KR20130048905A - Stress inducible transgenic plants - Google Patents

Abstract

PURPOSE: A stress inducible transgenic plant is provided to control the reproductive property of the plant by a promoter which expresses a foreign gene. CONSTITUTION: A stress inducible transgenic promoter which is specifically expressed in a flower of a plant contains a nucleotide sequence in sequence number 1. A vector contains the promoter, and a foreign gene which is operatively linked to the promoter. A transgenic plant is prepared by transforming with the vector. A method for producing the plant transformant comprises: a step of transforming a plant cell or a plant tissue with the vector; a step of selecting the transformed plant cell or tissue; and a step of re-differentiating a plant from the plant cell or plant tissue. The transgenic plant with enhanced abiotic stress resistance is produced by the method.

Description

Stress Inducible Transgenic Plants

The present invention relates to low-temperature inducible transgenic plants using the OsPOX1 promoter of rice.

Temperature is one of the major factors limiting the production of many crop species (Toenniessen 1991). During the reproductive stage, cold treatment of 12 ° C. with rice for 4 days induces damage (Satake and Hayase 1970). Low-temperature stress during pollen development leads to the death of pollen blasts because they cannot receive nutrients or to abnormalization of the tapetum such as male-sterility (Hoshikawa 1989). Because plants are not mobile, they need to change their metabolism to survive stress. These low temperature reactive mechanisms are associated with changes in the expression of kinases associated with signal transduction, accumulation of osmolyte, or changes in membrane lipid composition (Thomashow, 1999). cor (cold-regulated) genes, lea (late-embryogenesis abundant) genes, regulatory genes, antifreeze protein genes and genes encoding signal transduction proteins (Thomashow, 1998, 1999; Shinozaki & Yamaguchi-Shinozaki, 2000; Zhu, 2001 Many cold-reactive genes have been found in a variety of plant species.

Because low temperature sensitivity is important at the reproductive stage, several studies have been reported to identify the low temperature reactive genes / proteins present in rice flowers. Eight kinds of low temperature reactive genes of rice flower were identified using mRNA differential display (Kim et al., 2007). OsLti6b, which is homologous to arabidopsis RCI2, accumulates much in the anther wall due to cold stress (Kim et al., 2007). CDNA microarrays (Yamaguchi et al., 2004) and proteomics (Imin et al., 2006) were used to identify the cold-responsive genes and proteins of immature rice flowers subjected to cold stress. Further analysis of these clones is needed.

Because nonspecific expression of genes can lead to unintended phenotypes such as dwarfism, the need for tissue specific or developmental stage specific expression of specific genes is increasing to develop useful transformants. In rice and Arabidopsis, the C-repeat / Dehydration Responsive Element Binding (C / DREB) gene, regulated by the strong promoter and maize ubiquitin gene of the cauliflower mosaic virus 35S gene, is ectopically Expression. These transformants show upregulation of low temperature reactive genes, but often show severe dwarfism (Kasuga et al., 1999; Lee et al., 2004). Therefore, it may be necessary to find and study microregulatory promoters for the regulated expression of genes (Kasuga et al., 1999; Yi et al., 2010; Saad et al., 2010).

Peroxidase is a heme-containing glycoprotein that catalyzes the redox reaction between H ₂ O ₂ and various reducing agents (H ₂ O ₂ + AH ₂ → 2H ₂ O + A). Enzymes can be classified into three classes, Class I, II and III, based on the difference in primary structure (Welinder, 1992). Class Ⅰ peroxidase is an enzyme within the cell, a class Ⅱ peroxidase is lignin (lignin) peroxidase and Mn ^{2 +} - dependent enzymes, such as an extracellular peroxidase. Class III enzymes are plant-specific and externally secreted or vacuole transported enzymes (Hiraga et al., 2001). Classer III peroxidases play a role in lignification, suberization, cross-linking of cell wall proteins, auxin catabolism, defense against pathogens, salt resistance and senescence. I think. Genes encoding plant peroxidases of Arabidopsis and rice are composed of multigene families of 73 and 138, respectively (Valerio et al., 2004; Passardi et al., 2004). In vitro due to the presence and their low substrate specificity of many isozymes (isoenzyme) in (in vitro), it is not easy to define the precise function of each peroxy oxidase (Hiraga et al., 2001) .

Of the 42 independent rice POX Expression Sequence Tags (ESTs), 22 genes were identified and characterized (Sasaki et al., 2004). Recently, Sato et al. (2001) reported a rice transformant with improved low temperature resistance overexpressing OsAPXα, an ascorbate peroxidase gene.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors made diligent efforts to find a promoter having a function of regulating genes to be specifically expressed in the flowers of plants. As a result, OsPOX1, a peroxidase gene of rice ( O ryza s ativa) , was cloned and transformed with a fusion gene of the promoter and the GUS gene to confirm GUS activity in the reproductive organs of rice plants. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a stress inducible promoter that is specifically expressed in the flowers of plants.

Another object of the present invention is to provide a vector comprising a foreign gene operably linked with the promoter of the present invention.

Another object of the present invention to provide a transgenic plant.

Another object of the present invention to provide a method for producing a plant transformant.

It is another object of the present invention to provide a transgenic plant having enhanced resistance to abiotic stress.

Another object of the present invention is to provide a stress-inducing OsPOX1 protein.

Another object of the present invention is to provide a nucleic acid molecule encoding OsPOX1.

Another object of the present invention is to provide a recombinant vector comprising a nucleic acid molecule encoding OsPOX1.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a promoter consisting of the nucleotide sequence set forth in SEQ ID NO: 1 having the characteristic of expressing a gene specifically in a flower of a stress-inducing plant.

