KR100635230B1 - Nucleic acid molecule encoding an armadillo repeat protein and the use thereof - Google Patents

Abstract

The present invention relates to a gene encoding a protein designated as ARIA (armadillo repeat protein interacting with ABF2) comprising an amadillo repeat and a BTB / POZ domain. This protein is a novel ABA signaling component that affects plant ABA-regulated gene expression, seedling growth, ABA sensitivity and stress tolerance. Moreover, the present invention provides a method of making a salt-tolerant plant comprising introducing into an plant an expression cassette comprising an ARIA gene linked to a plant promoter.

ABF2, Interaction, Amadillo Repeat Protein, Arabidopsis, Salt-Resistant Plant

Description

Nucleic acid molecule encoding an armadillo repeat protein and the use according to the present invention             

1 shows a summary of a two-hybrid screen to isolate ABF2-interacting proteins. A: Schematic diagram of ABF2 and fragments used in a 2-hybrid screen. Conserved areas of ABF family members are represented by boxes. S and T represent serine and threonine residues, respectively, and are putative phosphorylation sites. Glutamine-rich (Q) and bZIP (bZIP) zones are also shown. Thick bars with amino acid position numbers in parentheses represent fragments used for bait constructs. Full-length ABF2 consists of 416 amino acid residues. B: Specificity of the interaction. The interaction between group 2 positive clones (clone 20) and ABF2 (amino acids 234-337), nuclear membranes, ABF3 (amino acids 274-373) or ABF4 (amino acids 265-352) was tested. Yeast containing each bait construct was transformed into positive clones, transformants were patched on SC-Leu medium, and growth was examined after 4 days to test LEU2 receptor activity. C: deduced amino acid sequence of ARIA. The arm repeat region is colored and the BTB / POZ domain is underlined. The expected nuclear position signal in the N-terminal region is shown in bold. The conserved motif is also shown below schematically. arm repetitions 1, 8 and 9 are less well preserved. D: In vitro interaction of ABF2 with ARIA. Left, full-length ARIA (Full), arm repeat region (ARM) (amino acids 1-518), or Kuma alone (GST) and GST-ARIA fusion proteins, including BTB domain (BTB) (amino acids 511-710), respectively Blue-dyed gel. Right, GST pulldown essay. Radiograph showing ³⁵ S-Met-labeled ABF2 retained by in vitro-interpreted, GST-ARIA fusion protein. The same amount of recombinant protein was used for the assay. Arrows indicate the location of the protein bands.

Figure 2 shows the expression pattern of ARIA. A: RNA gel blot analysis. RNA was isolated from seedlings treated with 100 μM ABA, 250 mM NaCl, cooling conditions (24 hours at 4 ° C.) or dehydration (holding water for 2 weeks). The bottom panel shows the ethidium bromide-stained gel. B: Histochemical GUS staining of transformed plants transformed with 2.1 kb ARIA promoter- GUS receptor construct. T2 or T3 generation plants were stained with X-gluc (5-bromo-4-chloro-3-indolyl-β-glucuronic acid) for 24 hours. a, 3-day old seedlings. Inset shows maturation embryos from dry silique. b, 2-week old seedlings. c. leaf. d, e, root. f, flower. g, with immature long shells. h, with mature longs. C: subcellular location of ABF2 and ARIA. The top panel shows optical microscopy images of onion cells instantaneously transformed with 35S-ABF2-GUS fusion constructs and stained with X-gluc (GUS) or 4 ', 6-diamino-2-phenylridol (DAPI). Indicates. The middle and bottom panels show confocal images of root cells of plants transformed with 35S-ARIA-GFP ( ARIA-GFP ) or 35S-GFP ( GFP ). Gfp, Gfp Channel. PI, cells stained with propidium iodide. Arrows mark nuclei.

3 is a phenotype of 35S-ARIA plants. A: ABA dose-response of germination. Seeds were cold-treated at 4 ° C. for 5 days and placed on sucrose-free MS medium containing various concentrations of ABA. Germination (full-appearance of young roots) was recorded after 3 days. The experiment was performed in three replicates (n = 36 each) and small bars represent standard errors. B: osmotic sensitivity of germination. Germination assays were performed as in (A) on MS medium containing varying concentrations of mannitol, glucose or NaCl, providing germination rates at 250 mM mannitol, 250 mM glucose and 125 mM NaCl. The experiment was performed in 3 replicates (n = 36 each). C: salt tolerance. Left, survival rate of seedlings under high salt conditions. Seeds were germinated and grown for 2 weeks on MS medium containing 100 mM or 125 mM NaCl, and growth rate was measured. The experiment was performed in 3 replicates (n = 36 each). Right, representative seedlings grown for 15 days in 125 mM NaCl.

