KR100893144B1 - Stress-inducible StGRX2 gene from potato - Google Patents

Abstract

The present invention potato ( Solanum tuberosum L. cv. Isolated three different GRX genes involved in tuberculosis of Desiree, potato-derived StGRX2 protein expressed during potato tuberification and corkation, and involved in various biological or abiotic stresses, genes encoding the protein, It relates to tissue-specific expression patterns of the genes in plants and to expression of the genes under various biological or abiotic stress conditions. StGRX2 gene isolated in the present invention can control the growth and development of the plant in response to plant development and various plant hormones.

StGRX2 Gene, Biological / Abiotic Stress, Glutaredoxin, Potato

Description

Stress-inducible StGRX2 gene from potato

1 is a potato ( Solanum tuberosum L. cv. Desiree) shows the alignment of the predicted amino acid sequence of three different StGRX genes. Alignment was done with the ClustalW program, with the asterisk residues indicating the conserved regions of the active moiety.

Figure 2 shows the results confirmed by Northern blot analysis of the expected number of copies of the StGRX gene through Southern blot analysis and the expression pattern of the StGRX gene in other tissues.

3 is a result of analyzing the expression patterns of three different StGRX genes under tuber formation and corkation condition of potatoes. Total RNA was wound at timed intervals, then tubers were introduced during corkation to cut potato nodes, isolated and analyzed by Northern blot. Total RNA isolated from the nylon membrane was hybridized with a probe of the StGRX gene and confirmed by chemiluminescence.

Figure 4 shows three StGRX in potato plants infected with P. infestans It shows the expression pattern of the gene. PR-2, PR-3 and StGRX gene Transcript levels were separated from the leaves of potatoes 30 days after pot cultivation and quantified by RNA gel-blot analysis.

5 shows three StGRXs under various stress conditions RNA gel blot analysis of the genes was shown, with stress conditions treated with 0, 100, 200, 300 mM NaCl, 0, 100, 200, 400 mM mannitol, low temperature and esepon. The results shown at the bottom of each treatment are the results of staining rRNA with ethidium bromide.

FIG. 6 shows three StGRXs under treatment with gibberellin (GA ₃ ), zeatin, abscis acid (ABA) and methyljasmonic acid (MeJA). RNA gel blot analysis of the gene is shown.

The present invention is potato derived separating the three different GRX genes for tuber screen of (Solanum tuberosum L. Cv. Desiree ) , and becomes expressed in potato tubers Chemistry and cork development process involved in a variety of biological or non-biological stress StGRX2 protein, gene encoding the protein, tissue specific expression patterns in the plant of the gene and expression of the gene under various biological or abiotic stress conditions.

Glutaredoxin (GRX) is a small ubiquitous protein of the thioredoxin group that catalyzes dithiol-disulfide exchange reactions or reduced protein-glutathione disulfide reduction. Glutaredoxin is known to be involved in the homeostasis of cellular redox reactions in bacteria, yeast and mammalian cells, but its function in plants is not yet clear. Proteins containing the thioredoxin superfamily contain Cys pairs by forming reversible disulfide bonds by the transfer of electrons to various reductases (Holmgren, 1989, J. Biol. Chem. 264, 13963-13966). Doxin is known to be specifically reduced by NADPH or peredoxin-dependent thioredoxin reductase. The related factor, glutaredoxin, is specifically reduced by glutathione (GSH), and the specificity of glutathione has been reported to be due to the glutathione binding portion of glutaredoxin without thioredoxin (Nikkola et al., 1991, J. Biol. Chem. 266, 16105-16112).