The present inventors have cloned a peroxidase (Peroxidase) gene OsPOX1 the sought results, rice (O ryza s ativa) in search of a promoter having a function to control so as to specifically expressed genes in the flowering of the plant, of the gene GUS activity was confirmed in the reproductive organs of rice plants by transforming with a fusion gene of promoter and GUS gene.

The term "specifically" as used herein means that the expression activity of the promoter is higher in a particular tissue than at least one or more other tissues in the same plant. The level of expression activity of a promoter is assessed by comparing the expression level of the promoter in tissues previously measured using those methods commonly used to those in other tissues. In general, the expression level of a promoter is measured by the amount of gene product expressed under the control of the promoter, for example, protein and RNA.

The term "promoter" as used herein refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Promoter sites are readily recognized by those skilled in the art. That is, an estimated start codon containing an ATG motif has been identified, and an upstream from the start codon is an estimated promoter site. The promoter consists of upstream and downstream elements. Adjacent elements typically include a TATA box that allows RNA polymerase II to initiate the synthesis of RNA at a suitable transcription initiation site. Far-field elements include regulatory sequences, also called enhancers, which are additional regulatory elements involved in tissue specific or time specific expression upstream of the TATA box. Enhancers are DNA sequences capable of promoting the activity of a promoter, and may be elements of the promoter or elements of heterologous that are inserted to enhance the tissue-specificity or expression level of the promoter. The promoter may be derived entirely from the original gene, or may be composed of different elements from different promoters found in nature, and may even include synthetic DNA. Those skilled in the art will appreciate that each of the different promoters can direct the expression of the gene in different tissues or cell types, at different stages of development, or in response to different environmental conditions. Promoters that express genes most of the time in most cell types are called "constitutive promoters." New types of new promoters that are useful in plant cells are constantly being found; A number of promoter examples are disclosed in compilations by Okamuro and Goldberg (1989, Biochemistry of Plants 15: 1-82). In most cases it is recognized that certain fragments of DNA fragments have the same promoter activity because the exact boundaries of the regulatory sequences are not fully defined.

According to another aspect of the present invention, there is provided a vector comprising a promoter consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a foreign gene operatively linked with the promoter.

As used herein, the term “operably linked” means a functional binding between a nucleic acid expression control sequence (eg, a promoter, a signal sequence, or an arrangement of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the regulation The sequence will control the transcription and / or translation of said other nucleic acid sequence.

The term "desired foreign gene" herein is not limited to a particular gene, and may be selected according to the desired application or phenotype to be achieved. In the present invention, it is preferable to select genes useful for improving the characteristics of flowers, for example, the reproductive properties of plants, the color or texture of flowers.

Vectors in the present invention can be constructed through a variety of methods known in the art, specific methods thereof are described in Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

Vectors of the invention can typically be constructed as vectors for cloning or expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts.

For example, when the vector of the present invention is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, a trp promoter, a lac promoter, a T7 promoter, a tac promoter, etc.) capable of promoting transcription, It is common to include ribosomal binding sites and transcription / detox termination sequences for initiation of translation. When E. coli is used as the host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158: 1018-1024 (1984)) and the leftward promoter of phage λ (pLλ) Promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14: 399-445 (1980)) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pGEX series, pET series and pUC19, etc.) which are often used in the art, phage (e.g. λgt4.λB , λ-Charon, λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

On the other hand, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from a genome of a mammalian cell (for example, a metallothionein promoter) or a mammalian virus Virus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may include an antibiotic resistance gene commonly used in the art as an optional marker, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin And resistance genes for tetracycline.

According to a preferred embodiment of the present invention, a promoter suitable for the present invention may be used any conventionally used in the art for the gene introduction of plants, for example, the ubiquitin promoter of corn, cauliflower mosaic virus (CaMV) 35S promoter, nopalin synthase (nos) promoter, pigwart mosaic virus 35S promoter, sugacran bacilli foam virus promoter, commelina yellow mottle virus promoter, ribulose-1,5-bis-phosphate carbide Photoinducible promoter of carboxylase small subunit (ssRUBISCO), rice cytosolic triosphosphate isomerase (TPI) promoter, adenine phosphoribosyltransferase (APRT) promoter of Arabidopsis and octopine synthase promoter do.

According to a preferred embodiment of the present invention, the 3'-non-detoxification site causing the polyadenylation suitable for the present invention is derived from the nopalin synthase gene of Agrobacterium turmeration (nos 3 'end) ( Bevan et al., Nucleic Acids Research, 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tuberculosis, of the protease inhibitor I or II gene of tomato or potato 3 'terminal portion, or CaMV 35S terminator.

Optionally, the vectors of the present invention additionally carry genes encoding reporter molecules such as luciferase and β-glucuronidase. In addition, the vectors of the present invention may be selected as antibiotic markers (e.g. neomycin, carbenicillin, kanamycin, spectinomycin, hygromycin, etc.) and resistance genes (e.g. neomycin phosphotransferase (nptII), or high). Gromycin phosphotransferase (hpt), and the like).

According to another aspect of the present invention, the present invention provides a method for producing a plant cell, comprising: (a) transforming a plant cell or a plant tissue with the vector of the present invention; (b) screening the transformed plant cell or plant tissue; And (c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant.

As used herein, the term “abiotic stress” refers to stress induced by abiotic factors such as low temperature, salt and drying, preferably low temperature and salt, most preferably low temperature.

According to another aspect of the present invention, there is provided a transformed plant transformed with the vector.

Host cells capable of stable and continuous cloning and expression of the vectors of the present invention are known in the art and can be used with any host cell, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. strains of the genus Bacillus, such as coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium, Serratia marcensons, and various Pseudomonas species. Enterobacteria and strains.

In the case of transforming a vector of the present invention into eukaryotic cells, as a host cell, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293 , HepG2, 3T3, RIN and MDCK cell lines) and plant cells and the like can be used. On the other hand, since the vector of the present invention is highly useful in plants, the transformants include not only cells but also callus derived from plant cells or tissues.

