WO2008069496A1 - Stress resistant plant introduced by stress - induced promoter and the gene encoding zeaxanthin epoxidase - Google Patents
Stress resistant plant introduced by stress - induced promoter and the gene encoding zeaxanthin epoxidase Download PDFInfo
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- WO2008069496A1 WO2008069496A1 PCT/KR2007/006120 KR2007006120W WO2008069496A1 WO 2008069496 A1 WO2008069496 A1 WO 2008069496A1 KR 2007006120 W KR2007006120 W KR 2007006120W WO 2008069496 A1 WO2008069496 A1 WO 2008069496A1
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- 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
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- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
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- C12Y114/15—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with reduced iron-sulfur protein as one donor, and incorporation of one atom of oxygen (1.14.15)
Definitions
- the present invention relates to a stress-resistant plant in which a stress-induced promoter and a zeaxanthin epoxidase (ZEP) gene are introduced. More particularly, the present invention relates to plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
- ZFP zeaxanthin epoxidase
- ABA abscisic acid
- ABA abscisic acid
- the amount of the plant hormone abscisic acid in cells is increased, and the expression of various osmotic stress- induced genes induced by abscisic acid is increased.
- abscisic acid induces stomatal closure to minimize the loss of water through transpiration (Hasegawa, P.M. et al, Annu. Rev. Plant Physiol. Plant MoI.
- abscisic acid plays an important role in embryo maturation and initiation of seed dormancy during seed development (Nambara, E. et al, Trends Plant Sd., 5:213, 2003).
- the biosynthetic pathways of abscisic acid playing an important role in plants as described above include two pathways, a direct pathway via C 15 farnesyl pyrophosphate and an indirect pathway via C 40 carotenoid (Taylor, I. B. et al, J. Exp. BoL, 51 : 1563, 2000; Schwartz, S.H. et al, Plant Physiol, 131 : 1591 , 2003).
- the main pathway of the biosynthetic pathways of abscisic acid was found to be the indirect pathway (Seo, M. et al, Trends Plant ScL 7: 41 , 2002).
- the first stage of the indirect pathway for the synthesis of abscisic acid is the epoxidation of zeaxanthin and antheraxanthin by zeaxanthin epoxidase (ZEP) and occurs in plastids (Xiong, L. et al., Plant Physiol, 133:29, 2003).
- Violaxanthin synthesized through the above modification is converted to 9-cis epoxicarotenoid, which is then converted to C 15 intermediate xanthoxin through oxidative cleavage by 9-cis epoxicarotenoid dioxygenase (NCED), and the xanthoxin moves to the cytoplasm, and then converted to abscisic acid.
- the xanthophyll cycle refers to the stoichiometric conversion of xanthophylls pigments, associated with the structural change among violaxanthin, antheraxanthin and zeaxanthin.
- Violaxanthin de-epoxidase has the activity to convert violaxanthin to antheraxanthin and zeaxanthin, and the reverse epoxidation is activated by ZEP.
- This xanthophyll cycle is important for the harvest of light, protects the photosynthetic system of plants from damage caused by excessive light and is involved in a process of converting excessive light to heat (Foyer, CH. et al, Physiol Plant., 92:696, 1994).
- ZEP is an enzyme important in both a stress reaction mediated by abscisic acid and a mechanism of protecting the photosynthetic system from excessive light.
- AtZEP which is the ZEP DNA of Arabidopsis thaliana
- the present inventors made many efforts to identify the biological function and environmental stress-associated in vivo function of ZEP and, as a result, found that ZisP-overexpressed plants have resistance to abiotic stress (Park, B. K., master's degree thesis, Pusan National University, 2006).
- the Z ⁇ P-overexpressed plants have problems in that they do not sufficiently grow, because the number of the seeds is rapidly reduced, unlike wild-type plants.
- the present inventors have made many efforts to develop plants, which have resistance to abiotic stress such as environmental stress and, at the same time, have good growth characteristics.
- a stress-induced promoter and a base sequence encoding ZEP are amplified in plants, it is possible to construct plants, which have resistance to abiotic stress and, at the same time, have growth characteristics almost similar to those of wild-type plants, thereby completing the present invention.
- the present invention provides plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
- FIG. 1 shows overexpression binary vector pFGL571 used for the overexpression of AtZEP.
- FIG. 2 shows a method for constructing an Arabidopsis thaliana plant in which AtZEP is overexpressed.
- FIG. 3 A shows the results of RT-PCR conducted to measure the expression level of AtZEP
- FIG. 3B is a graphic diagram showing the results of HPLC measurement for the content of zeaxanthin in a plant
- FIG. 4 is a graphic diagram showing the results of HPLC measurement for the content of zeaxanthin in a plant transformed with AtZEP, when the plant was treated with light stress at various light intensities (WT: wild type; and ZEP: plant transformed with AtZEP).
- FIG. 5 is a photograph showing that an ⁇ tZ£7 > -overexpressed transformant has resistance to NaCl.
- FIG. 6 is a photograph showing that an ⁇ tZEP-overexpressed transformant has resistance to LiCl.
- FIG. 7 is a photograph showing that an ⁇ tZ ⁇ P-overexpressed transformant has no resistance to KCl.
- FIG. 8 is a photograph showing that an ⁇ tZEP-overexpressed transformant has resistance to mannitol.
