. The recombinant expression vector of the present invention is very useful for the production of transformed plants having very strong resistance against multiple stresses caused by oxidative stress inducible herbicides, cold injury, high temperature, salt damage, or various environmental pollutions generating active oxygen, so that the vector can be a great contribution to the increase of productivity of agricultural crops or mass-production of useful components.

Description

Production have multiple stress tolerances plant recombinant expression vector and use it to prepare the method for multiple stress-tolerant plants

Technical field

The present invention relates to produce the recombinant expression vector of the plant with multiple stress tolerances; Transformed the multiple stress tolerances plant of above-mentioned carrier; With the method for the said plant of preparation, said recombinant expression vector prepares with expressing said gene in chloroplast(id) through the multiple stress tolerances gene is attached on the oxidative stress inducible promoter that comes from sweet potato.

Background technology

The most of biologies that comprise plant not only are subject to biology and coerce the influence such as pathogenic agent, insect, virus etc., and be subject to various environmental stresss such as high temperature, salt, arid, pollution, damage, frostbite, excessively illumination condition, ozone, sulfurous gas, over-exposure be in the influence of UV, osmotic shock etc.When plant received like the influencing of above-mentioned various environmental stresss, wherein life supported that essential oxygen is transformed into active oxygen radical, comprises ultra-oxygen anion free radical (O ^2-), hydrogen peroxide (H ₂O ₂), hydroxyl free radical etc., this causes serious physiologic derangement in vivo.Accurately, a spot of oxyradical is enough to conducted signal in cell in vivo, and in plant, induces the expression of the essential gene of egodefense (antioxidase, HSP etc.).Yet the increase of active oxygen radical causes physiologic derangement through dose-dependent manner, even causes necrocytosis.

Recently, many investigators are very interested in the signal transduction path of Mediated by Free Radicals in the plant.This is because the regulation and control of said approach make the expression increase of antioxidase and host-Buchner's bodies in the cell; And cause having development to the plant of the strong resistance of any environment-stress (Kovtun, etc., Proc.Natl.Acad.Sci.USA; 97 (6): 2940-2945,2000).Report according to nearest, play an important role in the signal transduction path of map kinase cascade (MARK cascade) by Mediated by Free Radicals in plant (Kovtun, etc., Proc.Natl.Acad.Sci.USA, 97 (6): 2940-2945,2000).

Superoxide-dismutase (SOD) is a kind of with ultra-oxygen anion free radical (O ^2-) change into hydrogen peroxide (H ₂O ₂) enzyme, and be divided into CuZnSOD, MnSOD and FeSOD according to the metal cofactor that is comprised in the enzyme.These enzymes distribute different in cell, and for example CuZnSOD finds in tenuigenin and chloroplast(id), and MnSOD finds in plastosome, and FeSOD is present in the chloroplast(id).SOD is a kind of important environmental resistance factor of eliminating the oxyradical that is produced by environment-stress in the organism, and it can be used to produce medical supply, food, makeup etc.And therefore; The transgenic plant that show strong active stress tolerance that preparation comprises sod gene cause having the development (PlantPhysiology to the plant of the strong resistance of environment-stress such as ozone, low temperature, weedicide etc.; 10:1049-1054,1995; US Patent No:5,538,878).

Ascorbate peroxidase enzyme (APX) is a kind of through using vitamins C as electron donor and with H ₂O ₂Change into the enzyme of water, and in plant and insect, find in large quantities.Known this kind of enzyme is present in (Free Rad.Biol.Med.23:473-479,1997) in tenuigenin, stroma and the thylakoid membrane in plant.

In the chloroplast(id) of plant, oxygen level is high relatively, and uses from the electric energy operating electronic movement system of utilizing the luminous energy water of decomposition to produce, so this organ is very responsive to various oxidative stresses.Therefore, the increase of chloroplast(id) resistance of oxidation possibly greatly help to keep the yield-power of plant under environment-stress.

Till now, in order to develop stress tolerant plants,, come construction of expression vector no matter the CaMV 35S promoter what condition can both constitutive expression is used with the assortment of genes that has to the resistance of specifically coercing widely.Therefore, the plant that obtains has only to the concrete resistance of coercing.In order to overcome this problem, need a kind of expression vector of preparation, it comprises and can coerce expression promoter and stress tolerance gene under the environment any, and preparation is with the transgenic plant of said carrier transfection.

In order to develop the transgenic crop that has to the tolerance of environmental disaster; The inventor has prepared the new expression vector that is used for Plant Transformation, its expressing said gene through multiple stress tolerances gene SOD (superoxide-dismutase) and APX (ascorbate peroxidase enzyme) being attached to the oxidative stress inducibility peroxidase promoter SWPA2 that comes from sweet potato and going up and in the chloroplast(id) of plant.Then, the inventor is through the conversion plant of tissue culture regeneration from yam, sweet potato and Festuca Arundinacea preparation.At last, the inventor accomplishes the present invention through confirming the multiple stress tolerances that transgenic plant of the present invention have an increase.

Disclosure

Technical problem

An object of the present invention is to provide a kind of recombinant expression vector that is used for Plant Transformation, it prepares with expressing said gene in chloroplast(id) through the multiple stress tolerances gene is attached on the oxidative stress inducible promoter that comes from sweet potato.

Another object of the present invention provides the multiple stress tolerances plant that has transformed above-mentioned expression vector, and about its preparation method.

Technical scheme

To achieve these goals; The present invention is provided for producing recombinant expression vector ' pSSA-K ' and ' pSSA-H ' of multiple stress tolerances plant, and it contains oxidative stress inducibility peroxidase promoter, tobacco etch virus (TEV) leader sequence, multiple stress tolerances gene, the transit peptide sequence that is used for chloroplast targeted expression, CaMV 35S transcription terminator, antibiotics resistance gene and T-DNA border sequence (T-DNA boarder sequence).

The present invention also provides with above-mentioned pSSA-K or pSSA-H expression vector transgenic plant transformed.

The present invention also provides the preparation method of multiple stress tolerances transgenic plant, and said method comprises the steps:

I) preparation is used for the expression vector of Plant Transformation, and said carrier comprises SWPA2 promotor, sod gene and APX gene;

Ii), above-mentioned expression vector prepares transformant through being inserted in plant or the culturing cell;

Iii) cultivate above-mentioned transformant; With

Iv) after the said transformant of tissue-cultivation, prepare transgenic plant through regeneration.

To describe the present invention in detail hereinafter.

The present invention is provided for producing recombinant expression vector pSSA-K and the pSSA-H of multiple stress tolerances plant, and it comprises oxidative stress inducibility peroxidase promoter, tobacco etch virus (TEV) leader sequence, the transit peptide sequence that is used for chloroplast targeted expression, multiple stress tolerances gene, CaMV 35S transcription terminator, antibiotics resistance gene and T-DNA border sequence.

In a preferred embodiment of the invention; Nucleotide sequence by SWPA2 (the sweet potato px negatively charged ion 2) promotor of SEQ.ID.No 11 representative; Preferably preferably be used as the multiple stress tolerances gene as the oxidative stress inducibility peroxidase promoter that comes from sweet potato with by the sod gene nucleotide sequence of SEQ.ID.No 12 representatives and by the APX gene nucleotide series that SEQ.ID.No 13 represents.

The inventor will be kept at the Korea S typical case culture collection center (Korean Collection for TypeCultures (KCTC)) of Korea Institute of Bioengineering (Korea Research Institute of Bioscience and Biotechnology (KRIBB)) by the inventor on November 7th, 2003 by the expression vector pSSA-K and the pSSA-H that are used to produce the multiple stress tolerances plant of above-mentioned preparation.The preserving number of pSSA-K expression vector is that the preserving number of KCTC 10536BP and pSSA-H expression vector is KCTC 10537BP.

