KR100282657B1 - A novel chitinolytic enzyme and a gene encoding this protein - Google Patents

Abstract

The present invention relates to a novel kitsase having excellent chitin resolving ability separated from amber and a nucleic acid encoding the protein.

Description

A novel chitinolytic enzyme and a gene encoding this protein

[TECHNICAL FIELD]

The present invention relates to a novel chitinase having strong chitin resolution and a nucleic acid encoding the protein.

BACKGROUND ART [0002]

While crops are attacked by numerous pathogens (fungi, bacteria, viruses, etc.) during their growing and harvesting processes, they are susceptible to a variety of diseases, but it is difficult for them to adapt to these environmental stresses . Therefore, when attacked by these pathogens, the crops will cause a decrease in growth and a decrease in yields, resulting in considerable economic loss to humans. In order to prevent such diseases, humans have been protecting crops from diseases by spraying various organic and inorganic chemical synthetic pesticides and herbicides for a long time (Sidebox NA ed ac., Environs. Health. Percept., 103 ), 1126-1134, 1995) Repeated administration over a long period of time and excessive administration due to the adaptability of pathogens not only induced fatal diseases caused by pesticide poisoning in humans, but also caused the accumulation of such synthetic pesticides in the soil (Sherman JD et al., Arch. Environ. Health., 52, 332-333, 1997), which is inadequate for mankind to live by bringing about environmental pollution and destruction of the natural environment. have. Particularly, people engaged in agriculture inhale toxic pesticides in the human body through the process of spraying pesticides and the growth of crops. As a result, new diseases and life threats are emerging as serious social problems. In addition, the accumulation of these pesticide residues in consumers who have absorbed agricultural chemicals that have been absorbed or accumulated in crops and have absorbed residual pesticide residues that have not been completely removed (London et al., Scan. J (2000)), and the results of this study are as follows.

BACKGROUND ART [0002]

In the meantime, it is known that wild plants produce various bio-defense proteins, effectively sterilize various pathogens and protect themselves (Bradley et al., Cell. 70, 21-30, 1992; Bovoles, DJ, Annu Rev. Biochem., 59, 873-907, 1990). (Antibiot., Somers et al., 40, 1751-1756, 1987), which has a strong bactericidal activity against pathogens by showing the ability to decompose strongly the chitin, a cell wall component of pathogenic bacteria, Enzymes are known.

The present invention relates to a novel chitinase enzyme which exhibits a potent sterilizing power against pathogenic bacteria in order to replace the oil and inorganic chemical synthetic pesticides which have been used during the cultivation process of the crops and accumulated in the soil and caused the destruction of the natural environment And a nucleic acid encoding the protein.

FIG. 1 is an electrophoresis experiment (coomassie staining) to examine the chitin degradation activity of various plants.

A: Coomassie staining of proteins separated by SDS electrophoresis

B: Measurement of chitin degradation activity by active staining electrophoresis (active staining SDS-PAGE)

In the diagrams A and B, the dotted line (---) ) Represents the absorbance A ₂₈₀ at ₂₈₀ nm.

S: standard molecular weight protein

No. 1: pepper leaves

No. 2: Tomato leaves

Number 3: Cucumber leaves

No. 4: Sesame leaves

Number 5:

No. 6: Pumpkin leaves

FIG. 2 is a chromatogram of chitinase (P-CH-kittinase) isolated from zucchini leaves with strong chitin resolution.

A: Chromatograms obtained by purifying water-soluble proteins of zucchini leaves fractionated with 80% ammonium sulfate by using a regenerated chitin column

B: Chromatogram obtained by eluting the protein bound to the chitin affinity column and purely separated using an anion exchange mono-Q column of high performance liquid chromatography (HPLC)

FIG. 3 shows the purity of P-CH chitinase purely isolated from the amber leaves.

S: standard molecular weight protein

A: coomassie staining

B: Antigen / antibody binding reaction using P-CH chitinase antibody

C: Purity verification by active dye electrophoresis

FIG. 4 is a sequence comparison and analysis of proteins with amino acid sequences similar to the N-terminal amino acid sequence of P-CH-ketiminase with high chitinolytic activity present on the amber leaves.

