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WO2006054815A1 - Vecteur d'expression recombine pour la production de vegetaux dotes de multiples tolerances au stress et procede de preparation de vegetaux dotes de multiples tolerances au stress au moyen dudit vecteur d'expression - Google Patents

Vecteur d'expression recombine pour la production de vegetaux dotes de multiples tolerances au stress et procede de preparation de vegetaux dotes de multiples tolerances au stress au moyen dudit vecteur d'expression Download PDF

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WO2006054815A1
WO2006054815A1 PCT/KR2005/000258 KR2005000258W WO2006054815A1 WO 2006054815 A1 WO2006054815 A1 WO 2006054815A1 KR 2005000258 W KR2005000258 W KR 2005000258W WO 2006054815 A1 WO2006054815 A1 WO 2006054815A1
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plant
expression vector
plants
pssa
vector
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PCT/KR2005/000258
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English (en)
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Sang-Soo Kwak
Suk-Yoon Kwon
Haeng-Soon Lee
Li Tang
Soon Lim
Byung-Hyun Lee
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Korea Research Institute Of Bioscience And Biotechnology
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Priority claimed from KR1020040093945A external-priority patent/KR100704751B1/ko
Application filed by Korea Research Institute Of Bioscience And Biotechnology filed Critical Korea Research Institute Of Bioscience And Biotechnology
Priority to US11/718,661 priority Critical patent/US7897835B2/en
Priority to CN2005800391729A priority patent/CN101061227B/zh
Publication of WO2006054815A1 publication Critical patent/WO2006054815A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

Definitions

  • the present invention relates to recombinant expression vectors for production of plants having multiple stress tolerance, which are prepared by attaching multiple stress tolerant genes to oxidative stress inducible promoter originated from sweetpotato to express the gene in chloroplasts, multiple stress tolerant plants transformed with the above vector, and a preparation method for the plants.
  • Superoxide dismutase is an enzyme that converts superoxide anion radical (O 2" ) into hydrogen peroxide (H 2 O 2 ) , and is divided into CuZnSOD, MnSOD and FeSOD according to metal cofactor included in the enzyme. These are located differently in cells, for example CuZnSOD is found in cytoplasm and chloroplasts, MnSOD is found in mitochondria and FeSOD exists in chloroplasts. SOD is an important environmental tolerant factor that eliminates oxygen free radicals generated in a living body by environmental stress, which can be useful for the production of medical supplies, food, cosmetics, etc.
  • Ascobate peroxidase is an enzyme that converts H 2 O 2 into water by using ascobate as an electron donor, and is largely found in plants and insects. This enzyme is known to exist in cytoplasm, stroma in chloroplasts and thylakoid membrane in plants (Free Rad. Biol. Med. 23:
  • chloroplasts of a plant oxygen content is relatively high and electron transport system is operating to utilize electronic energy produced from the decomposition of water with light energy, so this organ is very sensitive to various oxidative stresses.
  • the increase of anti-oxidative capability of chloroplast might be a great help to maintain productivity of a plant under environmental stresses.
  • CaMV 35S promoter which is constitutively expressed regardless of conditions, has been mostly used for the combination with a gene with resistance for specific stress to construct an expression vector. So, the resultant plant has resistance against only a specific stress. In order to overcome this problem, it is required to prepare an expression vector including a promoter being able to be expressed under any stress circumstances and a stress tolerant gene, and a transgenic plant transfected with the vector.
  • the present inventors prepared a novel expression vector for plant transformation by attaching multiple stress tolerant genes SOD (superoxide dismutase) and APX (ascorbate peroxidase) to oxidative stress inducible peroxidase promoter SWPA2 originated from sweetpotato so as to express the genes in chloroplasts of a plant. And then, the inventors regenerated transformed plants prepared from potato, sweetpotato and tall fescue by tissue culture. At last, the present inventors completed this invention by confirming that the transgenic plants of the invention have increased multiple stress tolerance.
  • SOD superoxide dismutase
  • APX ascorbate peroxidase
  • the present invention provides recombinant expression vectors 'pSSA-K' and 'pSSA-H' for the production of multiple stress tolerant plants containing oxidative stress inducible peroxidase promoter, tobacco etch virus (TEV) leader sequence, multiple stress tolerant genes, transit peptide sequence for chloroplast targeted expression, CaMV 35S transcription terminator, antibiotics resistant gene and T-DNA boarder sequence.
  • the present invention also provides a transgenic plant transformed with the above pSSA-K or pSSA-H expression vector.
  • the present invention also provides a preparation method for multiple stress tolerant transgenic plants comprising the following steps: i) Preparing expression vectors for plant transformation comprising SWPA2 promoter, SOD gene and APX gene; ii) Preparing a transformant by inserting the expression vector above into a plant or culture cells; iii) Culturing the transformant above; and iv) Preparing a transgenic plant by regeneration after tissue-culturing the transformant.
  • the present invention provides recombinant expression vectors pSSA-K and pSSA-H for the production of multiple stress tolerant plants containing oxidative stress inducible peroxidase promoter, tobacco etch virus
  • TMV TMV leader sequence
  • transit peptide sequence for chloroplast targeted expression TMV leader sequence
  • multiple stress tolerant genes TMV 35S transcription terminator, antibiotics resistant gene and T-DNA boarder sequence.
  • nucleotide sequence of SWPA2 sweetpotato peroxidase anionic 2 promoter, represented by SEQ. ID. No 11, is preferably used as oxidative stress inducible peroxidase promoter originated from sweetpotato
  • nucleotide sequence of SOD gene represented by SEQ. ID. No 12 and nucleotide sequence of APX gene represented by SEQ. ID. No 13 are preferably used as multiple stress tolerant genes.
