KR20150001701A - Composition for increasing gamma-aminobutyric acid content of grape comprising hydrogen peroxide as effective component - Google Patents

Abstract

The present invention provides a method for increasing the stilbene or gamma-aminobutyric acid (GABA) content in grape, which includes a step of treating grape with a signal transmission matter or hydrogen peroxide; grape with an increased stilbene or GABA content manufactured thereby; a method for increasing the resistance against fungal diseases of grape, which includes a step of treating grape with a signal transmission matter or hydrogen peroxide; and a composition for increasing the stilbene or GABA content in grape or the resistance against fungal diseases of grape, which includes the plant signal transmission matter or hydrogen peroxide as an active ingredient.

Description

Composition for increasing gamma-aminobutyric acid content of grape comprising hydrogen peroxide as effective component}

The present invention relates to a method of enhancing resistance to fungal diseases of grapes by treatment with a signal transducer, and more particularly, to a stilbene of grapes comprising the step of treating a signal transducer or hydrogen peroxide to grapes or A method of increasing the content of gamma-aminobutyric acid (GABA), grapes with increased stilbene or GABA content prepared by the method, and resistance to fungal diseases of grapes, comprising the step of treating the grapes with a signal transducer or hydrogen peroxide It relates to a method of increasing, a composition for enhancing the stilbene or GABA content of grapes containing a plant signal transducer or hydrogen peroxide as an active ingredient, or a composition for enhancing resistance to fungal diseases of grapes.

The disease defense mechanism of plants is induced not only by the invasion of pathogens, but also by abiotic stress such as ultraviolet treatment or signal transducer. Signal transducers that become such abiotic stressors include respiratory inhibitors, electrodeposits, antibiotics, growth regulators, Heavy metals and elicitors. The defense reaction of plants takes place through a complex network of signaling pathways. Endogenous signaling substances include salicylic acid (SA), jasmonic acid (JA), and ethylene (ET). , Salicylic acid, and hydrogen peroxide are important regulators that mediate disease resistance. Ethylene, methyljasmonic acid, and salicylic acid are important signaling substances that induce local or systemic disease resistance responses in plants, and artificial external processing of these signaling substances induces resistance-related genes that are activated by pathogen infection. In addition, phytoalexin is a small molecule secondary metabolite that is an antimicrobial substance that accumulates after infection with pathogens, and plants are also produced in response to abiotic stress such as _{pathogens, heavy metals, UV, AlCl 3 and the like.} Phytoalexins produced in grapes include simple stilbene resveratrol (trans-3,5,4'-trihydroxystilbene) and viniferins and terostilbene, trans-3, which are compounds biochemically related to glycosides. 5 dimethoxy-4'-hydroxystilbene).

Grapes are one of the most important fruit trees in the world, but when grown, they are susceptible to pathogens such as numerous bacteria, fungi, and viruses. Gray mold disease in grapes is caused by infection with mature fruits when the late rainy season or high relative humidity persists, and it develops even after harvesting, causing serious problems in various crops such as tomatoes, strawberries, cucumbers, bulbs, cut flowers, ornamental flowers, which are important agriculturally. .

The gray mold disease of grapes is caused by B. cinerea , and it mainly affects flowers and fruits, especially in the flowering period in facility houses, which is a factor that greatly affects grape production depending on the region. . It is expected that induction of disease resistance-related genes or accumulation of phytoalexin such as a stilbene compound by signal transducer treatment can enhance resistance to these pathogens. In the present invention, three signal transducers and hydrogen peroxide are used in Campbell Early. And it was treated on the leaves of the grapes to induce disease resistance-related genes and confirmed the effect of inhibiting the occurrence of gray mold disease by the accumulation of various stilbene compounds and GABA substances.

Korean Patent Laid-Open Publication No. 2003-0021976 discloses a resveratrol-fortified grape and a production method thereof, which amplifies the content of resveratrol, a natural anticancer substance derived from grapes, and Korean Patent No. 0582737 discloses grapes containing high resveratrol using environmental factors. Although a production method has been disclosed, a method for producing grapes with improved resistance to fungal diseases as in the present invention and grapes according thereto have not been disclosed.

The present invention was derived from the above requirements, and in the present invention, as a result of spraying a signal transducer or hydrogen peroxide of ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA) on grape leaves, stilbene of grapes ( stilbene) and GABA (gamma-aminobutyric acid) content was increased, and by confirming that resistance to gray mold disease of grapes was increased, the present invention was completed.