1) Based on these results, it was found that P-CH kitty or class was a kind of class III chitinase, and a PCR probe of C-terminal for gene coronation was prepared using a part which is kept relatively constant in this class of enzymes Respectively.

2) A N-terminal PCR probe for gene cloning was synthesized using the N-terminal amino acid sequence.

FIG. 5 shows the stability of pure P-CH chitinase to temperature change.

1) Determination of chitin degradation activity at various temperatures (20 ℃ -80 ℃)

2) The activity measurement was performed using a spectroscopic method.

FIG. 6 shows stability measurements for pH changes.

1) Activity is measured at various pH (2.0-8.0)

2) The activity measurement was performed using a spectroscopic method.

FIG. 7 is Northern blotting comparing the amount of transcriptional transcript in the amber.

FIG. 8 shows an antigen-antibody reaction to see the expression of P-CH chitinase by various stimuli.

A:

B: pumpkin stem

1: Experimental control (control)

2: 0.1% mercuric chloride

3: Neutrophil mycelium (Neurospore crassae hyphae)

4: UV-irradiation

5: 0.01% glycol chitin

6: 10 mM salicylic acid

FIG. 9 shows the expression of P-CH-chitinase by treatment with mercuric chloride and glycollicholine.

M.C: Brine treated with mercuric chloride

G.C: Ginkgo biloba treated with glycolchitin

A: After treatment with mercuric chloride and glycolchytine, RNA was isolated after various time periods to determine whether transcripts of P-CH-ketiminase were produced by Northern blotting.

B: The amount of time required for expression of P-CH kittinase by these stimuli through antigen-antibody reaction with antibody against P-CH chitinase by treating the mercuric chloride and glycollichtin and separating the protein after various time .

In FIG. 9, hr means hours (hours).

According to the present invention, a kittinase protein having an amino acid sequence represented by SEQ ID NO: 2 is provided, and a nucleotide sequence of a gene encoding these amino acids is provided.

Until now, the methods used to control diseases of crops have been toxic and have been applied to organic and inorganic chemical pesticides which are not decomposed in natural ecosystem. However, they have been exposed to residual toxicity, pesticide poisoning and fatal diseases caused by accumulation in living organisms. Serious social problems are being raised. Furthermore, environmental pollution and destruction of ecosystems for the overdose of these chemicals are causing irreparable human catastrophe. In order to overcome these serious problems, the research projects that have been started are the search and separation of biomaterials in living organisms and the development of pollution-free pesticides by their use (Kramer KJ et al., Insect. Biochem. -900, 1997). These studies have recently begun to be studied and include BT-toxin (Frankline SE et al., Mol. Gern. Genet., 256, 517-524, 1997) or a protease inhibitor et al., Biochemistry., 37, 4071-4079, 1998) and viral disease resistance crops using the viral skin protein (Von Schwedler UK et al., ENBO. J., 17, 1555-1568, Development is underway. Although these challenges remain in the baseline stage of research, a number of companies and research institutions are competing (Lorito M et al., MoI Biotechnol., 2, 209-217, 1994) And it can be expected to solve many problems. In addition, a defense mechanism that protects plants from invasion by pathogenic fungi includes the generation of phytoalexin, the strengthening of cell walls, and the protection of self by creating resistance proteins in plant cells that kill pathogens (Kauffman et al. EMBO J. 6, 6309-3212, 1987). One of the various defense mechanisms that kills pathogenic fungi is kitsinase (Shinshi et al., Proc. Natl. Acad Sci., 84, 89-93, 1987). In order to develop pollution-free pesticides which do not damage the ecosystem because they are rapidly degraded and destroyed in nature, they do not cause any damage to biological functions by the use of excessive amount or absorption into the living body. In this invention, wild plants are produced, By rapidly dissolving cell walls and sterilizing them, it is possible to isolate a protein called Kittinase, a bio-defense protein that shows resistance to these crop diseases, protects itself, and isolates the gene encoding this protein in order to mass-produce it. It is the analysis of the base sequence.

