KR20140040437A - Calcineurin b-like interacting protein kinase 15 promoter and use of the same - Google Patents

Abstract

The present invention relates to a calcineurin B-like interacting protein kinase 15 promoter and its use. According to one embodiment of the present invention, provided is a transformed plant which has resistance using a promoter and an expression cassette containing genes encoding CIPK 15 protein controlled by the promoter. According to another embodiment of the present invention, provided is a transformed plant in which CIPK 15 protein has nucleotide sequence of SEQ ID NO: 2.

Description

Calcineurin B-like interacting protein kinase 15 Promoter and use of the same}

The present invention relates to a calcineurin B-like interacting protein kinase 15 promoter and use thereof.

Oryza sativa is characterized by the ability to anaerobicly recruit endosperm to support energy metabolism of the non- photosynthetic embryo axis during anaerobic germination and post-germination growth (Guglielminetti L, Perata P, Alpi A. Plant Physiol 1995a; 108: 735-41.Guglielminetti L, Yamaguchi J, Perata P, Alpi A. Plant Physiol 1995b; 109: 1069-76). However, in order for the embryonic axis of submerged rice seeds to overcome the low energy efficiency of fermentation, anaerobic mobilization that produces a sufficient amount of sugar is crucial. Other cereals, such as barley and wheat, are not capable of anaerobic germination and degradation of endocrine stocks under immersion. Germination and post-germination growth under anaerobic conditions seem to reflect the convergence of many physiological and biochemical processes. However, the external supply of glucose or sugar suggests that anaerobic storage flow plays an essential role in the growth of oxygen-deficient grains such as rice and supports anaerobic germination of these grains (Perata P, Pozuetaromero J, Akazawa T, Yamaguchi). J. Planta 1992; 188: 611-8).

Among several hydrolase enzymes, L-amylase plays the most important role in the flow of endosperm stocks. L-amylase enzymes break down starch particles to produce glucose polymers in the form of amylose and amylopectin, which in turn are broken down into water-soluble sugars by other hydrolases. Interestingly, the expression of this L-amylase gene is increased and maintained under oxygen deficiency; Its anaerobic contribution to total L-amylase activity suggests that it plays an important role in oxygen deficiency storage flow (Hwang YS, Thomas BR, Rodriguez RL. Plant Mol Biol 1999; 40: 911-20).

Many studies on Amy3D sugar regulation have revealed sugar reactive cis-element (SRE), MybS1 (a transcriptional activator that binds to SRE), and SnRK1A (positive regulator for sucrose non-fermenting 1-related protein kinase; MybS1). (Hwang YS, Karrer EE, Thomas BR, Chen L, Rodriguez RL. Plant Mol Biol 1998; 36: 331-41; Lu CA, Lim EK, Yu SM. J Biol Chem 1998; 273: 10120-31; Lu CA, Ho TH, Ho SL, Yu SM.Plant Cell 2002; 14: 1963-80; Lu CA, Lin CC, Lee KW, Chen JL, Huang LF, Ho SL, et al.Plant Cell 2007; 19: 2484-99) .

As an internal secondary messenger, calcium ions mediate various cellular responses to various stimuli in plants and regulate a wide range of physiological processes. "Ca2 + signature", a combination of spatial and temporal changes in cellular Ca2 + concentration produced in response to a specific signal, is interpreted by an array of Ca2 + sensors (Boudsocq M, Sheen J. Stress signaling II: calcium sensing and signaling. Pareek A, Sopory SK, Bohner HJ, Govindjee, editors.Abiotic stress adaptation in plants: physiological molecular and genomic foundation. Dordrecht: Springer; 2010. p. 75-90). Calcineurin B-like (CBL) protein is a novel family of Ca2 + sensors similar to the neuronal Ca2 + sensor (NCS) in animals and a regulated beta-subunit calcineurin (Liu J, Zhu JK. Science 1998; 280: 1943-5). ). CBLs specifically target a novel class of protein kinases, CIPKs, that relay Ca2 + signals at the molecular level (Luan S. Trends Plant Sci 2009; 14: 37-42). Ca2 + has been proposed as an internal messenger of low oxygen in plants since plasma Ca2 + concentration shows a transient increase in submerged corn roots or in response to oxygen deficiency or hypoxic conditions in Arabidopsis (Subbaiah CC, Zhang J, Sachs MM. Plant Physiol 1994 105: 369-76). Glycolytic enzymes and alcohol dehydrogenases are also activated in Ca and Arabidopsis by Ca2 + signaling under low oxygen conditions (Subbaiah CC, Zhang J, Sachs MM. Plant Physiol 1994; 105: 369-76).