4 is a phenotype of aria knockout mutations. A: Schematic of T-DNA insertion mutation. Top, location of T-DNA insertion is provided. Bottom, expression level of ARIA and aria mutant (ARK10) plants in wild type (Col-0) as determined by RT-PCR. B: Germination Essay. Germination rate was measured as shown in FIG. 3A on MS medium (3 replicates, n = 36 each). C: growth of aria mutant seedlings. Left panel, seedlings grown on MS medium for 2 weeks. Right panel, Relative weight of atmospheric portion of soil-growing plants compared to Col-0 plants. Data points represent the average of six measurements (n = 6 each). D: ABA dose-response of germination. Germination assays were performed on sucrose-free MS medium containing various concentrations of ABA (3 replicates, n = 50 each). E: ABA dose-response of primary root kidney. Seeds were germinated on ABA-free MS medium for 3 days, transferred to medium containing various concentrations of ABA, and primary root elongation after migration was measured 5 days after migration (3 repetitions, n = 6 each). Control growth rates of Col-1 and ARK10 on ABA-free medium were 24.1 and 32.6 mm, respectively. F: glucose reaction. Seeds were germinated and grown on MS medium containing 3%, 4% or 5% glucose for 6 days before counting plants with green cotyledons (3 replicates, n = 30 each).

Figure 5 ABA-reactive gene expression in 35S-ARIA and aria plants. RNA levels were measured by RT-PCR using total RNA isolated from 2-week seedlings. The number of amplification cycles differs from other genes and overexpression and knockout lines.

The present invention provides a method for increasing salt resistance in plants using an expression cassette consisting of a nucleic acid molecule encoding an amadillo repeat protein (denoted "ARIA") and an ARIA gene operably linked to a plant promoter. It is about a method.

The arm repeat is the 42 amino acid protein-protein interaction motif (Peifer et al., 1994; Hatzfeld, 1999; Andrade et al., 2001). Repetition was first identified in the Drosophila segment polarity gene armadillo (Rigglelman, 1989) and then in many eukaryotic proteins involved in cell signaling or cell construction. Amidillo and its mammalian homologue β-catenin are components of the Wingless and Wnt signaling pathways that determine the pattern of Drosophila and embryonic body segments and mammalian cell death, respectively (Polakis, 2000). Initiation by Wingless or Wnt growth factor signaling otherwise stabilizes unstable amidillo / β-catenin, migrates into the nucleus, and activates the Wingless / Wnt target gene with the TCF / LEF subfamily of transcription factors. Β-catenin also plays a structurally important role in cell-cell adhesion by binding the transmembrane adhesion molecule cadherin to actin cytoskeleton.

Pfam (http://www.sanger.ac.uk/Software/Pfam/) and SMART (http://smart.emblheidelberg.de/) protein databases incorporate over 90 Arabidopsis arm repeat proteins . Based on these sequence homologues, these proteins can be classified into several different subfamily such as impotin-α, kinesin and U-box protein family (Coates, 2003). However, except for ARC1 and PHOR1, the functions of Arabidopsis and other plant arm repeat proteins have not been characterized in detail. ARC1 interacts with S-locus receptor kinase from Brassica (Gu et al., 1998) and has been demonstrated to be a positive regulator of self-incompatibility (Stone et al., 1999). Recent studies indicate that ARC1 enhances ubiquitination and proteasome degradation of affinity factors in pistils (Stone et al., 2003). In contrast, the potato arm repeat protein PHOR1 is involved in gibberellin (GA) signaling (Amador et al., 2001). Antisense inhibition of its expression decreases GA sensitivity and plant elongation while its overexpression increases GA sensitivity and intercutaneous length.

The BTB ( B R-C, t tk and b ab) domains are evolutionarily conserved protein-protein interaction domains (Bardwell and Treisman, 1994; Zollman et al., 1994). The ˜120 amino acid motif, also known as POZ (poxvirus and zinc finger, p oxvirus and z inc f inger) domains, contains poxvirus protein groups and Drosophila and zinc finger proteins, Broad-Complex (BR-C), Tramtrak (Ttk), and Bric. First identified in the -a-brac (bab) group. It was then discovered that BTB / POZ domains are present in some actin-binding proteins or ion channels within 5-10% of zinc finger transcription factors (Aravind and Koonin, 1998; Collins et al., 2001). Arabidopsis genome contains about 80 BTB domain proteins. However, only three of them are currently reported: NPH3 and RPT2 (Motchoulski and Liscum, 1999; Sakai et al., 2000), signal transducers of photoreactive responses, and NPR1 / NIMI (Cao et al., Regulator of gene expression during phylogenetic response). al., 1997; Ryals et al., 1997).

The plant hormone absic acid (ABA) regulates various aspects of plant growth and development (Finkelstein et al., 2002). It inhibits germination and growth after germination at high concentrations, but is required for normal seedling growth. It regulates the seed ripening process and prevents premature germination of the embryo. During nutrient growth, ABA plays an important role in adapting to various abiotic stresses such as drought, high salinity and cold (Xiong et al., 2002). Extensive genetic and biochemical studies have been conducted to identify various aspects of the regulatory component of the ABA response. As a result, many ABA signal components have been reported, including transcription factors, kinases / phosphatase, RNA-binding proteins, G-proteins and secondary messengers (Finkelstein et al., 2002; Xiong et al., 2002).

