KR20230153621A - Plant immunity inducing agent containing hopeaphenolas an active ingredient, and plant immunity validation method - Google Patents
Plant immunity inducing agent containing hopeaphenolas an active ingredient, and plant immunity validation method Download PDFInfo
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- 230000001939 inductive effect Effects 0.000 title claims description 9
- 239000004480 active ingredient Substances 0.000 title abstract description 4
- 238000010200 validation analysis Methods 0.000 title 1
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- YQQUILZPDYJDQJ-KGDQSQJYSA-N hopeaphenol Chemical compound C1=CC(O)=CC=C1[C@H](O1)[C@@H]2C3=CC(O)=CC(O)=C3[C@@H](C=3C=CC(O)=CC=3)[C@H]([C@@H]3C=4C=C(O)C=C5O[C@H]([C@@H](C=45)C4=CC(O)=CC(O)=C4[C@H]3C=3C=CC(O)=CC=3)C=3C=CC(O)=CC=3)C3=C2C1=CC(O)=C3 YQQUILZPDYJDQJ-KGDQSQJYSA-N 0.000 abstract description 26
- YQQUILZPDYJDQJ-PLGGFYDZSA-N hopeaphenol Natural products Oc1ccc(cc1)[C@@H]2Oc3cc(O)cc4[C@H]([C@@H]5[C@@H](c6ccc(O)cc6)c7c(O)cc(O)cc7[C@@H]8[C@H](Oc9cc(O)cc5c89)c%10ccc(O)cc%10)[C@@H](c%11ccc(O)cc%11)c%12c(O)cc(O)cc%12[C@@H]2c34 YQQUILZPDYJDQJ-PLGGFYDZSA-N 0.000 abstract description 26
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000219195 Arabidopsis thaliana Species 0.000 description 2
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 2
- 241000589615 Pseudomonas syringae Species 0.000 description 2
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- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical compound OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
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- 238000002955 isolation Methods 0.000 description 1
- 210000004901 leucine-rich repeat Anatomy 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
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- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- FJKROLUGYXJWQN-UHFFFAOYSA-N papa-hydroxy-benzoic acid Natural products OC(=O)C1=CC=C(O)C=C1 FJKROLUGYXJWQN-UHFFFAOYSA-N 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
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- 239000003375 plant hormone Substances 0.000 description 1
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- 230000037452 priming Effects 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/06—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings
- A01N43/12—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom five-membered rings condensed with a carbocyclic ring
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P21/00—Plant growth regulators
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Agronomy & Crop Science (AREA)
- Dentistry (AREA)
- Pest Control & Pesticides (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
본 발명은 호페아페놀을 유효성분으로 포함하는 식물 면역 유도제에 관한 것으로, 식물의 1차 면역반응인 PTI(PAMP-triggered immunity)와 2차 면역반응인 ETI(Effector-trigger immunity)를 유도할 수 있다. 본 발명의 식물 면역 유도제는 식물의 선천 면역 시스템을 증진시킬 수 있으며, 기존의 항생제를 대체할 수 있는 친환경적 식물 면역 유도제로 사용할 수 있다.The present invention contains hopeaphenol as an active ingredient. It relates to a plant immunity inducer, which can induce PTI (PAMP-triggered immunity), the primary immune response of plants, and ETI (Effector-trigger immunity), the secondary immune response. The plant immunity inducer of the present invention can enhance the innate immune system of plants and can be used as an eco-friendly plant immunity inducer that can replace existing antibiotics.
Description
본 발명은 호페아페놀(hopeaphenol)을 유효성분으로 포함하는 식물 면역 유도제 및 이를 이용한 식물 면역 유도방법에 관한 것이다.The present invention contains hopeaphenol as an active ingredient. It relates to plant immunity inducers and methods for inducing plant immunity using the same.
