KR101412077B1 - antiTobacco mosaic viral peptide and use thereof - Google Patents

Abstract

The present invention relates to a novel anti-tobacco mosaic virus peptide and provides a circular anti-tobacco mosaic virus peptide having a structural formula of cyclo(Ser-Thr-Val-Met-Pro-Leu-Ser-Leu-Gly) and a use thereof.

Description

Tobacco mosaic virus control peptide and use thereof [0002]

FIELD OF THE INVENTION The present invention relates to antiviral peptides and their uses, and more particularly to peptides exhibiting tobacco mosaic control effects and uses thereof.

Mosaic viruses are viral plant diseases that cause massive loss of greenhouses and crops. Drifted plant debris and long-lived viruses in the soil act as infectious agents that cause diseases in crops grown next (Lanter et al. Plant Dis. , 13, 552-555, 1982; Rabindran and Dawson, Virology. 284, 182-189, 2001). Therefore, plant viral disease control or inhibition is one of the major problems in crop protection during the growing season. In Korea, the incidence of viral diseases in greenhouse crops has increased markedly since the 1980s, and more than 90% of plant virus diseases have been caused by tobacco mosaic virus (TMV) (Kim et al. Plant Pathol. , 6, 125-132 1990).

Currently, various synthetic peptides for controlling plant virus diseases have been used. However, they are methods for inhibiting the migration or spread of viruses by controlling virus-mediated insects, and chemical treatment methods directly acting on TMV are known none. Therefore, this study focuses on methods to reduce or inhibit viral infections in order to control infestation and reinfection of viruses for TMV control.

Currently, compounds for effectively inhibiting plant viral diseases have been developed. For example, biological agents derived from plants and microorganisms (Yun et al., Tetrahedron Lett. , 41, 1429-1431, 2000; Kim et al. , Plant Pathol. , 20, 293-296, 2004; Wu et al., J. Agric. Food Chem ., 55, 1712-1717, 2007; Chen et al., J. Asian Nat. Prod. , 11 , 539-547, 2009). In addition, it has been studied that induced systemic resistance, one of the immune mechanisms against biological and non-biological stresses in plants, is involved in the suppression of viral diseases (Raupach et al., Plant Dis. , 80, 891-894, 1996;... Luo et al, FEMS Microbiol Lett, 313, 120-126, 2010), Pseudomonas chloro lapis O6 (Pseudomonas chlororaphis O6) is related to the induction resistance phenazine (phenazines) and 2R, 3R- It is known to produce bacterial determinants such as butanediol and to induce resistance to the mosaic virus disease of cucumber (Ryu et al., Eur. J. Plant Pathol. , 119, 383.390, 2007 ). However, the antiviral metabolites produced from P. chlororaphis O6 have not been identified.

However, the conventional P. chlororaphis O6 has been known to have a controlling effect against mosaic virus, but its active ingredient has not been identified.

The present invention is the first identification of an antiviral peptide from P. chlororaphis O6. It is intended to provide a peptide for controlling tobacco mosaic virus and its use. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention there is provided a cyclic peptide for the control of tobacco mosaic virus having the following structure:

cyclo (Ser-Thr-Val-Met-Pro-Leu-Ser-Leu-Gly).

In the cyclic peptide, the Ser, Thr, Val, Met, Pro, Leu, and Gly may independently be D-type or L-type amino acids, and the cyclic peptide may have the structure of FIG.

The cyclic peptide may be derived from Pseudomonas chlororaphis O6. The peptide was first identified by the present inventor and the control effect against mosaic virus was also proved for the first time.

According to another aspect of the present invention, there is provided a composition for controlling tobacco mosaic comprising the cyclic peptide as an active ingredient.

The composition for controlling composition may contain at least one active ingredient which exhibits the same or similar function as that of the cyclic peptide of the present invention. Examples of the composition include Serratia genus Gsm 01 (Korean Patent No. 0646669), Pseudomonas fluorescens A strain or a plant complex extract (Korean Patent Registration No. 0823085) which produces an antiviral active substance such as Oresense Gpf01 (Korean Patent No. 0545385) can be used together.