During trophic growth, ABA regulates many gene expressions associated with adaptive responses to drought and other abiotic stresses (Ramanulu and Bartels, 2002; Shinozaki et al., 2003). ABA-regulation of stress-responsive genes is mediated by cis-regulatory elements that share the CACGTGGC consensus. Prior to this, a small subfamily of basic leucine zipper (bZIP) class transcription factors interacting with the present element was identified (Choi et al., 2000; Uno et al., 2000). Factors named ABFs (ie ABF1-ABF4) or AREBs (ie AREB1-AREB3) have been shown to be involved in ABA and various abiotic stress responses (Kang et al., 2002; Kim et al., 2004). . In particular, ABF2 / AREB1, hereinafter referred to as ABF2, plays an important role in regulating seedling growth and preventing glucose-induced development. It also suggests that overexpressed phenotypes, such as altered ABA sensitivity and multiple stress tolerance, are involved in ABA and stress response. We aim to describe the ABA signaling pathway (s) that lead to ABF-dependent ABA / stress-responsive gene expression in trophic tissue. To this end, the inventors performed a 2-hybrid screen to isolate proteins that interact with ABF family members that regulate their activity. Here we describe arm repeats and BTB / POZ domain proteins that interact with ABF2. In vivo analysis of its function showed that the ABF2-interacting protein is a novel ABA signal component that regulates seed germination, seedling growth, glucose response and ABA / stress response. In particular, ARIA overexpression in Arabidopsis resulted in increased seedling survival under high salt conditions, indicating that it can be used to develop salt-tolerant plants.

The technical problem to be achieved in the present invention is a plant using an expression cassette consisting of a nucleic acid molecule encoding an amadillo repeat protein (denoted "ARIA") and an ARIA gene operably linked to a plant promoter. It is to provide a method for increasing salt resistance.

The present invention relates to a gene encoding a protein designated as ARIA (armadillo repeat protein interacting with ABF2) comprising an amadillo repeat and a BTB / POZ domain. This protein is a novel ABA signaling component that affects plant ABA-regulated gene expression, seedling growth, ABA sensitivity and stress tolerance. Moreover, the present invention provides a method of making a salt-tolerant plant comprising introducing into an plant an expression cassette comprising an ARIA gene linked to a plant promoter.

Isolation of ABF2 Interacting Proteins by Yeast 2-Hybrid Screen

We performed a yeast two-hybrid screen to isolate ABF2-interacting proteins (Chien et al., 1991; Gyuris et al., 1993). Since ABF2 has transcriptional activity (Choi et al., 2000), bait constructs were prepared using some fragments of ABF2 to reduce background activity (FIG. 1A). A cDNA expression library (Choi et al., 2000) showing RNA from ABA-treated Arabidopsis headings was used to transform yeast strains, including each bait construct. Five positive clones were recovered that interacted with various regions of ABF2 (amino acids 234-337). Insertion analysis of clones encodes transcription factors, two of which (group 1) will be reported elsewhere. The remaining three clones (group 2) encoded the arm repeat protein (see below). Group 2 clones did not interact with nuclear thin films or corresponding regions of ABF3 and ABF4 (FIG. 1B), which specifically interacted with ABF2.

The longest open reading frame (ORF) of group 2 clones encodes a protein containing 705 amino acid residues. ORF lost the start codon. Database search and subseparation / sequencing of full-length cDNA (SEQ ID NO: 1) indicated that the protein consists of 710 amino acid residues with an estimated molecular weight of 78 kD (SEQ ID NO: 2) (FIG. 1C). The ABF2-interacting protein, designated ARIA (Arpe Repeat Protein Interacting with ABF2), has 9 copies of arm repeats with 1, 8 and 9 arms less well-conserved in the N-terminal half. Furthermore it has a BTB / POZ domain in the C-terminal zone. The gene encoding ARIA (At5g19330) consists of 19 exons and ARIA shows the highest sequence identity (59%) for another Arabidopsis arm repeat protein (At5g13060) of unknown function.

Interaction of ARIA with ABF2 in vitro

The interaction between ARIA and ABF2 was confirmed by binding assays in the capillary. Recombinant proteins (FIG. 1D, lines 3-5) were prepared comprising fusion to glutathione- S -transferase (GST), including the entire ARIA coding region, arm repeat region or BTB domain. Their interaction with full-length ABF2 was measured by GST pulldown assays with translated ABF2 in vitro labeled with ³⁵ S. As shown in FIG. 1D, ABF2 was retained by the GST-full-length ARIA fusion protein, while not by GST alone (lane 6). Thus full-length ARIA interacted with ABF2. Similarly, fragments containing arm repeat regions or BTB domains also interacted with ABF2 (lanes 8 and 9). Stronger band intensities observed with the BTB domain (lane 9) indicated that ABF2 bound more strongly to the domain.