Previous studies have shown that the glycemic control of Amy3D is under the control of hexokinase (Umemura T, Perata P, Futsuhara Y, Yamaguchi J. Planta 1998; 204: 420-8), and is dependent on SnRK1 activity (Lu CA, Lin CC, Lee KW, Chen JL, Huang LF, Ho SL, et al. Plant Cell 2007; 19: 2484-99).

As a related prior art, Korean Patent Publication No. 1020080101883 discloses a controlled plant size, nutritional growth, number of organs, plant structure, growth rate, seedling vitality, growth rate, fruit and seed yield, powder and / or biomass characteristics in a plant. It is described with respect to this provideable isolated nucleic acid molecule and its corresponding encoded polypeptide.

In another related prior patent, Korean Patent Publication No. 1020080052606 provides a plant that is resistant to glyphosate and one or more ALS inhibitors and a method of using the same. Glyphosate / ALS inhibitor resistant plants are described as comprising polynucleotides encoding polypeptides that confer resistance to glyphosate and polynucleotides encoding ALS inhibitor resistant polypeptides.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a new promoter regulated by sugar and anoxic environment.

Another object of the present invention is to provide a transgenic plant having resistance to sugar and anoxic environments.

Another object of the present invention is to provide a method capable of screening sugar and anoxic environments of plants.

In order to achieve the above object, the present invention provides a CIPK [calcineurin-like interaction protein kinase] 15 promoter regulated by a sugar and anoxic environment consisting of the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequence complementary to the nucleotide sequence. 2.5 kB).

In another aspect, the present invention is the promoter; And an expression cassette comprising a gene encoding a CIPK [Calcineurin B-like (CBL) interacting protein kinase] 15 protein controlled by the promoter. Provide plants.

In one embodiment of the present invention, the CIPK 15 gene is preferably composed of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

The present invention also provides a method for producing a transgenic plant having resistance in a sugar deficient and oxygen deficient environment comprising the following steps:

(a) a step of obtaining a recombinant plant cell using the promoter of the present invention and an expression cassette comprising a gene encoding a protein of CIPK [Calcineurin B-like (CBL) interacting protein kinase] 15 controlled by the promoter ; And

(b) regenerating the plant cells in the plant.

In another aspect, the present invention provides an expression cassette for the production of a transgenic plant resistant in a sugar deficient and oxygen deficient environment comprising the following components:

(a) the promoter of claim 1; And

(b) a gene encoding a CIPK [Calcineurin B-like (CBL) interacting protein kinase] 15 protein controlled by said promoter.

The present invention also provides a method for screening sugar and oxygen deficiency states of plants comprising the following processes:

(a) an expression cassette comprising the promoter of claim 1 and a gene encoding the CIPK [Calcineurin B-like (CBL) interacting protein kinase] 15 controlled by said promoter and a gene capable of visualizing the expression of said gene Obtaining a recombinant plant cell using; And

(b) regenerating the plant cells in the plant.

In one embodiment of the present invention, a gene capable of visualizing the expression of the gene is preferably anthocyanin synthesis-related gene, but is not limited thereto.

As a vector for introducing into the plant for which the expression cassette of the present invention is intended, a vector for plant transformation is useful. The plant vector is not particularly limited as long as it has the ability to express the gene in plant cells and produce the protein. Examples of the plant vector include pBI221 and pBI121 (manufactured by Clontech) and vectors derived therefrom. have. In particular, in the transformation of monocots, pIG121Hm, pTOK233 (Heiei et al., Plant J., 6, 271-282 (1994)), pSB424 (Komari et al., Plant J., 10, 165-174 (1996)) , Superbinary vector pSB21 and vectors derived therefrom are exemplified.

The plant transformation vector preferably contains at least a promoter, a translation initiation codon, a desired gene (a DNA sequence of the present invention or a part thereof), a translation initiation codon and a terminator. The DNA encoding the signal peptide, the enhancer sequence, the untranslated regions on the 5 'and 3' sides of the desired gene, the selection marker region, and the like may be appropriately included. Examples of marker genes include luciferase gene, β-galactosidase, β-glucuronidase (tetracycline, ampicillin or kanamycin or neomycin, hygromycin or spectinomycin, etc.). GUS), green fluorescein protein (GFP), (beta) -lactamase, chloramphenicol acetyl transferase (CAT), etc. are mentioned.