  • the present inventors deposited pSSA-K and pSSA-H, expression vectors for the production of multiple stress tolerant plants, prepared by the inventors as the above at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB) , on November 7, 2003.
  • KCTC Korean Collection for Type Cultures
  • KRIBB Korean Research Institute of Bioscience and Biotechnology
  • the accession number of pSSA-K expression vector is KCTC 10536BP
  • the accession number of pSSA-H expression vector is KCTC 10537BP.
  • SWPA2 promoter is an oxidative stress inducible promoter isolated from sweetpotato (Ipomoea batatas) by the present inventors, which is very useful for the development of stress tolerant plants and plant cell lines for the production of other useful products (Korean Patent Publication No: 2001-51095; International Publication No: WO 01/31018 (applied on October 28, 2000); Kim, et al . , Plant MoI. Biol., 51: 831-838, 2003) .
  • SWPA2 is a gene whose expression is induced by oxidative stress.
  • the gene is specifically highly expressed in the late log phase in sweetpotato suspension culture cells and transformed tobacco suspension culture cells (International Publication No: WO 01/31018) .
  • the promoter showed at least 30-fold higher activity than CaMV 35S promoter in transient assay with GUS protein using tobacco protoplast.
  • the promoter is not expressed in leaves of a plant under normal conditions, but is expressed when they get oxidative stresses such as ozone, low temperature, wound, etc (Kim, et al . , Plant MoI. Biol., 51: 831-838, 2003) .
  • SWPA2 promoter of the present invention can be effectively used for the development of environmental stress tolerant plants and for the production of useful products using the transformed plant cells .
  • SWPA2 promoter of the present invention effectively induces the expression of a target gene by stress.
  • SWPA2 promoter of the invention includes factors recognizing outward stress generated by ABA (abscisic acid) , methyl jasmonate, wound, hypoxia, oxygen free radical, heat or nitrogen. Based on this characteristic of the promoter, SWPA2 promoter is useful for the construction of chimeric gene structure in which DNA sequence having a promoter activity is linked to a structural gene to work with the promoter sequence effectively.
  • Such chimeric gene structure enables the expression of a valuable factor under any environmental stress by the control of SWPA2 promoter owing to the linkage between a structural gene involved in the production of valuable factors and SWPA2 promoter, so that it can be effectively used for the preparation of transformants for the production of useful products.
  • a transformant having stress tolerance can be produced.
  • SOD gene used in this invention is separated from over 30 kinds of plants in general. And, in particular, CuZnSOD gene (mSODl) isolated first from cultured cells of cassava ⁇ Manihot esculenta) selected as SOD high- production cell line was used in the present invention (MoI. Gen. Genet. 262: 807-814, 1999) along with APX gene isolated from pea (Free Rad. Biol. Med. 23: 473-479, 1997) .
  • the present inventors constructed pSA vector by connecting SWPA2 promoter represented by SEQ. ID. No 15 to pRW20 vector, into which mSODl gene represented by SEQ. ID. No 16 was inserted to prepare pSS vector (see Fig.
  • SWPA2pro-TEV-TP-SOD-35S transcription terminator structure of pSS vector was inserted into an expression vector for plant transformation containing hygromycin resistant gene or kanamycin resistant gene, followed by the insertion of SWPA2pro-TEV-TP-APX-35S transcription terminator structure separated from pSA vector thereto.
  • pSSA-K and pSSA-H expression vectors for the production of multiple stress tolerant plants were constructed (see Fig.2) .
  • pSSA-K and pSSA-H expression vectors above include SWPA2 promoter, mSODl, APX gene, tobacco etch virus (TEV) leader sequence, transit peptide for chloroplast targeted expression and CaMV 35S transcription terminator in addition to hygromycin resistant gene or kanamycin resistant gene respectively, which makes the selection of multiple stress tolerant plants transformed with the vectors easy.
  • the present invention also provides a multiple stress tolerant plant transformed with pSSA-K or pSSA-H expression vector. All kind of plants are possibly used for the production of transgenic plants, but soybean, barley, corn, potato, sweetpotato or tall fescue is preferred and especially potato, sweetpotato or tall fescue is more preferred.
  • the present invention provides a multiple stress tolerant transgenic plants prepared by transforming with recombinant expression vector for the production of multiple stresses tolerant plants containing SWPA2 promoter, SOD gene and APX gene so as to express the SOD gene and APX gene massively in plants.
  • pSSA-K or pSSA-H expression vector constructed above was inserted into a plant, preferably in soybean, barley, corn, potato, sweetpotato or tall fescue and more preferably in potato, sweetpotato or tall fescue, to express SOD gene and APX gene.
  • the present inventors inserted the above expression vectors into young leaves or petiole sections of potato, sweetpotato or tall fescue by using Agrobacterium tumefaciens EHA105 or by means of particle bombardment, in order to prepare transformants .
  • the resultant transformants were tissue-cultured to induce regeneration into plants (see Fig. 3A, Fig. 4A and Fig. 5A) .
  • Genomic DNA was extracted from each regenerated transgenic potato, sweetpotato or tall fescue, from which SWPA2 promoter or APX gene was amplified by PCR to confirm the insertion of SWPA2 promoter or APX gene in plant genome (see Fig. 3B, Fig. 4B and Fig. 5B) .