In order to solve the above problems, the present invention provides a method of increasing the content of stilbene or gamma-aminobutyric acid (GABA) of grapes, including the step of treating the grape with a signal transducer or hydrogen peroxide.

In addition, the present invention provides a grape with an increased content of stilbene or GABA prepared by the above method.

In addition, the present invention provides a method of increasing the resistance to fungal diseases of grapes, comprising the step of treating the grape with a signal transducer or hydrogen peroxide.

In addition, the present invention provides a composition for enhancing the stilbene or GABA content of grapes containing a plant signal transducer or hydrogen peroxide as an active ingredient.

In addition, the present invention provides a composition for enhancing resistance to fungal diseases of grapes containing a plant signal transducer or hydrogen peroxide as an active ingredient.

The present invention is a result of spray treatment with a signal transducer of ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA) or hydrogen peroxide to grapes during the cultivation process. It was confirmed that it has an excellent effect on increasing resistance to resistance. Therefore, grapes produced by the production method of the present invention contain high levels of anticancer and antibacterial activity of stilbene and GABA, so that functional foods and pharmaceutical compositions using them as active ingredients can be prepared, which is very useful in the food industry and pharmaceutical industry It is an invention, and it is possible to provide a grape plant with improved resistance to grape fungal disease, and thus can greatly contribute to the increase in productivity of the grape industry.

, 대조구(물 처리),

1000 ppm 에테폰 처리,

10 mM H₂O₂ 처리,

0.5 mM 메틸자스몬산 처리,

1 mM 살리실산 처리. 수직 바는 SEs를 나타낸다 (n=9).
도 3은 신호전달물질을 처리한 거봉 포도나무 잎에서의 GABA 함량 변화를 나타낸다. Cont, 물 처리; MeJ, 메틸자스몬산; SA, 살리실산.1 shows the expression analysis of defense-related genes in Campbell-Early and Geobong vine leaves treated with signal transducers. After the signal transduction material was treated and a certain time elapsed, total RNA was isolated to synthesize cDNA, and RT-PCR analysis was performed. In order to confirm the same amount of RNA, the beta-actin gene was used as a control. (CHI; chalcone isomerase, CHS; chalcone synthase, CYP; cytochrome p450, FLS; flavonol synthase, GST; glutathione- S- transferase, TLP; thaumatine-like protein, PRP; proline rich protein, CI; cold induced protein, LOX; lipoxygenase, STSY; stilbene synthase, Cell wall; Cell wall protein, Mei5; meiosis 5, PGIP; Polygalacturonase-inhibiting protein, Sir; sirtuin)
Figure 2 shows the inhibition of the occurrence of gray mold disease in vine leaves inoculated with Botrytis cineria after treatment with a signal transducer and hydrogen peroxide. (A) Campbell Early, (B) Geobong.

, Control (water treatment),

1000 ppm Ethephon treatment,

10 mM H ₂ O ₂ process,

0.5 mM methyljasmonic acid treatment,

1 mM salicylic acid treatment. Vertical bars represent SEs (n=9).
Figure 3 shows the change of GABA content in the leaves of the grapevine treated with a signal transducer. Cont, water treatment; MeJ, methyl jasmonic acid; SA, salicylic acid.

In order to achieve the object of the present invention, the present invention provides a method of increasing the content of stilbene or gamma-aminobutyric acid (GABA) in grapes, including the step of treating the grape with a signal transducer or hydrogen peroxide.

In the method according to an embodiment of the present invention, the signal transducer may be ethylene, ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA), and preferably, ethephon, methyl jasmonic acid (MJ) or salicylic acid. (SA) may be, but is not limited thereto.

In the method according to an embodiment of the present invention, the treatment concentration of the signal transducer is preferably 450 to 1100 ppm ethephon, 0.08 to 0.6 mM methyl jasmonic acid (MJ) or 0.4 to 1.1 mM salicylic acid (SA), and hydrogen peroxide The treatment concentration of may be 4 ~ 11mM, but is not limited thereto.

In the method according to an embodiment of the present invention, the signal transducer or hydrogen peroxide may be sprayed or sprayed onto grape leaves, but is not limited thereto. The signal transducer or hydrogen peroxide may be sprayed or sprayed onto grape leaves using any method known in the art, and is not particularly limited to a specific method.