So far, a study on Kittinaje Boller. T, etc. However, since the enzymatic activity of these enzymes is very low and the amount of protein produced is low, the stability of protein is not good. Therefore, the use of the enzyme as a pest- (Cook RJ et al., Proc. Natl. Acad. Sci., 95, 1993-1995, 1998). However, in the present invention, a large amount of a chitinase protein is obtained from an amber leaf of a specific time, which is excellent in chitin degradation ability than the existing chitinase and particularly the expression amount of the protein is enormously large, and by cloning a gene encoding the protein, We have developed a method to solve various problems that have been raised in the process of pollution - free pesticide development. Accordingly, the present invention relates to a method for purifying chitinase from a plant by purely isolating the chitinase, which is a protein enzyme for cleavage of the chitinase, and isolating the gene encoding the enzyme and revealing the nucleotide sequence and gene characteristics, will be.

The cell wall of many fungi consists of chitin, a polymer of N-acetylglucosamine, which can resist mold attack by hydrolyzing chitin. In the present invention, a new plant protein having excellent chitinolytic activity is separated from the leaves of pumpkin and used as a biological countermeasure against pathogens, thereby causing harmful effects due to the use of existing pesticides and fungicides, that is, And the destruction of ecosystems caused by the use of pesticides indiscriminately.

The present invention will be described in more detail as follows.

1. Investigation of chitin degradation activity.

As a first step of the present invention, it was confirmed by the electrophoresis of various plants (pepper, tomato, cucumber, pumpkin, bean, soybean) Pure water was separated and named P-CH Kittinaja. Active measurement electrophoresis was performed by SDS-PAGE with glycol chitin as a substrate for 12-24 hours, followed by staining with calcofluor white M2R (Monreal, J et al., Can. J. Microbiol., 15, 689-696, 1969).

2. P-CH Chitinase pure separation.

The pure separation of P-CH chitinase was performed by applying a variety of chromatographic methods using the previously described properties of chitinase and isolating it by slight modification. P-CH chitinase was purified by using an anion exchange mono-Q column of a regenerated chitin column and affinity chromatography for chitinase and high performance liquid chromatography (HPLC).

3. Purity verification and molecular weight determination of purely isolated proteins.

(Laemmli, C., Nature, 227, 680-685, 1970) was used for the purity verification of pure P-CH chitinase using SDS-PAGE and the standard molecular weight substance (Biorad) And the movement distance was compared. As a result, it was confirmed that the molecular weight of the P-CH chitinase was 29, 000 Da (Da).

4. Preparation of Antibodies to P-CH Kittinase.

P-CH chitinase was injected into rabbits to prepare an antibody against this protein, which was found to react with a specific P-CH chitinase protein.

5. N-terminal protein sequence analysis.

The order of the 15 amino acids of the N-terminus constituting this protein from the purely isolated P-CH-kitty gene was determined by the Edman degradation method (Hewick et al., J. Biol. Chem., 256 , 7990-7997, 1981), comparing the amino acid sequence with other proteins registered in the gene bank, the P-CH kittinase was found to be a class III kitty or jade.

6. Gene cloning and gene sequencing.

Oligonucleotides derived from the N-terminal protein sequence and oligonucleotides that are constantly present in similar types of chitinases are synthesized and used for gene amplification (PCR) method to obtain a gene fragment that synthesizes this protein. This gene fraction was cloned by using a probe-filter-hybridization technique to clone the gene of P-CH chitinase. The nucleotide sequence of the cloned P-CH chitinase gene was determined by the method of Sanger et al., Proc. Natl. Acad. Sci., 74, 5463-5467, 1977) by the method of dideoxy DNA sequencing The overall base sequence was determined using an automated DNA sequencer (Applied Biosystems model 373A).

7. Measurement of stability of P-CH chitinase protein.

We have compared the changes in enzyme activity with changes in temperature and concentration (pH), which are important factors in determining the enzyme activity, to produce P-CH chitinase protein as an actual pesticide or drug. Activity measurements were performed spectrophotometrically with chitin as a substrate (Boller, T et al., Methods. Enzymol., 161, 430-435, 1988).

1) Stability of P-CH chitinase to temperature change.

The chitin degradation activity at various temperature conditions (20-80 ° C) was investigated. The activity was relatively high at 30-60 ° C and the maximum activity of P-CH-ketiminase was observed at 50 ° C.

2) Stability of P-CH chitinase to changes in pH.