The SWPA2 promotor be the inventor from the isolating oxidative stress inducible promoter of sweet potato (Ipomoea batatas), it is for exploitation stress tolerant plants and very useful (the korean patent publication No:2001-51095 of plant cell that produces other useful products; International publication No:WO01/31018 (in application on October 28th, 2000); Kim, etc., Plant Mol.Biol., 51:831-838,2003).Accurately, as the peroxidase promoter of expressing at the culturing cell camber, SWPA2 a kind ofly receives oxidative stress and the gene of abduction delivering.Especially, latter stage is highly expressed (international publication No:WO 01/31018) in logarithmic phase in said gene specific ground in the tobacco suspension culturing cell of sweet potato suspended culture cell and conversion.In the instantaneous detection of using tobacco protoplast to carry out with gus protein, said promotor shows the activity than at least 30 times of CaMV 35S promoter height.Under normal operation, said promotor is not expressed in the blade of plant, but when they receive oxidative stress such as ozone, low temperature, damage etc., its expression (Kim, etc., Plant Mol.Biol., 51:831-838,2003).Therefore, SWPA2 promotor of the present invention can be used for the development environment stress tolerant plants effectively and be used to use the plant transformed cell and produce useful product.

SWPA2 promotor of the present invention is effectively through coercing the expression of inducing target gene.SWPA2 promotor of the present invention comprises the external factor of coercing of identification by ABA (dormin), methyl jasmonate, damage, histanoxia, oxyradical, heat or nitrogen gas generation.Based on this characteristic of said promotor, the SWPA2 promotor is used for the chimeric genetic construct structure, and the dna sequence dna that wherein will have promoter activity is connected on the structure gene to work effectively in promoter sequence.Owing to participating in producing the gene as a result of the valuable factor and the connection between the SWPA2 promotor; Such mosaic gene structure can express the valuable factor through control SWPA2 promotor under any environment-stress, so that it can be used to prepare the transformant of producing useful products effectively.In addition, when the multiple stress tolerances gene gives the mosaic gene structure as structure gene, can produce transformant with stress tolerance.

Be used for sod gene of the present invention usually from separating more than 30 kind of plant.And; Especially; The at first isolating CuZnSOD gene of culturing cell (mSOD1) from the cassava (Manihot esculenta) selected as SOD height-production clone; Be used for the present invention (Mol.Gen.Genet.262:807-814,1999) together from the isolating APX gene of pea (Free Rad.Biol.Med.23:473-479,1997).

In a preferred embodiment of the invention; The inventor makes up the pSA carrier through being connected with the pRW20 carrier by the SWPA2 promotor of SEQ.ID.No 15 representatives, in the pRW20 carrier, inserts by the mSOD1 gene of SEQ.ID.No 16 representatives to prepare pSS carrier (referring to Fig. 1).Then; The SWPA2pro-TEV-TP-SOD-35S Transcription Termination minor structure of pSS carrier is inserted into the expression vector that is used for Plant Transformation that comprises hygromycin gene or kalamycin resistance gene, will be inserted into wherein from the isolating SWPA2pro-TEV-TP-APX-35S Transcription Termination of pSA minor structure then.As a result, made up pSSA-K and the pSSA-H expression vector (referring to Fig. 2) that is used to produce the multiple stress tolerances plant.Except comprising the hygromycin gene or kalamycin resistance gene that the multiple stress tolerances plant that makes with said carrier conversion is easy to select respectively; Above-mentioned pSSA-K and pSSA-H expression vector also comprise SWPA2 promotor, mSOD1, APX gene, tobacco etch virus (TEV) leader sequence, the transit peptide sequence that is used for chloroplast targeted expression and CaMV 35S transcription terminator, and this makes the selection to the multiple stress tolerance plant that has transformed said carrier be more prone to.

The present invention also provides the multiple stress tolerances plant that transforms with pSSA-K or pSSA-H expression vector.The plant of all kinds can be used to produce transgenic plant, but preferred soybean, Hordeum, Zea mays, yam, sweet potato or Festuca Arundinacea, and more preferably yam, sweet potato or Festuca Arundinacea especially.

The present invention provides the multiple stress tolerances transgenic plant; It prepares through the recombinant expression vector that conversion is used to produce the multiple stress tolerances plant; Said recombinant expression vector comprises SWPA2 promotor, sod gene and APX gene, with great expression sod gene in plant and APX gene.

In a preferred embodiment of the invention; The pSSA-K or the pSSA-H expression vector of above-mentioned structure are inserted in the plant; Preferably be inserted in soybean, Hordeum, Zea mays, yam, sweet potato or the Festuca Arundinacea; And more preferably be inserted in yam, sweet potato or the Festuca Arundinacea, to express sod gene and APX gene.Especially, the inventor perhaps is inserted into above-mentioned expression vector in the tender leaf or petiole section of yam, sweet potato or Festuca Arundinacea, with the preparation transformant through the method for particle bombardment through agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105.The transformant that obtains is carried out tissue culture, to induce the regeneration (referring to Fig. 3 A, Fig. 4 A and Fig. 5 A) in plant.From every kind of regenerated transgenic Rhizoma Solani tuber osi, sweet potato or Festuca Arundinacea, extract genomic dna; Through PCR from said genomic dna, increase SWPA2 promotor or APX gene; To confirm that SWPA2 promotor or APX gene are inserted into (referring to Fig. 3 B, Fig. 4 B and Fig. 5 B) in the Plant Genome.Confirmed successfully conversion through PCR after, also from every kind of transgenic plant, extract genomic dna, it is transferred on the film.Use SWPA2 promotor or APX gene to carry out southern blotting technique hybridization, to verify the insertion (referring to Fig. 3 C, Fig. 4 C and Fig. 5 C) in Plant Genome of SWPA2 promotor or APX gene once more.

In order to confirm stress resistance with pSSA-K or pSSA-H expression vector transgenic plant transformed; The inventor for example uses methyl viologen (MV) or hydrogen peroxide and induces oxidative stress the blade or the plant itself of the yam, sweet potato or the Festuca Arundinacea that transform; Study ionic conductance then (referring to Fig. 6; 7; 9,10 and 11), visible injury (referring to Fig. 8 A and Figure 11 C), photosynthetic efficiency (referring to Figure 11 A), leaf dry weight (referring to Fig. 8 B and Figure 11 B) and chlorophyll content (referring to Fig. 8 C).As a result, confirm to compare with unconverted plant, show fabulous oxidative stress tolerance with pSSA-K of the present invention or pSSA-H expression vector transgenic plant transformed.Confirm that also transgenic plant of the present invention can coerce like high temperature (referring to Figure 13 B～Figure 14 B), low temperature (referring to Figure 15 A and Figure 15 B) or SO at other ₂Coerce (referring to Figure 16 and Figure 17) and keep normal situation down.

Iii) cultivate above-mentioned transformant; With

At this moment; PSSA-K or pSSA-H are preferably as expression vector; And any plant can be used as above-mentioned plant or culturing cell, but preferred soybean, Hordeum, Zea mays, yam, sweet potato or Festuca Arundinacea, and more preferably yam, sweet potato or Festuca Arundinacea.

Above-mentioned transformant can pass through the conventional plant method for transformation (Horsch, etc., Cold SpringHarb Symp Quant Biol., 50:433-7,1985; Rogerset, etc., 1986) and prepare, attack and the generation transformant but preferably use particle in this article.