As a gene introduction method to plants, a method using Agrobacterium (Horsch et al., Science, 227, 129 (1985), Hiei et al .: Plant J., 6, 271-282 (1994)), leaf disk method (Horsch et al. : Science, 227, 1229-1231 (1985)), electroporation method (Fromm et al .: Nature, 319, 791 (1986)), PEG method (Paszkowski et al .: EMBO J., 3, 2717 (1984)), micro Injection methods (Crossway et al .: Mol. Gen. Genet., 202, 179 (1986)), micro water collision methods (McCabe et al .: Bio / Technology, 6, 923 (1998)), and the like. If it is a method of introducing a gene, it will not specifically limit. In these introduction methods, Preferably, a vector is transferred into Acrobacterium using a conjugation operation, etc., and this Acrobacterium is infected by a plant. Methods for infection are also well known to those skilled in the art. For example, how to injure part of a plant, infect bacteria there, infect germ tissue of a plant (including immature embryos), infect a callus, pro There is a method of coculturing toplast and bacteria, or a method of culturing small pieces of leaf tissue with bacteria (leaf disc method).

The obtained transformed cells can be selected from other cells depending on whether the appropriate marker is used as an index or whether the desired trait is expressed. By further transforming the transformed cells using conventional techniques, the transformed plant of interest can be obtained.

Analysis of the obtained transformant can be performed using various methods well known to those skilled in the art. For example, an oligonucleotide primer can be synthesized based on the DNA sequence of the introduced gene, and the chromosomal DNA of the transformed plant can be analyzed by PCR using the oligonucleotide primer. In addition, it can be analyzed by the presence or absence of expression of mRNA and protein corresponding to the introduced gene. In addition, the appearance of plants (e.g., the presence or absence of local necrotic plaques, or the size and number of local necrotic plaques, if a gene encoding a protein capable of producing local necrotic plaques), and disease resistance (e.g., For example, the presence or absence of resistance in contact with the pathogen, or the like) can be analyzed.

Hereinafter, the present invention will be described in detail.

CIPK15  And Amy3D  Comparison of Time-dependent Glycoregulation Patterns Between

It is assumed that the CIPK15 gene will be an upstream positive regulator for glucose-regulated Amy3D expression and will be under its own regulation of glycemic control, and the inventors have incubated rice embryos in medium with glucose or mannitol for up to 52 hours, while CIPK15 and Amy3D Kinetics of induction were compared. As shown in FIG. 1, CIPK15 transcript could be detected in 18 hours starved rice embryos; Their level peaked at 36 hours and remained unchanged up to 52 hours. MRNA transcripts of Amy3D not detected in cultures cultured for less than 18 hours reached maximum levels at 36 hours and then decreased. Interestingly, the CIPK15 gene exhibited a distinctive expression pattern that differed from the supposed role as an upstream positive regulator for the Amy3D gene. One of these examples, although the sugar inhibition of CIPK15 is leaky, the expression of the Amy3D gene showed stringent glycemic control. In addition, despite sufficient expression of the CIPK15 gene, Amy3D expression was drastically reduced in starved rice embryos for two days, indicating a significant discrimination between the expression patterns of the two genes.

CIPK15  Expression Hexokinase Mediated  Party conditioning

Several biochemical and genetic studies have shown that hexokinase triggers sugar signaling in many plants as a sugar sensor. Existing studies using transient expression systems have shown that hexokinase also mediates Amy3D sugar regulation (Umemura T, Perata P, Futsuhara Y, Yamaguchi J. Planta 1998; 204: 420-8).

Therefore, the present inventors conducted an experiment to test whether hexokinase-mediated sugar-regulated expression of CIPK15, a positive regulator for Amy3D. For this study, we first examined the effects of several sugars that triggered the inhibition of CIPK15 expression. FIG. 2 shows CIPK15 transcript levels from rice embryos cultured in media containing several hexasaccharides or glucose derivatives. Analysis of CIPK15 expression via RNA gel blots (FIGS. 2A and C) and real-time quantitative PCR (FIGS. 2B and D) shows that all hexoses that can be phosphorylated on hexokinase (glucose, fructose, galactose, mannose, and 2-deoxyglucose) successfully inhibit the expression of CIPK15 when compared to mannitol. Effective inhibition by mannose and 2-deoxyglucose (but not by 3-O-methylglucose or 6-deoxyglucose) indicates that the phosphorylation step by hexokinase is sufficient to trigger a series of events leading to suppressed CIPK15 expression.

Glucosamine, a hexokinase inhibitor, has been shown to effectively inhibit hexokinase mediated glucose signaling (Umemura T, Perata P, Futsuhara Y, Yamaguchi J. Planta 1998; 204: 420-8). In order to identify the important role of hexokinase in glucose suppression of the CIPK15 gene, we tested whether glucosamine could also inhibit the glycemic regulation of the CIPK15 gene. As shown in FIG. 3, the transcript level of CIPK15 in glucose-treated embryos was reduced to ˜15% of that of the mannitol control, whereas the CIPK15 transcript level in glucose-cultured embryos was that of the mannitol control co-treated with glucosamine. It reached about 90%. Taken together, hexokinase is involved in the glycemic control of CIPK15 expression and is consistent with the fact that CIPK15 is an upstream positive regulator of Amy3D expression under the control of hexokinase.

