Activity of AHPF protein of <i>Pseudomonas aeruginosa</i>, and use therefor

Abstract

An AhpF protein has thioredoxin reductase, peroxidase, and chaperone activities and is derived from Pseudomonas aeruginosa, and a use therefor. By using a novel activity of the AhpF of Pseudomonas aeruginosa according to the present invention, it is possible to produce a plant having strong resistance to various environmental stresses such as oxidative stress or heat stress, thereby making it possible to contribute to increasing crop productivity and mass production of useful constituents. In addition, it is possible to prevent desertification and environmental pollution through the development of transformed plants having resistance to high temperatures and drying.

Claims

1. A mutant AhpF protein of Pseudomonas aeruginosa in which cysteine residue at the position 342 in the amino acid sequence of SEQ ID NO: 1 is substituted with serine.

2. The mutant AhpF protein of claim 1, which is in a complex form, said complex having a molecular weight of 500 to 2000 kDa.

3. The mutant AhpF protein of claim 1, which is in a complex form, said complex having a molecular weight of 100 to 250 kDa.

4. A composition for improving resistance of a plant to oxidative stress or heat stress, the composition comprising a vector containing a polynucleotide encoding the mutant AhpF protein of claim 1.

5. An Agrobacterium transformed by a recombinant vector comprising a polynucleotide encoding the mutant AhpF protein of claim 1.

Description

DESCRIPTION OF DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIG. 1 is a mimetic view comparing activities of AhpF (PaAhpF) of Pseudomonas aeruginosa according to the present invention with activities of AhpF and AhpC of E. coli or Salmonella typhimurium known in the art.

(3) FIG. 2 illustrates a peroxidase activity depending on a concentration of the AhpF according to the present invention.

(4) FIG. 3A and FIG. 3B illustrate an influence of peroxidase activities of AhpF and AhpC of Pseudomonas aeruginosa.

(5) FIGS. 4A and 4B illustrate a holdase (FIG. 4A) and a foldase chaperone activity (FIG. 4B) depending on a concentration of AhpF according to the present invention.

(6) FIGS. 5A and 5B illustrate a thioredoxin reductase (TR) activity (FIG. 5A) and thioredoxin (Trx) activity (FIG. 5B) of AhpF according to the present invention.

(7) FIG. 6 is a mimetic view illustrating a structure of an AhpF protein according to the present invention, and illustrating a cloning method for producing mutants of amino acids included in respective terminals according to the present invention.

(8) FIG. 7 is a view illustrating functional switch of AhpF according to the present invention depending on formation structures of high and low molecular weight complex.

(9) FIGS. 8A and 8B are a graph (FIG. 8A) illustrating a result obtained by expressing a recombinant protein of AhpF of Pseudomonas aeruginosa and purifying AhpF using size exclusion chromatography in order to analyze a structure of AhpF according to the present invention and a result (FIG. 8B) obtained by confirming a fraction of each peak of fast protein liquid chromatography (FPLC) using native-polyacrylamide gel electrophoresis (PAGE).

(10) FIGS. 9A and 9B illustrate results of a peroxidase activity (FIG. 9A) and a holdase activity of the chaperone (FIG. 9B) depending on structures of a high molecular weight (HMW) complex and a low molecular weight (LMW) complex of AhpF.

(11) FIGS. 10A, 10B, and 10C, which illustrate a purification result of an AhpF protein using size exclusion chromatography when heat shock is applied to the AhpF protein, illustrate that there was a structural change in AhpF resulting from heat shock.

(12) FIG. 11 illustrates a structure of AhpF according to the present invention and a produced mutant. In FIG. 11, AhpF(PA) indicates AhpF of Pseudomonas aeruginosa according to the present invention, AhpF(PP) indicates AhpF of Pseudomonas putida.

(13) FIG. 12 is a graph illustrating that in a mutant of AhpF according to the present invention in which cysteine at the position 342 was mutated, a TR activity was increased 2 times or more as compared to wild type AhpF.