  • the present inventors induced oxidative stress like applying methyl viologen (MV) or hydrogen peroxide in leaf discs of transformed potato, sweetpotato or tall fescue or plant themselves, and then investigated ionic conductivity (see Figs. 6, 7, 9, 10 and 11), visual damage (see Fig. 8A and Fig. HC), photosynthetic efficiency (see Fig. HA), dry weight of leaf (see Fig. OB and Fig. HB) and chlorophyll content (see Fig. 8C) .
  • MV methyl viologen
  • HC visual damage
  • photosynthetic efficiency see Fig. HA
  • dry weight of leaf see Fig. OB and Fig. HB
  • chlorophyll content see Fig. 8C
  • transgenic plants transformed with pSSA-K or pSSA-H expression vector of the present invention showed excellent tolerance against oxidative stress tolerance than non-transformed plants. It was also confirmed that the transgenic plant of the present invention could maintain normal conditions under other stresses like high temperature (see Fig. 13B ⁇ Fig. 14B), low temperature (see Fig. 15A and Fig. 15B) or SO 2 stress (see Fig. 16 and Fig. 17) .
  • the present invention also provides a transgenic plant transformed with the above pSSA-K or pSSA-H expression vector.
  • the present invention also provides a preparation method for multiple stress tolerant transgenic plants comprising the following steps: i) Preparing expression vectors for plant transformation comprising SWPA2 promoter, SOD gene and APX gene; ii) Preparing a transformant by inserting the expression vector above into a plant or culture cells; iii) Culturing the transformant above; and iv) Preparing a transgenic plant by regeneration after tissue-culturing the transformant.
  • pSSA-K or pSSA-H is preferred as an expression vector, and any plant can be used as the plant or culture cells above but soybean, barley, corn, potato, sweetpotato or tall fescue is preferred, and potato, sweetpotato or tall fescue is more preferred.
  • the transformant above can be prepared by the conventional plant transformation method (Horsch, et al. , Cold Spring Harb Symp Quant Biol., 50: 433-7, 1985;
  • pSSA-K or pSSA-H is used as an expression vector since transgenic plants transformed with the vector can be easily selected because the vector contains SWPA2 promoter, SOD gene, APX gene, transit peptide sequence for chloroplast targeted expression, tobacco etch virus (TEV) leader sequence, CaMV 35S transcription terminator, and hygromycin or kanamycin resistant gene.
  • Fig. 1 is a schematic diagram showing the producing procedure of a vector of the present invention expressing SOD (superoxide dismutase) and APX (ascorbate peroxidase) genes simultaneously,
  • Fig. 2 is a schematic diagram showing a recombinant expression vector for the production of multiple stress tolerant plant containing SOD and APX genes
  • Fig. 3A is a photograph showing the morphology of each organ of a regenerated plant after tissue culturing of a transgenic potato (Variety: Superior) having kanamycin resistance produced by using a recombinant expression vector (pSSA-K) for the production of multiple stress tolerant plant,
  • Fig. 3B is an electrophoresis photograph confirming the amplification of SWPA2 promoter in transgenic potato plant transformed with the expression vector of the present invention (pSSA-K vector) by PCR using primers represented by SEQ. ID. No 1 and No 2,
  • Fig. 3C is a Southern blot photograph confirming that SOD and APX genes are presented on genome of transgenic potato plant prepared by using the expression vector of the present invention (pSSA-K vector) ,
  • Fig. 4A is a photograph showing the morphology of each organ of a regenerated plant after tissue culturing of kanamycin resistant transgenic sweetpotato (Variety: Yulmi) produced by using the expression vector of the present invention (pSSA-K vector)
  • Fig. 4B is an electrophoresis photograph confirming the amplification of SWPA2 in transgenic sweetpotato plant transformed with the expression vector of the present invention (pSSA-K vector) by PCR using primers represented by SEQ. ID. No 1 and No 2,
  • Fig. 4C is a Southern blot photograph confirming that SOD and APX genes are presented- on genome of transgenic sweetpotato plant transformed with the expression vector of the present invention (pSSA-K vector) ,
  • Fig. 5A is a set of photographs showing a regenerated plant obtained from tissue culture of hygromycin resistant transgenic tall fescue prepared by using the expression vector of the present invention
  • Fig. 5B is a PCR photograph confirming that SOD and APX genes are presented on genome of transgenic tall fescue plant transformed with the expression vector of the present invention (pSSA-H vector) ,
  • Fig. 5C is a Southern blot photograph confirming that SOD and APX genes are presented on genome of transgenic tall fescue plant transformed with the expression vector of the present invention (pSSA-H vector) ,
  • Fig. 6 is a set of graphs showing the membrane damage in leaf discs of NT and SSA potato plants after treating them in methyl viologen (MV) solution with different concentrations (0, 3, 5 and 10 ⁇ M) , which was measured by investigating ionic conductivities in the solution,
  • MV methyl viologen
  • Fig. 7 is a set of photographs showing the MV resistance levels in potato plants after 5 days from spray with MV solution at different concentration of 0, 150, 200 and 250 ⁇ M,
  • Fig. 8 is a set of graphs showing the visual damage in leaves of a potato plant (A) , relative dry weight of survived leaves (B) , and chlorophyll content (C) after 5 days from spray with MV solution at different concentrations of 0, 150, 200 and 250 ⁇ M,