Oxidative  Phosphorylated Glucose Controlled CIPK15  Suppression of expression

In Figure 4, we investigated anaerobic factors affecting glycemic control of the CIPK15 gene; We performed this investigation using several stages of metabolic inhibitors of oxidative phosphorylation, such as inhibitors of electron transport systems (KCN), uncouplers (DNP and dicoumarol), and ATP synthase inhibitors (DCCD and oligomycin A) in the cytochrome pathway. . All metabolic inhibitors tested effectively destroyed the glucose imposed inhibition of CIPK15 expression. Since all these metabolic inhibitors inhibit ATP synthesis in cells, a decrease in cellular energy state seems to negatively affect sugar regulation of CIPK15 expression. Taken together, cellular energy states can be involved in hexokinase-mediated sugar regulation and crosstalk.

In the transgenic cell line CIPK15  Glucose Regulation of Promoter Activity

As a first step to elucidate the molecular mechanisms involved in sugar-regulated changes in CIPK15 transcription, we investigated the activity of CIPK15 promoters isolated in the presence and absence of sugars. We constructed pCAMBIA-CIPK15 plasmid (FIG. 5A) comprising the CIPK15 promoter-driven GUS gene and used it for Agrobacterium-mediated transformation to produce a transformed rice cell line with the GUS reporter gene under the control of the CIPK15 promoter. . Analysis of GUS enzyme activity was performed on sugar starved or glucose treated transfected cells to investigate whether CIPK15 at the 5'-flanking site is capable of glycemic control. Fluorescence and histochemical analysis showed that GUS enzyme activity was significantly higher in glucose starved cells compared to glucose treated cells (FIGS. 5B and C, respectively). Thus the transcriptional activity of the CIPK15 promoter was markedly inhibited in the presence of glucose, while induced under glucose starved conditions. Interestingly, cells cultured in glucose containing media showed minimal levels of GUS enzyme activity, indicating that CIPK15 promoter activity was not completely inhibited even in the presence of sugar. In addition, histochemical staining confirmed very weak GUS expression in cells under starved conditions (data not shown). These results in GUS transformed cells are consistent with the results of the RNA gel blot analysis shown in FIG. 1, which indicates that the glucose inhibition of the CIPK15 gene is not as strong as that of Amy3D, and thus CIPK15 promoter activity (4-5 fold in response to sugar). ) Explain the relatively small differences. This GUS enzyme analysis indicates that the 2548-bp segment in CIPK15 at the 5'-flanking site may be mediated by glycemic control of the promoter, indicating that cis elements involved in glycemic control should be within this site. In addition to transcriptional regulation, post-transcriptional regulation may contribute to glycemic control of gene expression. For example, the 3'-untranslated region of the Amy3D gene is important for reducing prestitial stability (Chan MT, Yu SM. Proc Natl Acad Sci U S A 1998; 95: 6543-7). However, the 3 'UTR of the CIPK15 gene had no effect on transcript stability in the transient expression assay of the present invention (FIG. 5D).

As can be seen from the present invention, the regulation of CIPK15 expression was also mediated by hexokinase and interfered by impairment of mitochondrial ATP synthesis, which indicates that HXK-mediated sugar signaling is influenced by cellular energy state. Suggests that you can receive Therefore, the promoter of the present invention may be applied to a marker controlled by a sugar and anoxic environment or to screen such a plant state.