식물과 병원균의 상호작용을 연구하는 모델식물인 애기장대(Arabidopsis thaliana)의 면역 시스템은 1차 면역 반응과 이를 억제하는 병원균의 병원성 단백질을 인식할 수 있는 2차 면역 반응으로 분류된다. 1차 면역 반응은 병원균의 보존분자(conserved molecule)인 PAMP(pathogen-associated molecular pattern)을 인식하는 것으로 시작되는데, 식물의 세포 표면에 존재하는 수용체에 PAMP가 인식되고, 일련의 반응을 통해 1차 면역 반응이 활성화 된다. 이를 PTI (PAMP-triggered immunity)라 한다. 2차 면역 반응은 이러한 1차 면역 반응을 무력화시키려는 병원균의 병원성 단백질들을 식물의 2차 선천면역 수용체가 인식하여 이루어진다. ETI (Effector-trigger immunity) 라 불리는 2차 면역 반응은, 병원균이 분비하는 병원성 단백질인 이펙터 단백질을 식물에 존재하는 NLR(nucleotide-binding leucine rich-repeat) 면역 수용체가 직, 간접적으로 인식하면서 활성화된다. 이 두 가지의 면역 반응은 병원균에 감염된 국소적인 부분뿐만 아니라 감염되지 않은 식물 부위까지 면역 반응을 촉진하여 식물체 전반의 병저항성을 유도한다.The immune system of Arabidopsis ( Arabidopsis thaliana ), a model plant for studying the interaction between plants and pathogens, is divided into a primary immune response and a secondary immune response that can recognize pathogenic proteins of pathogens that suppress them. The primary immune response begins with recognition of PAMP (pathogen-associated molecular pattern), a conserved molecule of pathogens. PAMP is recognized by receptors present on the cell surface of plants, and through a series of reactions, the primary immune response The immune response is activated. This is called PTI (PAMP-triggered immunity). The secondary immune response occurs when the plant's secondary innate immune receptors recognize the pathogenic proteins of the pathogen that attempt to neutralize the primary immune response. The secondary immune response, called ETI (Effector-trigger immunity), is activated when NLR (nucleotide-binding leucine rich-repeat) immune receptors present in plants directly or indirectly recognize effector proteins, which are pathogenic proteins secreted by pathogens. . These two types of immune responses promote disease resistance throughout the plant by promoting immune responses not only in local areas infected with pathogens but also in uninfected plant parts.
식물이 외부 자극(미생물, 주변식물, 병원균의 이펙터 단백질, 천연물, 합성물 등)에 노출 시, priming이라는 반응을 통해 이후에 일어나는 생물적 또는 무생물적 스트레스에 대한 면역 능력이 국부적 또는 전신적으로 향상된다. 일반적으로 식물 뿌리에 서식하는 비병원성 세균이 식물체의 병저항성을 향상시키는 것으로 알려져 있으며, 살리실산(salicylic acid) 등의 식물 호르몬도 이러한 유형의 병저항성을 유도하는 것으로 알려져 있다.When plants are exposed to external stimuli (microorganisms, surrounding plants, pathogen effector proteins, natural products, synthetic substances, etc.), their immunity against subsequent biotic or abiotic stresses is improved locally or systemically through a response called priming. In general, non-pathogenic bacteria living in plant roots are known to improve disease resistance of plants, and plant hormones such as salicylic acid are also known to induce this type of disease resistance.
식물 면역 유도제는 항생제 저항성 식물 병원균의 출현 문제를 해결할 수 있을 뿐만 아니라, 천연물 유래 대사산물을 기반으로 하는 식물 면역유도제는 친환경적으로 식물을 보호할 수 있는 장점이 있다. 이에, 본 발명은 식물에서 분리한 천연물 대사산물의 식물 면역유도 효과를 확인하였으며, 본 발명에 따른 식물 면역유도제는 유기합성 항생물질을 대체하여, 항생제 저항성 문제뿐만 아니라 환경에 대한 부작용 우려를 대폭 낮출 수 있다.Not only can plant immunity inducers solve the problem of the emergence of antibiotic-resistant plant pathogens, but plant immunity inducers based on natural product-derived metabolites have the advantage of protecting plants in an environmentally friendly manner. Accordingly, the present invention confirmed the plant immunity-inducing effect of natural product metabolites isolated from plants, and the plant immunity inducer according to the present invention replaces organic synthetic antibiotics, significantly lowering not only the problem of antibiotic resistance but also concerns about side effects on the environment. You can.
본 발명은 식물의 선천 면역을 활성화시켜 식물병 방제제로서의 효과를 가지는 식물 면역 유도제를 제공하고자 한다.The present invention seeks to provide a plant immunity inducer that activates the innate immunity of plants and is effective as a plant disease control agent.
또한, 본 발명은 식물병 방제에 일반적으로 사용되는 항생제를 대체할 수 있는 친환경적인 식물 면역 유도제를 제공하고자 한다.In addition, the present invention seeks to provide an environmentally friendly plant immunity inducer that can replace antibiotics commonly used to control plant diseases.