Chitin resolution was investigated at various pH conditions. As a result, the highest activity was observed at pH 6, and the activity remained above 80% of the maximum activity even in the range of pH 3-7. This suggests that the P-CH kitty or P-CH is relatively stable against changes in pH or temperature, suggesting that it can be easily applied in terms of industrial applications.

8. Expression patterns of P-CH-chitinase.

1) Expression of P-CH-chitinase in Porphyra.

RNA was isolated from various organs (pistil, ovary, petal, calyx, and stomach) and subjected to nothern blotting (Watson, JC , Methods Enzynol. 118, 57-75, 1986). As a result, it was confirmed that the most abundant genes were present in the pistil and the stamen, and thus it was found that the chitinase was able to protect against pathogens in plant propagation and growth organs.

2) Investigation of expression inducer of P-CH chitinase.

P-CH Kitty and I have experimented with the amber leaves and stems to see how large quantities are produced under certain conditions. (2) 0.1% mercuric chloride, (3) Neurospore crassae hyphae, (4) UV irradiation, and (5) 0.01% (6) 10 mM salicylic acid, (7) 0.1% regenerated chitin, and then the protein was separated to examine the antigen-antibody reaction. As a result, In the leaves, the protein was expressed in large amounts under the conditions (2), (4) and (5) above and under the condition (7) in the stem.

3) Expression process of P-CH chitinase.

To determine the time required for the expression of P-CH chitinase protein after specific stimuli were applied to pumpkin, the pumpkin leaves were treated with the conditions (2) and (5) , 12, 24, 48 hours), samples were taken, and nodal blotting and west blasting were performed. (2), the amount of RNA was maximized at about 1 hour after treatment, and a large amount remained until 3 hours had elapsed. A large amount of RNA was expressed at about 24 hours after the treatment of protein And lasted up to 48 hours. On the other hand, when treated with the condition (5), the amount of RNA increased after 3 hours and continued until 12 hours. Protein expression pattern was similar in both cases.

Thus, P-CH chitinase, which is superior in chitinase activity to other chitinases found in other plants, maintains relatively high kitsin degradation activity even under changes in temperature and pH. Also, the site of production of chitinase is different, And showed the difference in appearance. Thus, various industrial applications using chitin degradation activity of this protein, such as development of pollution-free pesticide which is harmless to human body, development of a new breed with increased resistance to pathogens, development of health drink and food using chitin oligomer I think it is possible.

The present invention will now be described in detail with reference to the accompanying drawings.

Example 1. Investigation of chitin degradation activity using an active staining method.

Proteins were extracted from the leaves of various plants (pepper, tomato, cucumber, bean, bean, pumpkin) to obtain 12% (W / V) ESD electricity containing 0.01% (W / V) glycol chitin After performing SDS-PAGE, a 0.1M tris-hydrogen chloride pH 7.0 buffer (Tris-HCl pH 7.O buffer (pH 7.0) containing 1% (V / V) triton X- ) For 12-24 hours. The cultured gel was placed in a 0.5 M Tris-HCl pH 8.9 buffer solution (Tris-HCl pH 8.9 buffer) in which 0.1% calcofluor white M2R staining reagent was dissolved and incubated for 30 minutes in a dark state After staining, rinsed with sterile distilled water for 1 hour for 3-4 rinses, the chitin degraded portion was destained by the chitinase. The degradation activity was measured by the size of the decolorized portion of the UV transilluminator. The glycol chitin used as the substrate was acetylated with glycol chitosan (Monreal, J. et al, Can. J. Microbiol. 15, 689-696, 1969).

In addition, the same amount of protein was electrophoresed, stained with protein staining reagent, and compared with the results of the above active staining electrophoresis.

As a result, as shown in FIG. 1, the amount of the electrophoretic protein extracted from different plants was similar, but it was confirmed that there was a highly active chitinase at the position corresponding to 29 kDa of the zucchini leaves. Protein was isolated by pure separation.

Example 2: Pure separation of P-CH chitinase.