1 is a time-lapse analysis of sugar-regulated germination of CIPK15 and Amy3D in isolated rice embryos. Rice embryos were incubated in 60 mM glucose or mannitol for 0-52 hours. Total RNA was isolated and analyzed to determine the levels of Amy3D and CIPK15 transcripts by RNA gel block hybridization. Total RNA loading (40 μg) was determined by staining ribosomal RNA with ethidium bromide.
Figure 2 shows the effect of several sugars and glucose derivatives on CIPK15 expression. Rice embryos were cultured in a medium containing several types of hexose (top panel) or sugar derivatives (bottom panel). Transcription levels of CIPK15 were detected by RNA gel blot analysis (A and C) and real time quantitative PCR (qPCR) methods (B and D). For the latter, the ratio between CIPK15 expression and rice actin gene expression in 60 mM glucose medium was set to 1 as a control. Under other conditions the expression ratio is proportional to. Error bars represent standard deviation of the mean (n = 3).
Figure 3 shows the effect of hexokinase inhibitors on glucose-regulated CIPK15 expression. Rice embryos were incubated at 28 ° C. for 36 hours in 60 mM glucose or mannitol medium with or without 5 mM glucosamine. Levels of CIPK15 transcripts were analyzed and expressed as described in FIG. 2.
Figure 4 shows the interference of glucose-regulated CIPK15 expression by oxidative phosphorylation inhibitors. As described in rice embryos, (A) inhibitors on cytochrome pathway (1 mM KCN), (B) uncouplers (30 μM DNP; 5 μM dicoumarol), (C) ATP synthase inhibitors (75 μg / mL oligomycin A; 3 cultured for 36 hours in 60 mM glucose or mannitol medium with or without mM DCCD). Levels of CIPK15 transcripts were analyzed and expressed as described in FIG. 2.
5 shows the role of CIPK15 3 ′ UTR in glycemic control of gene expression and glycemic control of the CIPK15 promoter in transformed rice suspension cultured cells. (A) Figure 2548-bp fragment of CIPK15 5'-flanking site. (B) Fluorescence analysis of GUS activity in glucose-transformed rice cells. Transformed rice cells bearing the CIPK15 promoter driven GUS reporter gene were incubated for 24 hours in AA2 medium with 60 mM glucose or 60 mM mannitol (closed box) and used for fluorescence GUS analysis. (C) Figure showing histochemical staining of transformed rice cells in response to glucose. (D) Figure 3 shows the effect of the 3 'UTR of the CIPK15 gene on glycemic regulated gene expression. Plasmid, 35S :: RUC: NOS, served as a control and normalized firefly luciferase activity to Renilla luciferase activity from cotransfected protoplasts. Error bars represent standard deviation of the mean (n = 3).
6 is a model illustrating the interaction of sugar and energy signaling elements. In the presence of sugars, hexokinase triggers sugar signaling for CIPK15 regulation in the nucleus. The product of the hexokinase activity, glucose-6-phosphate (G6P), may block the SnRK1 reaction. High metabolic efficiency due to the mitochondrial respiration of sugars suppresses the cell's energy deficiency state. In the absence of sugars, the absence of inhibition of CIPK15 expression is induced by hexokinase, leads to the activity of Amy3D expression by MybS1, and induces transcriptional activators for the Amy3D gene. The energy deficiency state caused by sugar deficiency activates SnRK1, which coordinates the energy saving program through the SnRK1 pathway. Under stress, such as flooding and flooding, where the mitochondrial breathing of the sugar is disrupted, cells may be in an energy disorder. By sensing energy deficiency, the SnRK1 enzyme activates the SnRK pathway, which interferes with hexokinase-induced sugar signaling and allows expression of the CIPK15 gene even under conditions that are not starved. Direct and indirect associations are indicated by solid and dashed lines, respectively.

Example  1: plant material and paddy embryo treatment

Rice embryos manually isolated from whole rice seeds (Oryza sativa L. cv. Dongjin) were obtained from Hwang YS, Bethke PC, Cheong YH, Chang HS, Zhu T, Jones RL. Surface sterilization as described in Plant Physiol 2005; 138: 1347-58. About 150-200 surface sterilized embryos were placed on a single layer of 3 MM Whatman paper moistened with 10 mM calcium chloride (CaCl ₂ ) with specific compounds at the concentrations described in the growth chamber at 28 ° C. Glucosamine, 2,4-dinitrophenol (DNP), oligomycin A, N, N'-dicyclohexylcarbodiimide (DCCD), and dicoumarol (3,3'-methylene-bis [4-hydroxycoumarin]) were obtained from Sigma Aldrich (St. Louis, MO). , USA).

Example  2: Rice Suspension Cell Culture

Suspension cultured rice cells (O. sativa L. cv. Dongjin) were prepared using Huang N, Chandler J, Thomas BR, Rodriguez RL. Plant Mol Biol 1993; 23: 737-47, as established. These cells were maintained in AA2 culture medium (Thompson J, Abdullah R, Cocking E. Plant Sci 1986; 47: 123-33) and transferred 3-5 mL packed volumes of cells in 20 mL fresh AA2 medium, and Subcultured every 10 days with stirring at 150 rpm and 28 ° C. under conditions.

Example  3: RNA  Gel Blot  analysis

Total RNA isolation, preparation of [alpha- ³² P] -labeled DNA probes, and analysis of RNA gel blots are described in Park M, Yim HK, Park HG, Lim J, Kim SH, Hwang YS. It was performed as described in J Exp Bot 2010; 61: 3235-44. Probes specific for the CIPK15 gene were prepared by PCR using primer sets (Table 1). The membrane was overnight exposed on a Fuji imaging plate and detected using a PhosphorImager system (Fujifilm FLA-7000 imaging system; Fujifilm, Tokyo, Japan).