Expression patterns and subcellular locations of ARIA are similar to ABF2

ABA- and stress-induced ARIA expression was examined by RNA gel blot analysis. Similar to ABF2 induced by ABA and high salt (Choi et al., 2000), ARIA transcript levels were increased by ABA and high salt treatment (FIG. 2A). The GUS tissue of transgenic plants having a fusion construct is chemically β- glucuronidase in the (GUS) staining was carried out - ARIA promoter to specifically investigate the temporal and spatial expression patterns of the ARIA. Strong GUS activity was detected in the young roots of germinating seedlings (data not shown) and the roots of young seedlings (FIG. 2B, a). In more grown seedlings (FIG. 2B, b) the leaves showed stronger GUS activity than roots. In particular, catheter tissue and covariate cells were strongly stained (FIGS. 2B, c). In the roots of the older plants, GUS activity was detected mainly in the lateral roots rather than in the primary roots (Figure 2B, d). The catheter zone was stained stronger than the epidermal tissue (Figure 2B, e, top panel), and very strong GUS activity was observed in the basal root primordia and basal portions of the side roots (Figure 2B, e, bottom panel). . Anesthetization zones with anther, flower bed, ovule and immature long pole showed strong GUS activity in regenerative organs (FIG. 2B, fh). Embryos were also strongly stained (Figure 2B, a, inset). In summary, ARIA promoter activity was detected in the embryo and most nutritional and regenerative organs. The temporal and spatial expression patterns of ARIA are very similar to ABF2 . For example, the ABF2 promoter is very active in most vegetative tissues, especially in lateral roots, lobes and cosine cells. In addition, ABF2 is strongly expressed in anther, anther and stigma in flower organs.

ABF2 is a transcription factor and is located in the nucleus as shown in FIG. 2C (top panel). Note that the ARIA has a nuclear position signal near the N-terminus (FIG. 1C), suggesting that it is located in the nucleus. To measure the intracellular location of ARIA , transgenic plants were concealed to hide the ARIA-GFP (green fluorescent protein) fusion construct, and the location of the fusion protein was measured. 2C (center panel) shows that GFP is located in the nucleus, indicating that ARIA is nuclear-located. GFP was also detected around the cells. This indicates that ARIA is also located in the cell membrane.

Overexpression of ARIA Affects ABA and Osmotic Sensitivity During Germination

ARIA overexpression lines were generated and analyzed to investigate ARIA function in vivo. Transgenic Arabidopsis plants expressing ARIA under the control of the 35S promoter were generated (see method), and the ABA / stress-related phenotypes of the two representative lines were examined in more detail after preliminary analysis of seven T3 homozygote lines.

The ARIA overexpression line did not show a significant growth phenotype under normal conditions except for slightly delayed (˜1 hour) germination (data not shown). ARIA overexpression, however, affected ABA sensitivity during germination. ABA dose-response analysis (FIG. 3A) showed that germination of 35S-ARIA transformed seeds was more severely inhibited by medium concentrations (1 and 2 μM) of ABA, especially ABA, than wild type seeds. ARIA overexpression therefore increased ABA sensitivity during seed germination. Moreover, germination of the transgenic seeds was more sensitive to mannitol, glucose and NaCl (FIG. 3B), indicating that ARIA overexpression induced a hypersensitivity reaction.

We also examined the response of 35S-ARIA seedlings to various abiotic stresses and found that they were less sensitive to high salts. For example, the survival rate of wild type plants at 100 mM NaCl was 55%, while 35S-ARIA plants were 81% (AR40) and 72% (AR32), respectively (FIG. 3C). 38% (AR40) and 36% (AR32) of the transgenic plants survived at 125 mM NaCl, while the wild type survival was 11%. Thus the ARIA overexpression line was more resistant to high salinity conditions.

aria  Phenotype of mutation

To further investigate the in vivo function of ARIA, the aria mutant phenotype was analyzed. Mutations in which T-DNA was inserted into the promoter region of ARIA (FIG. 4A) were obtained from Arabidopsis stock center, and various phenotypes were recorded after confirmation of T-DNA insertion and ARIA expression abnormalities.

The germination assay (FIG. 4B) showed that the degree of difference was not large but mutated seeds germinated more effectively than wild type seeds under normal growth conditions. Post- germination growth of aria mutants was more effective; That is, aira seedlings were larger than wild type plants as shown in FIG. 4C. However, they developed normally and whole-grown mutant seedlings were similar in size to wild type plants, indicating that mutations only affected the growth of young seedlings. Together, the observations demonstrate that ARIA is a negative regulator of seed germination and young seedling growth.

The aira mutant also showed an altered ABA response. ABA dose-response analysis of germination (FIG. 4D) showed that mutant seed germination was less sensitive to ABA than wild type seeds at high concentrations of ABA, indicating that its germination was partially insensitive to ABA. Similarly, primary root elongation of aria plants was less sensitive to ABA-inhibition than wild type plants at high ABA concentrations (ie 2, 5 and 10 μM) (FIG. 4E).