In a preferred embodiment of the invention; Because said carrier contains SWPA2 promotor, sod gene, APX gene, the transit peptide sequence that is used for chloroplast targeted expression, tobacco etch virus (TEV) leader sequence, CaMV 35S transcription terminator and Totomycin or kalamycin resistance gene; So can easily select with said carrier transgenic plant transformed; Therefore, pSSA-K or pSSA-H are as expression vector.

Accompanying drawing is described

Fig. 1 shows that the present invention expresses the synoptic diagram of Production Flow Chart of the carrier of SOD (superoxide-dismutase) and APX (ascorbate peroxidase enzyme) gene simultaneously,

Fig. 2 shows to contain the synoptic diagram that SOD and APX gene are used to produce the recombinant expression vector of multiple stress tolerances plant,

Fig. 3 A is presented at the transgenic Rhizoma Solani tuber osi with kalamycin resistance that produces through the recombinant expression vector (pSSA-K) that use to produce the multiple stress tolerances plant (kind: Superior) carry out the photo of the form of regenerated all parts of a plant after the tissue culture

Fig. 3 B is to use the electrophoresis photo of the primer of SEQ.ID.No 1 and No 2 representatives through the amplification of PCR checking SWPA2 promotor in the transgenic Rhizoma Solani tuber osi plant that transforms with expression vector of the present invention (pSSA-K carrier),

Fig. 3 C is that checking SOD and APX gene are present in the expression vector (pSSA-K carrier) of the application of the invention and southern blotting technique photo in the genome of the transgenic Rhizoma Solani tuber osi plant for preparing,

Fig. 4 A be presented at the expression vector (pSSA-K carrier) of the application of the invention and the kalamycin resistance transgenic sweet potato that produces (kind: Yulmi) carry out the photo of the form of regenerated all parts of a plant after the tissue culture,

Fig. 4 B is to use the electrophoresis photo of the primer of SEQ.ID.No 1 and No 2 representatives through the amplification of PCR checking SWPA2 in the transgenic sweet potato plant that transforms with expression vector of the present invention (pSSA-K carrier),

Fig. 4 C is that checking SOD and APX gene are present in the southern blotting technique photo in the genome of the transgenic sweet potato plant of expression vector of the present invention (pSSA-K carrier) conversion,

Fig. 5 A is the one group of photo that shows the aftergrowth of the tissue culture of the hygromycin resistance transgenic tall fescue of preparation available from the expression vector (pSSA-H carrier) of the application of the invention,

Fig. 5 B is that checking SOD and APX gene are present in the PCR photo in the genome of the transgenic tall fescue plant of expression vector of the present invention (pSSA-H carrier) conversion,

Fig. 5 C is that checking SOD and APX gene are present in the southern blotting technique photo in the genome of the transgenic tall fescue plant of expression vector of the present invention (pSSA-H carrier) conversion,

Fig. 6 be presented at they with methyl viologen (MV) solution-treated of different concns (0,3,5 and 10 μ M) after one group of photo of membrane damage in NT and the SSA potato plants blade, it is measured through research effects of ion conductivity,

Fig. 7 is presented at different concns 0,150, one group of photo of the MV resistance level in the potato plants of MV spray solution after 5 days of 200 and 250 μ M,

Fig. 8 is presented at different concns 0,150, the relative dry weight (B) and the chlorophyll content (C) of the visible damage (A) in the potato plants blade of the MV spray solution of 200 and 250 μ M after 5 days, survival blade,

Fig. 9 is the set of diagrams table that shows with the membrane damage in the sweet potato blade of 72 hours the NT of MV solution (0,2.5,5 and 10 μ M) processing of different concns and SSA plant, and it detects through the ionic conductance of studying in the solution,

Figure 10 shows with different concns 0,100, one group of photo of the MV resistance level of the sweet potato plant of the MV spray solution of 150 and 200 μ M after 5 days,

Figure 11 is the sweet potato photosynthesis of plants efficient (A) of MV spray solution after 5 days, the set of diagrams table with respect to the visible damage (C) in heavy (B) and the blade of survival blade that shows different concns 0,100,150 and 200 μ M,

Figure 12 shows with 5 μ M MV solution (A) and 50mM H ₂O ₂The set of diagrams table of the membrane damage in the Festuca Arundinacea plant leaf that solution (B) is handled,

Figure 13 is the set of diagrams table that is presented at observed high temperature resistance in the blade of potato plants, accurately, and the ionic conductance after high temperature (25 ℃ (A) and 37 ℃ (B)) is handled 60 hours in the research film,

Figure 14 is one group of photo and the chart that shows potato plants high temperature resistance result of study.Accurately, research visible damage (A) that 42 ℃ of thermal treatments caused in 10 hours and 42 ℃ handle 10 hours, 20 hours after and after the pyroprocessing at the photosynthetic efficiency (B) of 25 ℃ of recoveries after 3 hours,

Figure 15 is one group of photo and the chart that shows sweet potato plant low temperature resistance result of study.Accurately, be presented at 4 ℃ of visible plant injuries (A) of handling after 24 hours, in the said back of handling at the photos (B) of 12 hours plants of 25 ℃ of recoveries with in 24 hours processes of 4 ℃ of subzero treatment and at the photosynthetic efficiency (C) of 25 ℃ of recoveries after 12 hours,

Figure 16 shows every day with 8 hours lasting photos of handling the sulfurous gas resistance of the SSA sweet potato plant that caused in 5 days of 500ppb sulfur dioxide treatment,

Figure 17 is the chart that shows NT and SSA sweet potato photosynthesis of plants efficient, and it afterwards obtains in 5 days processes of 500ppb sulfur dioxide treatment and adapting to normal condition (0ppb) after 5 days with sulfur dioxide treatment.

The invention pattern

As shown in the following embodiment, the present invention practical with embodiment preferred at present be exemplary.

Yet, should be appreciated that consider present disclosure, those skilled in the art can carry out change and the improvement within the term the spirit and scope of the present invention.

<Embodiment 1>Make up multiple stress tolerances expression carrier pSSA-K and pSSA-H

The inventor is through coding cassava CuZnSOD (the CuZn superoxide-dismutase with SEQ.ID.No 16 representatives; MSOD1) cDNA (GenBank registration number AF170297; Lee, etc., Mol.Gen.Genet.; 262:807-814,1999) and coding pea APX (the ascorbate peroxidase enzyme of SEQ.ID.No 17 representatives; Randy, etc., Free Rad.Biol.Med., 23:473-479,1997) cDNA be connected to oxidative stress inducibility SWPA2 promotor (the korean patent publication No:2001-51095 of SEQ.ID.No 15 representative; International publication No:WO 01/31018) on, and carrier construction.

Particularly, use every kind to carry out PCR by the primer of SEQ.ID.No 1 and No 2 representatives, 94 ℃ 1 minute, 51 ℃ 1 minute, 72 ℃ 1.5 minutes (this cycle repeats 30 times) is with amplification SWPA2 promoter sequence.And according to mfr's flow process, with the amplification the SWPA2 promoter sequence be cloned into pGEM-T Easy plasmid vector (Promega, USA) in.And analyze the nucleotide sequence of carrier, whether correctly increase with the target sequence that confirms the SWPA2 promotor.The nucleotide sequence of primer has Hind III and Xho I restriction site.Therefore, can be through handling with Xho I with Hind III and separating the SWPA2 promoter sequence from pGEM-T Easy plasmid vector.And, will for be delivered to the APX of pea (ascorbate peroxidase enzyme) the pRW20 carrier that chloroplast(id) makes up (Allen, etc., Free Rad.Biol.Med., 23:473-479,1997) with identical enzymic digestion, to eliminate enhancement type CaMV 35S promoter.Then, more previous isolating SWPA2 promotor is connected on the carrier, forms the structure (Fig. 1) of pSA carrier.