According to another aspect of the present invention, there is provided a method for controlling tobacco mosaic virus, comprising the step of treating the peptide with a plant, a seed of a plant, or a soil.

Since TMV inoculation with the peptide at the early stages of tobacco growth has proved to be effective in controlling TMV, it is possible to directly treat peptides in seeds or leaves of plants, It is possible to provide a control effect against the tobacco mosaic virus.

According to one embodiment of the present invention as described above, a novel cyclic peptide isolated from Pseudomonas chlororaphis O6 exhibiting an antiviral effect can be realized. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a schematic diagram showing MS fragmentation analysis results of an antiviral cyclic peptide isolated from Pseudomonas chlororaphis O6 according to an embodiment of the present invention. FIG.
FIG. 2 schematically shows an antiviral cyclic peptide according to an embodiment of the present invention. FIG.
FIG. 3 is a photograph (FIG. 3a) showing a TMV control effect of a peptamine which is an antiviral cyclic peptide according to an embodiment of the present invention and a graph (3b) showing a control rate.

The terms used in this document are defined as follows.

As used herein, " Pseudomonas chlororaphis O6" has been reported to exhibit inducible resistance activity against plant virus pathogens (Ryu et al., Eur. J. Plant Pathol ., 119, 383 -390, 2007), gacS self-regulation was published study is needed on the production induced resistance factors (determinant) (Han et al, Mol Plant Microb Inter, 19, 924-930, 2006;.... Kang et al. , J. Microbiol. Biotechnol. , 17, 586-593, 2007). However, the antiviral component of O6 is not known and was first identified by the present inventors.

As used herein, "peptamine" refers to an antiviral cyclic peptide isolated from P. chlororaphis O6, Q-TOF mass spectrometry results of C ₃₉ H ₆₇ N ₉ O ₁₂ S, Ser 2 (Met-Pro-Leu-Ser-Leu-Gly-Ser-Thr-Val), which contains one of each of Leu, Leu, Met, Val, Gly, Pro and Thr. do.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to fully inform the owner of the scope of the invention.

Example 1: Isolation of antiviral peptide

Pseudomonas chlororaphis O6 (O6) was cultured in a LB (Luria Bertani) medium (30 × 1 L) supplemented with 50 μg / ml ampicillin at 25 ° C until the color of the culture changed to orange in a stirred incubator. The change in color of the culture medium as described above means that a secondary product, phenazines, has been produced (Spencer et al., Physiol. , Palnt Pathology , 63, 27-34, 2003; Park et al. , J. Microbiol. Biotechnol. , 18, 1659-1662, 2008). Then, the culture was centrifuged at 8,000 × g for 20 minutes to remove cells, and the separated supernatant was extracted with the same amount of ethyl acetate as the supernatant. The organic phase was discarded and the aqueous layer was adjusted to pH 2.0 with 1 N HCl and extracted twice with the same volume of n-butanol. The n-butanol fraction was dried at 60 ° C in an evaporator to obtain 150 g of an extract. The extract was dissolved in ethyl acetate to perform column chromatography. An open glass column (33 mm id x 750 mm in length) was filled with a silica gel prepared using ethyl acetate, and the n-butanol extract was added to the column Lt; / RTI > The column was eluted with a mixture of ethyl acetate and methanol solvent and eluted with increasing concentration of methanol. Each fraction was evaporated in an evaporator at 50 < 0 > C and the antiviral effect was measured in the following experiments.

The antiviral active fractions (200 mg) were further purified by secondary silica gel column chromatography (16 mm i.d. × 900 mm in length). The column was then added with 40% (v / v) ethyl acetate in methanol to give 34 mg of the antiviral peptide.

Example 2: Preparation of TMV inoculum

The virus inoculum was prepared by modifying some of the methods of conventional studies (Kim et al., Plant Pathol. J. , 20: 293-296, 2004). To prepare the virus inoculum, TMV-infected N. tabacum cv. The leaves of the Samsun variety were milled in 10 ml of 0.1 M phosphate buffer (pH 7.2) containing 0.12 g of carborundum (Fisher, Philadelphia, Pa.), And then the sterilized cheesecloth cotton fabric was double- And filtered. The filtrate was centrifuged at 8,000 x g for 10 minutes to remove plant debris, and the supernatant was used as the TMV inoculum.