The method of carrying the vector of the present invention into a host cell is performed by the CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), when the host cell is a prokaryotic cell). ), One method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973); and Hanahan, D., J. Mol. Biol., 166: 557-580 (1983)) and electroporation methods (Dower, WJ et al., Nucleic. Acids Res., 16: 6127-6145 (1988)) and the like. In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell, 22: 479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology, 52: 456 (Wong, TK et al., Gene, 10: 87 (1980)), DEAE- (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and dextran treatment (Gopal, Mol. Cell Biol., 5: 1188-1190 ) Or the like into the host cell.

In the method of the present invention, the transformation of plant cells can be carried out according to a conventional method known in the art, which is electroporation (Neumann, E. et al., EMBO J., 1: 841 (1982) ), Particle bombardment (Yang et al., Proc. Natl. Acad. Sci., 87: 9568-9572 (1990)) and Agrobacterium-mediated transformation (US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011) Include. Of these, Agrobacterium-mediated transformation is most preferred.

It is known how to transform a dicotyledonous plant using Agrobacterium trimmers and obtain a transformed plant, and a method of obtaining a transformed plant for various other plants is known. See US Pat. Nos. 5,004,863 and 5,159135; See US Pat. Nos. 5,569,834 and 5,416011 for soy; Brassica is described in US Pat. No. 5,463,174; For peanuts, see Cheng et al. (1996) Plant Cell Rep . 15: 653-657, McKently et al. (1995) Plant Cell Rep . 14: 699-703; For papaya, see Ling, K. et al. (1991) Bio / technology 9: 752-758); and pea (Grant et al. (1995) Plant Cell Rep . 15: 254-258.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent (eg metabolic inhibitors, antibiotics and herbicides). Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, hygromycin phosphotransferase gene, glycophosphate tolerance gene and neomycin phosphotransferase (nptII) system.

Methods of developing or regenerating plants from plant protoplasts or various implants are well known in the art. The development or regeneration of plants containing foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (U.S. Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

In the present invention, the preferred transformation method is carried out using the Agrobacterium system, more preferably using the Agrobacterium tumefaciens-binary vector system.

Transformation of plant cells is carried out with Agrobacterium trimers containing Ti plasmids (Depicker, A. etal., Plant cell transformation by Agrobacterium plasmids.In Genetic Engineering of Plants, Plenum Press, New York (1983)). . More preferably, binary vector systems such as pBin19, pRD400, pRD320, pGA1611 and pGA1991 are used (An, G. et al., Binary vectors "In Plant Gene Res. Manual, Martinus Nijhoff Publisher, New York (1986) An et al., 1988 and Lee et al., 1999).

Binary vectors suitable for the present invention include (i) a promoter that operates on a plant; (Ii) structural genes operably linked to said promoter; And (iii) a polyadenylation signal sequence. Optionally, the vector additionally carries a gene encoding a reporter molecule (eg, luciferase and glucuronidase). Examples of promoters used in binary vectors include, but are not limited to, CaMV 35S promoter, 1 promoter, 2 promoter and nopalin synthase (nos) promoter.

Infection of implants with Agrobacterium trimmers includes methods known in the art. Most preferably, the infection process comprises coculture with an implant impregnated with a culture of Agrobacterium trimmers. As a result, Agrobacterium trimmers are infected into plants.

Explants transformed by Agrobacterium trimerization are re-differentiated in regeneration medium, which finally forms transgenic plants.

The transformed plants according to the present invention are confirmed to be transformed by methods known in the art. For example, PCR using DNA samples from tissues of transformed plants can identify foreign genes inserted into the genome of the transgenic plant. Alternatively, Northern or Southern blotting can be performed to confirm transformation (Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)).

According to another embodiment of the present invention, rice having the sequence set forth in SEQ ID NO: 4 sequence ( oryza satina ) provides a stress-inducing OsPOX1 protein.

Sequence Listing Second Sequence of the Invention is a nucleotide sequence encoding the OsPOX1 protein.

According to another aspect of the invention, there is provided a nucleic acid molecule encoding OsPOX1 having the amino acid sequence set forth in SEQ ID NO: 4.

According to another aspect of the present invention, there is provided a recombinant vector comprising a nucleic acid molecule encoding OsPOX1 of the present invention.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a stress-inducible novel gene promoter expressed in flowers.

(b) The present invention can be usefully used to control the reproductive properties of plants by providing a promoter capable of expressing a foreign gene in the flowers of the plant.