In higher plants, most of the research focuses on thioredoxin, but little is known about glutaredoxin via cDNA sequences that have been used for a long time (Minakuchi et al., 1994, FEBS Lett 337, 157-160; Szederket al., 2004, Planta 202, 349-356). In most organs, thioredoxin and glutaredoxin are major reducing molecules and are involved in most cellular processes. Recent genome analysis (Lemaire, 2004, Photosynthesis Research 79, 305318; Rouhier et al., 2004, Cell Mol Life Sci 61, 1266-1277) added three different forms of GRX, CPYC, CGFS group and so-called CC. GRX with CCXC or CCXS has been identified, the latter of which are known to have specificity in higher plants. Meanwhile, 31 GRX genes were identified in Arabidopsis; six genes with CPYC form were extensively studied in E. coli and yeast, and four genes in CGFS form were analyzed in yeast and human. GRX with active sites of type CPYC and CGFS in E. coli , yeast and humans have been found in lower and higher plants, but little has been reported about their function yet. Interestingly, the 21 GRX genes isolated from Arabidopsis are new and contain plant-specific CC-like positivities. Recently, the results of studies on the function and redox regulation of general CC type GRX have been reported. GRX is derived from TRX and PRX (Balachandran et al., 1997, Proc Natl Acad Sci USA 94, 14150-14155; Szederkenyi et al., 1997, Planta 202, 349-356; Rouhier et al., 2001, Plant Physiol 127, 1299 -1309) and is known to be abundantly present in phloem sap. Only biochemical data show that CPYC type GRX is obtained from poplar, and GRX is the oxidative stress response (Rouhier et al., 2001, Plant Physiol 127, 1299-1309; Rouhier et al., 2002, FEBS Lett 511, 145- 149) and GRX involved in reducing type II PRX. In addition, CC type glutathedoxin ROXY1 is known to be involved in petal development in Arabidopsis (Xing et al., 2005, Development 132, 1555-1565). However, despite these findings, the target for GRX is still not well known. Recently, the approach to GRX is similar to the method used to identify TRX targets, and the method of quantifying glutaredoxin targets in photosynthetic organs using affinity columns composed of GRX mutated from poplars has been used.

This approach has been demonstrated by identifying 94 estimated GRX targets from other higher plant tissues (Rouhier et al., 2005, Antioxidants & Redox Signaling 7, 919-929), but most of the targets identified in this study These were already present in the monocittic TRX column.

While studying glutaredoxin gene expression during potato tuberification and corkation, the inventors identified three different potato GRX sequences from cDNA library EST to isolate cDNA induced 6 hours after corkation. The present invention has been completed by performing licoscreening, of which the StGRX2 gene is expressed in the germ-line tissue of potato plants and reveals a specific role under plant development and various biological or abiotic stress conditions.

Accordingly, it is an object of the present invention to provide potato-derived StGRX2 protein whose expression is regulated during tuberculosis under various biological or abiotic stresses.

Another object of the present invention is to provide a gene encoding the StGRX2 protein.

Another object of the present invention is to provide an expression site in the potato plant of the gene.

Another object of the present invention is to provide expression patterns of the genes for various biological or abiotic stresses.

Another object of the present invention is to provide an expression pattern of the gene during potato in vitro tuber development.

In order to achieve the above object, the present invention provides a StGRX2 protein derived from potato having an expression pattern specific for various biological or abiotic stress, consisting of the amino acid sequence represented by SEQ ID NO: 2.

In order to confirm gene expression during corkization of potato tubers, in silico screening was performed from EST to separate cDNA expressed at 6 hours after corkation. As a result, in the present invention, three different GRX genes, StGRX1, StGRX2 and StGRX3, were identified, and the degree of expression and the position at which the isotype is expressed in the organs were measured by a virtual experiment.

Of the 1072 ESTs in the cDNA library, four ESTs were encoded with GRX with CxxC / S active portion, three ESTs with GRX with CCxC / S active portion, and no EST encoded with GRX with CGFS active portion was found. Did. In the present invention, GRX of type CCxC / S is named StGRX1, and two CxxC / S types of GRX are named StGRX2 and StGRX3, respectively.

The potato-derived StGRX1, StGRX2 and StGRX3 isolated from the present invention consist of 101, 125 and 108 amino acids, respectively (see FIG. 1), and the software program TargetP ( http://www.cbs.dtu.dk/services/TargetP). / ) Is expected to be located in the cytosol.