Example 3: Cultivation of tobacco

N. tabacum cv Xanthi-nc (Xanthi-nc) was used as a local lesion host in the following antiviral assay. The seeds of Xanthi-nc were immersed in a 70% (v / v) aqueous ethanol solution for 1 minute and then immersed in 3% (v / v) sodium hypochlorite solution for 15 minutes. To sterilize the seed surface, it was sterilized five times. Then, the sterilized seeds were washed several times with sterilized water, and an inorganic salt agar prepared from 0.5% (v / v) agar and 3% sucrose was placed on a 12-well plate on the bottom. The seeds were germinated in a growth chamber that provided a light period of 14 hours light cycle, a 10 hour dark cycle for 2 weeks, and a temperature condition of 25 占 폚. Thereafter, the germinated seedling was transferred into a commercial potted plant containing sterilized vermiculite in a growth chamber.

Experimental Example 1: Characterization of antiviral peptide

The antiviral peptide isolated in Example 1 was injected into a reversed phase semi-prep HPLC (Dionex P680 dual pump, Germering, Germany) equipped with a Dionex PDA-100 photodiode array detector at 190, 270 and 380 nm wavelength. The preparative HPLC column was a μ-Bondapack C18 stainless steel column, and the fluid phase of the HPLC consisted of 70% aqueous acetonitrile adjusted to pH 2.0 using trifluoroacetic acid, and 2 ml / min. < / RTI > The main peak detected was obtained except for the leading edge and tail portion of each peak to eliminate the possibility of contamination. The isolated compound exhibiting antiviral activity was recrystallized using methanol and was prepared in the form of a brown powder (6 mg). ESI-Q-TOF MS and NMR analyzes were then performed.

HPLC-MS (High performance liquid chromatography-mass spectrometry) analysis was performed on a Dionex model P680 HPLC (Germering, Germany) and Q- TOF MS (Quadruple time of flight mass spectrometer, Bremen, Germany). The HPLC column was a Nova-Pak C18 stainless steel column (2.0 id x 150 mm in length; Waters, Dublin, Ireland). At this time, the mobile phase consisted of 50% aqueous acetonitrile and had a flow rate of 0.5 ml / min. The HPLC system incorporates Q-TOF MX with electrospray inonization (ESI) in cationic mode. The capillary voltage and collision energy were 3 kV and 52 eV, respectively. Data were obtained using MRM (multiple reaction monitoring) at a range of 50-1,000 m / z . The sample was dried with nitrogen (N ₂ ) at a temperature of 210 ° C at 4.0 ml / min. ¹³ C was analyzed with Varian model Unity INFINITYplus 200 MNR (200 mHZ) (Agilent-Varian, California, USA) and Varian model Unity INOVA NMR (500 mHz) was used for ¹ H analysis .

The results of the analysis of the MS fragment of the separated antiviral peptide isolated in Example 1 are shown in FIG. As a result of Q-TOF mass spectrometry, the novel peptide according to one embodiment of the present invention has an (M + H) ⁺ peak at m / z 887.4242, which corresponds to C ₃₉ H ₆₇ N ₉ O ₁₂ S. MS / MS analysis of the ion peak at m / z 887.4242 showed that m / z (m / z ) due to the loss of methionine, Met, valine, and threonine The (M + H) ⁺ peak at 657.3180, 611.3124, and 556.2724 was detected as the major fragment ion. (M + H) ⁺ peak was detected at m / z 459.221, 345.187, and 171.113 at four times stronger intensity, followed by proline, pro, leucine, and leucine by subsequent ion fragmentation analysis. And two serines (Sers) were lost, leaving glycine (Gly) and Leu residues. Also, MS / MS / MS spectrum of the derived m / z 611.31 from MS / MS of the m / z 887.424 is the m / z 189.00 and 211.14 were generated by the ion product, which the Ser-Thr, and Leu-Pro, respectively internal fragment . The m / z 171.11 fragment of the MS / MS spectrum refers to a Gly-Leu bond. The formation of the partial structure of Ser-Gly-Leu-Ser-Leu was evidenced by the ionic products observed at m / z 459.22.