In the method according to an embodiment of the present invention, the grape may be a grape (Kyoho), a Delaware (Delaware), a Campbell Early (Campbell Early), a seridan, an MBA, a grapefruit or a green grape (Niagara), preferably Campbell Early Or it may be a grape grape, but is not limited thereto.

In the method according to an embodiment of the present invention, the stilbene may be resveratrol, piceatannol, or piceid, but is not limited thereto.

Stilbene is a kind of phytoalexin, one of the secondary metabolites derived from the phenylpropanoid pathway, and the physiological activity of the compound is related to its antioxidant activity, and the radical scavenging ability of stilbene is It is correlated with the number of hydroxyl groups (-OH) and also has a stronger antioxidant activity than resveratrol, which is a hydroxyl derivative of resveratrol.

In addition, the present invention provides grapes having an increased content of stilbene or gamma-aminobutyric acid (GABA) prepared by the above method.

GABA is a substance that induces a protective reaction against reactive oxygen stress in plants, plays a role in removing the accumulation of hydrogen peroxide water, and induces a mechanism to resist external stress such as attack by insects. Increasing the GABA content in the plant exhibits resistance to nematodes and insects, and the increase in free radicals produced by the invasion of pathogens in the plant is associated with an increase in the GABA content in the body.

The grapes according to an embodiment of the present invention may be Keobong (Kyoho), Delaware (Delaware), Campbell Early (Campbell Early), Seridan, MBA, berry or green grapes (Niagara), preferably Campbell Early or Geobong grapes May be, but is not limited thereto.

In addition, the present invention provides a method of increasing the resistance to fungal diseases of grapes, comprising the step of treating the grape with a signal transducer or hydrogen peroxide.

In the method according to one embodiment of the present invention, the fungal disease may be a gray mold disease caused by Botrytis cineria, but is not limited thereto.

In addition, the present invention provides a composition for enhancing the content of stilbene or gamma-aminobutyric acid (GABA) of grapes containing a plant signal transducer or hydrogen peroxide as an active ingredient. The composition includes a plant signaling substance or hydrogen peroxide as an active ingredient, and by spraying or spraying the plant signaling substance or hydrogen peroxide on grape leaves, the content of stilbene or GABA can be increased.

In the composition according to an embodiment of the present invention, the signal transducer may be ethylene, ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA), and preferably, ethephon, methyl jasmonic acid (MJ) or salicylic acid. (SA) may be, but is not limited thereto.

In addition, the present invention provides a composition for enhancing resistance to fungal diseases of grapes containing a plant signal transducer or hydrogen peroxide as an active ingredient. The composition contains a plant signal transducer or hydrogen peroxide as an active ingredient, and by spraying or spraying the plant signal transducer or hydrogen peroxide on grape leaves, the resistance of grapes to fungal diseases can be increased.

In the composition according to an embodiment of the present invention, the signal transducer may be ethylene, ethephon, methyl jasmonic acid (MJ) or salicylic acid (SA), and preferably, ethephon, methyl jasmonic acid (MJ) or salicylic acid. (SA) may be, but is not limited thereto.

In the composition according to an embodiment of the present invention, the fungal disease may be a gray mold disease caused by botrytis cineria, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Materials and methods

Processing of various signaling substances in vine leaves

_{Ethephon (500ppm, 1000ppm), H 2} O ₂ (5mM, 10 mM), methyljasmonic acid (MJ, 0.1mM, 0.5 mM), salicylic acid (SA, 0.5) on the leaves of Campbell-Early and Geobong vines growing in green houses mM, 1 mM) were divided into two concentrations and sufficiently sprayed. After spray treatment, grape leaves were sampled at 0, 1, 6, 24, 48 and 72 hours, respectively, and stored at -80°C to extract total RNA and used for RT-PCR. Ethephon, hydrogen peroxide and methyl jasmonic acid were dissolved in distilled water and diluted to respective concentrations, and salicylic acid was dissolved in ethanol and then diluted with distilled water.

gun RNA Separation and RT - PCR analysis

Total RNA was isolated from grape leaves, and the ^{first strand cDNA was synthesized from 1 μg of total RNA using PrimeScript TM} 1 ^st strand cDNA synthesis kit (TaKaRa Bio Inc., Japan) and used as a template for PCR reaction. Primers were prepared based on beta-actin and 16 defense reaction-related gene sequences (Table 1). PCR was performed with the first denaturation (94°C, 5 min), amplification reaction 35 times (denaturation 94°C, 45 sec; annealing 55°C, 45 sec; amplification 72°C, 1 min), final amplification (7 min, 72°C) The PCR product was performed and a band was confirmed on an agarose gel (1%).