  • Fig. 9 is a set of graphs showing the membrane damage in discs of sweetpotato leaves of NT and SSA plants treated with different concentrations of MV solution (0, 2.5, 5 and 10 ⁇ M) for 72 hours, which was measured by investigating ionic conductivities in the solution,
  • Fig. 10 is a set of photographs showing the MV resistance levels in sweetpotato plants after 5 days from spray with MV solution at different concentration of 0, 100, 150 and 200 ⁇ M,
  • Fig. 11 is a set of graphs showing the photosynthetic efficiency (A) , relative dry weight of survived leaves (B) and visual damage in leaves (C) of sweetpotato plant after 5 days from spray with MV solution at different concentrations of 0, 100, 150 and 200 ⁇ M,
  • Fig. 12 is a set of graphs showing the membrane damage in leaf discs of tall fescue plant treated with 5 ⁇ M of MV solution (A) and 50 mM of H 2 O 2 solution (B) ,
  • Fig. 13 is a set of graphs showing the high- temperature resistance observed in leaf discs of a potato plant, precisely, ionic conductivity in membrane was investigated after the treatment at high temperature (25°C
  • FIG. 14 is a set of photographs and a graph showing the result of the investigation on high temperature resistance of a potato plant. Precisely, visual damage (A) resulted from heat-treatment at 42°C for 10 hours, and photosynthetic efficiency (B) after treatment at 42°C for 10 hours, 20 hours and after 3 hour-recovery at 25°C after the treatment of high temperature were investigated,
  • Fig. 15 is a set of photographs and a graph showing the result of the investigation on low temperature resistance of a sweetpotato plant. Precisely, visual plant damage after 24 hour-treatment at 4°C (A), a photograph of plants which were recovered for 12 hours at
  • Fig. 16 is a photograph showing the sulfur dioxide resistance of SSA sweetpotato plant resulted from the treatment with 500 ppb of sulfur dioxide for 8 hours a day for 5 days,
  • Fig. 17 is a graph showing the photosynthetic efficiency of NT and SSA sweetpotato plants which were obtained during the treatment with 500 ppb of sulfur dioxide for 5 days and after the adaptation to normal condition (0 ppb) for 5 days after the treatment with sulfur dioxide.
  • Example 1 Construction of pSSA-K and pSSA-H, expression vectors for multiple stress tolerant genes
  • the present inventors constructed a vector- by ligating cDNA (GenBank accession number AF170297, Lee, et al., MoI. Gen. Genet., 262: 807-814, 1999) coding CuZnSOD (CuZn superoxide dismutase, mSODl) of cassava represented by SEQ. ID. No 16 and cDNA coding APX (ascorbate peroxidase; Randy, et al., Free Rad. Biol. Med., 23: 473- 479, 1997) of pea represented by SEQ. ID.
  • the nucleotide sequence of the vector was analyzed to confirm whether or not the target sequence of SWPA2 promoter was amplified correctly.
  • the nucleotide sequence of the primer had Hind Et and Xho I restriction sites.
  • pRW20 vector (Allen, et al. , Free Rad. Biol. Med., 23: 473-479, 1997) constructed for the delivery of APX
  • PCR was performed using primers each represented by SEQ. ID. No 3 and No 4 at 94°C for 1 minute, at 57°C for 1 minute and at 72°C for 1 minute (this cycle was repeated 30 times) to amplify mSODl gene. And the amplified mSODl was cloned into pGEM-T Easy plasmid vector
  • nucleotide sequence analysis was performed to confirm whether or not the target mSODl gene was correctly amplified.
  • the nucleotide sequence of the primer had Sal
  • mSODl gene was isolated from pGEM-T Easy plasmid vector in which mSODl gene was cloned earlier by the treatment of Sal I and Sac
  • the isolated mSODl gene was inserted into the above pSA vector digested with the same restriction enzymes .
  • the resultant vector was named pSS vector (Fig. 1) .
  • pSSA-K and pSSA-H vectors for plant transformation, were constructed to transform SOD and APX simultaneously by using pCAMBIA1300 plasmid (Center for Application of Molecular Biology to International Agriculture, Australia) containing hygromycin resistant gene and pCAMBIA2300 plasmid (Center for Application of Molecular Biology to International Agriculture, Australia) containing kanamycin resistant gene.
  • pCAMBIA1300 plasmid Center for Application of Molecular Biology to International Agriculture, Australia
  • pCAMBIA2300 plasmid Center for Application of Molecular Biology to International Agriculture, Australia
  • the above pSS vector was digested with Hind JR to obtain about 2.0 kb sized DNA fragment, which was inserted into PCAMBIA1300 plasmid and pCAMBIA2300 plasmid, pre-digested with the same restriction enzymes.
  • pSA was also digested with Pst I to obtain 2.3 kb sized DNA fragment, which was inserted into the above plasmids, resulting in the construction of expression vectors having both SOD and APX genes, pSSA-K vector (the DNA fragment was inserted into pCAMBIA2300) and pSSA-H vector (the DNA fragment was inserted into pCAMBIA1300) (Fig. 2) .
  • the present inventors deposited pSSA-K and pSSA-H constructed by the inventors as the above at Korean Collection for Type Cultures (KCTC) of Korea Research Institute of Bioscience and Biotechnology (KRIBB), on November 7, 2003.
  • the accession number of pSSA-K expression vector is KCTC 10536BP and the accession number of pSSA-H expression vector is KCTC 10537BP.
  • SWPA2 pro oxidative stress inducible promoter
  • E35S pro enhanced CaMV 35S promoter
  • TEV tobacco etch virus (TEV) leader sequence
  • TP signal sequence of pea CuZnSOD (Cu 1 Zn superoxide dismutase)
  • 35S 3' CaMV 35S transcription terminator
  • Amp antibiotics (ampicillin) resistant gene
  • H Hind III
  • P Pst I
  • Xh Xho I
  • R EcoR 1
  • N Nco 1
  • S Sal 1
  • B Bam ⁇ 1
  • X Xba 1
  • Sa Sac 1.