Example  4: Quantitative real time RT - PCR

First strand cDNA synthesis and real-time quantitative reverse transcription-PCR (qRT-PCR) were described by Park et al. As described in (2010). Accumulation of fluorescence PCR products was monitored using a Thermal Cycler Dice Real Time System (Takara Shuzo, Kyoto, Japan). All procedures were performed according to the manufacturer's instructions. Relative amplification of the rice actin gene was used as an internal control to normalize all data. To evaluate quantitative variation in each sample, three replicates of each sample were examined and each experiment was repeated at least twice. The gene specific primers used for quantitative PCR are listed in Table 1. When we compared the results of the real time qPCR (Tables 2B and D) with the results of the RNA gel blot analysis (FIGS. 2A and C), both showed similar results, which investigated the CIPK15 transcript levels through real time qPCR analysis. It was made.

Example  5: Plasmid  build

CIPK15 promoter :: beta-glucuronidase (GUS) construct, rice gene DNA (O. sativa L. et al.) Using the primer set of Table 1 to construct transformed cells with CIPK15 gene (2548 bp) at the 5'-flanking site. cv. Dongjin) was performed PCR amplification. To drive the GUS coding site, the CIPK15 gene of the 5'-flanking site was cloned into HindIII and BamHI restriction sites with the pCAMBIA1391Z vector (GenBank accession no. AF234312) to generate pCAMBIA-CIPK15. The firefly luciferase gene (LUC) coding site is cleaved with NheI and XbaI restriction enzymes from the pSP-luc + NF Fusion vector (Promega, Madison, WI, USA) and inserted in front of the GUS coding site of pBI221 in the XbaI site 35S: : LUC: GUS: nopaline synthase (NOS) vectors were prepared. The GUS coding site was removed by treatment with XbaI and SacI, and the product of the sticky terminus was self-ligated by blunting with T4 DNA polymerase to prepare a 35S :: LUC: NOS construct. To prepare the 35S :: LUC: CIPK15 3 'UTR construct, 35S :: LUC: GUS: NOS was treated with BamHI and SacI to remove GUS coding sites, the sticky ends of which were blunted with T4 DNA polymerase to self-lye Gated. NOS terminator was replaced with CIPK15 3 'UTR fragment flanked by BamHI and SacI; This fragment was removed from the pMD20 T vector containing the PCR-amplified 3 'UTR. Renilla luciferase (RUC) fragments were removed from pRL-TK vector (Promega) by NheI and XbaI restriction and inserted into pBI221 vector to prepare 35S :: RUC: GUS: NOS. 35S :: RUC: NOS vectors were prepared by treatment with XbaI and SacI to remove GUS coding sites, blunting sticky ends with T4 DNA polymerase, and religating. This final 35S :: RUC: NOS vector was used as an internal control in the transient expression assay.

Example  6: Transgenic Rice Cell Line

PCAMBIA1391Z vector with CIPK15 promoter :: GUS was used to transform Agrobacterium tumefaciens LBA4404 with pAL4404 Ti plasmid and Agrobacterium-mediated rice transformation was performed by Hiei Y, Ohta S, Komari T, Kumashiro T. Plant J 1994; 6 As described by: 271-82. The transgenic rice calli was treated with 2N6-CH solid medium (N6 salts and vitamins, 1 g / L casamino acid, 30 g / L sucrose, 2 mg / L 2,4-D, 2.0 for 3 weeks at 28 ° C. under cancer conditions). g / L phytagel, 250 mg / L cefotaxime, and 50 mg / L hygromycin B, pH 5.8). Hygromycin-resistant rice calli were subcultured several times and used in the experiment.

Example  7: GUS Assay

For fluorescent GUS assay, packed cell volume (100 μL) of cultured cells was transferred to 100 μL of GUS extraction buffer (50 mM NaH 2 PO 4, pH 7.0, 10 mM beta-mercaptoethanol, 10 mM Na 2 EDTA, 0.1% sodium lauryl sarcosine, And 0.1% Triton X-100), using a pestle. Mold and GUS assays are Hwang YS, Karrer EE, Thomas BR, Chen L, Rodriguez RL. It was performed as described in Plant Mol Biol 1998; 36: 331-41. Fluorescence was monitored at excitation wavelength 355 nm and emission wavelength 460 nm using a spectrofluorometer Victor 3 (PerkinElmer, Wellesley, Mass., USA). Fluorescent GUS activity was normalized to the amount of protein in the cell extracts determined by Bradford protein assay (Bradford, 1976). Histochemical GUS assays are described in Jefferson et al. (1987).

Example  8: Protoplast  Separation and transient  Expression analysis

Preparation and transformation of rice protoplasts is described in Hwang et al. As described by (1998). protoplasts were resuspended in Kao and Michayluk medium (Kao KN, Michayluk MR. Planta 1974; 115: 355-67) containing 0.06 M glucose / 0.34 M mannitol or 0.4 M mannitol and 24 h at 28 ° C. under dark conditions without agitation. Incubated. After incubation, the protoplasts were collected by centrifugation (8 min at 600 rpm) and lysed in Reporter Lysis buffer (Promega). Firefly luciferase and Renilla luciferase activities were measured using a Glomax 20/20 luminometer (Promega) with Dual-GloTM Stop & Glo® Reagent (Promega).