In order to replace the APX gene in the pSA carrier with the mSOD1 gene, use respectively primer to carry out PCR by SEQ.ID.No 3 and No 4 representatives, 94 ℃ 1 minute, 57 ℃ 1 minute with 72 ℃ 1 minute (this cycle repeats 30 times), with the mSOD1 gene that increases.And according to mfr's flow process, with the amplification mSOD1 be cloned into pGEM-T Easy plasmid vector (Promega, USA) in.Whether carry out nucleotide sequence analysis is correctly increased with checking target mSOD1 gene.The nucleotide sequence of primer has Sal I and Sac I restriction site.Therefore, the mSOD1 gene is separated from pGEM-T Easy plasmid vector, through with Sal I and Sac I processing earlier with the mSOD1 gene clone in said carrier.Then, isolating mSOD1 gene is inserted in the above-mentioned pSA carrier with identical Restriction Enzyme digestion.And the carrier called after pSS carrier (Fig. 1) that obtains.

At last; PCAMBIA1300 plasmid (the Centerfor Application of Molecular Biology to International Agriculture that contains hygromycin gene through use; Australia) and contain pCAMBIA2300 plasmid (the Center for Applicationof Molecular Biology to International Agriculture of kalamycin resistance gene; Australia); Structure is used for the carrier pSSA-K and the pSSA-H of Plant Transformation, to transform SOD and APX simultaneously.Particularly, above-mentioned pSS carrier with Hind III digestion, to obtain the dna fragmentation of about 2.0kb size, is inserted in the pCAMBIA1300 plasmid and pCAMBIA2300 plasmid that digests in advance with identical enzyme.Also pSA is digested with Pst I; To obtain the dna fragmentation of 2.3kb size; Be inserted in the above-mentioned plasmid, cause being built into and have SOD and APX expression carrier: pSSA-K carrier (dna fragmentation is inserted among the pCAMBIA2300) and pSSA-H carrier (dna fragmentation is inserted among the pCAMBIA1300) are (Fig. 2).The inventor will be kept at Korea S typical case's culture collection center of Korea Institute of Bioengineering by the inventor on November 7th, 2003 by the pSSA-K of above-mentioned structure and pSSA-H.The preserving number of pSSA-K expression vector is that the preserving number of KCTC10536BP and pSSA-H expression vector is KCTC 10537BP.In Fig. 1 and Fig. 2, SWPA2pro: oxidative stress inducible promoter, E35S pro: enhancement type CaMV 35S promoter, TEV: tobacco etch virus (TEV) leader sequence; TP: the signal sequence of pea CuZnSOD (CuZn superoxide-dismutase), 35S 3 ': CaMV 35S transcription terminator, Amp: microbiotic (penbritin) resistant gene; H:Hind III, P:Pst I, Xh:Xho I; R:EcoR l, N:Nco l, S:Sal l; B:BamH l, X:Xba l and Sa:Sac l.

According to An ' s method (An, Meth.Enzymol., 153:292-305; 1987); Through the expression vector that is used for Plant Transformation (pSSA-K or pSSA-H) that preceding text are made up be inserted into agrobacterium tumefaciens EHA105 prepare the transgenic Rhizoma Solani tuber osi plant (Hood, etc., Trans.Res.; 2:208-218,1993).

And in addition, the plasmid Maxi test kit through using Qiagen Co (U.S.A) to provide from intestinal bacteria isolated plasmid dna (pSSA-K carrier), then, is used to transform sweet potato through particle bombardment, and this will make an explanation among the embodiment 3 hereinafter.

<Embodiment 2>Preparation has transformed the transgenic Rhizoma Solani tuber osi of pSSA-K expression vector

<2-1>Preparation transgenic Rhizoma Solani tuber osi plant

The inventor prepares the transgenic Rhizoma Solani tuber osi plant through the blade wafer of cultivating potato plants altogether and the agrobacterium tumefaciens EHA105 that contains the pSSA-K carrier that in preceding text embodiment 1, makes up.

Particularly, prepare the potato plants (Solanumtuberosum L.) that this paper is used to transform through in incubator, cultivating the kind Superior that the most extensively cultivates in the world with the kind Atlantic that is used to handle.Potato plants is cultivated in the MS substratum that has replenished 3% sucrose (Murashige and Skoog, Physiol.Plant., 15:473-497,1962), in 25 ℃ of culturing room at cold white fluorescent (40 μ molm ^-2Sec ^-1) cultivate under hour dark optical condition in following 16 hours illumination/8.After cultivating for 2 weeks, with petiole with separate at bud vertical second or the 3rd leaf, and as the material of Plant Transformation.

With the 5ml LB substratum that has replenished the 50mg/l kantlex (bactopeptone 10g/l, yeast extract 5g/l and NaCl 10g/l) inoculation Agrobacterium, in 28 ℃ of wave and culture casees, cultivated 1 day then.The blade and the petiole sheet of 100 μ l Agrobacterium culture solution and potato plants are mixed in the petridish that contains the basic liquid nutrient medium of 10ml MS fully, cultivated altogether 2 days at 25 ℃ in the dark then.Agrobacterium with the basic liquid nutrient medium washing of MS, is eliminated available from the moisture in the blade of cultivating potato plants altogether and the petiole sheet with aseptic filter paper then.Then, place selection to cultivate on (containing the 2mg/l zein, 0.01mg/l NAA (naphthylacetic acid), 0.1mg/l GA3 (Plant hormones regulators,gibberellins), the MS substratum of 300mg/l claforan and 100mg/l kantlex) in blade and petiole sheet.Per 3 weeks are transferred to said disk in the fresh new substratum, carry out subculture then.After week, observe the bud and the callosal generation of kalamycin resistance at the 3-4 that begins from cultivation.

When growing one or two leaf, bud is transferred in the root induction substratum (the MS minimum medium that contains 300mg/l claforan and 100mg/l kantlex) to induce root.Root is well induced from said bud, and will have the plantlet of root from culture vessel, to take out, and is exposed to outer 4-5 days (adapting to culturing room), transfers to then in the flowerpot of seedbed, greenhouse, in incubator, further cultivates then (Fig. 3 A).As a result, form microtubule (Fig. 3 A) in the potato plants in being grown in incubator.On the form between transgenic Rhizoma Solani tuber osi plant of carrying alien gene and the unconverted potato plants, there is not significant difference.Simultaneously, when petiole is used as converting material, than when blade is used to transform, forming more bud.Although, through the PCR checking not conversion of great majority in them.

<2-2>Through PCR and southern blotting technique check transformant

Whether correctly transform in order to study the potato plants that in the foregoing description 2-1, prepares; The regenerated plant is at first selected in the substratum that has replenished kantlex; Auele Specific Primer with the SEQ.ID.No 5 and the SWPA2 promotor of No 6 representatives carries out PCR then, 94 ℃ 1 minute, 53 ℃ of 1 minute and 72 ℃ 1 minute (these cycle repeats 30 times); With amplification SWPA2 promoter sequence, carry out the selection (Fig. 3 B) of transformant.As a result, from the increased fragment of 0.5kb of the plant of carrying alien gene, this shows and has inserted the SWPA2 promotor.In Fig. 3 B, M: the big tick marks of molecular weight, NT: unconverted potato plants, 1-5: kalamycin resistance potato plants, and P: positive control dna.