StGRX2 has an N-terminal dilator so that proteins are likely to be directly associated with organelles, but not directly with pigments, vesicles and mitochondria.

The StGRX2, consisting of 125 amino acid sequences (SEQ ID NO: 2), is a Medica Truncatula (72% homogeneity, Genbank ABE93576), Arabidopsis thaliana , 66% homogeneous, Genbank BAB08846) and tomato extract ( Solanum lycopersicum . 64% homogeneity, Genbank CAA77130) and is shown to have a high homology with glutardoxin.

In the present invention, the stress may be dry, low temperature, wound, methyljasmonic acid, gibberellin, esepone, osmotic, hydrogen peroxide, salicylic acid, chitosan, salt, cadmium, ultraviolet irradiation, protein kinase inhibitor, ozone or plant pathogen infection However, the present invention is not limited thereto.

The plant pathogens of the present invention represent potato toxin ( Phytophthora. Infestans ).

 The present invention also provides a gene encoding the StGRX2 protein. The gene of the present invention includes both genomic DNA and cDNA encoding StGRX2 protein. Preferably, the gene of the present invention may include a nucleotide sequence represented by SEQ ID NO: 1.

In the present invention, to determine the number of copies of the StGRX gene, potato ( Solanum tuberosum L. cv. Southern blot analysis was performed with genomic DNA extracted from the leaves of Desiree). When three different restriction enzymes were used, one or two strong bands were identified in StGRX2, indicating that the gene exists as a small family of genes with one or two copies.

In addition, the present invention provides a plant expression site of the gene.

There is much interest in the distribution of GRX in other plant organs, but little has been reported for intracellular location. As a result of confirming the intracellular expression positions of the three StGRX genes isolated in the present invention, it was confirmed that the StGRX1 transcripts were accumulated and accumulated in tissues of trophic growth stages such as leaves, stems, and roots. On the other hand, as shown in Figure 2, the StGRX2 transcript was found to have a high expression pattern in the reproductive stage tissue such as flowers and tubers of potato plants opposed to StGRX1. Therefore, it can be predicted that the accumulation of glutaredoxin mRNA from their intracellular expression is related to the reproductive stage during the potato life cycle. In the Northern blot analysis of FIG. 2, the samples colored with ethidium bromide were similar, indicating that the total amount of RNA extracted from leaves, roots, stems, and flowers was about the same.

In addition, the present invention provides the expression pattern of glutaredoxin transcript during potato in vitro tuber development using the gene.

In the present invention, the expression of glutaredoxin transcript was confirmed during the development of potato bovine tubers using StGRX1, StGRX2 and StGRX3 as probes. As can be seen in Figure 3, the expression of StGRX1 mRNA was reduced during tuber development, but the expression of StGRX2 mRNA was increased up to 4 days after tuber introduction, so that expression was maintained during tuber maturation. there was. On the other hand, although the expression of StGRX3 showed similar results to StGRX1, it was observed that the expression was slightly weak.

Expression of StGRX2 transcripts was confirmed to accumulate in reproductive stage tissues such as flowers and tubers (see FIG. 2). These results indicate that StGRX2 is associated with and induced by the development of the reproductive organs.

In the present invention, the expression patterns of the StGRX genes were wound during potato cork during various time intervals of 0, 6, 12, 24, 36, 72 and 120 hours. In FIG. 3, the expression of StGRX1 mRNA was decreased immediately after wounding, but increased until 36 hours after wounding. The expression of StGRX2 mRNA was increased up to 24 hours after wounding and then decreased during corkation. StGRX3 was hardly expressed before wounding, but it was confirmed that expression was induced after wounding. These results suggest that StGRX3 is involved in wound response and is expected to be activated by ethylene or methyljasmonic acid.

In addition, the present invention provides patterns of expression of these genes under various biological or abiotic stress conditions.