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 이성질체를 포함하는 식물 면역 유도제를 제공한다.The present invention provides a plant immunity inducer comprising a compound represented by the following formula (1) or an isomer thereof.
[화학식 1][Formula 1]
본 발명에 있어서, 상기 화합물 또는 이의 이성질체는, 바람직하게는 식물 잎에서 활성산소를 발생시키는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the compound or its isomer may preferably generate active oxygen in plant leaves, but is not limited thereto.
본 발명에 있어서, 상기 화합물 또는 이의 이성질체는, 바람직하게는 식물 잎에서 이온을 발생시키는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the compound or its isomer may preferably be one that generates ions in plant leaves, but is not limited thereto.
본 발명에 있어서, 상기 화합물 또는 이의 이성질체는, 바람직하게는 세균성 반점병에 대한 면역을 유도하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the compound or an isomer thereof may preferably induce immunity against bacterial spot disease, but is not limited thereto.
본 발명에 있어서, 상기 화합물 또는 이의 이성질체는, 바람직하게는 포도 뿌리로부터 분리된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the compound or its isomer may preferably be isolated from grape roots, but is not limited thereto.
또한, 본 발명은 상기 식물 면역 유도제를 식물체 또는 토양에 처리하는 단계를 포함하는 식물 면역 유도방법을 제공한다.In addition, the present invention provides a method for inducing plant immunity, comprising the step of treating the plant or soil with the plant immunity inducer.
본 발명은 호페아페놀을 유효성분으로 포함함에 따라, 식물의 1차 면역반응인 PTI(PAMP-triggered immunity)와 2차 면역반응인 ETI(Effector-trigger immunity)를 통해 국부적, 전신적 저항성을 유도하는 식물 면역 유도제를 제공할 수 있다.The present invention contains hopeaphenol as an active ingredient, thereby inducing local and systemic resistance through PTI (PAMP-triggered immunity), the plant's primary immune response, and ETI (Effector-trigger immunity), the secondary immune response. A plant immunity inducer can be provided.
또한, 본 발명의 식물 면역 유도제는 식물의 선천 면역 시스템을 증진시킬 수 있으며, 기존의 항생제를 대체할 수 있는 친환경적 식물 면역 유도제로 사용할 수 있다.In addition, the plant immunity inducer of the present invention can enhance the innate immune system of plants and can be used as an eco-friendly plant immunity inducer that can replace existing antibiotics.
도 1은 본 발명의 일 실시예에 있어서, 호페아페놀의 분자량을 확인한 도이다.
도 2는 본 발명의 일 실시예에 있어서, 호페아페놀의 구조 분석 결과를 보여주는 1H-NMR 스펙트럼을 나타낸 도이다.
도 3은 본 발명의 일 실시예에 있어서, 호페아페놀 처리에 따른 활성산소 발생량을 확인한 도이다.
도 4는 본 발명의 일 실시예에 있어서, 호페아페놀 처리에 따른 이온 전도도를 확인한 도이다.
도 5는 본 발명의 일 실시예에 있어서, 호페아페놀 처리에 따라 면역이 유도된 식물 모델에서 병원균의 밀도를 확인한 도이다.Figure 1 is a diagram confirming the molecular weight of hopeaphenol in one embodiment of the present invention.
Figure 2 is a diagram showing a 1 H-NMR spectrum showing the results of structural analysis of hopeaphenol in one embodiment of the present invention.
Figure 3 is a diagram confirming the amount of active oxygen generation according to hopeaphenol treatment in one embodiment of the present invention.
Figure 4 is a diagram confirming the ionic conductivity according to treatment with hopeaphenol in one embodiment of the present invention.
Figure 5 is a diagram confirming the density of pathogens in a plant model in which immunity is induced by treatment with hopeaphenol, in one embodiment of the present invention.
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 이성질체를 포함하는 식물 면역 유도제를 제공한다.The present invention provides a plant immunity inducer comprising a compound represented by the following formula (1) or an isomer thereof.
[화학식 1][Formula 1]
또한, 본 발명은 상기 식물 면역 유도제를 식물체 또는 토양에 처리하는 단계를 포함하는 식물 면역 유도방법을 제공한다.In addition, the present invention provides a method for inducing plant immunity, comprising the step of treating the plant or soil with the plant immunity inducer.