The potato plants of selecting at random to through the PCR checking carries out southern blotting technique.Accurately, through using Dneasy Plant Maxi test kit (QIAGEN Co.), from the blade of the every strain Atlantic that incubator, grows and Superior plant, extract genomic dna.The genomic dna of 30 μ g is digested with Restriction Enzyme EcoRI, on sepharose, carry out electrophoresis then.To transfer on the Zeta Probe film (Bio-Rad Co.) at the genomic dna on the gel, use as probe then ³²The DNA that the 0.5kb dna fragmentation hybridization of the SWPA2 promoter sequence of P mark is shifted.In case accomplish hybridization, film washed, and be exposed to X-ray film, with test strip.As a result,, in Atlantic that transforms and Superior plant, observe 3 bands, this means that the SWPA2 promotor stably is inserted in the genome of yam (Fig. 3 C) although in the non-transgenic control plant, do not observe band.In Fig. 3 C, NT: unconverted potato plants, A1 and A2: the Atlantic plant of conversion, and S1 and S2: the Superior plant of conversion.

<Embodiment 3>Through using the pSSA-K expression vector to prepare transgenic sweet potato plant

<3-1>Preparation transgenic sweet potato plant

The inventor uses the pSSA-K carrier that in the foregoing description 1, makes up to transform sweet potato embryo generation corpus callosum through particle bombardment.

Sweet potato (the Ipomoea batatas Lam. that will cultivate particularly, in Korea S; Kind: embryo generation corpus callosum Yulmi) is as converting material of the present invention.More accurately, the embryo generation corpus callosum of sweet potato is induced and is kept, with set up sweet potato plant regeneration system (Kwon, etc., Korean J.PlantBiotechnol., 29:189-192,2002).

Embryo generation corpus callosum is cut into diameter 1-2mm by cell cluster.With cell cluster be placed at replenished 1mg/l 2,4 dichloro benzene ethoxyacetic acid (2, on MS solid medium 4-D) in the center ring of diameter 2cm, it was cultivated 1 day, carry out particle then and attack (Fig. 4 A).More accurately, be prepared in the DNA that is used for Plant Transformation pSSA-K carrier that makes up among the embodiment 1.Then, (diameter 1 μ m) encapsulates with said DNA with gold particle, and then 1, the 9cm distance uses PDS-1000/He particle delivery system (BioRad Co.) to carry out particle bombardment under the 100psi pressure.After particle bombardment, plant was in the dark cultivated 3 days at 25 ℃, then from having replenished 1mg/l 2,4-D and 100mg/l kantlex MS select the embryo generation corpus callosum of substratum selection kalamycin resistance.In the subculture process, select to continue 5-6 month with the timed interval in 3 weeks.Kalamycin resistance embryo generation corpus callosum transferred to only replenished the 100mg/l kantlex and do not have 2, in the MS minimum medium of 4-D.And in incubator at 25 ℃ at 40 μ μ mol m ^-2.sec ^-1Under the cold white fluorescent condition, embryo generation corpus callosum is changed into somatic embryo, this causes the regeneration (Fig. 4 A) of plant.

<3-2>Through PCR and southern blotting technique checking transformant

In order to verify whether the transgenic sweet potato plant that in the foregoing description 3-1, prepares is correctly transformed, and the regenerated plant is at first selected in containing the substratum of kantlex.Use the Auele Specific Primer of the SEQ.ID.No 7 and the SWPA2 promotor of No 8 representatives or the Auele Specific Primer of the SEQ.ID.No 9 and the APX gene of No 10 representatives; Said plant is carried out PCR; 94 ℃ 1 minute; 56 ℃ of 1 minute and 72 ℃ 1 minute (these cycle repeats 30 times) with amplification SWPA2 promotor or APX gene, are carried out electrophoresis to select transformant (Fig. 4 B) then.The result; When using the Auele Specific Primer of SWPA2 promotor; Obtain the 1kb dna fragmentation and when using the APX gene-specific primer, obtain the dna fragmentation of about 0.5kb from wherein increasing from the plant amplification of inserting foreign gene, this shows that SWPA2 or APX gene are stably inserted.In Fig. 4 B, M: molecular size mark, NT: unconverted sweet potato plant, 1-9: kalamycin resistance sweet potato plant, and P: positive control dna.

The sweet potato plant of selecting at random to through the PCR checking carries out southern blotting technique.Particularly, through using Dneasy Plant Maxi test kit (QIAGEN Co.), from the blade of the sweet potato plant that incubator, grows, extract genomic dna.The genomic dna that 30 μ g are obtained digests with Restriction Enzyme EcoR I, on sepharose, carries out electrophoresis then.To transfer on the Zeta Probe film (Bio-Rad Co.) at the genomic dna on the gel.With the dna fragmentation of the DNA hybridization 0.5kb that shifts, said dna fragmentation is the part of mSOD1, uses ³²The P mark is as probe.In case accomplish hybridization, film is washed, and be exposed to X-ray film, to find band.As a result, in kalamycin resistance sweet potato plant, stably inserted mSOD1, and in control plant, do not observed band (Fig. 4 C) more than 2 copies.In Fig. 4 C, NT: unconverted sweet potato plant, and T1-T4: the sweet potato of conversion (kind: Yulmi) plant.

<Embodiment 4>Through using the pSSA-H expression vector to prepare the transgenic tall fescue plant

<4-1>Preparation transgenic tall fescue plant

The inventor cultivates agrobacterium tumefaciens EHA105 and the Festuca Arundinacea section that contains the pSSA-H carrier (DNA) that in the foregoing description 1, makes up altogether, to induce its conversion.

The Kenturky-31 that will cultivate mainly as animal-feed particularly, is used for transforming Festuca Arundinacea (alta fascue (Festuca arundinacea Schreb.)).To be used for the corpus callosum that Festuca Arundinacea transforms in order preparing, seed to be sterilized, and remove the epidermis of seed.Then, seed was cultivated for 4 weeks to induce corpus callosum in corpus callosum inducing culture [containing 9mg/l 2,4-D, 0.1mg/l BA (phenmethyl VITAMIN B4), 30g/l sucrose, the MS substratum of 5g/l gelite].Will be centrifugal to obtain bacterial cell 28 ℃ of Agrobacterium culture solution of in the YEP liquid nutrient medium that has replenished the 50mg/l kantlex (10g/l bacterium usefulness-peptone, 10g/l yeast extract and 5g/l NaCl), cultivating 2 days.Then, solution is suspended in has replenished 100 μ M Syringylethanones, 20mg/l xitix and 5mg/l Silver Nitrate (AgNO ₃) induce in the callosal liquid nutrient medium, up to OD ₆₀₀Reach 1.Under vacuum, said corpus callosum is immersed in the agrobacterium suspension 30 minutes with inductive infection.Then, remaining Agrobacterium is removed, and corpus callosum is transferred in the common substratum (containing 100 μ M Syringylethanones, 20mg/l xitix and 5mg/l Silver Nitrate), further cultivated 3 days at 28 ℃ then.The corpus callosum that infects is transferred to cultivation back substratum (contain 5mg/l 2,4-D, 1mg/l BA, 140mg/l FeNaEDTA, 70mg/l inositol, 25mM proline(Pro), 0.4mM Thioproline, 50mM K ₂SO ₄, 2g/l yeast extract, the MS substratum of 30g/l sucrose and 5g/l gelite) in, further cultivated for 1 week then.Then, said corpus callosum (is contained 0.5mg/l 2,4-D, 2mg/l BA, 140mg/lFeNaEDTA, 70mg/l inositol, 25mM proline(Pro), 0.4mM Thioproline, 50mMK at elementary selection substratum again ₂SO ₄, 2g/l yeast extract, 30g/l sucrose, the N6 minimum medium of 5g/l gelite and 25mg/l Totomycin) and middle the cultivation for 2 weeks.The corpus callosum that in elementary selection substratum, survive and regenerated bud are transferred to second and are selected to cultivate then 3 days, with the regeneration of transgenic plant in the substratum (first selects substratum+50mg/l Totomycin).The bud of aftergrowth is downcut, be transplanted to then in the 1/2MS solid medium that has replenished 50mg/l Totomycin and 30g/l sucrose, to induce the growth of root.Only select those to show the individuality of hygromycin resistance.After conforming, these individualities are transplanted in the flowerpot and are cultivated (Fig. 5 A).