To 2 kg of zucchini leaves collected in an early autumn niger were added 0.1 M Tris-HCl pH 7.09 buffer (0.1 M Tris-HCl pH 7.0 buffer (pH 7.4)) containing 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF , Hereinafter referred to as Buffer A) was added, and the mixture was pulverized with a polytron and centrifuged at 8,000 xg to obtain a supernatant. To the supernatant is added 80% ammonium sulfate to precipitate the protein, centrifugation to obtain a precipitate, and then a minimum amount (about 250 ml) of buffer solution A is added and dissolved. This was put on a dialysis membrane and dialyzed against a large amount of buffer solution A 3-4 times. The protein solution was passed through a regenerated chitin affinity chromatography column (3 x 30 cm), and buffer solution A was shed to remove nonspecific proteins from chitin. The protein bound to the chitin was eluted using a 20 mM sodium hydroxide (NaOH) pH 12 solution, and the protein solution was concentrated and injected into an anion exchange Mono-Q column of high performance liquid chromatography (HPLC) And eluted with a salt concentration gradient (0-0.4 M NaCl). The P-CH chitinase was purified from the amber leaf by the same method and the chromatographic results are summarized in FIG. Based on the fact that the chitinase binds to the substrate chitin in the process of separating the protein having excellent chitin cleavage activity from the amber leaf, the chitin affinity chromatography was introduced at the early stage, and most of the chitin- By removing the protein, the P-CH chitinase could be separated pure through a two step chromatography.

Example 3. Purity verification and molecular weight measurement

In order to measure the purity of the P-CH chitinase of the zucchini leaves purified by the same method as in Example 2, Essex electrophoresis was carried out to separate the proteins according to the combination of polarity and molecular weight, coomassie-brilliant blue) was used to stain the separated proteins. As a result, only one protein band was stained as shown in FIG. 3 (A), thereby proving that the protein was purely isolated. Approximately 360 μg of pure protein was obtained from 2 kg of zucchini leaves. (3- (S)). By comparing the migration distance of these proteins with the migration distance of purely separated proteins, the molecular weight of the P-CH chitinase isolated from the amber leaves was 29,000 Dalton. At the same time, using the method (westhern blotting, 3- (B)) and active staining electrophoresis (3- (C)) using an antigen-antibody reaction using an antibody against P-CH chitinase as described in Example 4 The purely isolated protein proved to be Kittina.

Example 4. Antibody production against P-CH < RTI ID = 0.0 >

200 μg of protein and 300 μg of Freund's adjuvant were mixed thoroughly to prepare an antibody against the P-CH chitinase protein purely isolated from the amber leaf, and then injected into the subcutaneous tissue of the rabbit three times at 3-week intervals. In the fourth injection, only 200 μg of protein was injected after one month, and after 3 days, blood was collected from the heart of the rabbit and centrifuged to obtain the antibody. In order to measure the reactivity between the antibody and the P-CH chitinase protein, 10 μg of P-CH chitinase was separated by electrophoresis and electrotransferred at 100 ° C. for 2 hours at 4 ° C. The nitrocellulose membrane was coated with an 8% skim milk solution and then the antigen-antibody reaction was confirmed. At this time, the confirmation of the reaction was confirmed by binding a peroxidase-conjugated secondary antibody to the primary antibody and then adding a substrate for peroxidase to search for a fluorescent substance (ECL- detection method). As a result, as shown in Fig. 3- (B), it was confirmed that an antigen-antibody reaction appears at a position coincident with a protein band stained with a protein staining reagent. This antibody was also used in experiments to identify substances that induce the production of P-CH-ketiminase in leaves and stems of amber.

Example 5. N-terminal protein sequence analysis.

In order to determine the N-terminal amino acid sequence of the P-CH chitinase protein isolated from the amber leaf in the same manner as in Example 2, the chitin degradation activity fraction eluted from the HPLC on a Mono-Q column was subjected to 12% SDS- PAGE. The separated proteins were transferred to a PVDF membrane and stained with a ponceu-red dye to remove only the P-CH chitinase protein band. In this band, the P-CH chitinase protein was isolated and the N-terminal 15 amino acid sequence of the P-CH chitinase protein was determined using the Ednan degradation method (Terras et al., J. Biol. Chem., 267, 15301-15309, 1992). Comparing the amino acid sequence revealed with other proteins registered in the gene bank, P-CH-chitinase shows high similarity in amino acid sequence with class III chitinase, so that P-CH-chitinase can also be deduced as a class III kitty or jasmine (Fig. 4).

Example 6. Gene cloning and gene sequence analysis.