In order to confirm the role of the StGRX gene in the defense against phytopathogenic infection, the present invention provides RNA gel-regulatory levels of transcript levels of PR-2, PR-3 and StGRX genes in the leaves of potato plants infected with potato blight. Blot analysis was performed. Expression of the two protective marker genes was induced 3 hours after infection and decreased after 12 hours (see FIG. 4). However, although the expression of the StGRX2 gene did not change significantly, it was confirmed that the expression of the StGRX1 gene immediately decreased when infected. Therefore, the StGRX1 gene was found to be negatively involved in the defense against pathogen infection. On the other hand, as can be seen in Figure 4, StGRX2 and StGRX3, unlike StGRX1, the expression was not immediately reduced when exposed to the pathogen.

On the other hand, under salt and osmotic stress conditions, the entire RNA of the StGRX gene and dihydrin genes was extracted from the salt and mannitol treated plants and the expression patterns were confirmed by RNA gel-blot analysis. The dihydrin gene, an expression marker gene, was induced by treatment with 200 mM salt and 100 mM mannitol for 24 hours (see FIG. 5). As a result , no significant change in the expression of the StGRX1 , StGRX2 and StGRX3 genes was observed upon treatment with salt and mannitol. However, the StGRX2 gene was found to decrease slightly after 24 hours after 200 mM salt treatment.

Thus, the StGRX1 gene was found to be not resistant to salt stress. In addition, no significant changes were observed in the expression of StGRX1 , StGRX2 and StGRX3 genes for cold stress treatment. On the other hand, when treated with Esepon, expression of StGRX1 gene was decreased, but expression of StGRX2 was increased after 6 to 24 hours after treatment. In case of StGRX3, it increased slightly after 6 hours and gradually decreased.

Three potato glutaredoxins isolated in the present invention responded differently to the treatment of plant hormones. In general, StGRX2 responds very sensitively to plant hormones. When treated with gibberellin and zeatin, only StGRX2 genes were found to be expressed at significantly higher levels than other StGRX genes. In addition, no significant change was observed in the expression of StGRX1 in various plant hormone treatments. In the case of the StGRX3 gene, expression was weakly increased after 10 hours of gibberellin treatment and 1 hour after treatment with methyljasmonic acid.

Among the alleged potato GRX genes, the plant-specific CCXC / S type GRX, the StGRX1 gene, is induced by all organs of potato and various biological and abiotic stresses and hormones, and is expressed similarly to genes expressed in all cells. house keeping gene).

On the other hand, CxxC / S type GRX, StGRX2, was induced when treating various hormones and wounds, but was structurally expressed against pathogen invasion and various abiotic stresses.

Transcript expression of StGRX3 appeared somewhat similar to StGRX2, but expression levels were observed lower than the other two genes. The three StGRX genes show different expression patterns, indicating that their intracellular function is different.

Of the three StGRX genes, only StGRX2 is expected to contain the membrane target domain (see FIG. 1). The StGRX2 gene was found to be located in the mitochondria or chloroplasts, two major organelles involved in the production of reactive oxygen species (ROS) during photosynthesis and cell respiration (Rhoads et al., 2006, PLANT PHYSIOLOGY 141, 357-366 ). StGRX2 transcript levels were upregulated by hormones such as gibberellin (GA ₃ ) and cytokinin (zeatin), while StGRX1 was structurally expressed for hormonal treatment.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example  1: plant material and growth conditions

Potatoes ( Solanum tuberosum L. cv. Desiree plants have been tested in vitro to identify their manifestation. in vitro ) . The stem portion of the potato was incubated for 16 hours at 24 ℃, light conditions in MS medium containing 3% (w / v) sucrose. The leaves and stems were slightly modified in vitro, resulting in corkation and in-flight tubers. Sections were cut and transferred to medium with increased sucrose to 90 g / L and incubated. Growth temperature conditions were maintained at 18 ° C. for in vitro corkation and various biological or abiotic stress treatments were performed. Tubers were grown in greenhouses and used for corkation experiments.