<4-2>Through PCR checking transformant

Use genomic dna to carry out PCR and southern blotting technique, whether be inserted in the transgenic tall fescue plant that in the foregoing description 4-1, prepares with the checking target gene.The genomic dna that will from the contrast of the wild-type Festuca Arundinacea that during containing the selection substratum of Totomycin, shows the genomic dna that extracts in the Festuca Arundinacea plant of strong resistance and grow up to from normal rudiment, extract simultaneously, is as template.And in addition; In the nucleotide sequence of pSSA-H carrier, select by the forward primer of SEQ.ID.No11 representative and the reverse primer of representing by SEQ.ID.No 12; And be used for PCR, to verify the insertion of target gene through the said expression vector specificity nucleotide sequence district that increases.PCR carries out 30 circulations, and each circulates in 94 ℃/1 minute, 52 ℃/1 minute and 72 ℃/1 minute.As a result, amplification obtains the target fragment of about 0.5kb size, and this shows that the APX gene correctly inserts.In Fig. 5 B, Mw: molecular size mark, P: positive control dna, NT: unconverted Festuca Arundinacea plant, 1-3: hygromycin resistance Festuca Arundinacea plant.

Every kind of genomic dna with Restriction Enzyme Hind III digestion, carries out electrophoresis then then from hygromycin resistance Festuca Arundinacea plant and the wild-type Festuca Arundinacea plant extract that grows up to from normal rudiment on sepharose.DNA is transferred on the nylon membrane.The APX gene fragment (426bp) that the primer that use is represented by SEQ.ID.No 13 and No14 passes through pcr amplification is carried out southern blotting technique as probe.As a result, in having the Festuca Arundinacea plant of strong hygromycin resistance, observe a specific band.On the other hand, in control plant, do not detect band (Fig. 5 C).In Fig. 5 C, 2,3 and 4: the Festuca Arundinacea plant of conversion.

<Embodiment 5>The environmental resistance of SSA transgenic plant

<5-1>The oxidative stress resistance

<5-1-1>The oxidative stress resistance of potato plants blade

In order to study SSA transgenic Rhizoma Solani tuber osi (kind: oxidative stress resistance Atlantic), growth unconverted plant (NT plant) and SSA plant in the greenhouse.The blade (from the 5th or the 7th blade at top) of big plant is taked 10 blade wafers (diameter 8mm) from 7 weeks, it is swum in contain 0,3, in the 5ml 0.4M sorbitol solution of 5 and 10 μ M methyl viologens (MV).They were in the dark cultivated 12 hours, so that they absorb MV.After this, they were cultivated 48 hours at the light place again.Then, measure the ionic conductance of solution through using mhometer (Orion, Model 162), this causes the mensuration (Fig. 6) to blade injury.Fig. 6 A shows that with 0 μ MMV process result 6B shows that with 3 μ M MV process result 6C shows with 5 μ M MV process result and 6D and shows with 10 μ M MV process result.In the situation of not handling, the ionic conductance of NT and SSA plant leaf keeps being stabilized in 20% and continues 48 hours.In the situation that MV handles, the specific conductivity of solution that contains the SSA plant leaf is more much lower than the specific conductivity of the solution that contains the NT plant leaf.From handling back 12 hours, in the NT plant, begin to observe serious blade injury with 3,5 and 10 μ M MV.Especially, observe cell injury in processing after back 48 hours greater than 80%.Yet in processing back 36 hours, the cell injury in the SSA plant was merely about 40%.These results show that strong resistance that the SSA plant has is the twice of the resistance that had of NT plant.That is, wherein SOD and the APX transgenic Rhizoma Solani tuber osi plant of in chloroplast(id), expressing simultaneously has the resistance to the increase of the oxidative stress that is caused by MV.In Fig. 6, NT: unconverted plant and SSA: the plant of carrying the pSSA-K carrier.

<5-1-2>The oxidative stress resistance of potato plants

In order to study the resistance capacity of plant to oxidative stress; Through using spraying cabinet (ModelSB-6, DeVries Manufacturing, Hollandale; MN); NT plant, EV plant (carrying the plant of pCAMBIA2300 carrier) and SSA plant are handled with the MV solution (containing 0.1% polysorbas20) of 70ml different concns 0,150,200 and 250 μ M.Spraying MV solution after 5 days, the visible damage in the research plant leaf.As a result, when spraying 150 μ M MV, in the blade of NT and EV plant, observe part and wilt, but in the SSA plant, do not observe damage.Damage in NT and the EV plant leaf increases along with the increase of MV concentration.Use the concentration of 250 μ M, the blade injury 90% of plant, and just damage (Fig. 7) slightly of the blade of SSA plant.In Fig. 7, NT: non-transformant, EV: the plant and the SSA that carry the pCAMBIA2300 carrier: the potato plants that carries the pSSA-K carrier.

Detect visible damage, leaf dry weight and chlorophyll content, with the MV resistance of research SSA plant.When 150 μ M MV spray solutions on every strain NT plant and EV plant leaf the time, are observed the blade injury of 20-40% in every strain plant, but the damage in the SSA plant is less than 20% (Fig. 8 A).Simultaneously, the dry weight of the normal blade of the survival of NT and EV plant reduces along with the increase of MV concentration after MV handles.When with 250 μ M MV solution-treated, the dry weight of blade reduces by 50% (Fig. 8 B).On the contrary, in the situation of transgenic plant, almost the dry weight with untreated blade is identical for the dry weight of the blade of survival after handling, and is irrelevant with MV concentration.Result of study about chlorophyll content is similar with the result that dry weight detects.The chlorophyll content of the transgenic plant that untreated plant and MV handle is about 40mg/cm ²(Fig. 8 C).In Fig. 8 A-8C, NT: non-transformant, EV: the plant and the SSA that carry the pCAMBIA2300 carrier: the potato plants that carries the pSSA-K carrier.

<5-1-3>The oxidative stress resistance of sweet potato plant leaf

(kind: oxidative stress resistance Yulmi) is grown in non-transgenic plant (NT plant) and every kind 3 strain of SSA plant in the greenhouse in order to study SSA transgenic sweet potato.Take 10 blade wafers from the blade (top the 3rd or the 4th leaf) of plant in 8 weeks of growing, then through with embodiment 5-1-1 in used same procedure with 0,2.5, the processing of 5 and 10 μ M methyl viologens (MV).After MV handles, measure different concns MV and handle the damage (Fig. 9) in the blade wafer that causes.Fig. 9 A shows the damage that 0 μ M MV handles, and B shows the damage that 2.5 μ M MV handle, and C shows the damage that 5 μ M MV handle, and D shows the damage that 10 μ M MV handle.Compare with the specific conductivity of the solution of the blade wafer that contains the NT plant, the specific conductivity of solution that contains SSA plant leaf disk is very low.And after MV handles 12 hours, in the blade wafer of NT plant, observe cell injury greater than 50%, cell injury is lower than 30% in the blade wafer of SSA plant.Especially, compare with the NT plant, 5 μ M MV handle and in the blade wafer of SSA plant, cause 45% cell injury.In Fig. 9, NT: unconverted plant and SSA: the sweet potato plant of carrying the pSSA-K carrier.