1 shows the genomic structure of OsPOX1 and its cDNA sequence. Figure 1a is the genomic structure of OsPOX1 of chromosome 1, ATG and arrows indicate the direction of initiation codon and translation, respectively. OsPOX1 gating portions are shown in black boxes and introns are shown in white boxes. Bars indicate the 0.5 kb ddOs319 fragment portion of the OsPOX1 gene. 1B shows the nucleotide and putative amino acid sequence of OsPOX1. The forward and reverse primers of nested PCR are underlined.
Figure 2 shows the results confirmed by the Northern blot the response of OsPOX1 to various stresses. Figure 2a is a Northern analysis of the OsPOX1 response of the reproductive stage to cold stress (12 ℃, 4 days). Total RNA from stressed untreated leaves (L) or flowers (F) and cold-treated leaves (CL) or flowers (CF) was isolated on agarose gels and blotted onto nylon membranes followed by radiolabeled OsPOX1 cDNA. Hybridized. EtBr-stained rRNA bands are shown. Figure 2b is a result of Northern analysis of the response of OsPOX1 of the reproductive stage to cold stress (12 ℃, 4 days). Total RNA was isolated from a variety of stressed seedlings such as cold (4 ° C.), physical wounds, 100 μM abscisic acid (ABA), dried and 250 mM salts, isolated on agarose gels, blotted onto nylon membranes, and then radioactivity- Labeled OsPOX1 cDNA was hybridized.
Figure 3 shows the structure of the OsPOX1 promoter and P _{OsPOX1 -GUS} . 3A shows the OsPOX1 promoter ca. A nucleotide sequence of 1.8 kb portion is shown. Presumably CAAT, TATA box and initiation codons are underlined. 3B shows the chimeric gene construct of GUS under OsPOX1 promoter. The ps228 was prepared by fusion of the OsPOX1 promoter portion with the GUS gene of the binary vector and introduced into the rice genome through Agrobacterium -mediated coculture. RB is the right border of T-DNA, P _OsPOX1 is the promoter of OsPOX1, GUS is the β-glucloronidase gene, T _NOS is the nopaline synthase terminator, and P _35S is CaMV 35S promoter, hph is a hygromycin phosphotransferase ( hygromycin phosphotransferase ) gene, 7 'indicates the transcription7 termination portion of pTiA6 and LB indicates the left border of T-DNA.
4 is a result of analyzing the histochemical location of GUS activity in PsPOX1-GUS transgenic plants. 4A shows germinating seeds under normal growth conditions. 4B shows germinated seeds treated with cold stress (4 ° C., 6 hours). 4C shows ca. under normal growth conditions. Show seedlings grown 10 days. 4D shows the flowers of the early young vesicle stage under normal growth conditions. 4E shows the flower of the pollen stage with a liqueur under normal growth conditions. Figure 4f shows the flower of the pollen stage with vacuoles treated with cold stress (12 ° C., 4 days). 4F is a cross-sectional view of the upper part of FIGS. 4G and 4G '(an enlarged view of the red box portion of FIG. 4G), and a cross-sectional view of the middle part of FIG. 4F is a cross section of FIG. 4H and 4H' (of FIG. 4H). Magnification of the red box portion), and the cross-sectional view of the lower portion of FIG. 4F transversely shows FIGS. 4i and 4i '(an enlargement of the red box portion of FIG. The bars of FIGS. 4A-4F represent 1 mm, FIGS. 4G-4I represent 0.5 mm, and FIGS. 4G-4-4 show 0.2 mm.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and methods

Plant Samples and Bacteria Strains

Plant-growing rice ( Oryza sativa cv. Dongjin) was treated as described by Kim et al. (Kim et al., 2007). After cold-stress treatment, plant samples were immediately frozen with liquid nitrogen and stored at -70 ° C. For GUS analysis, various tissues of different stages were immediately immersed in X-glu solution.

E. coli strains XL-1 Blue MRF 'and MC1000 were used as hosts for molecular cloning. F1 helper phage R408 was used to delete pBluescript phagemid from the λ UNI-Zap XR vector (Stratagene, USA).

Full sequence of OsPOX1

To obtain the overall cDNA of ddOs319, reverse transcription-PCR was performed using the specific primer designed based on the ddOs319 sequence and the corresponding sequence in the NCBI database. Total RNA was isolated as described by Koh (2007) et al., And 5 μg, 0.5 mM dNTP, 0.1 μM primers (POX-A1, 5′-TAG AAC AAC CAA ATG AAC TC) in total RNA of cold-treated flowers. -3 ') and 200 U Superscript ^TM (II) reverse transcriptase (invitrogen, USA) were used to synthesize cDNA. Reverse transcription-PCR was performed with 2 μl cDNA, 0.2 μM specific primers (POX-S1, 5'-AGT GCA GTG TGC AGT GC-3 '; POX-A1), 0.2 mM dNTPs and 5 units Ex-Taq DNA polymerase (Takara, Japan) was carried out with 50 μl reaction solution. The reaction proceeded to an initial 94 ° C. 3 minute denaturation step, PCR 30 cycles (94 ° C. 1 minute-54 ° C. 1 minute 30 seconds-72 ° C. 1 minute 30 seconds), and a final 72 ° C. 10 minute extension step. . 0.2 μM specific primers (POX-S2, 5'-GCG AAG GTT TAC TTG GCG AC-3 '; POX-A2, 5'-TGA ACT CAT TTT ATA TAT TG-3'), 0.2 Mixed with mM dNTPs and 5 units Ex-Taq DNA polymerase (Takara, Japan) was used for nested PCR reactions. The reaction proceeded to an initial 94 ° C. 3 minute denaturation step, PCR 30 cycles (94 ° C. 1 minute-47 ° C. 1 minute 30 seconds-72 ° C. 1 minute 30 seconds) and a final 72 ° C. 10 minute extension step. .

Northern blot analysis

10 μg RNA was denatured and isolated via 1.3% agarose gel electrophoresis and blotted onto nylon membrane. ³² P-labeled OsPOX1 probes were hybridized at 42 ° C. for 16-24 hours (Sambrook et al., 1989). The nylon membrane was washed at room temperature with 0.1 × SSC (Saline-Sodium Citrate), 0.1% Sodium Dodecyl Sulfate (SDS) and photosensitive on a hyperfilm ^TM MP film (Amersham, UK) or a phosphorous image plate (Buji 1500) (Fuji, Japan). It was.