Glucose inhibits chute development (ie cotyledon greening, cotyledon expansion and actual leaf formation) at high concentrations, and the inhibition process is dependent on ABA (Jang et al., 1997; Leon and Sheen, 2003). To determine whether ARIA is involved in this process, glucose sensitivity of aria plants was measured. 4F shows that cotyledon greening of wild-type plants was gradually inhibited with increasing glucose concentration in the medium. In addition, aria mutant plants responded to glucose in a similar manner, but the degree of inhibition was lower than that of wild type plants. No differential response at mannitol was observed, ie there was no osmotic response (data not shown). The results demonstrate that ARIA is an essential component for glucose-inhibition of chute development.

ARIA Affects ABA-Reactive Gene Expression

To investigate whether ARIA affects ABF2-regulated gene expression, the expression levels of many ABF2-reactive genes in 35S-ARIA plants were measured. Bound reverse transcription and polymerase chain reaction (RT-PCR) (FIG. 5) is down-regulated by ABF2 under normal conditions but up-regulated under high salt conditions and rd29A (Yamguchi-Shinozaki and Shinozaki, 1994) and CHS (Feinbaum) and Ausubel, 1988) have higher RNA levels in 35S-ARIA plants. In contrast, SUS1 (Martin et al., 1993) and ADH1 (de Bruxelles et al., 1996) expression levels down-regulated by ABF2 under normal conditions were somewhat lower than wild type levels. While CHS RNA levels were decreased in aria mutant plants, SUS1 RNA levels were increased, suggesting a regulatory role of ARIA in these expressions. Thus overexpression or under-expression of ARIA altered the expression of some ABF2-regulated genes, indicating that it is involved in the ABF2-dependent gene regulation process.

debate

We describe an arm repeat protein named ARIA that specifically interacts with ABF2. In animals, arm proteins are involved in a variety of cellular functions such as cell contact, signal transduction, tumor suppression, and nuclear inclusion (Hatzfeld, 1999; Andrade et al., 2001). Their function in plants is little known except for some genes. ARC1 and PHOR1 are regulators of self-incompatibility and GA signals, respectively, as described above (Stone et al., 1999; Amador et al., 2001). The tobacco arm repeat protein NtPUB4, which interacts with receptor-like kinases, has been proposed as a developmental regulator (Kim et al., 2003). Most Arabidopsis arm repeat proteins, including ARC1 and PHOR1, include U-box motifs found in subclasses of the E3 ubiquitin ligase (Coates, 2003; Mudgil et al., 2004). Thus most of them participate in ubiquitin-dependent proteolysis. ARIA, however, does not contain U-box motifs. Instead it has another conserved sequence motif, the BTB / POZ domain. Sequence comparison indicates that only two Arabidopsis proteins, including ARIA, have both arm repeat and BTB / POZ domains. BTB / POZ domains have been found in many transcription factors in animals and some actin-binding proteins in animals (Aravind and Koonin, 1998; Collins et al., 2001). Most recent studies indicate that some BTB domain proteins are substrate-specific adapters for CUL-3-based E3 ubiquitin ligase (van den Heuvel, 2004). arm repeats and BTB domain proteins play a variety of roles, but the basic function of the two motifs is to mediate protein-protein interactions. ARIA therefore has the potential to complex with other proteins or function as a scaffold.

The physiological relevance of the ABF2-ARIA interaction was supported by their similar expression pattern. Expression of ABF2 and ARIA is induced by ABA and high salts (FIG. 2) (Choi et al., 2000). Both are highly expressed in trophic tissues (especially lateral roots, leaf conduit tissues and covariate cells) and in regenerative organs (ie, anther, anther, stigma and breakaway zone). Moreover, both proteins have ARIA found in the cell membrane or cell wall zone but located in the nucleus (FIG. 2).

Data on the in vivo function of ARIA further support the physiological significance of ABF2-ARIA interactions. ARIA overexpression increases ABA and osmotic sensitivity in the germination stage. During subsequent seedling growth this increases salt tolerance. In contrast, cleavage of this expression enhances germination / plant growth and impairs the glucose response. Some 35S-ARIA and aria mutant phenotypes are similar to 35S-ABF2 and abf2 plants. For example, delayed germination of overexpression lines, faster germination / growth of mutant seedlings, salt tolerance of overexpression lines and glucose insensitivity of knockout mutants were also observed in ABF2 (Kim et al., 2004). Furthermore, altered expression of some ABF2-reactive genes in 35S-ARIA and aria plants was observed (FIG. 5), indicating that ARIA affects ABF2-regulated gene expression.