<110> Konkuk University Industrial Cooperation Corp. <120> Calcineurin B-like interacting protein kinase 15 Promoter and use          of the same <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 2562 <212> DNA <213> Artificial Sequence <220> <223> PROMOTER <400> 1 caccaagctt cgctctcatg aaaggcttga ttgatgcaaa ccaattgagc cgtcatcggc 60 tatctgtaca ttatctgtac agctgccaag tttgtattga tcggttttct ttgaggttaa 120 tttgtaagta tgtttttctt ctagtaactt aagtaattat ttttaaatct aaggtcaata 180 tgcataacaa catattctga aaaacttatg gataagctga ttattcattg gtaaaatata 240 gacaggccga tgtcaataaa aaccaatgat gcctgaccga aacaaatact acatgtctac 300 atccatgcta aaattaaata agttcagttc tatacatgaa tttgactata ttcatattta 360 tgtaaaaaat aaatttattt ttgaatggag gaaataagta tatacggtga taattgaata 420 ccgctcacca aagatccagc tagtagcaca cggataaatg gctgcaagtc accaaccatt 480 tccggtggtc caatacctga agaacagcca acccattgat gagtcagcaa aggatcagat 540 catgagagat gagccaatgc catgcatttg agtaactcca aaccaaatta aacacaacat 600 cacatggaaa actactctaa caaatgcctc aattaacatt ggttcctcag aaaataaaag 660 ttgcagttaa cggaatcaga ttaaagaagg aaacaagtgc tcattttctg taagacagca 720 tcacagcctt cagatctata aataggatta ggcaaggtga ttagtggtac attatgtgct 780 gtacctgttt caagaacaca atgatgagct gtaccatcat caattgtgaa ataattaatt 840 aaattatcat taagccccaa gccaagccaa accaatccaa gccaagccaa atgaatgatt 900 gctgtggtga agggcgacta gtcaggggtg agtcggatgg atatgctctt tatgcgatgc 960 ggtgcccagg aaacgcggcg cagccaaatc agggtcatta acgtcaaatc aatctctgct 1020 aataatgcaa ggcaccactg tttcttctcc aactggcggc tgcctcctct cctcttcttg 1080 atcggttccc cctgtataaa agagaacccc gtcttctcgc tgtcctcgca tccactcctg 1140 atactaccac tcaaaaactc tctctctcta cccaaacagg tgttgagaaa gaagtgaggg 1200 acagatccaa aaagttcacc ttttgttacc tgaatctcct cttcttcctg tcaccggaag 1260 gtatgcagtt cctctctcca tatcatcttc tagcgtttct ttctactgat tcttgattgc 1320 tcgctttttt ccattattct cttttgctcc aaggtagggg taggggcaca tcagttggac 1380 aagtagaaat tgatggcaca tcaattgagt agatgcggaa atttatggtt tttagttaaa 1440 gtttagacca agccgatgac aaagtgctgc cgtttaatcg aatacgacga tatgtactct 1500 tgcaatggca tgtgtcatga ggtgtttggg tttggcgagg ctcagttttc attaatttcc 1560 cctccttccc atggccgtat caaacctaat ctttgaaaaa tattcaattc atgtgggtgc 1620 ggaatcacta gatgcagctt aactccatgc aaatcctaac tcctgccatg tgcccatgtg 1680 cctgtccctt atcctgattt gcatctgaat tcatttgctg agtaatttgc ttgttcagtg 1740 cctaatctgc tagacgtcca aacaaagttt taattatgag caaaagatct agtaatgtca 1800 ttttttctaa attcgtaatg ttttggtctc tgaattcgta attgactagt tttattaatc 1860 acctttctga acttgaacga aagatgtaga ataattcaca tcggttgcaa attttaattc 1920 gtaattgact agttttattt tgttagcttc tgatttactg tcctagattt ctgataagta 1980 cttagttgaa gttccttatt atattaatta tctattacat aacacgttga actcatgtaa 2040 gttctgattg attctaggga acacttatat tatataagta gtaggtacaa aaatgcattt 2100 agtattacaa taagccttca aaattcttcg cattaacttt tgtttggtta aagttcttac 2160 agactttcag attattatac gtaccttgtt agcacttgag tgcatatcct aggacattca 2220 attactaaat ctgatgcctg gttttgttta tacaggggaa tcaataaaaa aaacaagcag 2280 tgaaagcctc aaggaatgct gaaagatgtg cttgcataga agaaattttc cgtgtgagat 2340 aaaacagatt gctggttcac cagtgctact gatctactca agtcttgatg tgagtgatca 2400 actttcctgc actaattcta caccatttag actccgaaac gacgatgata taagaatgtt 2460 gttctccatc cacgatcatg accttgggga ggatggctaa agaattgcag tccatctgat 2520 tggtggtcaa aacatagtgc tgagatttat atctagaggg gg 2562 <210> 2 <211> 1305 <212> DNA <213> Oryza sativa <400> 2 atggagagta gagggaagat tctaatggag aggtatgagt tggggagatt gttggggaaa 60 ggaacatttg gcaaggtgca ctatgcaagg aatctggagt caaaccagag tgtggccata 120 aagatgatgg acaaacagca gatattgaag gtcgggcttt cggagcagat cagacgtgag 180 atcacaacca tgcggttggt ggctcataag aacattgttc agcttcatga ggtcatggca 240 acacggaaca agatctactt tgtgatggag tatgtgaaag gtggtgagct atttgaaaag 300 gttgcaaagc gtggaaagct tacagaggtt gttgcacata agtatttcca gcaactcatt 360 agtgcagtgg attactgcca cagtcgaggt gtgtatcacc gggacttgaa gcctgagaac 420 ctactgttgg atgagaatga gaacctgaaa gtctcagact ttggattgag tgcgctttca 480 gagtcgaaga ggcaagatgg cttactccat accacctgtg gaacacctgc atatgtagct 540 ccagaggtga ttagcaagat aggctatgat ggtgcaaagt cagatatttg gtcttgtggt 600 gttatcctgt ttgttcttgt tgctggttac cttcctttcc agggcccaaa cttgatggaa 660 atgtatcgga agatacaaca cggtgaattc aggtgccccg gttggttttc acgcaaactc 720 cagaagttgt tgtacaagat catggacccc aacccaagca caaggatttc aatccagaag 780 ataaaggagt ctacctggtt ccggaaaggt cctgaggaga accgtatttt gaaggaaaga 840 actttgaatg aaaacaccac caaaaatgtt gctccggtgc ttggtgtgag acgcaagaaa 900 aatgctcatg aagatgtgaa gcccatgtca gtgacaaact taaatgcttt tgaaattatc 960 tctttctcca agggatttga tctctctggc atgttcattg taaaggaatg gagaaatgag 1020 gcaaggttca cttcagataa atctgcctca accataatct caaagctaga agatgtagca 1080 aaggcgctaa atctcagggt aaggaagaaa gacaatggtg tagtgaagat gcaagggagg 1140 aaggagggaa ggaatggtgt tcttcagttt gacatagaga tatttgaggt taccacttcc 1200 tatcatatca tcgagatgaa acaaacaagt ggcgattcat tggagtaccg acagctactg 1260 gaggagggca tccggccagc tctgaaggac attgtcttgg cctag 1305