(14) FIGS. 13A, 13B, and 13C are photographs illustrating states of T.sub.3 generation Arabidopsis thaliana transformed by a vector including an AhpF gene and wild-type Arabidopsis thaliana after 7 days from heat treatment at a high temperature (42 C.) for 2 hours, which illustrates that Arabidopsis thaliana overexpressing the AhpF protein according to the present invention had resistance to heat.

(15) FIGS. 14A, 14B, 14C, and 14D are photographs illustrating whether or not the transgenic Arabidopsis thaliana overexpressing the AhpF had thermotolerance after heat stress at 37 C. for 4 days to wild-type Arabidopsis thaliana and transgenic Arabidopsis thaliana overexpressing the AhpF according to the present invention.

BEST MODE

(16) Hereinafter, the present invention will be described in detail through Examples. The present invention may be modified in various different forms, and is not limited to limited to Examples described below.

<Example 1> AhpF Gene Cloning of Pseudomonas aeruginosa

(17) A PaAhpF gene was cloned from genome DNA of Pseudomonas aeruginosa PAO1 into a pGEM T-easy vector corresponding to a cloning vector using polymerase chain reaction (PCR), and the gene was confirmed using sequencing analysis. PCR was performed under the following conditions for cloning the PaAhpF gene. A reaction of a mixture of genome DNA (10 ng), dNTP (0.2 M), a forward primer (20 pmol), a reverse primer (20 pmol), Taq polymerase (1 unit), and distilled water (20 l) was performed under the conditions: one cycle of pre-denaturation (94 C., 1 minute), 35 cycles of denaturation (94 C., 30 second), annealing (50 C., 45 second), extension (72 C., 45 second), and one cycle of extension (72 C., 10 minutes). In this case, the forward primer was PaAhpF-F(NdeI) 5-AAGCTCATATGTTGGACGCCAATC-3 (SEQ ID No: 2), and the reverse primer was PaAhpF-R(BamH1) 5-GGGATCCTCACTCCGGCGCG-3(SEQ ID No: 3).

<Example 2> Separation and Purification of AhpF Protein of Pseudomonas aeruginosa

(18) In order to separate and purify the AhpF protein, the PaAhpF gene cloned into the pGEM T-easy vector in <Example 1> was sub-cloned into pET28a vector using a restriction enzyme site. Sub-cloning of pET28a::PaAhpF was confirmed through sequencing analysis. The PaAhpF protein was over-expressed in E. coli (BL21 (DE3)) by adding 0.2 mM IPTG using a T7 promoter system in the pET28a vector. The over-expressed PaAhpF protein to which six histidines were tagged was separated and purified using a Ni-NTA chelate resin.

(19) A purification process of the protein was performed as follows: A 1/100 dilution of a seed culture (pET28a::PaAhpF in BL21(DE3)) was inoculate into a 2 L Erlenmeyer flask containing 500 mL of LB, and then cultured at 30 C. and 120 rpm. After culturing the diluted seed culture until an OD.sub.600 reached 0.5, finally, 0.2 mM IPTG was added thereto, and expression of the PaAhpF protein was induced at 30 C. and 120 rpm for 4 hours. Cells were obtained by centrifugation (6000 rpm, 10 minutes, 4 C.) and lysed using a PBS buffer (137 mM NaCl, 27 mM KCl, 10 mM Na.sub.2HPO.sub.4, 2 mM KH.sub.2PO.sub.4). The cells were lysed using a sonicator, and the cell lysate was subjected to centrifugation (15000 rpm, 40 minutes, 4 C.), thereby separating only a supernatant. The over-expressed paAhpF protein to which histidine was tagged was bound to a resin by adding the cell lysate to a NTA-chelate resin previously equilibrated by affinity chromatography, and then treated with thrombin, thereby separating and purifying the PaAhpF protein. The separated and purified protein was moved to a membrane tube, and then subjected to repetitive dialysis three times in 1 L of 50 mM HEPES (pH8.0) buffer, followed by cryopreservation. Enzyme activities were analyzed using the protein separated and purified as described above.