The results indicate that ARIA is a positive component of the ABA signal. ABA sensitivity was increased by its overexpression and impaired by knockout mutations. Germination is delayed by its overexpression and enhanced by its mutation. Other ABA-related processes, such as osmotic sensitivity and sugar response, are also positively and negatively affected by ARIA exaggeration and mutations thereof, respectively. Two observations are sufficient to discuss the role of ARIA in the ABA response. First, most of the ARIA overexpression and knockout phenotypes are consistently observed and are relatively weak and partial (FIGS. 3 and 4). As discussed above, there is an arm repeat / BTB domain protein in the Arabidopsis genome, which is highly homologous to ARIA in terms of the amino acid sequence as well as the gene structure (data not shown), and thus functional excess between the two proteins. Can be guessed. Another observation is that ARIA only affects a subset of ABA-dependent processes. ABA sensitivity and young seedling growth during germination were affected by ARIA. However, other ABA-dependent processes, such as abiotic stress responses other than pore closure and salt tolerance, were not significantly affected (data not shown).

Altered expression of some ABF2-regulated genes (FIG. 5) suggests that ARIA affects ABF2-dependent gene expression. We currently do not know the biochemical mechanism of ARIA function. However, it can be inferred to function as a coactivator or inhibitor of ABF2. In animals, the arm protein, β-catenin, has been demonstrated as a transcriptional co-activator; That is, they move into the nucleus and form complexes with transcription factors in response to hormonal signals to activate target gene expression (Polakis, 2000). In contrast, the BTB / POZ domain is known to mediate transcriptional inhibition by recruiting histone deacetylases to recruit transcriptional co-inhibitors and eventually inhibit transcription (Collins et al., 2001). The BTB / POZ domain is also involved in protein degradation (van den Heuvel, 2004). ARIA is thus involved in regulating the stability of ABF2 or other proteins possibly related thereto. Since ARIA has two protein-protein interaction domains, another possibility is to function as an adapter for ABF2 to form a protein complex.

Regardless of how the ARIA biochemical mechanism (s) function, the data indicate that ARIA overexpression increases salt tolerance of Arabidopsis plants. The results can be designed to increase the plant's salt tolerance, thus making it possible to develop salt-tolerant plants using the ARIA gene.

(Example)

Example 1 DNA Manipulation and RNA Gel Blot Analysis

DNA manipulation and RNA gel blot analysis were performed according to standard methods (Sambrook and Russel, 2001). DNA sequencing was performed with ABI 310 Genetic Analyzer (Applied Biosystems). RNA was isolated by the method of Chomczynski and Mackey (1995) and further purified by LiCl precipitation and ethanol precipitation. ABA, salt, cooling and drought treatment of Arabidopsis seedlings were performed as described (Choi et al., 2000). For RNA gel blot analysis, 25 μg of total RNA was separated on a 1.1% formaldehyde agarose gel and transferred to a nylon membrane (Hybond-XL, Amersham Pharmacia Biotech) and immobilized using Stragagene's UV Crosslinker (Model 2400). . Hybridization was carried out at 18-24 at 65 ° C. in Rapid-hyb buffer (Amersham Pharmacia Biotech) using ³² P-labeled DNA fragments containing the less-conserved region of the ARIA (amino acid positions 336-554) as probes. Was performed for hours. The filter was washed successively as follows: twice at room temperature for 10 min in 2X SSC (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate), twice at room temperature at 0.2X SSC for 10 minutes at 0.2X SSC 65 ° C. Twice for 10 minutes. Exposure was carried out at -70 ° C. RT-PCR was performed by treating 0.5 μg total RNA using the Access RT-PCR System (Promega) according to the manufacturer's instructions. Primer sets containing Actin primers used in the control reaction (Arabidopsis actin-1 gene, accession number M20016) have been described above (Kang et al., 2002). RNA samples were confirmed to be free of DNA contamination by using a set of actin primers that span introns and, where possible, a set of primers that span intron (s). The number of PRC cycles varied with the particular gene within the first range of PCR amplification (typically 20-30 cycles). The results of RT-PCR were confirmed by some independent reactions.