  • a transgenic potato plant was prepared by inserting the expression vector for plant transformation constructed above (pSSA-K or pSSA-H) into Agrobacterlum tumefaclens EHA105 (Hood, et al. , Trans. Res., 2: 208-218, 1993) according to An' s method (An, Meth. Enzymol., 153: 292-305, 1987) .
  • Plasmid Maxi kit provided by Qiagen
  • the present inventors prepared a transgenic potato plant by co-culturing leaf discs of a potato plant and Agrobacterium tumefaciens EHA105 containing pSSA-K vector constructed in the above example 1.
  • the potato plant (Solanum tuberosum L.) used for the transformation herein was prepared by culturing Superior, the most world-widely cultivated variety, and Atlantic, a variety for processing, in an incubator.
  • the potato plant was cultured in MS medium (Murashige and Skoog, Physiol.
  • leaf and petiole discs were placed on the selection medium (MS medium containing 2 mg/l of zeatin, 0.01 mg/l of NAA (naphthaleneacetic acid), 0.1 mg/l of GA3 (gibberellin) , 300 mg/l of claforan and 100 mg/l of kanamycin) .
  • the discs were transferred to a fresh new medium every three weeks, followed by sub-culture. The generation of kanamycin resistant shoot and callus was observed after 3 - 4 weeks from the beginning of culture.
  • the shoot was transferred to a root inducing medium (MS basic medium containing 300 mg/l of claforan and 100 mg/l of kanamycin) to induce roots. Roots were well induced from the shoot, and the small plant with roots was taken out of the culture container to be exposed outside for 4 - 5 days (acclimated in incubating room) , then transferred to horticultural bed soil flowerpot, followed by further culture in an incubator (Fig. 3A) . As a result, microtuber was formed in a potato plant grown in the incubator (Fig. 3A) . There was no significant difference in morphology between a transgenic potato plant harboring a foreign gene and a non-transformed one. In the meantime, when petioles were used as transformation material, more shoots were formed than when leaf discs were used for the transformation. Though, most of them were not transformed, confirmed by PCR.
  • a root inducing medium MS basic medium containing 300 mg/l of claforan and 100 mg/l of kanamycin
  • genomic DNA was extracted from leaves of each Atlantic and Superior plants that were growing in an incubator by using Dneasy Plant Maxi kit (QIAGEN Co.) . 30 ⁇ g of genomic DNA was digested with restriction enzyme EcoRI, followed by electrophoresis on agarose gel. The genomic DNA on the gel was transferred to Zeta Probe membrane (Bio-Rad Co.), followed by hybridization of the transferred DNA with 0.5 kb DNA fragment of SWPA2 promoter sequence labeled with 32 P as a probe.
  • Fig. 3C Upon completion of hybridization, membrane was washed and then exposed on X- ray film to detect a band. As a result, while no band was observed in a non-transgenic control plant, more than 3 bands were observed in transformed Atlantic and Superior plants, meaning SWPA2 promoter was stably inserted in the genome of the potato (Fig. 3C) .
  • NT non- transformed potato plant
  • Al and A2 transformed Atlantic plants
  • Sl and S2 transformed Superior plants.
  • the present inventors transformed sweetpotato embryogenic callus by particle bombardment using pSSA-K vector constructed in the above example 1.
  • embryogenic callus of sweetpotato ⁇ Ipomoea batatas Lam.; Variety: Yulmi) being cultivated in Korea was used as transformation material in this invention. More precisely, embryogenic callus of sweetpotato was induced and maintained to establish sweetpotato plant regeneration system (Kwon, et al . , Korean J. Plant Biotechnol. , 29: 189-192, 2002) .
  • the embryogenic callus was cut by cell clusters into 1-2 mm in diameter.
  • the cell clusters were placed within a central circle in 2 cm in diameter on MS solid medium supplemented with 1 mg/l of 2, 4-dichlorophenoxy acetic acid (2,4-D), which was cultured for one day, followed by particle bombardment (Fig. 4A) .
  • plasmid DNA of pSSA-K vector for plant transformation constructed in the example 1 was prepared.
  • gold particles (1 ⁇ m in diameter) were coated with the plasmid DNA, followed by particle bombarding using PDS-1000/He particle delivery system (BioRad Co.) under 1,100 psi pressure with 9 cm distance.
  • kanamycin resistant embryogenic callus was selected from MS selection medium supplemented with 1 mg/l of 2,4-D and 100 mg/l of kanamycin. The selection proceeded for 5 - 6 months, at 3 weeks interval, during sub-culture.
  • the kanamycin resistant embryogenic callus was transferred to MS basal medium supplemented with only 100 mg/l of kanamycin without 2,4-D.
  • the embryogenic callus was converted to somatic embryo in incubator under 40 ⁇ mol • m- 2 • sec -1 cool-white fluorescent condition at 25°C, leading to regeneration to a plant (Fig. 4A) .
  • regenerated plant was selected first from kanamycin containing medium. PCR was performed with the plant using specific primers for SWPA2 promoter represented by SEQ. ID. No 7 and No 8 or specific primers for APX gene represented by SEQ. ID. No 9 and No 10, at
  • Southern blotting was performed with randomly selected sweetpotato plants confirmed by PCR. Particularly, genomic DNA was extracted from leaves of sweetpotato plant that was being grown in an incubator by using Dneasy Plant Maxi kit (QIAGEN Co.) . 30 ⁇ g of the obtained DNA was digested with restriction enzyme EcoR I, followed by electrophoresis on agarose gel. The genomic DNA on the gel was transferred to Zeta probe membrane
  • the present inventors co-cultured Agrobacterium tumefaciens EHA105 containing pSSA-H vector (plasmid DNA) constructed in the above example 1 and tall fescue section to induce transformation of the same.