N-terminal oligonucleotide probes for use in gene amplification (PCR) were synthesized from the amino acid sequences disclosed in Example 5. Oligonucleotide primers to be used at the C-terminus were synthesized using a portion common to the class III chitinase. In addition, a gene template necessary for cloning a specific gene by the gene propagation method is obtained by separating the mRNA from the amber leaf and synthesizing a complementary DNA for the mRNA by using a reverse transcriptase, (Lagrimini et al., Proc. Natl. Acad Sci., 84, 7542-7546, 1987). A gene that determines the amino acid sequence of P-CH chitinase is amplified by a PCR method using a heat-resistant gene synthetase (taq-polymerase) using the thus-synthesized genetic primer and N-terminal and C-terminal bidirectional gene primers A part of the gene coding for P-CH chitinase was obtained and a part of this gene was used as a probe necessary for cloning whole genes. The gene coding for P-CH chitinase was cloned by a filter-hybridization method with a ³² P-containing gene probe (Sambrook et al., Cold Spring Harbor Lab, 1989). The nucleotide sequence of the cloned gene was determined by the dideoxy chain termination method of Sanger, using the USB sequenase kit. The deduced amino acid sequence deduced from the amino acid sequence of the determined nucleotide sequence was exactly the same as that of the N-terminal amino acid sequence determined using purified P-CH chitinase protein. As a result, it was confirmed that the gene cloned in this experiment and the gene coding for P-CH chitinase. As can be seen from SEQ ID NO: 1, the gene coding for P-CH chitinase present on the amber leaf is composed of 1019 nucleotides, 27 in the N-terminus and 27 in the C-terminus, and 71 non-coding genes And there were 60 nucleic acids corresponding to the signal peptide at the N-terminus. The amino acid deduced from the nucleic acid sequence of this gene was 267 including 6 cysteine residues which were constantly present. The molecular weight of the protein was 29,370 daltons and the protein was directly separated and analyzed by SDS electrophoresis When compared with the confirmed 29,000 daltons, it was very similar.

Example 7. Stability of P-CH < RTI ID = 0.0 > chitinase < / RTI >

1) Stability against temperature change

To evaluate the stability of P-CH-chitinase in industrial applications, we measured the stability of this protein against temperature changes. This experiment was carried out by using pure P-CH chitinase protein, and the change of chitin degradation activity to temperature change between 20 ° C and 80 ° C was measured by spectroscopic method. A predetermined amount of chitinase and 1 mg of swollen chitin were mixed with 100 mM sodium acetate pH 5.2 buffer (100 mM sodium acetate pH 5.2 buffer), and the mixture was incubated at various temperatures for 1 hour. After centrifugation at 10,000 xg for 10 minutes Separately, 150 mu l of supernatant was obtained. 15 μl of potassium phosphate pH 7.1 solution and 10 μl of protoplast-forming enzyme isolated from snake gut were mixed and reacted at room temperature for 30 minutes to obtain chitooligosaccharides ). After reacting the resulting monomeric N-acetylglucosamine with p-dimethylaminobenzaldehyde (DMAB), the absorbance was measured and the change of chitin degradation activity with temperature was observed. As a result, as shown in FIG. 5, the chitinolytic activity of the P-CH chitinase protein showed the best results when reacted at 50 ° C., and maintained not less than 90% activity at 40 ° C. and 60 ° C., , And showed 70% or more chitin degradation activity even when reacted at 70 ° C, so that P-CH chitinase protein was relatively stable to temperature change.

2) Stability against pH change

In order to measure the stability of the P-CH chitinase protein according to the change in pH, the pure protein isolated with Swell chitin as a substrate was dissolved in 20 mM acetic acid at pH 2.0, 20 mM sodium acetate at pH 3.0-6.0 sodium acetate) and 20 mM sodium bicarbonate buffer solution at a pH of from 8.0 to 9.0. Then, the activity of chitin degradation was measured in the same manner as in Example 7. As a result, as shown in FIG. 6, the maximum activity was observed at pH 6.0, and it was found that various pH changes, particularly, high activity were maintained under acidic conditions. In Examples 7 and 8, the P-CH chitinase protein can adapt to various environmental changes and maintain its function, suggesting that it is due to the structural stability of the protein.