Claims (10)

A CIPK (calcineurin-like interacting protein kinase) 15 promoter regulated by a sugar and anoxic environment consisting of the nucleotide sequence set forth in SEQ ID NO: 1 and a complementary nucleotide sequence of the nucleotide sequence. The promoter of claim 1; And a transformed plant having resistance in a sugar deficient and oxygen deficient environment by transforming with an expression cassette comprising a gene encoding a CIPK 15 protein controlled by the promoter. According to claim 2, wherein the CIPK 15 gene is a transgenic plant, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2. A method for producing a transgenic plant that is resistant in a sugar deficient and oxygen deficient environment comprising the following steps:
(a) obtaining a recombinant plant cell using the expression cassette comprising the promoter of claim 1 and a gene encoding the CIPK 15 protein controlled by the promoter; And
(b) regenerating the plant cells in the plant. According to claim 4, wherein the CIPK 15 gene is a method for producing a transgenic plant, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2. Expression cassettes for the production of transgenic plants resistant in a sugar deficient and oxygen deficient environment comprising the following components:
(a) the promoter of claim 1; And
(b) a gene encoding a CIPK 15 protein controlled by said promoter. According to claim 6, wherein the CIPK 15 gene is an expression cassette, characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2. Methods for screening sugar and oxygen deficiency states of plants, including the following processes:
(a) obtaining a recombinant plant cell by using the expression cassette comprising the promoter of claim 1 and the gene encoding the CIPK 15 protein controlled by the promoter and a gene capable of visualizing the expression of the gene; And
(b) regenerating the plant cells in the plant. The method of claim 8, wherein the CIPK 15 gene is characterized in that consisting of the nucleotide sequence of SEQ ID NO: 2. The method of claim 8, wherein the gene capable of visualizing the expression of the gene is an anthocyanin synthesis related gene.

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