이하, 실시예 및 실험예를 통하여 본 발명을 보다 자세히 설명한다. 다만, 하기 실시예 및 실험예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다.Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are provided as examples of the present invention, and if it is judged that a detailed description of a technique or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description is omitted. It can be done, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.
<실시예 1> 호페아페놀(hopeaphenol) 분리 및 활성물질 분석<Example 1> Isolation of hopeaphenol and analysis of active substances
1-1. 호페아페놀 분리 및 정제1-1. Separation and purification of hopeaphenol
포도 뿌리를 가루형태로 제분하여, 포도 뿌리 분말 1 g 당 20 mL의 99.5 %(v/v) 메탄올에 침지시킨 후 24시간 동안 실온에서 추출하였으며, Whatman no. 2 여과지를 이용하여 여과하고, 감압농축기를 이용하여 38℃에서 농축하였다. 이후, C18 sep-pak 크로파토그래피를 수행하였다. 각 분획은 물과 메탄올을 일정비율로 혼합하여 전개하였으며, 그 중 활성분획을 모아 감압 농축한 후, 유속 2 mL/min, UV 검출기 254 nm, 용매 0.1% 포름산을 포함한 10~80% 메탄올을 이용하는 분취 조건으로 C18 역상 컬럼을 이용한 HPLC 시스템을 통해 분리 정제하였다.Grape roots were milled into powder, immersed in 20 mL of 99.5% (v/v) methanol per 1 g of grape root powder, and extracted at room temperature for 24 hours. Whatman no. 2 It was filtered using filter paper and concentrated at 38°C using a vacuum concentrator. Afterwards, C18 sep-pak chromatography was performed. Each fraction was developed by mixing water and methanol in a certain ratio, and the active fractions were collected and concentrated under reduced pressure, using 10-80% methanol with a flow rate of 2 mL/min, UV detector 254 nm, and 0.1% formic acid as a solvent. It was separated and purified through an HPLC system using a C18 reverse-phase column under preparative conditions.
1-2. 분리된 활성물질의 분석1-2. Analysis of isolated active substances
분리된 활성물질의 분자량 및 스펙트럼 분석을 통해 본 발명에서 분리된 물질이 호페아페놀임을 확인하였다.Through molecular weight and spectrum analysis of the isolated active substance, it was confirmed that the substance isolated in the present invention was hopeaphenol.
HPLC 시스템을 통해 분리된 활성물질의 분자량을 측정하기 위해 고분해능 질량분석법을 사용하였다. 유속 0.4 mL/in, UV 256 nm, 용매 0.1% 포름산을 포함한 20~100% 아세토나이트릴을 이용하는 분취 조건으로 C8 역상 컬럼을 이용한 UPLC-Q/TOF Mass spectrometer를 통해 활성물질의 분자량을 측정하였다.High-resolution mass spectrometry was used to measure the molecular weight of the active material separated through the HPLC system. The molecular weight of the active substance was measured using a UPLC-Q/TOF mass spectrometer using a C8 reversed-phase column under preparative conditions using a flow rate of 0.4 mL/in, UV 256 nm, and 20 to 100% acetonitrile containing 0.1% formic acid as a solvent.
도 1에서 보듯이, 활성물질의 분자량을 negative mode에서 m/z 905.2593임을 확인하였다.As shown in Figure 1, the molecular weight of the active material was confirmed to be m/z 905.2593 in negative mode.
활성물질의 구조를 분석하기 위해 핵자기공명법을 사용하였다. HPLC 시스템을 통해 정제한 활성물질을 감압농축기를 이용하여 38℃에서 농축한 후, 10 mg의 활성물질을 600 μL의 아세톤에 용해시키고, 500 MHz의 1H-NMR 분석을 진행하였다.Nuclear magnetic resonance was used to analyze the structure of the active material. The active material purified through the HPLC system was concentrated at 38°C using a vacuum concentrator, then 10 mg of the active material was dissolved in 600 μL of acetone, and 1H-NMR analysis at 500 MHz was performed.
도 2에서 보듯이, 활성물질의 1H-NMR의 chemical shift값이 기존 문헌에 보고된 호페아페놀과 동일한 값을 갖는 것을 확인하였다.As shown in Figure 2, it was confirmed that the 1H-NMR chemical shift value of the active material was the same as that of hopeaphenol reported in the existing literature.