- FIG. 9 is a photograph showing an ⁇ tZi ⁇ '/'-overexpressed transformant has resistance to drought stress.
- FIG. 10 is a graphic diagram showing that, in a transgenic plant in which AtZEP is expressed in a stress-induced manner, a negative effect on seed development in the continuous expression of AtZEP is inhibited.
- FIG. 1 1 is an RT-PCR photograph showing that the expression of stress-induced genes RD29A and Rabl8 in an y4/Z£'/ J -overexpressed transformant is increased under normal conditions and salt stress conditions.
- FIG. 12 shows a process for constructing an Arabidopsis thaliana plant in which AtZEP is overexpressed in a stress-induced manner.
- FIG. 13 is a RT-PCR photograph showing that the expression of AtZEP is increased due to salt stress in an Arabidopsis thaliana plant in which AtZEP is overexpressed in a stress-induced manner.
- ⁇ /ZisP-overexpressed transformants show resistance to salts, such as NaCl and LiCl, and drought stress.
- salts such as NaCl and LiCl
- drought stress the ⁇ tZ£P-overexpressed transformants have a problem in that they do not sufficiently grow, because the number of the seeds is rapidly reduced, unlike wild-type plants.
- this negative effect occurring in the continuous expression of AtZEP was solved through a stress-induced promoter.
- the present inventors have found that a plant transformed with an expression vector, containing a stress-induced promoter and AtZEP, has not only resistance to stress, but also growth characteristics almost similar to those of wild- type plants.
- the present invention relates to plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
- the stress-induced promoter is preferably an RD29A promoter, a Rabl8 promoter, a COR15A promoter, an RD22 promoter, a LOS5 promoter, an SDIRl promoter, an RD 17 promoter, a P5CS promoter, a CBF3 promoter or an RCI2A promoter
- said zeaxanthin epoxidase preferably has an amino acid sequence of SEQ ID NO: 2.
- the base sequence encoding zeaxanthin epoxidase is preferably a base sequence of SEQ ID NO: 1 or a base sequence having a homology of 80% with the base sequence of SEQ ID NO: 1.
- the abiotic stress is preferably one or more selected from the group consisting of osmotic stress, high-salt stress and drought stress.
- the high-salt stress is preferably stress induced by NaCl or LiCl, and the plants are preferably monocotyledonous plants or dicotyledonous plants.
- RD29A as a stress- specific promoter
- an Rabl ⁇ promoter a CORl 5 A promoter
- an RD22 promoter an LOS5 promoter
- an SDIRl promoter an SDIRl promoter
- an RD 17 promoter a P5CS promoter
- a CBF3 promoter a CBF3 promoter or an RCI2A promoter
- Example 1 Construction of ⁇ tZEP-overexpressed transformant and examination of resistance thereof
- Arabidopsis thaliana seeds (Arabidopsis Biological Resource Center, USA) were sterilized with 70% ethanol and 10% Clorox, and then subjected to vernalization at 4 ° C in a dark condition.
- the treated seeds were germinated in solid Murashige and Skoog (1962) media at 22 °C in a short-day condition of 8-hr light/16-hr dark.
- 10- 12-day-old seedlings were transferred to soil, and then cultivated in a long-day condition of 16-hr light/8-hr dark.
- a plant expression vector comprising a full-length CaMV35S promoter has a characteristic in that it can overexpress genes at specific plant regions, particularly the shoot apical region and the root apical region, whereas an expression vector comprising only the B domain and minimal promoter (core) of the CaMV35S promoter can overexpress genes in all the regions in a plant (Philip, N.B. et al, EMBO J., 9: 1685, 1990).
- the B domain and minimal promoter (core) of the CaMV35S promoter was amplified by PCR and cloned into the HmdIII ⁇ PstI site of dicot binary vector pPZP211 (Peter ⁇ . et al, Plant Molecular Biology, 25:989, 1994).
- the constructed vector was named "pFGL571" (FIG. 1).
- AtZEP which is the ZEP gene of Arabidopsis thaliana
- the binary vector pFGL571 (FIG. 1), constructed in Example 1-2 above, was used.
- the full-length AtZEP of pdaO8179 which is an RIKEN Arabidopsis full-length (RAFL) cDNA clone of SEQ ID NO: 1
- RAFL RIKEN Arabidopsis full-length
- the cloned plasmid (pFGL612) was transformed into Agrobacterium (GV3101), and the plant was transformed with the vector according to the floral-dipping method (Clough and Bent, Plant J., 16:735, 1998), thus obtaining the T 0 seeds of an Arabidopsis thaliana plant with overexpression of AtZEP (FIG. 2).
- T 1 plants were primarily screened using kanamycin resistance, and the total RNA of the screened transformant leaves was isolated using TRI-reagent (Invitrogen, USA). 5 ⁇ g of the isolated RNA was treated with RNase-free DNaseI (Promega, USA), and then treated with MMLV-reverse transcriptase (Promega, USA) to synthesize primary cDNA. Then, RT-PCR for an AtZEP gene was performed using the synthesized primary cDNA as a template. Herein, as an internal control, a GAPc (glyceraldehyde-3 -phosphate dehydrogenase C subunit) gene was used. On the basis of the results of RT-PCR, an AtZEP- overexpressed plant was screened, and primers used in RT-PCR are shown in Table 1 below.