Isolation of OsPOX1 Promoter

To obtain the proboter portion of OsPOX1, PCR of genomic DNA was performed using primers designed based on the genome sequence of the NCBI database. 0.1 μg rice genomic DNA, 0.2 μM primer (POX1-P5, 5′-GAT CTA CAT CAG AAC AAG G-3 ′; POX1-P7, 5′-AGC ATC AAC AGG CAA CC-3 ′) in 50 μl reaction solution ), 0.2 mM dNTPs and 2.5 units Ex-Taq DNA polymerase (Takara, Japan) were mixed to perform the first PCR. The reaction proceeded to an initial 94 ° C. 5 minute denaturation step, PCR 30 cycles (94 ° C. 1 minute-53 ° C. 1 minute 30 seconds-72 ° C. 2 minutes 10 seconds) and a final 72 ° C. 10 minute extension step. . The PCR reaction product was diluted 20-fold and the nested PCR reaction was carried out with 1 μl. 0.2 μM primers (POX1-P6, 5′-GTT CTA GAG CTA TAC ACG-3 ′; POX1-P8, 5′-AAG GTA CCG GAA GTC GCC AAG-3 ′), 0.2 mM dNTPs in 50 μl of reaction solution and 2.5 units were mixed with Ex-Taq DNA polymerase (Takara, Japan). The reaction was followed by 26 cycles of PCR (94 ° C. 1 minute-59 ° C. 1 minute) after the initial 94 ° C. 5 minute denaturation step and PCR 4 cycles (94 ° C. 1 minute-42 ° C. 1 minute 30 seconds-72 ° C. 2 minutes 10 seconds). 30 seconds-72 ° C. 2 minutes 10 seconds) and a final 72 ° C. 10 minute extension step.

DNA sequencing

Both strands of the PCR amplified promoter and cDNA were sequenced via didioxynucleotide chain termination (Sanger et al., 1977) with an ABI-PRISM auto sequencer (Perkin-Elmer, USA). Sequences were compared to sequences in the NCBI database using the Blast program (Altschul et al., 1990) and nucleotide / amino acid sequences were compared using ClustalX (Thomspson et al., 1997) and GeneDoc (Nicholas et al., 1997) software. Analyzed.

Rice Transformation Vector Construct

Nested PCR reaction products of the OsPOX1 promoter were purified using PCR clean UP-M ^™ kit (Viogene, USA) and subjected to restriction enzyme treatment with Xba I and Kpn I. PSK228 was prepared by ligation of a 1.8 kb OsPOX1 promoter to a promoter-deficient plant transformation binary vector comprising a GUS reporter gene. The vector was transferred to the Agrobacterium fungus LBA4404 via the freeze-thaw method (An et al., 1988).

Rice transformation

Rice was transformed through the Agrobacterium -mediated cocultivation method (Hiei et al., 1994; Lee et al., 1999). All transformed plants were incubated in 40 mg L- ¹ hygromycin B-containing medium and then in greenhouses. To confirm that the plants were transformed, GUS was PCR amplified using primers (GUS5 ', 5'-CTA CAC CAC GCC GAA CAC CT-3'; GUS3 ', 5'- GAC GCA CAG CAC ATC AAA GA-3 '). After harvesting the seeds, 15 seeds were sown in each furrow. Seeds of the T2 generation were germinated on day 3 in Petri dishes containing SDW (Sterilized Distilled Water) and then on day 4 in medium containing Hygrosin B medium. After an additional week, hygromycin resistant plants were transferred to Magenta-box with non-hygromycin medium. The seedling plants were then transplanted into the greenhouse for GUS analysis.

GUS  analysis

GUS analysis was performed in the same manner as in Jefferson et al. (1997). 5-10 days prior to emergence, the various tissues of the transgenic plants were washed with 0.1% (w / v) 5-bromo-4-chloro-3-indoryl-β-D-glucuronide (X-Gluc), 10 Under light blocking at 37 ° C. in 100 mM sodium phosphate buffer (pH 7.0) containing mMethylenediaminetetraacetic acid (EDTA), 0.5% (v / v) Triton X-100, 5 mM potassium ferrocyanide and 10% methylol Incubated for -13 hours. In order to improve the permeability of the substrate, the sample was placed under vacuum in the X-Gluc solution for 2 minutes. After staining, tissues were washed several times with 50 ° C. ethanol and observed under a dark field illumination (Leica, Germany).

result

OsPOX1 cDNA Separation and analysis

To study the cold-response mechanism of rice in the pre-flowering stage, cold-reactive genes were isolated using mRNA fractionation display technology (Kim et al., 2007). For further analysis, the ddOs319 fragment, a transcript that exhibits flower-specific cold-induced expression, was selected for further analysis (Kim et al., 2007). As a result of NCBI database search, ddOs319 is a gene which comprises four types of exons of chromosome 1 (FIG. 1A). Since ddOs319 is a partial clone of 0.5 kb, nested PCR was performed using the primer of ddOs319 to isolate the whole cDNA to obtain the entire sequence. Nested PCR fragment 1.2 kb was cloned to confirm the entire sequence of the selected clone Os319 (FIG. 1B). Referred Os319 protein has an open reading frame (ORF) of 335 amino acids, rice, Arabidopsis, asparagus (accession No. BAA94962), tobacco (accession No. AAK52085) and soybean (accession No. ACG44598.1). ), 77-98% similarity with peroxidase. Maize peroxidase 72 (Accession No. ACG44598.1) showed 94% similarity to Os319 protein. Structural analysis of the Os319 protein revealed a 21 amino acid N-terminal signal peptide, a distal heme domain, a central conserved domain, a proximal heme-binding domain, 8 conserved Cys residues, and an acid / base catalyst. It was expected to have 3 His residues involved in action and heme stabilization (Welinder, 1992). On the basis of homology with other peroxedases and the presence of conserved domains within the Os319 protein, OsPOX1 ( O ryza s ativa p er o xidase gene 1 ). The molecular mass of OsPOX1 protein is 36 kDa and the pI value is expected to be 7.9. The location of OsPOX1 protein was expected to be apoplast according to PSORT ( http://psort.hgc.jp/form.html ). Northern analysis confirmed that OsPOX1 cDNA exhibits cold-reactivity in flowers (FIG. 2A). However, OsPOX1 in seedlings was invariably expressed following the diversified stressor treatment shown in FIG. 2B.