<5-1-4>The oxidative stress resistance of sweet potato plant

In order to study the resistance capacity of eucalyptus plant to oxidative stress; To in the greenhouse, grow NT plant and the SSA plant in 4 weeks with 70ml different concns 0; The MV solution of 100,150 and 200 μ M (containing 0.1% polysorbas20) is handled, similar described in treatment process and the embodiment 5-1-2.MV handled back 5 days, observed the visible damage of plant leaf.When spraying 100-150 μ M MV, the blade of NT plant suffers from albinism and wilts.Yet, in the blade of SSA plant, only observe the part damage.Simultaneously, when spraying 200 μ M MV, the blade of NT plant has almost been wilted, and spends phenomenon (Figure 10) in vain but only observe in some part of the blade of SSA plant.In Figure 10, NT: non-transformant, EV: the sweet potato plant and the SSA that carry the pCAMBIA2300 carrier: the sweet potato plant of carrying the pSSA-K carrier.

The MV resistance of SSA plant is also studied through measuring photosynthetic efficiency, leaf dry weight and visible blade injury.After MV handles 2 days, take the 3rd leaf in plant top to detect photosynthetic efficiency.When spraying 100 μ M MV, with handle before compare, the photosynthetic efficiency in NT and the SSA plant drops to 0.7 (Figure 11 A) a little.When spraying 150 μ M and 200 μ M MV, photosynthetic efficiency is greater than 0.4, and this is the twice height of NT photosynthesis of plant efficient.After MV handles 5 days, also measured leaf dry weight.As a result, before processing, the about 400mg of dry weight of NT plant and SSA plant, but the dry weight of blade reduces along with the increase of MV concentration in the NT plant.Accurately, when handling with 200 μ M MV, compare with the plant of not handling with MV, dry weight reduces 90%.Simultaneously, the dry weight of SSA plant leaf reduces 60%, and this shows that resistance exceeds 3 times (Figure 11 B) than NT plant.And in addition, handle the back visible damage of observing plant in 5 days at MV.As a result, between the NT plant and SSA plant of handling, in visible damage, there are not a lot of differences with 100 μ M MV.Yet the blade 85% of NT plant is damaged by 200 μ M MV, and the blade 40% of SSA plant or still less be damaged this means with the resistance of NT plant and compare that the MV resistance doubles (Figure 11 C) in the SSA plant.In Figure 11 A-11C, NT: non-transformant, SSA4 and SSA5: the sweet potato plant of carrying the pSSA-K carrier.

<5-1-5>The oxidative stress resistance of the blade wafer of Festuca Arundinacea plant

In order to study the oxidative stress resistance of SSA transgenic tall fescue plant, growth unconverted plant (NT plant) and SSA plant in the greenhouse.The blade (from the 7th blade of 5-at top) of big plant is taked 10 blade wafers (diameter 8mm) from 7 weeks; Then it is swum in the 5ml 0.4M sorbitol solution of having replenished 5 μ M methyl viologens (MV); In the dark cultivated then 12 hours, so that said disk absorbs MV.After with dark processing, they were cultivated 48 hours at the light place again.Then, measure the ionic conductance of solution through using mhometer (Orion, Model 162), to measure blade injury (Figure 12 A).After MV handles, compare with the NT plant, the specific conductivity of solution that contains SSA plant leaf disk is very low.Handled back 12 hours from MV, in the serious damage of blade wafer appearance of NT plant, and from handling back 48 hours, cell injury is greater than 75% in the NT plant.Handled back 36 hours up to MV, the cell injury in the SSA plant just is 40%.These results show, compare with the NT plant, and the SSA plant has the 2 times higher resistance to MV inductive oxidative stress.To use the blade wafer of the Festuca Arundinacea plant of above-mentioned same procedure growth to use the 50mM hydrogen peroxide treatment, further cultivate then 48 hours.Through the ionic conductance of mhometer mensuration solution, with research blade injury (Figure 12 B).After with hydrogen peroxide treatment, to compare with the NT plant, the cell injury in the SSA plant is very low.More accurately, in processing back 12 hours, the cell injury in the NT plant was greater than 65%.On the contrary, up to handling back 48 hours, cell injury is less than 25% in the SSA plant.In a word, compare with the NT plant, the SSA plant has at least 2.6 times higher resistance to the oxidative stress that is caused by hydrogen peroxide.Confirm that from The above results the SSA plant has the higher antibody that is caused oxidative stress by NV and hydrogen peroxide that is directed against than NT plant.That is, SOD and the APX transgenic tall fescue plant of in chloroplast(id), expressing simultaneously has the resistance to the oxidative stress that is caused by MV and hydrogen peroxide of increase.In Figure 12 A-12B, NT: unconverted plant and SSA: the plant of carrying the pSSA-H carrier.

<5-2>The resistance of coercing to temperature

<5-2-1>The heat stress resistance of potato plants blade wafer

In order to study SSA transgenic Rhizoma Solani tuber osi plant (kind: heat stress Atlantic) (high temperature) resistance, growth NT plant (unconverted plant) and SSA plant in the greenhouse.The blade (from the 7th blade of 5-at top) of big plant is taked 10 blade wafers (diameter 8mm) from 7 weeks, and it is swum in the 5ml 0.4M sorbitol solution, then, cultivates 60 hours at 37 ℃.Control plant is cultivated under the same terms except 25 ℃ temperature.Measured the ionic conductance of solution in per 12 hours, with the research blade injury.When not giving high temperature stress (25 ℃ of treatment group), 60 hours (Figure 13 A) of the stable maintenance of the ionic conductance of NT plant and SSA plant.Yet when the high temperature stress that gives 37 ℃, the SSA plant significantly increases (Figure 13 B) to the pyritous stress resistance.After 12 hours, beginning detects stress resistance in the SSA plant, and is respectively 42% and 52% at 36 and 48 hours ionic conductance in pyroprocessing.And after handling 60 hours, resistance doubles.In Figure 13 A and Figure 13 B, NT: unconverted plant and SSA: the plant of carrying the pSSA-K carrier.

<5-2-2>The heat stress resistance of potato plants

(kind: Atlantic) to the resistance of coercing that is caused by high temperature, (kind: NT plant Atlantic), EV plant and SSA plant are exposed to 42 ℃ temperature 10 hours with the growth potato plants in 4 weeks in order to study transgenic Rhizoma Solani tuber osi.As a result, NT plant and EV plant wilt are but survive in SSA plant health ground (Figure 14 A).Compare before pyroprocessing and the photosynthetic efficiency after the pyroprocessing.As a result, in the NT plant,

pyroprocessing

10 and 20 hours reduce 15% and 30% with photosynthetic efficiency respectively, but in the SSA plant, even pyroprocessing 20 hours is also only with photosynthetic efficiency minimizing 6% (Figure 14 B).After the pyroprocessing, plant was recovered 3 hours at 25 ℃.As a result, in NT plant and EV plant, compare with the photosynthetic efficiency before handling, photosynthetic efficiency recovers 50%.Simultaneously, photosynthetic efficiency almost returns to initial photosynthetic efficiency in the SSA plant.In Figure 14 A and Figure 14 B, NT: non-transformant, EV: carry the plant of pCAMBIA2300 carrier, and SSA: the plant of carrying the pSSA-K carrier.