Example 2 Yeast Technology and 2-Hybrid Screening

Yeast growth and transformation were according to standard techniques (Guthrie and Fink, 1991). Two-hybrid screens were performed using the MATCHMAKER LexA 2-Two Hybrid System (Clontech) in some variations. The bait construct was prepared by cloning some fragments of ABF2 with pGilda (Clontech), which performed the LexA DNA binding domain under the control of the GAL1 promoter and HIS3 marker gene. ABF2 fragments spanning amino acid residues 65-162 (conserved region) and 234-337 (variable region), respectively, were PCR (primer set, 5'-GCTAGTGGTGTGGTTCCAGTT C-3 '(SEQ ID NO: 3) and 5'- gaga gctcgagCTGAGCTCTTGCAGCAACCTG-3 '(SEQ ID NO: 4) and 5'-CCAATCATGCCTAAGCAGCC-3' (SEQ ID NO: 5) and 5'-gagagctcgagCTCTACAAC TTTCTCCACAGTG-3 '(SEQ ID NO: 6) and digested with Xho I It was then ligated to pGilda and eventually produced by Bam HI digestion, Klenow fill-in reaction and Xho I digestion. The bait construct was then introduced into the receptor yeast, EGY48 (MATα , his3, trp1, URA3 :: LexA _{op (x8)} -LacZ, LexA _{op (x6)} -LEU2 ) by transformation . The EGY48 strain carries two receptor genes, LEU2 and LacZ , integrated within the chromosome. Large-scale transformation for screening was performed as described (Choi et al., 2000). Receptor yeast was transformed with library plasmid DNA representing the cDNA of ABA / salt-treated Arabidopsis seedlings (Choi et al., 2000). Transformed yeasts were grown on Gal / Raf / CM-His-Leu-Trp-Ura medium for 5-7 days and positive colonies were identified by colony lift β-galactosidase assay. Leu ⁺ / LacZ ⁺ positive colonies were purified by streaking on the same selection medium before becoming another round of β-galactosidase assay. For each receptor yeast 6.6 million transformants were screened and five positive clones were obtained from mutable zone baits, whereas no positive clones were obtained from conserved zone baits. The specificity of the positive clone interactions was tested by re-transforming the receptor yeast with plasmid DNA reclaimed from the clone.

Plasmid recapture and insertion DNA analysis was performed as described (Choi e tal., 2000). Sequencing of plasmid DNA recaptured from positive clones showed that three of them (clones 12, 20 and 24) encoded the arm repeat protein (At5g19330), and two of them (clones 17 and 27) encoded transcription factors. It was. The longest arm protein clone lost the first five amino acid residues. The full-length gene was subjected to PCR using a primer set, 5'-GGATCGTCTTTTACTTTGTGAACG-3 '(SEQ ID NO: 7) and 5'-CATTCAA GAC CGA TTG TGATCAG - 3' (SEQ ID NO: 8) and 1 μg of library DNA. Separated by. PCR products, including the entire coding region and 5 '(208 base) and 3' (24 base) additional sequences, were cloned into the Zero Blunt TOPO PCR Cloining Kit (Invitrogen) and were sequenced throughout. The accuracy of its nucleotide sequence was confirmed by comparing it with genomic sequences on Arabidopsis databases.

Example 3 In Vitro Binding Essay

GST-ARIA fusion constructs were prepared by cloning PCR fragments of various parts of ARIA (full-length, amino acids 1-518 and amino acids 511-710) into the Sma I site (Amersham Pharmacia Biotech) of pGEX-6P-2. The construct was used to transform BL21 cells, and the transformed cells were grown overnight in 2 × YT medium containing 50 μg / ml ampicillin. Cultures were diluted 100-fold and grown to 30 ° C. or 37 ° C. at A ₆₀₀ of 0.6 (full-length and ARM constructs). Expression of the recombinant protein was induced for 3 hours with 0.5 mM isopropyl-β-D-thiogalactopyranoside. After termination of induction, the cells were pelleted down by centrifugation, resuspended and sonicated in 6 ml of PBS (0.14 M NaCl, 2.7 mM KCl, 10.1 mM Na ₂ HPO ₄ , 1.8 mM KH ₂ PO ₄ , pH 7.3). . Lysates were cell debris removed by centrifugation and further purified according to the supplier's instructions. For in vitro translation of ABF2, full-length ABF2 cloned into pCITE (Novagen) was treated with a TNT in vitro translation kit (Promega) in the presence of ³⁵ S-Met according to the manufacturer's instructions.

For binding assays, GST-ARIA fusion protein (0.5 μg) was bound to binding buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM PMSF) at 4 ° C. for 1 hour. Incubated with glutathione-Sepharose 4B resin in the interior. Thereafter, in vitro-translated ³⁵ S-labeled ABF2 was added and incubation continued for 2 hours with constant rotation. The resin was washed five times with binding buffer and resuspended in SDS-polyacrylamide gel electrophoresis sample buffer. Proteins were separated on 15% SDS-polyacrylamide gels and visualized by radiophotography.

Example 4 Histochemistry GUS Staining

2.1 promoter fragments were prepared by PCR using a primer set, 5'-GATCCGAAGAAGAGGAGAGATC-3 '(SEQ ID NO: 9) and 5'-GCCACGCTGTCTTCTTTCACTACACTAAAAAATACAGC-3' (SEQ ID NO: 10), Hind III- of pBI101.2 Xba I was cloned into the site. Teuneun constructs were introduced into the transgenic Arabidopsis thaliana (Arabidopsis) (L er) by transformation, T2 or T3 generation plant has been used for GUS activity assay. GUS staining was performed according to Jefferson et al. (1987). The whole plant or tissue was a 1 mM 5-bromo-4-chloro-3-indolyl-β-glucuronic acid (X-gluc) solution in 100 mM sodium phosphate, pH 7.0, 0.1 mM EDTA, 0.5 mM ferricyan ( ferrycyanide), 0.5 mM ferrocyanide and 0.1% Triton X-100 inundated for 24 hours at 37 ℃. Chlorophyll was removed from tissue by ethanol series: 35%, 50% and 70%.