OsPOX1  Isolation and Analysis of Promoter Sequences

Since OsPOX1 is up-regulated in cold-treated flowers, the promoter portion of OsPOX1 was cloned for further analysis. 1.8 kb of nested PCR fragment was sequenced (FIG. 3A). The putative CAAT box and TATA box sequences were expected to be located at -265 and -240, respectively, of the transcription initiation point, and other putative regulatory motifs are shown in Table 1 (FIG. 3A). Four types of ABA-responsive element (ABRE), cold-reactive dehydration-responsive element / C-repeat and three pollen-specific regulatory elements were found in the promoter (Dolferus et al., 1994, Baker et al., 1994, Yamaguchi-Shinozaki and Shinozaki, 1994, Weterings et al., 1995). ^A of Table 1, Guess observed in the 1,787 bp upstream of the translation initiation point of the OsPOX1 St. cis (cis) - and element, ^b is A conservative nucleotide sequence reported from a promoter of another plant, ^c is Refers to a sequence found in reverse orientation compared to the orientation of OsPOX1 ORF.

ABA-response element (ACGT) ^b Pollen-specific regulatory elements (AAATGA) DRE / CRT (CCGAC) ^b
AC ACGT AGC
(-1774 to -1766)
TA ACGT TTC
(-1297 to -1289)
TG ACGT TTG
(-1132 to -1124)
TA ACGT TCT
(-631 to -623) TT AAATGA GC
(-1255 to -1246)
AA AAATGA AG ^c
(-403 to -394)
TC AAATGA CT
(-335 to -326) TCT CCGAC CAT
(-1434 to -1424)

Structure of Rice Transformation Vector and Preparation of Transgenic Plant

To understand the regulatory role of the OsPOX1 promoter, a 1.8 kb OsPOX1 promoter portion (P _OsPOX1 ) was fused with the GUS gene to prepare a plant transformation binary vector pSK228. Fusion genes were introduced into rice blastoccal callus by Agrobacterium -mediated transformation (Hiei et al., 1994, Lee et al., 1999). After regeneration, transgenic plants were identified by PCR using primers of the GUS gene. A total of 14 primary transgenic plants were cultured from paddy to maturity and seed harvested. After amplification of progeny plants, 100% hygromycin resistance of T2 plants was confirmed by GUS analysis.

P _OsPOX1 - GUS   Phosphorus drilling of transgenic rice GUS  analysis

Histochemical GUS analysis of transgenic rice grown under normal growth or cold-stress conditions was performed. 100% germination was shown for hygromycin media in three independent lines of T2 generation. In germinating seeds, GUS expression was weakly detected mainly in the vascular system of germinating roots and slightly induced by cold stress (FIG. 4A). In seedlings, GUS expression was detected in lamina joints and veins (FIG. 4C). Cold-stress treatment showed no detectable change in GUS expression. The inventors treated the germinating seeds and seedlings with other stresses, such as salt and drying, but found no GUS expression. Little GUS activity was observed in the shoots and roots of the growth stage.

During flowering, GUS analysis of the vesicle development stage was performed from the initial microspore stage to the vacuolated pollen stage. GUS activity was detected in reproductive tissue at the initial vesicle stage (FIG. 4D). However, although weak expression was detected in the veins of the flowers, little GUS activity was detected in the reproductive tissues of the pollen phase with late vesicles and vacuoles. This refers to developmental specific expression of OsPOX1.

In addition, transgenic plants were subjected to cold stress to test the effect of stress on the activity of the OsPOX1 promoter. Whole transformed plants before flowering were transplanted into a chamber for plant growth and subjected to GUS analysis after treatment at 12 ° C. for 4 days. Interestingly, when treated with cold stress, the flowers showed enhanced GUS expression in the reproductive tissues and vascular bundles of palea and lemma (FIG. 4F). To investigate which tissues exhibit cold-induced GUS expression, flowers were cross-sectioned to examine GUS staining. As shown in Fig. 4g and 4i, the upper and lower portions of the flower, respectively, GUS expression was detected mainly in the leaf veins. In the middle of the flower, GUS was detected in the endothecium, the vesicles, and the vascular bundle of anther.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