<Example 3> Analysis of Enzymatic Function of AhpF Protein According to the Present Invention

(20) <3-1> Measurement of Peroxidase Activity

(21) In order to measure a peroxidase activity, an Ahp reductase system was used. Scavenging of hydrogen peroxide (H.sub.2O.sub.2) was indirectly measured by measuring a degree of oxidation of NADH to NAD.sup.+ through a change in absorbance at 340 nm. A change in absorbance of a total of 500 l of a reaction solution (0.3 mM NADH, PaAhpF (14 M), PaAhpC (14 M), 1 mM H.sub.2O.sub.2) was measured at 340 nm for 10 minutes.

(22) <3-2> Measurement of Thioredoxin Reductase (TR) Activity

(23) In order to measure a thioredoxin reductase (TR) activity, reduction of Di-thio-bisnitrobenzoic acid (DTNB) was used. In detail, at the time of measuring thioredoxin reductase (TR) activity, first, a reduction rate of DTNB to two molecules of 2-nitro thiobenzoate (TNB) anion was measured at OD 412 nm in an AhpF-free state as a control. A change in absorbance of a total of 500 l of a reaction solution (50 mM potassium phosphate buffer (pH 8.0), 2 mM EDTA, 5 mM DTNB, 0.3 mM NADH, AhpF (0.11 M)) was measured at 412 nm for 5 minutes. Yeast thioredoxin reductase (yTR) was used as a positive control.

(24) <3-3> Measurement of Thioredoxin (Trx) Activity

(25) In order to measure a thioredoxin (Trx) activity, insulin reductase was used. In general, insulin exists in a state in which and chains are linked to each other by disulfide bonds, but in a case in which thioredoxin (Trx) reduces the disulfide bond, the chain is denatured, thereby forming an aggregate. A degree of formation of the aggregate as described above was determined by measuring absorbance at 650 nm. A change in absorbance of a total of 500 l of a reaction solution (100 mM potassium phosphate buffer (pH 7.5), mM EDTA, 500 g insulin, 2 mM DTT, PaPrx (110 M)) was measured at 650 nm for 30 minutes.

(26) <3-4> Measurement of Molecular Chaperone

(27) In general, a molecular chaperone activity is divided into a holdase activity and a foldase activity. In the present experiment, both of the holdase activity and the foldase activity were analyzed. First, the principle for analyzing the holdase activity was as follows: when malate dehydrogenase (MDH), which is sensitive to heat stress, is heated to 43 C., malate dehydrogenase (MDH) is denatured to thereby be aggregated, such that absorbance is increased. However, in a state in which malate dehydrogenase (MDH) coexists with a protein having a molecular chaperone activity, denaturation of MDH is prevented, and thus formation of aggregates is suppressed, such that absorbance is not increased. In the present invention, a change in absorbance of a total reaction solution (50 mM Hepes buffer (pH 7.5), 52 g MDH, various concentrations of PaAhpF) was measured at 340 nm for 15 minutes using the principle as described above.

(28) In order to measure the foldase activity, after glucose-6-phosphate dehydrogenase (G6PDH) protein was treated with guanidine-HCl to thereby be denatured, a recovered G6PDH activity was measured. A degree of refolding of the total reaction solution was measured. In detail, after 40 M G6PDH was chemically denatured in a denaturation buffer (50 mM Tris-HCl (pH 7.5), 4 M guanidine-HCl) at room temperature for 2 hours 30 minutes, the denatured G6PDH was diluted 50 times and reacted for 6 hours, 12 hours, and 24 hours in a renaturation buffer (50 mM Tris-HCl (pH 7.5), at various concentrations of AhpF protein or GroEL protein, 10 mM ATP, 10 mM KCl, 2.5 mM MgCl.sub.2). Thereafter, an activity of the renatured G6PDH was measured. In detail, NADPH formed in a total of 500 l of a reaction solution (50 mM Tris-HCl (pH 7.5), 1 mM NADP, 2 mM glucose-6-phosphate (Glucose-6-P), 4 nM renatured G6PDH) was calculated by measuring absorbance at 340 nm for 5 minutes. GroEL was used as a positive control.