  • pSSA-H vector Plasmid DNA
  • Kenturky-31 mainly cultured as animal feed, was used for the transformation of tall fescue ⁇ Festuca arundinacea Schreb.
  • a callus inducing medium [MS medium containing 9 mg/l of 2,4-D, 0.1 mg/l of BA (benzyl adenine) , 30 g/l of sucrose, 5 g/l of gelite] for 4 weeks to induce callus .
  • Agrobacteria culture solution which was cultured in YEP liquid medium (10 g/l of bacto- peptone, 10 g/l of yeast extract and 5 g/l of NaCl) supplemented with 50 mg/l of kanamycin at 28°C for 2 days, was centrifuged to obtain bacteria cells. Then, the solution was suspended in callus inducing liquid medium supplemented with 100 ⁇ M acetosyringone, 20 mg/l of ascorbic acid and 5 mg/l of silver nitrate (AgNO 3 ) until OD 600 was reached to 1. The callus was dipped in Agrobacteria suspension for 30 minutes under vacuum to induce infection.
  • callus inducing medium containing 100 ⁇ M acetosyringone, 20 mg/l of ascorbic acid and 5 mg/l of silver nitrate
  • further culture at 28°C for 3 days.
  • the infected callus was transferred to after- culture medium (MS medium containing 5 mg/l of 2,4-D, 1 mg/l of BA, 140 mg/l of FeNaEDTA, 70 mg/l of myo-inositol, 25 mM proline, 0.4 mM thioproline, 50 mM K 2 SO 4 , 2 g/l of yeast extract, 30 g/l of sucrose and 5 g/l of gelite) , followed by further culture for 1 week.
  • MS medium containing 5 mg/l of 2,4-D, 1 mg/l of BA, 140 mg/l of FeNaEDTA, 70 mg/l of myo-inositol, 25 mM proline, 0.4 mM thioproline, 50 mM K 2 SO 4 , 2 g/l of yeast extract, 30 g/l of sucrose and 5 g/l of gelite
  • the callus was cultured again in the primary selection medium (N ⁇ basic medium containing 0.5 mg/l of 2,4-D, 2 mg/l of BA, 140 mg/l of FeNaEDTA, 70 mg/l of myo-inositol, 25 mM proline, 0.4 mM thioproline, 50 mM K 2 SO 4 , 2 g/l of yeast extract, 30 g/l of sucrose, 5 g/l of gelite and 25 mg/l of hygromycin) for 2 weeks.
  • the survived callus in the primary selection medium and regenerated shoots were transferred to the second selection medium (first selection medium + 50 mg/l of hygromycin) , followed by culture for 3 weeks to regenerate the transgenic plant.
  • the shoots of the regenerated plant were cut off and then explanted in 1/2 MS solid medium supplemented with 50 mg/l of hygromycin and 30 g/l of sucrose to induce the development of roots. Only those individuals showing hygromycin resistance were selected. After acclimation, those individuals were transplanted into a flowerpot and cultivated (Fig. 5A) .
  • genomic DNA extracted from a tall fescue plant showing a strong resistance in a selection medium containing hygromycin and from a control that is wild type tall fescue grown up from normal germination were used as templates.
  • a forward primer represented by SEQ. ID. No 11 and a reverse primer represented by SEQ. ID. No 12 were selected among nucleotide sequences of pSSA-H vector and used for PCR to confirm the insertion of a target gene by amplifying a specific nucleotide sequence region of the expression vector.
  • PCR was performed with 30 cycles of 94°C/1 minute, 52°C/1 minute, and 72°C/1 minute. As a result, about 0.5 kb sized target fragment was amplified, suggesting that APX gene was correctly inserted.
  • Mw molecular size marker
  • P positive control DNA
  • NT non- transformed tall fescue plant
  • 1-3 hygromycin resistant tall fescue plants.
  • Each genomic DNA was extracted from a hygromycin resistant tall fescue plant and a wild type tall fescue plant that was grown up from normal germination, and then digested with restriction enzyme Hind III, followed by electrophoresis on agarose gel. The DNA was transferred to nylon membrane.
  • a non-transformed plant (NT plant) and SSA plant were grown in a greenhouse.
  • 10 leaf discs (8 mm in diameter) were taken from a leaf of 7 week old plant (the 5 th or the 7 th leaf from the top), which were floated on 5 ml of 0.4 M sorbitol solution containing 0, 3, 5 and 10 ⁇ M of methyl viologen (MV) . They were cultured for 12 hours under darkness to let them absorb MV. After that, they were incubated again for 48 hours under the light. Then, ionic conductivity of the solution was measured by using conductivity meter (Orion, Model 162), leading to the measurement of leaf damage (Fig.
  • Fig. 6A shows the result of the treatment with 0 ⁇ M of MV
  • 6B shows the result of the treatment with 3 ⁇ M of MV
  • 6C shows the result of the treatment with 5 ⁇ M of MV
  • 6D shows the result of the treatment with 10 ⁇ M of MV.
  • ionic conductivities of NT and SSA plant leaves were maintained steady as 20% for 48 hours.
  • electrical conductivity of the solution containing SSA plant leaf discs was much lower than that of the solution containing NT plant leaf discs.
  • Serious leaf damage began to be observed in NT plants from the 12 hours after treatment of 3, 5, and 10 ⁇ M of MV. In particular, over 80% cell damage was observed after 48 hours after treatment.