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<110> Industry-University Cooperation Foundation Sogang University <120> Stress Inducible Transgenic plants <130> PN110612 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 1790 <212> DNA <213> OsPOX1 promoter <400> 1 gttctagagc tatacacgta gcatgcatac ctgtgactgt ctctgtgagc ctgtgactgt 60 aacaaccgcc atgctgcttc cttcaagaaa cggccactga tatgtgcttc acagaagaac 120 aaggctgccg gtgacaaagc cttggaacag ttgaagaacc tgctttgtga caagaatgta 180 ggccatgcaa gacacctaac gccgaatcat catcacgcac cgtgtgttga gagttggtac 240 atggcacatc tcgtttgacc tgatcgaccg atcccccatc tccatgcatg tgttagctag 300 gatatcagca cctgtgttgt acaacaaaat gcaccatggt actacacatc ctctctccga 360 ccattgatga gccatcagaa ctcccagttg atttgatcgc tgcaacaaca tacaaaaggt 420 aacttagttt ggcagtacaa ttatacaagg acaacagcat atgaaaaatg ttggatatta 480 tcctttgata taacgtttct actaataagt atacggaatg tagcatcata ctttaaatga 540 gccatcaaaa atgccagttt gatcgctgca ataacataga aaaggtaact tagtttggtg 600 gtatctacaa ggacaacaga atatatgaag aaaccttaga gattatactg tgatatgacg 660 tttgtagtaa taaatatacg gcaatgtagc atcatacttt tgatccccaa agacagggat 720 acgaatccaa gcttgatgac caccaacaat agaaaacatt catgtgaata gtccagcata 780 cataaggtat tgcattaaac aaagccgtat gatctgtagt gacacttgtg tataaccaaa 840 gtagtaggat taaaacggaa gcaaaacctt acttttgtct acgcttttgt gctttgtcag 900 tttgatttca cacatgatgt gcaaattatc atgtgttttg tgaggttaaa ttttggatat 960 ggtccaggga atatatatat atacatacta gtacaacctg tcaggtgtgg gcctactatg 1020 aggcaaaaag gttaaagtga gaggaacgac tgatatatct ttgtgcatat gcattgcctc 1080 attatggtaa tttaaaacaa catctctaac gttctgatac aggagggtat aatataatat 1140 tatataatcc atcaaaggtt tcgataatga gtagaaagat aatgtgtagt gtacaggggc 1200 agattatgct gccttctagt ttttcatagt acaagcagtt atatgaatat ttaattggaa 1260 tgcttatttt taagtagcat tttctgtttc atcagaccat tttatcctct ctccaactgc 1320 taagcaatat tgagcttcat ttttgagaga ttatcactta tcactccatc tacacttgtt 1380 gacaatgaca agcattaaat cttcaaatga ctttgctaaa attaaagccc caaacaaaac 1440 aataagacga ccgaaaagcc ttgcaagcaa gttggagtta ctattagctg ataattgatg 1500 gaattactga agcagttgag cacaatgcgt agctagctga cttattttat acgcatcacc 1560 ctaacaaccc aagctgccct gccgcgtctc atctcactgt cctctgctcc aaaccaccct 1620 tcctacatgc acccccccac aggtgctata tatgccaccc catgcccgcc tcttctcgcc 1680 acccacaacc aagagaagta gaaacaaaca gagagcaatt ctcttctcct acctagcaac 1740 ctagtgcagt gcagtgcagt gcagcgaagg tttacttggc gacttccatg 1790 <210> 2 <211> 1194 <212> DNA <213> OsPOX1 <220> <221> CDS <222> (25) .. (1029) <400> 2 gcgaaggttt acttggcgac ttcc atg ggt tgc ctg ttg atg ctc tgc 48                                  Met Gly Cys Leu Leu Met Leu Cys                                    1 5 ttg gtt tct ccc ctc ctc ctc gcc acc tct gtc cac ggc aac ccg tgg 96 Leu Val Ser Pro Leu Leu Leu Ala Thr Ser Val His Gly Asn Pro Trp      10 15 20 tat ggg tat ggg tat ggc ttg ttc ccg cag ttc tac gac cac tcg tgc 144 Tyr Gly Tyr Gly Tyr Gly Leu Phe Pro Gln Phe Tyr Asp His Ser Cys  25 30 35 40 ccc aag gcg aag gag atc gtg cag tcc atc gta gca cag gcg gtg gcc 192 Pro Lys Ala Lys Glu Ile Val Gln Ser Ile Val Ala Gln Ala Val Ala                  45 50 55 agg gag acc agg atg gcg gca tcc ttg gtc agg ctg cat ttt cat gac 240 Arg Glu Thr Arg Met Ala Ala Ser Leu Val Arg Leu His Phe His Asp              60 65 70 tgc ttt gtc aag ggg tgt gac gcg tct gtg ctc ctg gac aac agc acc 288 Cys Phe Val Lys Gly Cys Asp Ala Ser Val Leu Leu Asp Asn Ser Thr          75 80 85 acc atc atc agt gag aag ggg tca aac cct aac atg aac tcc ctc agg 336 Thr Ile Ile Ser Glu Lys Gly Ser Asn Pro Asn Met Asn Ser Leu Arg      90 95 100 ggt ttc gag gtc gtc gac gag atc aag gcc gcc ctc gaa gca gct tgc 384 Gly Phe Glu Val Val Asp Glu Ile Lys Ala Ala Leu Glu Ala Ala Cys 105 110 115 120 ccc ggc acc gtc tcc tgc gcc gac ata ctc gcc ctc gct gca cgc gat 432 Pro Gly Thr Val Ser Cys Ala Asp Ile Leu Ala Leu Ala Ala Arg Asp                 125 130 135 tcc act gtc ctc gtt ggt ggc ccg tac tgg gat gtg cca ctt ggc cgg 480 Ser Thr Val Leu Val Gly Gly Pro Tyr Trp Asp Val Pro Leu Gly Arg             140 145 150 agg gac tca ctg ggt gcc agc atc cag ggc tcc aac aac gac atc cca 528 Arg Asp Ser Leu Gly Ala Ser Ile Gln Gly Ser Asn Asn Asp Ile Pro         155 160 165 gct ccc aac aac acc ctc ccc acc atc atc acc aag ttc aag cgc cag 576 Ala Pro Asn Asn Thr Leu Pro Thr Ile Ile Thr Lys Phe Lys Arg Gln     170 175 180 ggc ctt aac atc gcc gac gtc gta gcc ctc tca ggt ggc cac acc att 624 Gly Leu Asn Ile Ala Asp Val Val Ala Leu Ser Gly Gly His Thr Ile 185 190 195 200 ggc atg tct cgg tgc acc agc ttc cgt cag agg ctc tac aat cag agc 672 Gly Met Ser Arg Cys Thr Ser Phe Arg Gln Arg Leu