<실시예 2> 호페아페놀의 1차 면역 유도 효과 확인<Example 2> Confirmation of the primary immunity-inducing effect of hopeaphenol
본 실험에서는 식물의 1차 면역 반응을 정량적으로 확인할 수 있는 활성산소 측정법을 수행하여 호페아페놀로부터 식물의 1차 면역이 유도되는지 확인하였다 (Marcec et al., “Crosstalk between calcium and ROS signaling during flg22-triggered immune response in Arabidopsis leaves”, Plants 11.1 (2021): 14).In this experiment, we performed an active oxygen measurement method that can quantitatively confirm the plant's primary immune response to confirm whether the plant's primary immunity is induced by hopeaphenol (Marcec et al., “Crosstalk between calcium and ROS signaling during flg22 -triggered immune response in Arabidopsis leaves”, Plants 11.1 (2021): 14).
호페아페놀을 100 μM 농도로 희석한 희석액을 발아 4주 후의 애기장대 잎에 주사기를 이용하여 주입하였다. 대조군으로 물을 주입하였다. 주입 16시간 후, 잎을 일정한 크기로 잘라내어 96 well plate에 각각 넣은 뒤, 활성산소 측정용 용액(Pseudomonas aeruginosa에서 분리한 flg22 단백질 100 nM, Luminol 30 μg /mL, Peroxidase 20 μg/mL)을 200 μL씩 분주하여 Luminescence 측정기에서 1시간 동안 2분 간격으로 활성산소 발생량을 측정하였다.A diluted solution of hopeaphenol to a concentration of 100 μM was injected into Arabidopsis leaves 4 weeks after germination using a syringe. As a control group, water was injected. 16 hours after injection, the leaves were cut to a certain size and placed in a 96 well plate, and then 200 μL of active oxygen measurement solution (flg22 protein isolated from Pseudomonas aeruginosa 100 nM, Luminol 30 μg/mL, Peroxidase 20 μg/mL) was added. The amount of active oxygen generated was measured at 2-minute intervals for 1 hour using a Luminescence meter.
도 3에서 보듯이, 호페아페놀을 처리한 경우, 대조군에 비해 활성산소 발생이 증가하여 1차 면역이 유도된 것을 확인하였다.As shown in Figure 3, when treated with hopeaphenol, it was confirmed that primary immunity was induced by increasing the generation of reactive oxygen species compared to the control group.
<실시예 3> 호페아페놀의 2차 면역 유도 효과 확인<Example 3> Confirmation of secondary immunity-inducing effect of hopeaphenol
본 실험에서는 식물의 2차 면역 반응을 정량적으로 확인할 수 있는 이온 전도도 측정법을 수행하여, 호페아페놀로부터 식물의 2차 면역이 유도되는지 확인하였다(Menna et al., “Elevated temperature differentially influences effector-triggered immunity outputs in Arabidopsis”, Frontiers in plant science 6 (2015): 995).In this experiment, ion conductivity measurement, which can quantitatively confirm the plant's secondary immune response, was performed to confirm whether the plant's secondary immunity is induced by hopeaphenol (Menna et al., “Elevated temperature differentially influences effector-triggered”) immunity outputs in Arabidopsis”, Frontiers in plant science 6 (2015): 995).
호페아페놀을 100 μM 농도로 희석한 희석액을 4 엽기까지 자란 담배식물 잎에 주사기를 이용하여 주입하였다. 대조군으로 물을 주입하였다. 주입 3시간 후, 동일한 위치에 슈도모나스 시린게 pv. 토마토 DC3000 균주 현탁액(106 CFU/mL)을 주입하였다. 주입 40시간 후, 식물의 잎을 일정한 크기로 잘라내어 물 6 mL이 들어있는 테스트튜브에 넣고 잎에서 유출되는 이온을 2시간 간격으로 12시간 측정하였다.A diluted solution of hopeaphenol to a concentration of 100 μM was injected into the leaves of tobacco plants grown to the 4th leaf stage using a syringe. As a control group, water was injected. Three hours after injection, Pseudomonas syringae pv was placed in the same location. Tomato DC3000 strain suspension (10 6 CFU/mL) was injected. 40 hours after injection, the leaves of the plant were cut to a certain size, placed in a test tube containing 6 mL of water, and the ions flowing out of the leaves were measured at 2-hour intervals for 12 hours.