Investigation of inhibition of gray mold disease by treatment of signal transducers

Gray mold pathogens (Botrytis) cultured in PDA for 5 days by alternately irradiating with fluorescent lamps for 12 hours cinerea ) conidia were collected. ^{After preparing Botrytis cineria spore suspension (10 6} spores/ml) in 0.24% potato dextrose broth (containing 0.8% NaCl), 20 µl of spore suspension on the back of the wounded vine leaves evenly using a pencil tip. Was inoculated onto the wound on the back side of grape leaves using an Eppendorf repeater (pipette). In addition, for mycelial inoculation, agar blocks containing the mycelium of gray mold pathogens cultured in PDA were placed on the wound and inoculated. Grape leaves inoculated with pathogens were measured for changes in the size of necrotic spots after 4 days in a humid room at 25°C, and 9 leaves of untreated and signal transducer treated leaves were investigated.

GABA (γ- aminobutyric acid ) And HPLC analysis

GABA extraction was carried out by adding liquid nitrogen to 1 g of treated vine leaves, followed by grinding with 10 ml of 3% TCA (trichloroacetic acid) for 3 minutes, and centrifuging the obtained extract at 25,000xg for 20 minutes. I did. The supernatant was taken and stored at -20°C and injected into HPLC to analyze the GABA content. The qualitative and quantitative analysis of GABA was performed using HPLC/FLD.

Stillben ( Stilbene ) Extraction of compounds and HPLC analysis

To extract the stilbene compound, add liquid nitrogen to 1 g of treated vine leaves to completely grind it, add 4 ml of 80% (v/v) methanol, and grind for 3 minutes. The resulting extract was added at 25,000xg for 20 minutes. Centrifuged. The supernatant was taken and stored at -20°C and injected into HPLC to analyze the stilbene content. The stilbene compound was analyzed using an HPLC-mass spectrophotometer (model 2695 HPLC, model 3100 MS, Waters, USA), and the analysis method was Choi et al. (2011 Kor. J. Hort. Sci. Technol. 29:374-381 The experiment was conducted under the same conditions as in ).

Example 1. Comparison of expression of genes related to grape defense by signal transducer treatment

RT-PCR was performed using a total of 38 primers in Table 1. To find a gene that is specifically expressed, cDNA was first mixed and RT-PCR was performed to select a gene, and then individual RT-PCR was performed again. In Campbell-Early grapes, when etephon was treated among a total of eight selected genes, the expression of CYP (cytochrome p450) and TLP (thaumatin-like protein) increased, and decreased in CHS (chalcone synthesis), and methyl jasmine was treated. After treatment, gene expression such as chitinase-like protein (CLP), lipoxygenase (LOX), proline-rich protein (PRP), and thaumatin-like protein (TLP) was increased, and CHS (chalcone synthesis) when salicylic acid was treated. , PGIP (polygalacturonase-inhibiting protein), sirtuin, etc. gene expression was increased. Among the 14 selected genes in the grape grapes, CHI (chalcone isomerase), CHS (chalcone synthesis), CYP (cytochrome p450), FLS (flavonol synthase), GST (glutathione- S- transferase), Gene expression such as TLP (thaumatin-like protein) showed a tendency to increase, and PRP (proline-rich protein) showed a decrease in gene expression (FIG. 1). When methyljasmonic acid was treated, the expression of three genes: CI (cold induced protein), LOX (lipoxygenase), and STSY (stilbene synthase) was increased, and when salicylic acid was treated, Mei5 (cell wall protein, meiosis 5) , PGIP (polygalacturonase-inhibiting protein), STSY (sirtuin, stilbene synthase) and the like gene expression was increased (Fig. 1). Overall, genes such as CYP, sultuin, STSY and TLP tended to be strongly expressed.