Tyr Asn Gln Ser                 205 210 215 ggc aat ggc atg gct gac tac aca ctg gat gtg tcc tac gcg gca cag 720 Gly Asn Gly Met Ala Asp Tyr Thr Leu Asp Val Ser Tyr Ala Ala Gln             220 225 230 ctg agg cag gga tgc ccc cgt tct ggt ggt gac aac aac ctc ttc ccg 768 Leu Arg Gln Gly Cys Pro Arg Ser Gly Gly Asp Asn Asn Leu Phe Pro         235 240 245 cta gac ttt gtc agc ccc gca aag ttc gac aac ttc tac ttc aag aac 816 Leu Asp Phe Val Ser Pro Ala Lys Phe Asp Asn Phe Tyr Phe Lys Asn     250 255 260 atc ctg tct ggc aag ggc ctt ctc agc tct gat cag gtc ctg ctt acc 864 Ile Leu Ser Gly Lys Gly Leu Leu Ser Ser Asp Gln Val Leu Leu Thr 265 270 275 280 aag agc gct gaa aca gcg gcg ctc gtg aag gcg tat gct gat gat gtc 912 Lys Ser Ala Glu Thr Ala Ala Leu Val Lys Ala Tyr Ala Asp Asp Val                 285 290 295 aac ctc ttc ttc aag cac ttc gca cag tca atg gtg aat atg ggc aac 960 Asn Leu Phe Phe Lys His Phe Ala Gln Ser Met Val Asn Met Gly Asn             300 305 310 atc tcg cct ctg act gga tca cag ggg gag atc agg aag aac tgc agg 1008 Ile Ser Pro Leu Thr Gly Ser Gln Gly Glu Ile Arg Lys Asn Cys Arg         315 320 325 agg ctc aac aac tac tac cac t gaggttgtct tgtgtgctga gttttacaag 1060 Arg Leu Asn Asn Tyr Tyr His     330 335 gtggtggtag tggcatgttt tgtttaaata agtaaagctg gttgtaatgc gtcaaggctg 1120 tttgatcatt tgtgttattg tgattgcgtt gcaaaacgtg tatgtttaat aattcaatat 1180 ataaaatgag ttca 1194 <210> 4 <211> 351 <212> PRT <213> OsPOX1 <400> 4 Met Glu Thr Gly Cys Leu Leu Met Glu Thr Leu Cys Leu Val Ser Pro   1 5 10 15 Leu Leu Leu Ala Thr Ser Val His Gly Asn Pro Trp Tyr Gly Tyr Gly              20 25 30 Tyr Gly Leu Phe Pro Gln Phe Tyr Asp His Ser Cys Pro Lys Ala Lys          35 40 45 Glu Ile Val Gln Ser Ile Val Ala Gln Ala Val Ala Arg Glu Thr Arg      50 55 60 Met Glu Thr Ala Ala Ser Leu Val Arg Leu His Phe His Asp Cys Phe  65 70 75 80 Val Lys Gly Cys Asp Ala Ser Val Leu Leu Asp Asn Ser Thr Thr Ile                  85 90 95 Ile Ser Glu Lys Gly Ser Asn Pro Asn Met Glu Thr Asn Ser Leu Arg             100 105 110 Gly Phe Glu Val Val Asp Glu Ile Lys Ala Ala Leu Glu Ala Ala Cys         115 120 125 Pro Gly Thr Val Ser Cys Ala Asp Ile Leu Ala Leu Ala Ala Arg Asp     130 135 140 Ser Thr Val Leu Val Gly Gly Pro Tyr Trp Asp Val Pro Leu Gly Arg 145 150 155 160 Arg Asp Ser Leu Gly Ala Ser Ile Gln Gly Ser Asn Asn Asp Ile Pro                 165 170 175 Ala Pro Asn Asn Thr Leu Pro Thr Ile Ile Thr Lys Phe Lys Arg Gln             180 185 190 Gly Leu Asn Ile Ala Asp Val Val Ala Leu Ser Gly Gly His Thr Ile         195 200 205 Gly Met Glu Thr Ser Arg Cys Thr Ser Phe Arg Gln Arg Leu Tyr Asn     210 215 220 Gln Ser Gly Asn Gly Met Glu Thr Ala Asp Tyr Thr Leu Asp Val Ser 225 230 235 240 Tyr Ala Ala Gln Leu Arg Gln Gly Cys Pro Arg Ser Gly Gly Asp Asn                 245 250 255 Asn Leu Phe Pro Leu Asp Phe Val Ser Pro Ala Lys Phe Asp Asn Phe             260 265 270 Tyr Phe Lys Asn Ile Leu Ser Gly Lys Gly Leu Leu Ser Ser Asp Gln         275 280 285 Val Leu Leu Thr Lys Ser Ala Glu Thr Ala Ala Leu Val Lys Ala Tyr     290 295 300 Ala Asp Asp Val Asn Leu Phe Phe Lys His Phe Ala Gln Ser Met Glu 305 310 315 320 Thr Val Asn Met Glu Thr Gly Asn Ile Ser Pro Leu Thr Gly Ser Gln                 325 330 335 Gly Glu Ile Arg Lys Asn Cys Arg Arg Leu Asn Asn Tyr Tyr His             340 345 350

Claims (9)

A stress-inducible promoter specifically expressing in a flower of a plant consisting of the nucleotide sequence described in SEQ ID NO: 1.
A vector comprising (a) the promoter of claim 1 and (b) a foreign gene operatively linked to the promoter.
Transgenic plant transformed by the vector of claim 2.
A method for producing a plant transformant comprising the following steps of transforming a plant with the vector of claim 2 to express the foreign gene in the plant:
(a) transforming a plant cell or plant tissue with the vector of claim 2;
(b) screening the transformed plant cell or plant tissue; And
(c) regenerating the plant from the transformed plant cell or plant tissue to obtain a transformed plant.
A transgenic plant having improved resistance to abiotic stress produced by the method of claim 4.
Rice has the amino acid sequence shown in Sequence Listing SEQ ID NO: 4 (oryza satina ) stress-induced OsPOX1 protein.
A nucleic acid molecule encoding OsPOX1 having the amino acid sequence set forth in SEQ ID NO: 4.
8. The nucleic acid molecule of claim 7, wherein the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO: 2.
Recombinant vector comprising the nucleic acid molecule of claim 7 or 8.

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