Example  2: cDNA Library  And sequencing design

Grown potato tubers were used for total RNA extraction. Total RNA was extracted by the phenol / SDS method and approximately 5 μg of mRNA was used to link the EcoRI and XhoI restriction enzyme portions for direct cloning into Uni-ZAP XP vectors using cDNA synthesis and cDNA Library Kit (Stratagene). . More than 1200 pBluescript phagemids were isolated from phagemid libraries according to Stratagene's method and cDNA inserts were isolated using T3 primers using the DYEnamic ET Terminator Cycling Sequencing Kit (Amersham Biosciences) with an automatic sequencer (RISA384, Shimadzu).

(5'-AAT TAA CCC TCA CTA AAG GG-3 '(SEQ ID NO: 3)) was sequenced. Base and amino acid sequences were sorted by ClustalW and analyzed using NCBI Blast X search (Altschul et al., 1997, Nucleic Acids Res 25, 3389-3402).

Example  3: biological and Abiological  Stress condition

Responses to potato blight infection stress were performed by well-known analytical methods (Doke, 1975, Physiological Plant Pathology 7, 1-7). Potatoes grown in pots for one month were infected with 20 mL of follicular sac suspension (1 × 10 ³ spore sac / mL) on the leaf surface, and then incubated for 3 days at 100% humidity and 21 ° C. temperature. DNA probes were subjected to PCR with PR-2 and PR-3 genes of pepper cDNA adopted from the EST database ( http://plant.pdrc.re.kr/ks200201/pepper.html ).

The stressed plants used seedlings grown in vials for 5 days. For salt stress, seedlings were transferred to 0, 100, 200 and 300 mM NaCl secondary culture medium and left for 24 hours. Osmotic stress The seedlings were also transferred to 0, 100, 200 and 400 mM mannitol secondary culture medium and left for 24 hours. Dihydrin probes were amplified by performing PCR from cDNA of potato dihydrin gene (GenBank accession No. AAB53203). On the other hand, in the case of low temperature stress, it was left for 0, 1, 3, 6, 12 and 24 hours while maintaining the 0 ℃ in the ice box. In the case of Esepon, the cells were transferred to a secondary culture medium containing 25 mg / L of Esepon and left for 0, 6, 12, 24, and 48 hours. Various hormone treatments were left at 0, 1, 3, 6, 12 and 24 hours in culture media of gibberellin 1 mg / mL, zeatin, abscis acid (ABA), methyl jasmonic acid (MeJA). Each plant sample was treated with different stresses and frozen with liquid nitrogen for RNA extraction.

Example  4: three different isolates from potatoes StGRX  Southern and Northern Genes Blot  analysis

Southern blot analysis of potatoes ( Solanum tuberosum L. cv. Genomic DNA extracted from the leaves of Desiree). The extracted genomic DNA was digested with a suitable restriction enzyme, and the DNA was electrophoresed on a 0.7% agarose gel and transferred to a nylon membrane. Three different GRX probes were subjected to PCR using cloned potato cDNA with primers designed from potato GRX1, 2 and 3 gene sequences (Genbank accession No. EF635995, EF635996 and EF635997).

Nylon membranes were hybridized to probes labeled with digogeninin (DIG) using the PCR DIG probe synthesis kit. Hybridization was continued for 16 hours at 42 ° C. in hybridization buffer, and the membrane was washed twice with 2 × SSC, 0.1% (w / v) SDS for 5 minutes at room temperature. Then, the cells were washed with 0.1 × SSC, 0.1% SDS at 68 ° C. for 15 minutes, and the target DNA was identified using a DIG emission detection kit by a method suggested by the manufacturer (Roche Molecular Biochemicals). Total RNA was extracted from the samples using the phenol / SDS method. 25 μg of total RNA was isolated by electrophoresis on a 1% agarose gel containing 2.2 M formalin and transferred to a nylon membrane. Nylon membranes were hybridized to probes labeled with digogeninin (DIG) using the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals), and hybridization and detection were performed in the same manner as Southern blot analysis.