- HPLC as pigment analysis was performed. Specifically, the rosette leaf of Arabidopsis thaliana cultivated for
- Example 1-1 60 days according to the method of Example 1-1 above was frozen with liquid nitrogen and finely powdered using a mixer-mill (Qiagen, USA), and then plastids were extracted from the powder using 100% acetone. The extracted plastids were separated using a spherisorb ODS-I column and an HPLC system
- FIG. 3 the AtZEP gene was overexpressed in the transgenic plants of line Nos. 4 and 5, and the screened transformants contained little or no zeaxanthin under light for growth and also contained a relatively small amount of antheraxanthin.
- T 2 plants obtained by treating the screened ⁇ tZEP-overexpressed transformant with light intensities of 300, 600 and 1200 /imol-m "2 ⁇ "1 for 30, 60 and 120 minutes, were measured for the amount of zeaxanthin (FIG. 4).
- the content of zeaxanthin was lower in the AtZEP- overexpressed transformant than in a wild-type plant at all the time periods and light intensities. That is, it can be seen that, in the T 2 transformants, the amount of zeaxanthin was reduced due to the overexpression of AtZEP.
- a T 3 homo- line was screened using kanamycin resistance and tested for various stresses.
- Arabidopsis thaliana wild-type and the transgenic seedling screened in Example 1-4 above were cultured in MS media for 7 days. Then, the plants were subjected to various stress conditions in MS media for 7 days, and the phenotypes of the above-ground portion (leaf and shoot) and below-ground portion (root) of the wild-type and transgenic plants were observed.
- NaCl, LiCl and KCl were used.
- the ⁇ tZEP-overexpressed transformant in the 10 mM LiCl-containing media had high LiCl resistance compared to the wild-type plant.
- the plants were treated with mannitol showing the effect of drought stress.
- the seedlings, cultured in MS media in normal conditions for 7 days were transferred and cultured in MS media, containing 0 mM, 100 mM, 200 mM, 300 mM and 400 mM mannitol, for 7 days, and the phenotypes thereof were compared with each other.
- the grown plants were treated with drought stress.
- the wild-type plant and the ⁇ /Zi ⁇ '/'-overexpressed transformant, cultured in soil for 3 weeks, were treated with drought stress (not watered) for 3 weeks, and then watered for 3 days, and the phenotypes thereof were observed.
- the size of the rosette leaf of the v4tZ£7 3 -overexpressed transformant was larger than that of the wild-type plant, and the overall plant size was also larger in the case of ⁇ tZ ⁇ '.P-overexpressed transformant. That is, it could be seen that the AtZEP- overexpressed transformant had higher resistance to drought stress compared to the wild-type plant.
- the effect of AtZEP overexpression on seed development was examined.
- the seed number of the transformant with continuous overexpression of AtZEP was greatly reduced compared to that of the wild-type plant. That is, it could be seen that the overexpression of AtZEP had a negative effect on seed development.
- Example 2 Construction of transformant in which AtZEP is overexpressed in a stress-induced manner and characteristics thereof
- the expression patterns of stress-induced genes RD29A and Rabl8 in the vltZ£'/- ) -overexpresed transformant were examined.
- the wild- type plant and the ⁇ /ZiiP-overexpressed transgenic plant were germinated in normal MS medium conditions. After 2 weeks, the plants were transferred into 300 mM NaCl liquid and treated with high-salt stress for 5 hours, and then total RNA was extracted from the plants in the same manner as in Example 1-4, and cDNA was synthesized by PCR using the primers shown in Table 2 below. Then, the expression levels of RD29A and Rabl8 were examined. As an internal control in the RT-PCR, a GAPc gene was used.
- an RD29A promoter was cloned into a pPZP211 vector comprising a Nos terminator.
- the RD29A promoter was amplified by PCR using Arabidopsis thaliana genomic DNA as a template and primers of SEQ ID NOs: 11 and 12.
- SEQ ID NO: 11 5'-GCGAAGCTTGGTGAATTAAGAGGAGAGAGAGGAGG-S'
- SEQ ID NO: 12 5'-ACACTGCAGTGAGTAAAACAGAGGAGGGTCTCAC-S'
- the AtZEP gene was treated with BamR ⁇ and Kpnl and cloned between the RD29A promoter and Nos terminator of the prepared vector.
- the cloned vector was transformed into Agrobacterium (GV3101), and then a plant was transformed with the Agrobacterium according to the floral-dipping method (Clough and Bent, Plant J., 16:735, 1998), thus obtaining the T 0 seeds of an Arabidopsis thaliana plant with overexpression of AtZEP (FIG. 12).
- T 1 plants were primarily screened using kanamycin resistance, and in order to examine whether the expression of the screened transformants would be increased in a stress-induced manner, the transformants were treated with salt stress.
- the seedlings of the T 1 transformants were placed on a filter, wetted with 300 mM NaCl, for 5 hours.
- the total RNA of the transformants was isolated using TRI-reagent (Invitrogen, USA). 5 ⁇ g of the isolated RNA was treated with RNase-free DNaseI (Promega, USA), and then treated with MMLV-reverse transcriptase (Promega, USA) to synthesize primary cDNA.
- RT-PCR for the AtZEP gene was performed using the synthesized cDNA as a template.
- a GAPc gene was used as an internal control.
- transgenic plants, in which AtZEP has been overexpressed in a stress-induced manner were screened, and primers used in the RT-PCR are shown in Table 1.