  • SSA plant has twice as strong resistance as NT plant does. That is, a transgenic potato plant in which SOD and APX are expressed simultaneously in chloroplasts has increased resistance against oxidative stress caused by MV.
  • NT non-transformed plant
  • SSA plant harboring pSSA-K vector.
  • NT plant, EV plant (a plant harboring pCAMBIA2300 vector) and SSA plant were treated with 70 ml of MV solution (containing 0.1% tween 20) at different concentrations of 0, 150, 200 and 250 ⁇ M by using a spray booth (Model SB-6, DeVries Manufacturing, Hollandale, MN) .
  • MV solution containing 0.1% tween 20
  • 5 days after spraying MV solution visual damage in plant leaves was investigated.
  • 150 ⁇ M of MV solution was sprayed, partial wilting was observed in leaves of NT and EV plants, but no damage was observed in SSA plant.
  • the damage in NT and EV plant leaves was increased with the increase of MV concentration.
  • the leaves of the plants were damaged by
  • NT non-transformant
  • EV plant harboring pCAMBIA2300 vector
  • SSA potato plant harboring pSSA-K vector.
  • Chlorophyll contents of non-treated plant and MV treated transgenic plant were about 40 mg/cm 2 (Fig. 8C) .
  • NT non-transformant
  • EV plant harboring pCAMBIA2300 vector
  • SSA potato plant harboring pSSA-K vector.
  • Fig. 9A shows the damage by the treatment of 0 ⁇ M of MV
  • B shows the damage by the treatment of 2.5 ⁇ M of MV
  • C shows the damage by the treatment of 5 ⁇ M of MV
  • D shows the damage by the treatment of 10 ⁇ M of MV.
  • Oxidative stress resistance of a sweetpotato plant In order to investigate resistant capacity against oxidative stress of a sweetpotato plant, NT plant and SSA plant that were grown for 4 weeks in a greenhouse were treated with 70 ml of MV solution (containing 0.1% tween 20) at different concentrations of 0, 100, 150 and 200 ⁇ M in the analogy to the procedure as described in example 5- 1-2. 5 days after MV treatment, visual damage in leaves of a plant was observed. When 100-150 ⁇ M of MV was sprayed, leaves of NT plant were wiited with albinism. However, only partial damage was observed in leaves of SSA plant.
  • NT non-transformant
  • EV a sweetpotato plant harboring pCAMBIA2300 vector
  • SSA a sweetpotato plant harboring pSSA-K vector.
  • MV resistance of SSA plant was also investigated by measuring photosynthetic efficiency, dry weight of leaves and visual leaf damage. The third leaf from the top of a plant was taken to measure photosynthetic efficiency 2 days after MV treatment. When 100 ⁇ M of MV was sprayed, photosynthetic efficiencies in NT and SSA plants were a little decreased to 0.7, comparing to before the treatment
  • Fig. HA When 150 and 200 ⁇ M of MV was sprayed, photosynthetic efficiency was over 0.4, which was twice as high as that of NT plant. Dry weight of leaves was also measured 5 days after MV treatment. As a result, dry weights of NT plant and SSA plant were about 400 mg before MV treatment, but dry weight of leaves in NT plant was decreased with the increase of MV concentration. Precisely, when 200 ⁇ M of MV was treated, dry weight was 90% reduced, comparing to MV-non treating plant. In the meantime, dry weight of SSA plant leaves was 60% reduced, showing three-fold higher resistance than NT plant (Fig. HB) . And also, visual damage in a plant was observed 5 days after MV treatment.
  • NT plant As a result, there was not much difference in visual damage between NT plant and SSA plant treated with 100 ⁇ M of MV. However, leaves of NT plant were 85% damaged by 200 ⁇ M of MV and leaves of SSA plant were 40% or less damaged, meaning that MV resistance in SSA plant became doubled, comparing to that of NT plant (Fig. HC) .
  • Figs. HA - HC NT: non-transformant
  • SSA4 and SSA5 sweetpotato plants harboring pSSA-K vector.
  • NT plant and SSA plant were grown in a greenhouse.
  • 10 leaf discs (8 mm in diameter) were taken from a leaf of a plant at 7 weeks (the 5 th - the 7 th leaf from the top) , and then floated on 5 ml of 0.4 M sorbitol solution supplemented with 5 ⁇ M of methyl viologen (MV), followed by culture for 12 hours under darkness to let the discs absorb MV. After the treatment with darkness, they were cultured again for 48 hours under the light. Then, ionic conductivity of the solution was measured by using electrical conductivity meter (Orion, Model 162) to measure leaf damage (Fig. 12A) .
  • Orion electrical conductivity meter
  • SSA plant had at least 2.6-fold higher resistance against oxidative stress caused by hydrogen peroxide than NT plant. From the above result was confirmed that SSA plant had higher resistance against oxidative stresses caused by MV and hydrogen peroxide than NT plant. That is, transgenic tall fescue plants in which SOD and APX were expressed simultaneously in chloroplast had increased resistance against oxidative stress caused by MV and hydrogen peroxide.
  • NT non- transformed plant
  • SSA a plant harboring pSSA-H vector.
  • NT plant non-transformed plant
  • SSA plant a SSA plant
  • 10 leaf discs 8 mm in diameter
  • 10 leaf discs were taken from a leaf of 7 week old plant (the 5 th - the 7 th leaf from the top) and floated on 5 mi of 0.4 M sorbitol solution, followed by incubate at 37°C for 60 hours.