Then, it was used to place all of the proton and carbon chemical shifts of the separated the ¹ H-, ¹³ C-NMR spectrum with gHSQC and gHMBC through mechanical analysis of metabolites, the ¹ H- and ¹³ C-NMR Chemical shifts and coupling constants are summarized in Table 1: ESI-Q-TOF MS, m / z 887.4242 (M + H) ⁺ , 756.3859, 657.3180, 611.3124, 556.2724, 459.2208, 345.1873, 171.1130; ¹ H- and < ¹³ > C-NMR.

^{The 1} H-NMR spectrum showed 9 amide signals between δ 7.62-8.1 (s) and the ¹³ C-NMR spectrum (CD 3 OD, d 4) showed δ C 172.8, 173.0, 173.1, 173.2, 173.3 ), 173.7, 174.3 (2) and 174.4 showed 9 amide carbonyl groups, and these results indicate the peptide structure of the separated compounds.

According to the results of the MS / MS analysis and 2D ¹ H- ¹ H COZY, HMBC and HSQC analysis, the peptide according to one embodiment of the present invention is composed of 7 amino acids and has 2 Ser, 2 Leu, , Gly, Pro and Thr, respectively.

The presence of two leucines (Leu) means that the chemical shifts of two methylene groups at δC 41.2 and 49.5 in the four methyl and ¹³ C-NMR spectra at C 22.1 and 24.0, and the chemical shifts of ¹ H- ¹ H COZY And the HMBC spectrum.

In addition, the presence of valine (Val) was evidenced by methyl groups having the same β-proton (δH 2.21) in the ¹ H - ¹ H COZY spectrum with two methyl groups at δ C 18.9 and 20.0.

In addition, the presence of threonine (Thr) leads to a proton of oxidized carbon of δH 4.4 in the ¹ H- ¹ H COZY spectrum and a δH 1.25 d ( J = 6.5 Hz ). ≪ / RTI >

One S-CH ₃ group was placed in the HSQC through the attachment of the carbon of the methyl group of δC 15.8 and the proton singlet of δH 2.07. Also, in HMBC, the relationship between protons of δH 1.95, 1.97, 2.04, and 2.21 and carbon of methyl groups was observed. These results indicate that methionine (Met) is present in the chemical structure.

The presence of proline (Pro) also correlates the proton signal of δC 60.1 C-α with three methyl groups of δC 26.1, 29.6 and 48.2 and four amide carbonyl of HMBC δC 173.2 Based.

In addition, the presence of two serines (Ser) was revealed by a decrease in molecular weight between fragments of m / z 345.18 and 171.11 observed in the MS / MS fragmentation pattern. These results were also reaffirmed in relation to carbon signals and C-α of methylene protons and HMBC at δ C 61.5 (CH-α) and 49.2 (CH ₂ ).

The proton (H-alpha) of delta H 4.63 (delta C 51.2) associated with the carbon of the HMBC spectra 174.3, 173.1 and 41.2 in Leu-2 implies peptide linkage between leucine and serine.

That is, the mass spectrometry and NMR analysis results show that the metabolite of P. chlororaphis O6 isolated through one embodiment of the present invention can be identified as a cyclic peptide of a nonamer consisting of 7 amino acids And the present inventor named it 'peptamine'.

δc
(multi)
δH
(multi,
J in Hz) HMBC
( J = 8 Hz)
δc
(multi)
δH
(multi,
J in Hz) HMBC
( J = 8 Hz) Gly-1 Met-6 CO 173.7 CO 174.3 alpha 49.9
3.32 m, 3.31 m
alpha 60.1 4.61m 174.3 Leu-2


beta 30.5 1.97 m, 1.95, 60.1, 15.8 CO 173.1 gamma 26.2 2.21 m, 2.04 m 60.1, 15.8
alpha 51.2 4.63m 174.3, 173.1, 41.2, 25.9 S-CH3 15.8 2.07s beta
41.2
1.53-1.59m
173, 51.2, 24, 22.1 Val-7



gamma 25.9 2.02m 173.1 CO 17.28 Me 22.1 0.95d (6.0) 41.2, 25.9 alpha 62.1
4.51m Me 24 0.95d (6.0) 41.2, 25.9 beta 30.5 2.21 m, 2.04 m 62.1
Ser-3 Me 18.9 0.95d (6.6) 30.6 CO 174.3a Me 20 0.95d (6.6) 30.6 alpha 61.5 4.47m Thr-8 beta 49.2 3.65-3.9m
61.5
CO
173.3