- AtZEP gene was overexpressed when the transgenic plants with stress-induced overexpression of AtZEP underwent stress, compared to when the plants did not undergo stress (FIG. 13). On the transformants with stress-induced overexpression of AtZEP, various stress tests were conducted.
- the effect of the stress-induced AtZEP overexpression on seed development was examined.
- Example 1-1 were collected and the number thereof was counted. As a result, it can be seen that the seed number was reduced in the transformant with continuous overexpression of AtZEP, the same as in Example 1 -7. However, it could be observed that the seed number of the transgenic plant, prepared in Example 2-2 above and containing the stress-induced promoter and AtZEP, was much larger than that of the transformant with continuous overexpression of AtZEP, and that the transformant with stress-induced overexpression of AtZEP restored growth to levels of wild-type Arabidopsis thaliana (FIG. 10). Accordingly, it can be found that the use of the stress- induced promoter can minimize the effect of AtZEP overexpression on plant growth.
- the present invention provides useful plants having not only resistance to abiotic stress, but also growth characteristics similar to those of wild-type plants, wherein a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are amplified. According to the present invention, useful plants having resistance to a variety of abiotic stresses can be grown in a simple and systematic manner.
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Abstract
The present invention relates to stress-resistant plants in which a stress-induced promoter and a zeaxanthin epoxidase (ZEP) gene are introduced. More specifically, relates to plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified. According to the present invention, useful plants having resistance to a variety of abiotic stresses can be obtained in a simple and systematic manner.
Description
Stress Resistant Plant Introduced by Stress — Induced Promoter and the Gene Encoding Zeaxanthin Epoxidase
TECHNICAL FIELD
The present invention relates to a stress-resistant plant in which a stress-induced promoter and a zeaxanthin epoxidase (ZEP) gene are introduced. More particularly, the present invention relates to plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
BACKGROUND ART
Because plants grow and develop in various environments, they are tolerant to high salt, drought, high temperature and osmotic stress. Particularly, a typical hormone associated with the osmotic and drought stress of plants is abscisic acid (ABA). When plants undergo osmotic stress, the amount of the plant hormone abscisic acid in cells is increased, and the expression of various osmotic stress- induced genes induced by abscisic acid is increased. Also, when water is deficient, abscisic acid induces stomatal closure to minimize the loss of water through transpiration (Hasegawa, P.M. et al, Annu. Rev. Plant Physiol. Plant MoI. Biol, 52:463, 2000; Bray, Plant Cell Environ., 25:153, 2002; Finkelstein, R.R. et al, Plant Cell, 14:S15, 2002). In addition to this function, abscisic acid plays an important role in embryo maturation and initiation of seed dormancy during seed development (Nambara, E. et al, Trends Plant Sd., 5:213, 2003).
The biosynthetic pathways of abscisic acid playing an important role in plants as described above include two pathways, a direct pathway via C15 farnesyl pyrophosphate and an indirect pathway via C40 carotenoid (Taylor, I. B. et al, J.
Exp. BoL, 51 : 1563, 2000; Schwartz, S.H. et al, Plant Physiol, 131 : 1591 , 2003). Through recent research results of abscisic acid-deficient mutant and the identification of genes in the mutants, the main pathway of the biosynthetic pathways of abscisic acid was found to be the indirect pathway (Seo, M. et al, Trends Plant ScL 7: 41 , 2002).
The first stage of the indirect pathway for the synthesis of abscisic acid is the epoxidation of zeaxanthin and antheraxanthin by zeaxanthin epoxidase (ZEP) and occurs in plastids (Xiong, L. et al., Plant Physiol, 133:29, 2003). Violaxanthin synthesized through the above modification is converted to 9-cis epoxicarotenoid, which is then converted to C15 intermediate xanthoxin through oxidative cleavage by 9-cis epoxicarotenoid dioxygenase (NCED), and the xanthoxin moves to the cytoplasm, and then converted to abscisic acid.
Meanwhile, the xanthophyll cycle refers to the stoichiometric conversion of xanthophylls pigments, associated with the structural change among violaxanthin, antheraxanthin and zeaxanthin. Violaxanthin de-epoxidase has the activity to convert violaxanthin to antheraxanthin and zeaxanthin, and the reverse epoxidation is activated by ZEP. This xanthophyll cycle is important for the harvest of light, protects the photosynthetic system of plants from damage caused by excessive light and is involved in a process of converting excessive light to heat (Foyer, CH. et al, Physiol Plant., 92:696, 1994). Thus, it can be seen that ZEP is an enzyme important in both a stress reaction mediated by abscisic acid and a mechanism of protecting the photosynthetic system from excessive light.
In a recent experiment in which RD29A promoter activity was measured using a luciferase gene as a reporter gene, it was found that the expression of RD29A is increased due to environmental stress and abscisic acid. On the basis of this finding, mutants, in which the expression of the RD29A gene is changed by
osmotic stress, low temperature and abscisic acid, were isolated (Xiong, L. et al, J. Biol. Chem., 277:8588, 2002). Among the mutants isolated using this method, Ios6 showed a phenotype in which the accumulation of abscisic acid was reduced, and this phenotype was the same as in abal, which is an ABA-deficient mutant. Also, it was reported that the expression of Rabl8 is increased due to a signaling process mediated by an increase in the concentration of abscisic acid (Matthieu, M. et al, Plant Physiol, 130:265, 2002).