<5-2-3>The sweet potato plant is to the resistance of coercing that is caused by low temperature

In order to study transgenic sweet potato (kind: Yulmi) be directed against the resistance of coercing that causes by low temperature, the NT plant in 4 weeks of will in the greenhouse, growing and the temperature that the SSA plant is exposed to 4 ℃ 24 hours.As a result, the NT plant wilt, but the SSA plant keeps normal (Figure 15 A).After the subzero treatment 24 hours, plant was recovered 12 hours at 25 ℃.As a result, although the NT plant almost can not recover, remain wilting, the SSA plant almost returns to original state (Figure 15 B).Before subzero treatment, NT plant and SSA photosynthesis of plants efficient all are 0.8.After the subzero treatment 6 hours, their photosynthetic efficiency and those photosynthetic efficiencies that before processing, obtain are big-difference very not, but from that time, and photosynthetic efficiency time-dependency ground reduces.Therefore, after 24 hours, in the NT plant, photosynthetic efficiency reduces greater than 30%, and in the SSA plant, reduces greater than 20%.After the subzero treatment 24 hours, plant was recovered 12 hours at 25 ℃, and then the research photosynthetic efficiency.As a result, after recovery, NT photosynthesis of plants efficient still reduces to 60%.Simultaneously, SSA photosynthesis of plants efficient returns to the level before handling, although between individuality, there is difference (Figure 15 C) a little.In Figure 15 A-15C, NT: unconverted sweet potato plant, SSA4 and SSA5: the plant of carrying the pSSA-K carrier.

<5-3>The sulfurous gas resistance

In order to study the sulfurous gas (SO of SSA transgenic sweet potato plant ₂) resistance, (kind: NT plant Yulmi) and SSA plant 8 hours every days, continue 5 days with the sulfur dioxide treatment of 500ppb to the sweet potato plant in 4 weeks of will in the greenhouse, growing.As a result, compare with the SSA plant, the blade of NT plant is owing to sulfurous gas is wilted, and plant-growth also stops.Simultaneously, it is normal and healthy that the SSA plant keeps, and shows luxuriant growth (Figure 16).In Figure 16, NT: non-transformant, and SSA: the sweet potato plant of carrying the pSSA-K carrier.

The sulfurous gas resistance of SSA plant is studied through measuring photosynthetic efficiency.From second day of sulfur dioxide treatment, measure the photosynthetic efficiency of the 3rd blade in plant top.As a result, the photosynthetic efficiency of NT plant (SSA plant) from 0.80 (before handling) reduce to gradually the 5th day 0.47.On the contrary, in the SSA plant, photosynthetic efficiency chance behind the 5th day of handling does not reduce (still being 0.76), and this shows with the NT plant compares, SSA plant keep fit (Figure 17).After plant is with sulfur dioxide treatment, they were grown in the greenhouse 5 days, in process of growth, detect photosynthetic efficiency.As a result, NT photosynthesis of plants efficient is 0.32, and this shows that it has almost lost the photosynthesis function.Simultaneously, in the SSA plant, photosynthetic efficiency is 0.75, and this shows that it has almost recovered from sulfurous gas damage.In a word, the SSA plant shows the resistance of the increase of the sulfurous gas that is directed against one of most typical pollution substance.

Industrial applicability

As explaining hereinbefore, the recombinant expression vector transgenic plant transformed that produces the multiple stress tolerances plant with the present invention shows the strong resistance to the various environment-stress of oxidative stress inducibility weedicide, cold damage, high temperature, salt damage or generation oxyradical.Therefore, carrier of the present invention can be for the yield-power that increases farm crop or the mass-produced great contribution of useful component.

Sequence list text none

SEQ.ID.No 1 and No 2 are the used primer sequences of SWPA2 promotor PCR that in embodiment 1, carry out.

SEQ.ID.No 3 and No 4 are the used primer sequences of mSOD1 PCR that in embodiment 1, carry out.

SEQ.ID.No 5 and No 6 are the used primer sequences of SWPA2-1PCR that in embodiment 2-2, carry out.

SEQ.ID.No 7 and No 8 are the used primer sequences of SWPA2-2PCR that in embodiment 3-2, carry out.

SEQ.ID.No 9 and No 10 are the used primer sequences of APX PCR that in embodiment 3-2, carry out.

SEQ.ID.No 11 is the used forward primer sequences of mSOD1 PCR of in embodiment 4-2, carrying out.

SEQ.ID.No 12 is the used reverse primer sequences of CaMV 35S terminator PCR of in embodiment 4-2, carrying out.

SEQ.ID.No 13 and No 14 are described primer sequences of southern blotting technique of the APX that in embodiment 4-2, carries out.

SEQ.ID.No 15 is the nucleotide sequences that come from the oxidative stress inducibility SWPA2 promotor of sweet potato (Ipomoea batatas).

SEQ.ID.No 16 is cDNA sequences of coding cassava (Manihot seculenta) CuZnSOD.

The SEQ.ID.No 17 is cDNA sequences of coding pea (Pisum sativum) APX.

It should be appreciated by those skilled in the art, in aforementioned description disclosed notion and specific embodiments can be easily as improving or the basis of other embodiment of the identical purpose of design implementation the present invention.Those skilled in the art be also to be understood that the embodiment of such equivalence does not deviate from spirit and scope that the present invention proposes in the claim of enclosing.

The microbial preservation budapest treaty that is used for the international recognition of patented procedure

International form

Microbial preservation proves

According to detailed rules and regulations Rule 7.1 distribution

Cause: KWAK, Sang-Soo

Korea Institute of Bioengineering,

#52，Oun-dong，Yuseong-gu，Daejeon，305-333，

Korea S

International form

Microbial preservation proves

According to detailed rules and regulations Rule 7.1 distribution

Cause: KWAK, Sang-Soo

Korea Institute of Bioengineering

#52，Oun-dong，Yuseong-gu，Daejeon，305-333，

Korea S

Claims

1. recombinant expression vector that is used to produce the multiple stress tolerances plant, the multiple stress tolerances gene that it comprises oxidative stress inducibility peroxidase promoter, antibiotics resistance gene, tobacco etch virus leader sequence, the transit peptide sequence that is used for chloroplast targeted expression, CaMV 35S transcription terminator, T-DNA border sequence and is present in said oxidative stress inducibility peroxidase promoter downstream.

2. the expression vector that proposes of claim 1, wherein said oxidative stress inducibility peroxidase promoter is the nucleotide sequence of the sweet potato px negatively charged ion 2 of SEQ.ID.No 15 representatives.

3. the expression vector that proposes of claim 1, wherein said multiple stress tolerances gene are the ascorbate peroxidase enzyme genes that superoxide dismutase gene and the SEQ.ID.No 17 of SEQ.ID.No 16 representatives represents.

4. the expression vector that proposes of claim 1, wherein said carrier are included in to be deposited in the pSSA-K in the intestinal bacteria at Korea S typical case's culture collection center or to be included in preserving number KCTC 10537BP with preserving number KCTC10536BP and are deposited in the pSSA-H in the intestinal bacteria at Korea S typical case's culture collection center.

5. preparation method about the multiple stress tolerances transgenic plant, said method comprises the steps:

I) preparation is used for the expression vector of Plant Transformation, and said carrier comprises SWPA2 promotor, sod gene and APX gene, and said carrier is the carrier described in the claim 4;

Iii) cultivate above-mentioned transformant; With