  • a control plant was incubated under the same conditions except 25°C temperature. Ionic conductivity of the solution was measured every 12 hours to investigate leaf damage. Ionic conductivities of NT plant and SSA plant were steadily maintained for 60 hours (Fig.
  • NT non-transformed plant
  • SSA plant harboring pSSA-K vector.
  • NT non-transformant
  • EV plant harboring pCAMBIA2300 vector
  • SSA plant harboring pSSA-K vector.
  • NT plant and SSA plant grown for 4 weeks in a greenhouse were exposed on the temperature of 4°C for 24 hours.
  • NT plant was wilted, but SSA plant was maintained as normal (Fig. 15A) .
  • SSA plant was maintained as normal (Fig. 15A) .
  • Fig. 15B Photosynthetic efficiencies of NT plant and SSA plant were both 0.8 before the low-temperature treatment.
  • NT plant and SSA plant of sweetpotato (Variety: Yulmi) grown for 4 weeks in a green house were treated with 500 ppb of sulfur dioxide, 8 hours a day, for 5 days.
  • leaves of NT plant were wilted by sulfur dioxide and the growth of the plant was also retarded, comparing to a SSA plant.
  • SSA plant stayed normal and healthy, showing vigorous growth (Fig. 16) .
  • NT non- transformant
  • SSA sweetpotato plant harboring pSSA-K vector.
  • Sulfur dioxide resistance of SSA plant was investigated by measuring photosynthetic efficiency.
  • Photosynthetic efficiency in the third leaf from the top of a plant was measured from the next day of sulfur dioxide treatment.
  • photosynthetic efficiency of SSA plant was gradually decreased from 0.80 (before treatment) to 0.47 on the fifth day.
  • photosynthetic efficiency in SSA plant was hardly decreased after the fifth day from the treatment (still 0.76), suggesting that SSA plant was maintained as healthy, comparing to NT plant (Fig. 17) .
  • photosynthetic efficiency of NT plant was 0.32, suggesting that it almost lost photosynthetic function.
  • photosynthetic efficiency in SSA plant was 0.75, indicating that it was almost recovered from damage by sulfur dioxide.
  • SSA plant showed increased resistance against sulfur dioxide, one of the most representative pollutants.
  • a transgenic plant transformed with a recombinant expression vector of the present invention for the production of a multiple stress resistant plant shows a strong resistance against oxidative stress inducible herbicides, cold injury, high temperature, salt damage, or various environmental stresses generating oxygen free radicals. Therefore, the vector of the present invention can be a great contribution to the increase of productivity of agricultural crops or mass-production of useful components.
  • the SEQ. ID. No 1 and No 2 are primer sequences used for the PCR of SWPA2 promoter performed in Example 1.
  • the SEQ. ID. No 3 and No 4 are primer sequences used for the PCR of mSODl performed in Example 1.
  • the SEQ. ID. No 5 and No 6 are primer sequences used for the PCR of SWPA2-1 performed in Example 2-2.
  • the SEQ. ID. No 7 and No 8 are primer sequences used for the PCR of SWPA2-2 performed in Example 3-2.
  • the SEQ. ID. No 9 and No 10 are primer sequences used for the PCR of APX performed in Example 3-2.
  • the SEQ. ID. No 11 is a forward primer sequence used for the PCR of mSODl performed in Example 4-2.
  • the SEQ. ID. No 12 is a reverse primer sequence used for the PCR of CaMV 35S terminator performed in Example 4- 2.
  • the SEQ. ID. No 13 and No 14 are primer sequences used for the Southern blot of APX performed in Example 4-2.
  • the SEQ. ID. No 15 is a nucleotide sequence of oxidative stress inducible SWPA2 promoter originated from Ipomoea batatas.
  • the SEQ. ID. No 16 is a cDNA sequence coding CuZnSOD of Manihot seculenta.
  • the SEQ. ID. No 17 is a cDNA sequence coding APX of Pisum sativum.

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Abstract

La présente invention se rapporte à un vecteur d'expression recombiné pour la production de végétaux dotés de multiples tolérances au stress obtenu en fixant des gènes dotés de multiples tolérances au stress à des promoteurs inductibles de stress oxydant; elle se rapporte également à des végétaux dotés de multiples tolérances au stress transformés par ledit vecteur d'expression et à un procédé de préparation des végétaux, ou, plus précisément, à un vecteur d'expression recombinant pour la production de végétaux dotés de multiples tolérances au stress obtenu en combinant un promoteur de peroxydase inductible de stress oxydant (SWPA2) extrait de la patate douce à des gènes dotés de multiples tolérances au stress (SOD (superoxyde dismutase) et APX (ascorbate peroxydase)) afin d'exprimer les gènes dotés de multiples tolérances au stress dans les chloroplastes; enfin l'invention se rapporte à des végétaux dotés de multiples tolérances au stress transformés par ledit vecteur d'expression et à une méthode de préparation desdits végétaux. Le vecteur d'expression recombinant objet de la présente invention est très utile pour la production de végétaux transformés dotés d'une très forte résistance à de multiples agressions causées par des herbicides inductibles de stress oxydant, les lésions dues au froid, les températures élevées, les dommages dus au sel ou diverses pollutions environnementales générant de l'oxygène actif, de sorte qu'il peut contribuer dans une large mesure à accroître la productivité agricole ou la production en masse de composants utiles.
PCT/KR2005/000258 2003-11-07 2005-01-28 Vecteur d'expression recombine pour la production de vegetaux dotes de multiples tolerances au stress et procede de preparation de vegetaux dotes de multiples tolerances au stress au moyen dudit vecteur d'expression WO2006054815A1 (fr)

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