Meanwhile, transgenic plants with overexpression of AtZEP (LOS6/ABA1), which is the ZEP DNA of Arabidopsis thaliana, showed high RD29A promoter activity in osmotic stress conditions. From this result, it can be seen that ZEP plays an important role in a stress reaction mediated by abscisic acid. However, the in vivo biological function and environmental stress-associated function of ZEP have not yet been clearly identified.
Accordingly, the present inventors made many efforts to identify the biological function and environmental stress-associated in vivo function of ZEP and, as a result, found that ZisP-overexpressed plants have resistance to abiotic stress (Park, B. K., master's degree thesis, Pusan National University, 2006).
However, the Z^P-overexpressed plants have problems in that they do not sufficiently grow, because the number of the seeds is rapidly reduced, unlike wild-type plants. Thus, in the art to which the present invention pertains, there is an urgent need to develop useful plants, which have resistance to abiotic stress and, at the same time, show good growth characteristics, like wild-type plants.
Accordingly, the present inventors have made many efforts to develop plants, which have resistance to abiotic stress such as environmental stress and, at the same time, have good growth characteristics. As a result, the present inventors have found that, when a stress-induced promoter and a base sequence encoding
ZEP are amplified in plants, it is possible to construct plants, which have resistance to abiotic stress and, at the same time, have growth characteristics almost similar to those of wild-type plants, thereby completing the present invention.
SUMMARY OF THE INVENTION
It is an object of the present invention to provide plants having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase is introduced or amplified.
To achieve the above objects, the present invention provides plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows overexpression binary vector pFGL571 used for the overexpression of AtZEP.
FIG. 2 shows a method for constructing an Arabidopsis thaliana plant in which AtZEP is overexpressed.
FIG. 3 A shows the results of RT-PCR conducted to measure the expression level of AtZEP, and FIG. 3B is a graphic diagram showing the results of HPLC measurement for the content of zeaxanthin in a plant.
FIG. 4 is a graphic diagram showing the results of HPLC measurement for the content of zeaxanthin in a plant transformed with AtZEP, when the plant was treated with light stress at various light intensities (WT: wild type; and ZEP: plant transformed with AtZEP).
FIG. 5 is a photograph showing that an ΛtZ£7>-overexpressed transformant has resistance to NaCl.
FIG. 6 is a photograph showing that an ΛtZEP-overexpressed transformant has resistance to LiCl.
FIG. 7 is a photograph showing that an ΛtZϋP-overexpressed transformant has no resistance to KCl.
FIG. 8 is a photograph showing that an ΛtZEP-overexpressed transformant has resistance to mannitol.
FIG. 9 is a photograph showing an ΛtZiϊ'/'-overexpressed transformant has resistance to drought stress.
FIG. 10 is a graphic diagram showing that, in a transgenic plant in which AtZEP is expressed in a stress-induced manner, a negative effect on seed development in the continuous expression of AtZEP is inhibited.
FIG. 1 1 is an RT-PCR photograph showing that the expression of stress-induced genes RD29A and Rabl8 in an y4/Z£'/J-overexpressed transformant is increased under normal conditions and salt stress conditions.
FIG. 12 shows a process for constructing an Arabidopsis thaliana plant in which AtZEP is overexpressed in a stress-induced manner.
FIG. 13 is a RT-PCR photograph showing that the expression of AtZEP is increased due to salt stress in an Arabidopsis thaliana plant in which AtZEP is overexpressed in a stress-induced manner.
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENT
In the previous studies, the present inventors found that Λ/ZisP-overexpressed transformants show resistance to salts, such as NaCl and LiCl, and drought stress. However, the ΛtZ£P-overexpressed transformants have a problem in that they do not sufficiently grow, because the number of the seeds is rapidly reduced, unlike wild-type plants.
In the present invention, this negative effect occurring in the continuous expression of AtZEP was solved through a stress-induced promoter. Specifically, the present inventors have found that a plant transformed with an expression vector, containing a stress-induced promoter and AtZEP, has not only resistance to stress, but also growth characteristics almost similar to those of wild- type plants.
The present invention relates to plants or plant cells having resistance to abiotic stress, in which a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
In the present invention, the stress-induced promoter is preferably an RD29A promoter, a Rabl8 promoter, a COR15A promoter, an RD22 promoter, a LOS5 promoter, an SDIRl promoter, an RD 17 promoter, a P5CS promoter, a CBF3 promoter or an RCI2A promoter, and said zeaxanthin epoxidase preferably has an amino acid sequence of SEQ ID NO: 2. Also, the base sequence encoding
zeaxanthin epoxidase is preferably a base sequence of SEQ ID NO: 1 or a base sequence having a homology of 80% with the base sequence of SEQ ID NO: 1.
In the present invention, the abiotic stress is preferably one or more selected from the group consisting of osmotic stress, high-salt stress and drought stress. The high-salt stress is preferably stress induced by NaCl or LiCl, and the plants are preferably monocotyledonous plants or dicotyledonous plants.
Examples
Hereinafter, the present invention will be described in further detail with reference to examples. It will be apparent to one skilled in the art that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.