In the present invention, the StGRX2 gene derived from potato and the StGRX2 protein expressed from the gene are expressed in reproductive stage tissues such as flowers and tubers of potato plants, and are also induced by various biological or abiotic stresses and hormones. It was clarified. Transformation with the gene can produce a transgenic plant that is expression specific and has increased resistance to stress.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Stress-inducible StGRX2 gene from potato <160> 3 <170> KopatentIn 1.71 <210> 1 <211> 619 <212> DNA <213> Solanum tuberosum <400> 1 aatttataca gacaccacta tcaaagaaaa aaaatattag gacgaggaat ccttagccaa 60 ttggattgtg aaagtgcata aaataaacgc aattgatgcc atctccgttt tcttgattaa 120 aaagtcaata caagagagca gaagtgtcat ttctggaaca cgaagcaaaa cccagcttca 180 aacaccttgt taagcttatt cggctatctt tgattgtgtt aataaaggaa ggaggcaatg 240 ggatcaatgt tcagttcacc tcagtttacc aaagaacaaa tggagatcgc tattaccaaa 300 gccaagcaga tcgtttcctc taatcctgtt gttgtattca gtaaaacata ctgcggatat 360 tgcacgaggg tgaagcaact tctttcccag cttggtgcta cttttaaagt tattgaactt 420 gaccaggaaa gtgatggaga tgaggtacaa caagcattac tagaatggac taggcagagg 480 actgtgccta acgtgtttat tgggggagaa catgttggtg gctgcgacag cgtattagag 540 aagcatcaac agggaaagct acttcccatg ctgaaggatg ccgctgctat tcccaacaat 600 cctgccaaag tctagaaaa 619 <210> 2 <211> 125 <212> PRT <213> Solanum tuberosum <400> 2 Met Gly Ser Met Phe Ser Ser Pro Gln Phe Thr Lys Glu Gln Met Glu   1 5 10 15 Ile Ala Ile Thr Lys Ala Lys Gln Ile Val Ser Ser Asn Pro Val Val              20 25 30 Val Phe Ser Lys Thr Tyr Cys Gly Tyr Cys Thr Arg Val Lys Gln Leu          35 40 45 Leu Ser Gln Leu Gly Ala Thr Phe Lys Val Ile Glu Leu Asp Gln Glu      50 55 60 Ser Asp Gly Asp Glu Val Gln Gln Ala Leu Leu Glu Trp Thr Arg Gln  65 70 75 80 Arg Thr Val Pro Asn Val Phe Ile Gly Gly Glu His Val Gly Gly Cys                  85 90 95 Asp Ser Val Leu Glu Lys His Gln Gln Gly Lys Leu Leu Pro Met Leu             100 105 110 Lys Asp Ala Ala Ala Ile Pro Asn Asn Pro Ala Lys Val         115 120 125 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> T223 primer <400> 3 aattaaccct cactaaaggg 20  

Claims (7)

A StGRX2 protein from potato derived from tuberculosis under biological or abiotic stress conditions, consisting of the amino acid sequence represented by SEQ ID NO: 2. The method of claim 1, wherein the stress is dry, low temperature, wound, methyljasmonic acid, gibberellin, esponone, osmotic, hydrogen peroxide, salicylic acid, chitosan, salt, cadmium, ultraviolet irradiation, protein kinase inhibitors, ozone or plant pathogens Characterized by proteins. The protein of claim 2, wherein the plant pathogen is Phytophthora . Infestans . The gene encoding the StGRX2 protein of claim 1. The gene according to claim 4, comprising a nucleotide sequence represented by SEQ ID NO: 1. The gene according to claim 4, which is expressed in flowers and tubers of potato plants. The gene of claim 4, wherein the expression is induced by the plant hormone gibberellin (GA3), zetin or abscis acid (ABA).

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