Example 2. Suppression of the occurrence of grape gray mold disease by treatment of signal transducers

In order to check the effect of treatment of various signal transducers on the occurrence and inhibition of gray mold disease, Ethephon, hydrogen peroxide, methyl jasmine or salicylic acid were sprayed on the leaves of Campbell-Early and Geobong vines at two concentrations, and botrytis cineria was inoculated. After the lesion formation and the size of the lesion were investigated. It was found that the occurrence of gray mold disease was suppressed by the treatment of signal transducers in Campbell-Early and Geobong varieties (FIG. 2).

It was found that both inoculation of spore suspension or colony with or without wounds in Campbell Early and Geobong suppressed mycelial growth. Compared to the case where there was no wound in the plant that was wounded and inoculated with the spore suspension, the size of the lesion was 1.4-9 times larger in Campbell Early and 1.2-7 times larger in Geobong. The inoculation tended to be 1.2-3 times higher in mycelial growth than the free wound inoculation, but the mycelial growth was suppressed in the Geobong, regardless of the presence of wounds, compared to the untreated group.

Example 3. Stillben Compounds and GABA Content analysis

Ethephon, hydrogen peroxide, methyl jasmonic acid, or salicylic acid were sprayed on grape leaves at low and high concentrations, respectively, and collected by time to analyze the content of stilbene compounds. Each compound was confirmed by using the retention time as an indicator in the SIR chromatogram of the corresponding m/z, and the content of each compound was compared with trans-resveratrol, which can secure a standard, and the peak area Calculated by relative ratio (Choi, 2011 Kor. J. Hort. Sci. Technol. 29:374-381). The content of stilbene compounds in Campbell Early and Geobong was slightly higher in piceatannol, which has higher antioxidant activity than resveratrol, and in particular , the content of trans- Piceid was the most detected among the five compounds. Methyl jasmonic acid treatment with high growth inhibitory effect was detected a lot (Tables 2 and 3). In the present invention, it is determined that the increase of the stilbene compound by the treatment of the signal transducer is related to the inhibition of mycelial growth in gray mold disease.

In the present invention, the contents of the three signal transducers of low and high concentration and the stilbene compound by hydrogen peroxide treatment are shown in Tables 2 and 3. Among the stilbene compounds in both Campbell-Early and Geobong varieties, trans/cis-piside, a glycoside form with lower physiological activity than resveratrol or piseataol, showed a relatively higher content, and trans/cis-resveratrol, aglycone form. Was detected in trace amounts. In particular, the trans-type of Pisade was higher than that of the cis-type, and Campbell Early's trans-pisade content was higher than that of Geobong. Resveratrol was detected in trace amounts in both Campbell-Early and Geobong cultivars, but in Geobong, piseataol, a hydroxyl derivative of resveratrol, was detected more than Campbell-Early. It was reported that inpiceataol had stronger antioxidant activity than resveratrol.

Changes in GABA content by treatment with signal transducers are shown in FIG. 3. The GABA content was higher than that of the untreated group in all signal transducer treatments, and the GABA content after 24 hours of treatment was higher than that after 48 hours. In particular, in the case of ethephon treatment, it was found that the GABA content increased sharply after 48 hours than after 24 hours. Among the three signal transducers and hydrogen peroxide treatment, the GABA content was generally higher in the salicylic acid treatment group.

In the present invention, when the plant was treated with a signal transducer and hydrogen peroxide, the content of GABA was increased in vine leaves, and the occurrence of grape gray mold disease was decreased in the plants with increased content. That is, it is thought that resistance to diseases is increased by activation of various defense reaction genes, accumulation of stilbene compounds, and accumulation of GABA by treatment with signal transducers and hydrogen peroxide.

The increase in the content of stilbene compounds with high antioxidant activity after treatment with signal transducers in grape leaves is thought to be of great help in enhancing the functionality of grapes and also improving disease resistance by these phytoalexin substances. In the present invention, by treating grape leaves with three signaling substances and hydrogen peroxide, expression of disease resistance-related genes was induced, and the content of stilbene phytoalexin was increased, thereby confirming the effect of inhibiting the mycelial growth of grape gray mold disease. It is believed that such signal transduction substance treatment can inhibit the occurrence of various pathogens in grapes, or increase the content of stilbene compounds having antioxidant activity can be used as a health functional substance.

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Claims (1)

A composition for enhancing GABA (gamma-aminobutyric acid) content of grapes containing hydrogen peroxide as an active ingredient.

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