Example 5 Subcellular Position

The entire coding region of ARIA was prepared by PCR to produce 35S-ARIA GFP fusion construct and cloned into the same site of pCAMBIA1302 (CAMBIA) after digestion with Nco I-Spe I. Constructs were introduced into Arabidopsis (Col-1) by transformation and T1 plants were used for GFP site analysis. Nuclei were visualized by propidium iodide (PI) -staining. Roots of 10-day-old transgenic seedlings were used for green (GFP position) and red (PI) fluorescence analysis using confocal microscopy (Leica, TCS-NT).

To examine the ABF2 position, the coding region of ABF2 was inserted in front of the GUS coding region of pBI221 in frame. Onion epidermal cells were then instantaneously transformed into ABF2-GUS constructs by particle bombardment using PDS 1000 (Bio-Rad). GUS activity was measured by X-gluc (5-bromo-4-chloro-3-indolyl-β-glucuronic acid) staining after 24 hours at 23 ° C. Nuclei were visualized by 4 ', 6-diaminodino-2-phenylindole (DAPI) staining and observed under fluorescence microscopy (Olympus BX51).

Example 6 Overexpression and Knockout Mutation Lines

The coding region of AIRA was prepared using primers 5'cgcggatccATGGACCAACAACCGGAGAGG-3 '(SEQ ID NO: 11) and 5'-gcgggatcc CAACCTCAAG CTTTG CAGGTT TG-3' (SEQ ID NO: 12) to prepare the 35S-ARIA construct, After digestion with Bam HI, it was cloned into the Bam HI site of pBI121 without the GUS coding region. Arabidopsis thaliana transformant of (L er) is A. followed the vacuum infiltration method using tumefaciens strain GV3101 (Bechtold and Pelletier, 1998) . Seven homology lines were recovered and after a preliminary analysis two representative lines (T4) were selected for detailed analysis.

Four putative ARIA knockout mutation lines were obtained from Arabidopsis stock center to establish aria mutation lines. Stock seeds were sown and grown in soil, and seeds were harvested from individual plants. The sequestration ratio of kanamycin resistance (Kan ^R ) was tested to select a T-DNA insertion line with a single integration, and the homozygous subline was established from the segregation at a 3: 1 ratio of Kan ^R : Kan ^S. Genomic DNA was isolated from the subline and integration of T-DNA at the annotated sites was confirmed by sequencing the PCR fragments. One insertion line (SALK_143439) with a single T-DNA insertion was identified at the annotated site out of the five putative lines. T-DNA is inserted at -379 from the translation start site. Expression analysis by RT-PCR showed that ARIA expression was eliminated within the insertion line. Two sublines (ARK5 and ARK10) were used for phenotypic analysis. The same results were obtained from them and those from AR10 are present.

Example 7 Phenotypic Analysis

Arabidopsis thaliana ecotypes Landsberg erecta (L er ) and Columbia (Col-0) were used. The plants were grown on MS plates or mixtures of vermiculite, pearlite, and moss of 1: 1 at 1: 1 ° C under long-term conditions (16 hours light condition / 8 hours dark condition cycle). Soil-growing plants were irrigated once a week with 0.1% Hyponex. For normal sterile growth, seeds were sterilized for 5 minutes in 70% ethanol and 5 minutes in 30% household bleach, washed 5 times with sterile water, added 1% sucrose and coagulated with 0.8% Phytoagar (Murashige). and Skoog, 1962).

Understanding Germination Tests Seeds collected at the same time were placed on MS medium added with 1% sucrose and other additives (ie ABA, mannitol, glucose and NaCl), unless otherwise noted, and the appearance of young roots varied. It was investigated at that point. Sucrose was omitted from the medium for the ABA dose response analysis of germination. Phenotypic analyzes other than germination assays were performed on MS medium with 1% sucrose and ABA, glucose or mannitol added as shown in the figure. In the case of root kidney assays, the plants were grown in a vertical position.

The effect of the present invention is directed to a gene encoding a protein designated as ARIA (armadillo repeat protein interacting with ABF2) comprising the amadillo repeat and BTB / POZ domains. The present invention also provides a method of making a salt-tolerant plant comprising introducing an expression cassette comprising an ARIA gene linked to a plant promoter into the plant.

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Claims (6)

An isolated nucleic acid molecule encoding Amadillo Repeat Protein (ARIA) having the nucleotide sequence of SEQ ID NO: 1 Recombinant DNA molecule comprising the polynucleotide of claim 1 operatively linked to a promoter functioning in a plant cell A method for producing a salt-tolerant plant comprising transforming plant cells with the recombinant DNA molecule of claim 2 Plant cell comprising the recombinant DNA molecule of claim 2 delete delete

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