(29) ##STR00001##

(30) As a result of analyzing the enzymatic activities of AhpF, it may be confirmed that AhpF of Pseudomonas aeruginosa had all of the thioredoxin reductase, thioredoxin, peroxidase, and chaperone activities, as illustrated in FIGS. 2, 3A, 3B, 4A, 4B, 5A, and 5B.

<Example 4> Structural Change of AhpF Protein According to the Present Invention and Analysis of Activity Depending Thereon

(31) In order to analyze a structure of the AhpF protein, size exclusion chromatography (SEC) was performed. In detail, the AhpF protein and 10 mM Tris-HCl (pH 8.0) buffer solution were passed through a Superdex 200 10/300 GL column at a constant rate (0.5 ml/min) using FPLC (Amersham Biosciences; AKTA). 0.5 ml of the buffer solution and 0.5 ml of the AhpF protein passed through the column were collected at each time. In addition, the collected AhpF protein was largely divided and fractionated into three groups (F1, F2, and F3) depending on protein peaks detected at OD 280 nm. Each of the fractionated proteins was concentrated in order to verify activities of the protein.

<Example 5> Production of Mutant of AhpF Protein and Analysis of Activity Thereof

(32) Mutants of a TR domain (AhpF_C), and a Trx domain (AhpF_N) of the AhpF protein and various mutants (C128S, C131S, C342S, C344S, C347S, C342A, C128/131S, C342/344/347S, All C to S_AhpF) in which an active cysteine residue was substituted with serine were produced as illustrated in FIGS. 6 and 11. Each of the proteins from these mutants was separated and purified using affinity chromatography as in <Example 2> and an activity of each of the enzymes was measured by the same method as in <Example 3>.

(33) As a result, the TR activity was decreased in other mutants as compared to a wild type AhpF protein, but in C342S and C342A mutants, the TR activity was increased by two times or more as compared to the wild type AhpF protein (see FIG. 12).

<Example 6> Production of Arabidopsis thaliana Transformed by Recombinant Vector into which AhpF Gene is Introduced and Confirmation of Resistance to Heat Shock

(34) In order to produce transgenic Arabidopsis thaliana overexpressing PaAhpF, a PaAhpF gene was constructed into a pCAMBIA1302 vector corresponding to a transformation vector, and this construct (pCAMBIA1302:PaAhpF) was transformed into Agrobacterium. Thereafter, Arabidopsis thaliana was transformed. In order to confirm a transformant, a transformant having resistance was selected in hygromycin selective media, and a third-generation (T.sub.3) transformant was secured through repetitive selection. Further, PaAhpF protein over-expressed was confirmed in the T.sub.3 transformant through a western blotting test.

(35) Thermotolerance of the selected transgenic Arabidopsis thaliana overexpressing PaAhpF was verified in a LB media and soil. A test of thermotolerance in the LB media, after Arabidopsis thaliana cultivated for 10 days was subjected to heat shock at 42 C. for 2 hours, recovery of Arabidopsis thaliana was observed at 22 C. for 3 to 7 days again. A test of thermotolerance in the soil, after Arabidopsis thaliana cultivated at 22 C. for 3 weeks was subjected to heat shock at 37 C. for 4 days, recovery of Arabidopsis thaliana was observed at 22 C. for 5 days again. The transgenic Arabidopsis thaliana overexpressing PaAhpF had resistance to heat in the soil as well as in the LB media (see FIGS. 13A, 13B, and 13C, and FIGS. 14A to 14D).

(36) Although Examples of the present invention have been illustrated and disclosed, those skilled in the art will appreciate that various modifications are possible, without departing from the scope and spirit of the invention, and the scope of the invention will be disclosed by the accompanying claims.