Example 8. Expression of P-CH < RTI ID = 0.0 >

1) Expression of P-CH2Kitinase.

Experiments were performed to see if a large amount of P-CH chitinase transcripts were produced in a flower's organs. Replaces the stigma of pumpkins harvested in open field, ovaries, petals, sepals, the RNA 20㎍ separated from surgery to 1.8% agarose gel (agarose gel) nylon membrane (nylon membrane) after it has been deployed on a ³² p attached to it P-CH < / RTI > kittinase gene probe for 18 hours to remove non-specific portions. As shown in FIG. 7, a small number of transcripts in the pistil and a large amount of transcripts in the operation were observed on the X-ray film. As a result, a large amount of P-CH kitty It was found that the protein was produced.

2) Investigation of expression inducer of P-CH chitinase.

In plants, Kittinaja is a protein that functions like an animal 's immune system. It does not always exist in large quantities, but shows a large - scale expression by specific stimuli. In this example, various treatments of zucchini leaves were conducted to examine the induction of a large amount of P-CH chitinase protein on a stimulus. (2) 0.1% mercuric chloride, (3) Neurospore crassae hyphae, (4) UV irradiation, (3) fungal hyphae, and 5) After treatment of 0.01% glycol chitin, 6 mM salicylic acid, and 7 regenerated chitin, RNA was isolated from the leaves and stems to obtain P-CH As shown in Fig. 8, the most P-CH chitinase transcripts were observed under the conditions of (2), (4) and (5) , It was found that the most P-CH kinetinase transcript was present under the condition (7). This indicates that the substances inducing the expression of P-CH chitinase protein in the leaves and stems are different from each other, which means that the response patterns against pathogen invasion or sudden changes in the external environment are different from each other.

3) Expression process of P-CH chitinase.

In the above experiment, mercury chloride and glycollchicine, which were found to effectively induce the expression of P-CH chitinase in the zucchini leaves, were treated with leaves, samples were taken after various periods of time, proteins and RNA were separated, We examined the time used to induce the expression of CH-chitinase. As shown in FIG. 8, a large amount of RNA was present after one hour of treatment with mercuric chloride, and the amount of RNA was reduced after 3 hours. The expression of the protein was markedly increased after 24 hours, and the amount of the protein was maintained until 48 hours. On the other hand, when treated with glycollichtin, the amount of RNA was remarkable at 3 hours after treatment, and the amount of RNA was longer than that of mercuric chloride, and continued until 12 hours after the treatment, As in the case of mercury treatment, it was increased over 24 hours and maintained for 48 hours. It was also observed that the time required to generate proteins in response to different external stimuli was also different.

As described above, the mass production and industrial application of the P-CH chitinase of the present invention, which is a protein having an excellent chitinolytic activity over other chitinases present in other plants, can reduce the environmental pollution by replacing the use of existing pesticides, It is expected that the development of various health foods and health drinks will be possible by producing a large amount of chitin oligomer by controlling the pathogen by a non-existent biological method and further utilizing the excellent chitin degrading activity.

[Sequence List]

SEQ ID NO: 1

Sequence length: 1,019

Sequence type: nucleic acid

Number of chains: 1 chain

Topology: Linear

Molecular type: cDNA

origin

Name of creature: Amber (Cucubita maxima)

Type of tissue: Leaf

Characteristics of sequence

Symbol for characterization: sig peptide

Present location: 28..87

How to determine features: S

Symbol indicating characteristics: mat peptide

Present location: 88..891

How to determine features: S

[SEQ ID NO: 1]

SEQ ID NO: 2

Length of sequence: 287

Sequence type: Amino acid

Number of chains: 1 chain

Torroji: Linear

Molecular type: Protein

origin

Name of creature: Amber (Cucubita maxima)

Type of tissue: Leaf

Characteristics of sequence

Symbol for characterization: sig peptide

Present location: 1..20

How to determine features: S

Symbol indicating characteristics: mat peptide

Present Location: 21..287

How to determine features: S

[SEQ ID NO. 2]

Claims (3)

A protein having an amino acid sequence represented by SEQ ID NO: 2. A DNA encoding the protein of claim 1. 3. The method of claim 2, Wherein the DNA has the nucleotide sequence represented by SEQ ID NO: 1.