도 4에 호페아페놀 주입 결과를 나타내었다. 도 4에서 보듯이, 호페아페놀을 주입한 잎은 이온 발생량이 증가하였다.Figure 4 shows the results of hopeaphenol injection. As shown in Figure 4, the amount of ions generated in leaves injected with hopeaphenol increased.
<실시예 4> 호페아페놀의 방제 효과 확인<Example 4> Confirmation of the control effect of hopeaphenol
본 실험에서는 호페아페놀에 의해 사전에 식물 면역이 활성화된 애기장대 잎의 병저항성을 확인하였다.In this experiment, the disease resistance of Arabidopsis leaves whose plant immunity was previously activated by hopeaphenol was confirmed.
혼합토양(피트모스, 펄라이트, 버미큘라이트가 4:3:3의 부피비로 혼합된 토양)이 담긴 플라스틱 포트에 애기장대 종자를 파종하고 암처리 4일, 광처리 7일 후에 36-플라스틱 포트에 옮겨심어 3주간 재배하였다. 호페아페놀을 100 μM 농도로 희석한 희석액을 잎에 주입하고 대조군으로는 물을 주입하였다. 16시간 후에 슈도모나스 시린게 pv. 토마토 DC3000 균주 현탁액(105 CFU/mL)을 주입하였다. 접종 당일과 3일 후의 애기장대 잎을 일정한 크기로 잘라 2 mm 크기의 비드가 들어있는 튜브에 옮겨담고 잎을 파쇄하였다. 물 1 mL를 넣어 1/10 연속희석법으로 희석하고, 10 μL 씩 King’s B 배지에 점적하였다. 24시간 후, 배지에서 자란 세균의 콜로니 개수를 계산하여 병 피해도를 평가하였다.Arabidopsis seeds were sown in plastic pots containing mixed soil (peat moss, perlite, and vermiculite mixed in a volume ratio of 4:3:3), and after 4 days of dark treatment and 7 days of light treatment, they were transplanted into 36-plastic pots and grown for 3 weeks. It was cultivated. A diluted solution of hopeaphenol to a concentration of 100 μM was injected into the leaves, and water was injected as a control group. After 16 hours, Pseudomonas syringae pv. Tomato DC3000 strain suspension (10 5 CFU/mL) was injected. On the day of inoculation and 3 days after, Arabidopsis leaves were cut into uniform sizes, transferred to a tube containing 2 mm-sized beads, and the leaves were crushed. Add 1 mL of water and dilute by 1/10 serial dilution, and drop 10 μL onto King's B medium. After 24 hours, the number of bacterial colonies growing in the medium was calculated to evaluate disease damage.
도 5에서 보듯이, 호페아페놀을 처리한 애기장대 잎에서의 세균 CFU 값은 면역유도제를 처리하지 않은 대조군에 비해 유의한 수준으로 적었으며, 이에 따라 본 발명의 호페아페놀 처리시 면역이 효과적으로 유도되는 것을 확인하였다.As shown in Figure 5, the bacterial CFU value in the Arabidopsis thaliana leaves treated with hopeaphenol was significantly lower than the control group that was not treated with an immune inducer, and therefore, the immunity was effectively treated with the hopeaphenol treatment of the present invention. It was confirmed that it was induced.
Claims (6)
[화학식 1]
A plant immunity inducer comprising a compound represented by the following formula (1) or an isomer thereof:
[Formula 1]
상기 화합물 또는 이의 이성질체는,
식물 잎에서 활성산소를 발생시키는 것인, 식물 면역 유도제.According to paragraph 1,
The compound or its isomer,
A plant immunity inducer that generates active oxygen in plant leaves.
상기 화합물 또는 이의 이성질체는,
식물 잎에서 이온을 발생시키는 것인, 식물 면역 유도제.According to paragraph 1,
The compound or its isomer,
A plant immunity inducer that generates ions in plant leaves.
상기 화합물 또는 이의 이성질체는,
세균성 반점병에 대한 면역을 유도하는 것인, 식물 면역 유도제.According to paragraph 1,
The compound or its isomer,
A plant immunity inducer that induces immunity against bacterial spot disease.
상기 화합물 또는 이의 이성질체는,
포도 뿌리로부터 분리된 것인, 식물 면역 유도제.According to paragraph 1,
The compound or its isomer,
A plant immunity inducer isolated from grape roots.
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