Leu-4 alpha 60.2 4.32m CO 173 beta 68.2 4.2m alpha 56.2
4.56m

gamma
20.6
1.25d (6.5) 68.2, 60.2
beta 49.5
3.84 m, 3.72 m 25.9
Ser-9



gamma 26.3 2.05m 173 CO 174.4a Me 22.1 0.95d (6.0) 26.3 alpha 61.5 4.47m Me 24 0.95d (6.0) 26.3 beta 49.2 3.65-3.9m 61.5 Pro-5 CO 173.2 alpha
60.1
4.69dd
(8.4, 5.4) 174.3, 173.3, 29.6, 26.1



beta
29.6
2.39 m, 2.01 m 26.1



gamma
26.1
2.01m, 1.96m 29.6




delta
48.2
3.65 m, 3.62 m 29.6, 26.1

Experimental Example 2: Antiviral effect analysis

Antiviral efficacy assays were performed using the spray method described in the following literature (Luo et al., FEMS Microbiol. Lett. , 313, 120-126, 2010). The antiviral peptide isolated in Example 1 was dissolved in a 20% (v / v) methanol aqueous solution at a concentration of 10 μg / ml to 1,000 μg / ml, and the solution was mixed with the TMV inoculum prepared in Example 2 at a ratio of 1: By weight. 15 ml of the mixture was sprayed onto the leaf placed at the lower end of the cigarette (Xanthi-nc) grown in Example 2. In this case, the lower part means the third to fifth leaves from the top in 8 to 9 leafed cigarettes, and a small hand spray (DIA-7570, Furupla, Tokyo, Japan) was used. For comparison, 20% (v / v) aqueous methanol solution without antiviral peptide and TMV inoculum were mixed and sprayed on tobacco leaves. The inhibition rate (%) of plant virus disease was calculated using the following equation (1) when 5 days passed after the injection. In the following Equation 1, C means the number of lesions occurring in the control leaf, and T means the number of lesions in the tobacco leaf treated with the antiviral peptide according to one embodiment of the present invention.

As a result, as shown in Fig. 3, the peptide (peptamine) according to one embodiment of the present invention showed antiviral activity against TMV disease. The tobacco leaf sprayed with 100 μg / ml peptamine and 10 μg / ml TMV inoculum according to an embodiment of the present invention showed a 40% to 45% infection rate lower than that of the control, It is meant that the peptamine according to the embodiment inhibits mosaic disease. In addition, TMV infection rate was inhibited by 95% or more in tobacco leaves sprayed with 1,000 μg / ml peptamine and 10 μg / ml TMV inoculum. On the other hand, spots similar to staining were observed on the control tobacco leaves sprayed with solvent and TMV inoculum instead of peptamine, and were infected by TMV.

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> Industry Foundation of Chonnam National University <120> anti-Tobacco mosaic viral peptide and use thereof <130> PD13-0675 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> PRT <213> Pseudomonas chlororaphis O6 <400> 1 Ser Thr Val Met Pro Leu Ser Leu Gly   1 5

Claims (5)

A cyclic peptide for the control of tobacco mosaic virus having the following structure:
cyclo (Ser-Thr-Val-Met-Pro-Leu-Ser-Leu-Gly). The method according to claim 1,
Wherein said Ser, Thr, Val, Met, Pro, Leu and Gly are independently L- or D-type cyclic peptides for controlling tobacco mosaic virus. The method according to claim 1,
A cyclic peptide for the control of tobacco mosaic virus, having the structure:

A composition for controlling tobacco mosaic comprising the cyclic peptide of claim 1 as an active ingredient. A method for controlling tobacco mosaic comprising the step of treating the cyclic peptide of claim 1 to a plant, plant seed, or soil.

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