Particularly, although the following examples illustrated only RD29A as a stress- specific promoter, it will be obvious to those skilled in the art that it is possible to use an Rabl δ promoter, a CORl 5 A promoter, an RD22 promoter, an LOS5 promoter, an SDIRl promoter, an RD 17 promoter, a P5CS promoter, a CBF3 promoter or an RCI2A promoter, and the scope of the present invention is not limited thereto.
Example 1 : Construction of ^tZEP-overexpressed transformant and examination of resistance thereof
1-1 : Germination and cultivation of plant
Arabidopsis thaliana seeds (Arabidopsis Biological Resource Center, USA) were sterilized with 70% ethanol and 10% Clorox, and then subjected to vernalization at 4 °C in a dark condition. The treated seeds were germinated in solid
Murashige and Skoog (1962) media at 22 °C in a short-day condition of 8-hr light/16-hr dark. After the germination of the Arabidopsis thaliana seeds, 10- 12-day-old seedlings were transferred to soil, and then cultivated in a long-day condition of 16-hr light/8-hr dark.
1-2: Construction of binary vector (pFGL571) for overexpression of plant
A plant expression vector comprising a full-length CaMV35S promoter has a characteristic in that it can overexpress genes at specific plant regions, particularly the shoot apical region and the root apical region, whereas an expression vector comprising only the B domain and minimal promoter (core) of the CaMV35S promoter can overexpress genes in all the regions in a plant (Philip, N.B. et al, EMBO J., 9: 1685, 1990). On the basis of this fact, in order to construct a vector for plant overexpression, which can overexpress a target gene in all the regions in a plant and is easy to operate, the B domain and minimal promoter (core) of the CaMV35S promoter was amplified by PCR and cloned into the HmdIIIλPstI site of dicot binary vector pPZP211 (Peter Η. et al, Plant Molecular Biology, 25:989, 1994). The constructed vector was named "pFGL571" (FIG. 1).
1-3: Construction of ΛtZEP-overexpressed transformant
For the overexpression of AtZEP, which is the ZEP gene of Arabidopsis thaliana, the binary vector pFGL571 (FIG. 1), constructed in Example 1-2 above, was used. Meanwhile, the full-length AtZEP of pdaO8179, which is an RIKEN Arabidopsis full-length (RAFL) cDNA clone of SEQ ID NO: 1 , was treated with EcoRl and Sad, and the EcoRl site of the pdaO8179 was modified by treatment with klenow and cloned into the Sacl-Smal site of the pFGL571 vector (FIG. 1). The cloned plasmid (pFGL612) was transformed into Agrobacterium (GV3101), and the plant was transformed with the vector according to the floral-dipping method
(Clough and Bent, Plant J., 16:735, 1998), thus obtaining the T0 seeds of an Arabidopsis thaliana plant with overexpression of AtZEP (FIG. 2).
1-4: Screening of AtZEP overexpression line
From the T0 seeds constructed in Example 1-3 above, T1 plants were primarily screened using kanamycin resistance, and the total RNA of the screened transformant leaves was isolated using TRI-reagent (Invitrogen, USA). 5 μg of the isolated RNA was treated with RNase-free DNaseI (Promega, USA), and then treated with MMLV-reverse transcriptase (Promega, USA) to synthesize primary cDNA. Then, RT-PCR for an AtZEP gene was performed using the synthesized primary cDNA as a template. Herein, as an internal control, a GAPc (glyceraldehyde-3 -phosphate dehydrogenase C subunit) gene was used. On the basis of the results of RT-PCR, an AtZEP- overexpressed plant was screened, and primers used in RT-PCR are shown in Table 1 below.
Table 1
In order to examine whether the amount of zeaxanthin in the selected plant was actually changed by overexpressed AtZEP, HPLC as pigment analysis was performed. Specifically, the rosette leaf of Arabidopsis thaliana cultivated for
60 days according to the method of Example 1-1 above was frozen with liquid nitrogen and finely powdered using a mixer-mill (Qiagen, USA), and then plastids were extracted from the powder using 100% acetone. The extracted plastids were separated using a spherisorb ODS-I column and an HPLC system
(HPl 100 series; Hewlett Pacard, Waldbronn, Germany) (FIG. 3).
As a result, as can be seen in FIG. 3, the AtZEP gene was overexpressed in the transgenic plants of line Nos. 4 and 5, and the screened transformants contained little or no zeaxanthin under light for growth and also contained a relatively small amount of antheraxanthin.
Also, T2 plants, obtained by treating the screened ΛtZEP-overexpressed transformant with light intensities of 300, 600 and 1200 /imol-m"2^"1 for 30, 60 and 120 minutes, were measured for the amount of zeaxanthin (FIG. 4). As a result, as can be seen in FIG. 4, the content of zeaxanthin was lower in the AtZEP- overexpressed transformant than in a wild-type plant at all the time periods and light intensities. That is, it can be seen that, in the T2 transformants, the amount of zeaxanthin was reduced due to the overexpression of AtZEP. From the screened ΛtZϋT^-overexpressed transformants of line Nos. 4 and 5, a T3 homo- line was screened using kanamycin resistance and tested for various stresses.
1-5: Examination of resistance to salt stress
Arabidopsis thaliana wild-type and the transgenic seedling screened in Example 1-4 above were cultured in MS media for 7 days. Then, the plants were subjected to various stress conditions in MS media for 7 days, and the phenotypes of the above-ground portion (leaf and shoot) and below-ground portion (root) of the wild-type and transgenic plants were observed. To examine the response of the ΛtZiiP-overexpression transformant to salt stress, NaCl, LiCl and KCl were used.
In the case of NaCl, the seedlings, cultured in MS media in normal conditions for
7 days, were transferred and cultured in MS media, containing each of 0 mM, 100 mM, 150 mM and 200 mM NaCl, for 7 days, and then the phenotypes thereof were compared with each other. As a result, the Arabidopsis thaliana plants,
cultured in the MS media containing 0-100 mM NaCl, showed little or no difference in phenotype between the wild-type plant and the transformant, but in the case of 150 mM and 200 mM NaCl, the above ground portion of the plants developed, suggesting that the ^tZEP-overexpressed seedling had resistance higher than that of the wild-type plant. In addition, the results of culture of the wild-type and v4/Zis/J-overexpressed transformant plants in the MS media containing 150 mM NaCl are shown in FIG. 5. As shown in FIG. 5, it could be seen that the ΛtZZΪT'-overexpressed transformant had much higher resistance compared to the wild-type plant.
In the case of LiCl, the seedlings, cultured in MS media in normal conditions for 7 days, were transferred and cultured in MS media, containing 0 mM, 5 mM and 10 mM LiCl, for 7 days, and then the phenotypes thereof were compared with each other. As a result, as can be seen in FIG. 6, the ^tZEP-overexpressed transformant in the 10 mM LiCl-containing media had high LiCl resistance compared to the wild-type plant.
However, in the case in which the seedlings were treated with each of 0 mM, 100 mM and 200 mM KCl, the difference in phenotype between the plants did not appear at all the concentrations of KCl (FIG. 7). From the fact that there was no difference between the wild-type plant and the ΛtZE/'-overexpressed transformant, even though the KCl concentration was high, it could be seen that the salt stress resistance of the ΛtZZsP-overexpressed transformant was not the influence of Cl", but resistance to Na+ and Li+.
1-6: Examination of resistance to drought stress
In order to examine the response of the ΛtZEP-overexpressed transformants to drought stress, the plants were treated with mannitol showing the effect of drought stress. In the same manner as in the case of salt stress, the seedlings,
cultured in MS media in normal conditions for 7 days, were transferred and cultured in MS media, containing 0 mM, 100 mM, 200 mM, 300 mM and 400 mM mannitol, for 7 days, and the phenotypes thereof were compared with each other. As a result, as shown in FIG.8, it could be seen that the Arabidopsis thaliana plants, cultured in the MS media containing 0-300 mM mannitol, showed no difference in phenotype between the wild-type and the transformant, but in the case of 400 mM mannitol, the above-ground portion of the AtZEP- overexpressed transformant showed higher resistance, and the lateral roots of the below-ground region developed more.
In order to re-confirm the above-confirmed drought stress resistance, the grown plants were treated with drought stress. Specifically, the wild-type plant and the Λ/Ziϊ'/'-overexpressed transformant, cultured in soil for 3 weeks, were treated with drought stress (not watered) for 3 weeks, and then watered for 3 days, and the phenotypes thereof were observed. As a result, as can be seen in FIG. 9, the size of the rosette leaf of the v4tZ£73-overexpressed transformant was larger than that of the wild-type plant, and the overall plant size was also larger in the case of ΛtZϋ'.P-overexpressed transformant. That is, it could be seen that the AtZEP- overexpressed transformant had higher resistance to drought stress compared to the wild-type plant.
1-7: Examination of the effect of AtZEP overexpression on seed development
The effect of AtZEP overexpression on seed development was examined. The seeds of each of the wild-type plant and the ΛtZisP-overexpressed transformant, cultured for about 60 days according to the method of Example 1-1 above, were collected and the number thereof was counted. As a result, as can be seen in FIG. 10, the seed number of the transformant with continuous overexpression of AtZEP was greatly reduced compared to that of the wild-type plant. That is, it
could be seen that the overexpression of AtZEP had a negative effect on seed development.
Example 2: Construction of transformant in which AtZEP is overexpressed in a stress-induced manner and characteristics thereof
2-1 : Examination of expression of stress-induced genes
In order to examine whether the stress resistance of the ΛtZZsP-overexpressed transgenic plant, shown in Examples 1-5 and 1-6 above, was induced by abscisic acid, the expression patterns of stress-induced genes RD29A and Rabl8 in the vltZ£'/-)-overexpresed transformant were examined. For this purpose, the wild- type plant and the Λ/ZiiP-overexpressed transgenic plant were germinated in normal MS medium conditions. After 2 weeks, the plants were transferred into 300 mM NaCl liquid and treated with high-salt stress for 5 hours, and then total RNA was extracted from the plants in the same manner as in Example 1-4, and cDNA was synthesized by PCR using the primers shown in Table 2 below. Then, the expression levels of RD29A and Rabl8 were examined. As an internal control in the RT-PCR, a GAPc gene was used.
Table 2
As a result, as can be seen in FIG. 1 1, the expression levels of RD29A and Rabl8 in the ΛtZEP-overexpressed transformant were higher than in the Arabidopsis thaliana wild-type in all the stress condition and the normal condition. This
directly demonstrates that the stress resistance of the overexpressed transformant is mediated by abscisic acid.
2-2: Construction and screening of transgenic plant in which AtZEP is overexpressed in stress-induced manner
For the stress-induced overexpression of AtZEP, an RD29A promoter was cloned into a pPZP211 vector comprising a Nos terminator. The RD29A promoter was amplified by PCR using Arabidopsis thaliana genomic DNA as a template and primers of SEQ ID NOs: 11 and 12.
SEQ ID NO: 11: 5'-GCGAAGCTTGGTGAATTAAGAGGAGAGAGGAGG-S' SEQ ID NO: 12: 5'-ACACTGCAGTGAGTAAAACAGAGGAGGGTCTCAC-S'
The AtZEP gene was treated with BamR\ and Kpnl and cloned between the RD29A promoter and Nos terminator of the prepared vector. The cloned vector was transformed into Agrobacterium (GV3101), and then a plant was transformed with the Agrobacterium according to the floral-dipping method (Clough and Bent, Plant J., 16:735, 1998), thus obtaining the T0 seeds of an Arabidopsis thaliana plant with overexpression of AtZEP (FIG. 12).
From the T0 seeds constructed as described above, T1 plants were primarily screened using kanamycin resistance, and in order to examine whether the expression of the screened transformants would be increased in a stress-induced manner, the transformants were treated with salt stress. The seedlings of the T1 transformants were placed on a filter, wetted with 300 mM NaCl, for 5 hours. The total RNA of the transformants was isolated using TRI-reagent (Invitrogen, USA). 5 μg of the isolated RNA was treated with RNase-free DNaseI (Promega, USA), and then treated with MMLV-reverse transcriptase (Promega, USA) to synthesize primary cDNA. RT-PCR for the AtZEP gene was performed using the synthesized cDNA as a template. Herein, as an internal
control, a GAPc gene was used. On the basis of the results of RT-PCR, transgenic plants, in which AtZEP has been overexpressed in a stress-induced manner, were screened, and primers used in the RT-PCR are shown in Table 1.
As a result, it could be seen that the AtZEP gene was overexpressed when the transgenic plants with stress-induced overexpression of AtZEP underwent stress, compared to when the plants did not undergo stress (FIG. 13). On the transformants with stress-induced overexpression of AtZEP, various stress tests were conducted.
2-3: Stress resistance of transformant with stress-induced overexpression of AtZEP
The phenotypes of the above-ground portion (leaf and shoot) and below-ground portion (root) of Arabidopsis thaliana wild-type and the transgenic seedling, constructed in Example 2-2 above, were observed in various stress conditions according to the methods of Examples 1-5 and 1-6. As a result, the transgenic seedling showed the same stress resistance as that of the ΛtZEP-overexpressed transformant.
2-4: Examination of effect of stress-induced AtZEP overexpression on seed development
The effect of the stress-induced AtZEP overexpression on seed development was examined. The seeds of each of the wild-type plant, the transformant with continuous overexpression of AtZEP and the transformant with stress-induced overexpression of AtZEP, cultured for about 60 days according to the method of
Example 1-1, were collected and the number thereof was counted. As a result, it can be seen that the seed number was reduced in the transformant with continuous overexpression of AtZEP, the same as in Example 1 -7.
However, it could be observed that the seed number of the transgenic plant, prepared in Example 2-2 above and containing the stress-induced promoter and AtZEP, was much larger than that of the transformant with continuous overexpression of AtZEP, and that the transformant with stress-induced overexpression of AtZEP restored growth to levels of wild-type Arabidopsis thaliana (FIG. 10). Accordingly, it can be found that the use of the stress- induced promoter can minimize the effect of AtZEP overexpression on plant growth.
INDUSTRIAL APPLICABILITY
As described in detail above, the present invention provides useful plants having not only resistance to abiotic stress, but also growth characteristics similar to those of wild-type plants, wherein a stress-induced promoter and a base sequence encoding zeaxanthin epoxidase are amplified. According to the present invention, useful plants having resistance to a variety of abiotic stresses can be grown in a simple and systematic manner.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims
1. Plants or plant cells having resistance to abiotic stress, in which a stress- induced promoter and a base sequence encoding zeaxanthin epoxidase are introduced or amplified.
2. The Plants or plant cells having resistance to abiotic stress according to claim 1 , wherein the stress-induced promoter is RD29A promoter or Rablδ promoter
3. The Plants or plant cells having resistance to abiotic stress according to claim 1 , wherein said zeaxanthin epoxidase has an amino acid sequence of SEQ ID NO: 2.
4. The Plants or plant cells having resistance to abiotic stress according to claim 1 , wherein the base sequence encoding zeaxanthin epoxidase is a base sequence of SEQ ID NO: 1 or a base sequence having a homology of 80% with the base sequence of SEQ ID NO: 1.
5. The Plants or plant cells having resistance to abiotic stress according to claim 1, wherein the abiotic stress is one or more selected from the group consisting of osmotic stress, high-salt stress and drought stress.
6. The Plants or plant cells having resistance to abiotic stress according to claim 5, wherein the high-salt stress is stress induced by NaCl or LiCl.
7. The Plants or plant cells having resistance to abiotic stress according to any one claim among claims 1-6, wherein said plants are monocotyledonous plants or dicotyledonous plants.
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WO2017066894A1 (en) * | 2015-10-23 | 2017-04-27 | Universidad De Talca | Synthetic promotor induced by abiotic stress, genetic construct containing same and plant cells transformed therewith |
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