JP2008143864A - Anti-plant virus agent - Google Patents
Anti-plant virus agent Download PDFInfo
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- JP2008143864A JP2008143864A JP2006334851A JP2006334851A JP2008143864A JP 2008143864 A JP2008143864 A JP 2008143864A JP 2006334851 A JP2006334851 A JP 2006334851A JP 2006334851 A JP2006334851 A JP 2006334851A JP 2008143864 A JP2008143864 A JP 2008143864A
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
本発明は、抗植物ウイルス剤及びその使用に関する。 The present invention relates to an anti-plant virus agent and use thereof.
植物の病原体の分類と割合をいうと、約80%が菌類であり、約10%は細菌、さらに残りの約10%はウイルス、ウイロイド、ファイトプラズマ、線虫などである。植物ウイルスは病原体の中でわずかな割合しか占めていない。しかし、植物ウイルス病は殻類や野菜類、果樹類などの重要作物に深刻な被害を与えている。植物細胞に侵入したウイルスは、全面的に細胞の代謝に依存して増殖するため、抗ウイルス剤の場合には攻撃目標とする特異的な代謝経路を見つけることが容易ではない。 In terms of the classification and proportion of plant pathogens, about 80% are fungi, about 10% are bacteria, and the remaining 10% are viruses, viroids, phytoplasmas, nematodes, and the like. Plant viruses account for only a small percentage of pathogens. However, plant viral diseases cause serious damage to important crops such as shells, vegetables and fruit trees. Viruses that have invaded plant cells grow entirely depending on the metabolism of the cells, so in the case of antiviral agents, it is not easy to find a specific metabolic pathway targeted for attack.
植物ウイルスは、傷や媒介虫により植物細胞内に侵入すると外被タンパク質の一部が壊れ、粒子内部のゲノム核酸が裸出する脱外被を行う。その後、宿主植物の代謝経路を利用してゲノムRNAの複製や各種タンパク質の発見を行い、細胞間移行と長距離移行を経て植物全体に広がっていく。ウイルス増殖阻害する同時に植物にも薬害を及ぼす(久能ら、1998)。植物ウイルスは各種の代謝過程を完全に宿主植物に依存しているため、植物の代謝経路を正常に保ったままウイルスの増殖のみを特異的に阻害することは困難である。このため、植物ウイルス病は難防除病害とされている( Francki et al,1979 )。 When a plant virus enters a plant cell by a wound or a vector insect, part of the coat protein is broken, and the genome nucleic acid inside the particle is uncovered. After that, replication of genomic RNA and discovery of various proteins are performed using the metabolic pathway of the host plant, and it spreads throughout the plant through cell-to-cell and long-distance transfer. Inhibits virus growth and at the same time causes phytotoxicity to plants (Kunou et al., 1998). Since plant viruses completely depend on the host plant for various metabolic processes, it is difficult to specifically inhibit only the growth of the virus while keeping the plant metabolic pathway normal. For this reason, plant viral diseases are regarded as difficult to control diseases (Frankki et al, 1979).
植物病害のうちで、ウイルス病に対する防除薬剤を開発することはきわめて難しい。抗ウイルス剤には増殖阻害剤と感染阻害剤の2種が考えられ、前者はブラストサイジンS(BcS)、リボソーム不活性化タンパク(ribosome inactivating proteins:RIPs)などがあるが、残念ながら薬害もある。後者は予防剤としてスキムミルクやアルギン酸ソーダなどがあるが、感染後のウイルス増殖を阻害する効果はない。そのため実用的な抗ウイルス剤は現在まで開発されていない。 Among plant diseases, it is extremely difficult to develop a control agent for viral diseases. There are two types of antiviral agents: growth inhibitors and infection inhibitors. The former includes blasticidin S (BcS) and ribosome inactivating proteins (RIPs), but unfortunately there are also phytotoxicity is there. The latter includes skim milk and sodium alginate as preventive agents, but has no effect of inhibiting virus growth after infection. Therefore, a practical antiviral agent has not been developed so far.
最近、マイタケから抗ウイルス性タンパクが抽出された報告があった。マイタケの干し物を磨り潰し、硫安塩析で抽出した。その抽出物をゲル濾過でさらに精製すると、Grifola frondosa antiviral protein(GFAP)が得られた。GFAPはTMV感染阻害タンパクであり、Cysを含有し、熱に安定であることがわかった(Chenら、2004)。 Recently, there was a report that antiviral protein was extracted from maitake. Maitake mushrooms were ground and extracted by ammonium sulfate salting out. When the extract was further purified by gel filtration, Grifola frondosa antiviral protein (GFAP) was obtained. GFAP was a TMV infection inhibitory protein, containing Cys and found to be heat stable (Chen et al., 2004).
従って、本発明は、実用的な、抗植物ウイルス剤を提供しようとするものである。 Therefore, the present invention seeks to provide a practical anti-plant virus agent.
実用的な抗ウイルス物質を探索するために、本研究ではキノコ菌糸抽出物を候補物質として用い、その選抜と作用機構の解析に試みた。候補物質から抗ウイルス活性を示すものをキュウリモザイクウイルス(Cucumber mosaic virus:CMV)とササゲを用いて選抜した結果、マツタケ80%硫安塩析抽出物が高い感染阻害効果を示した。植物ウイルスの一つであるCMV は、Bromoviridae 科 Cucumovirus 属に属し、直径28~30nmの粒子形態をとっている。CMVは40科191種以上の植物に感染し、植物ウイルスの中でもっとも広い宿主範囲を持つウイルスの一つである( Francki et al,1979 )。 In order to search for practical antiviral substances, this study used mushroom mycelium extracts as candidate substances, and tried to select them and analyze the mechanism of action. As a result of selecting a candidate substance exhibiting antiviral activity using Cucumber mosaic virus (CMV) and cowpea, matsutake 80% ammonium sulfate salting out extract showed a high infection inhibitory effect. CMV, one of the plant viruses, belongs to the genus Cucumovirus of the Bromoviridae family and takes a particle form with a diameter of 28-30 nm. CMV infects more than 191 plants in 40 families and is one of the broadest host range of plant viruses (Frankki et al, 1979).
従って、本発明は、マツタケ菌を培養して菌糸を形成させ、当該菌糸を破砕して上清を得、この上清を80%飽和の塩析にかけ、それにより生ずる沈澱物から回収される、マツタケの80%硫安塩析抽出物を含んでなる抗植物ウイルス剤を提供する。
本発明はまた、上記のマツタケの80%硫安塩析抽出物を植物に適用することを特徴とする、ウイルス感染から植物を防護する方法を提供する。
Therefore, the present invention cultivates matsutake fungi to form mycelia, crush the mycelia to obtain a supernatant, subject the supernatant to salting out with 80% saturation, and recover from the resulting precipitate. An anti-plant virus agent comprising an 80% ammonium sulfate salting out extract of matsutake is provided.
The present invention also provides a method for protecting a plant from virus infection, characterized in that the above-mentioned 80% ammonium sulfate salting out extract of matsutake is applied to the plant.
本発明は更に、前記のマツタケの80%硫安塩析抽出物の製造方法において、
(1)マツタケ菌を培養して菌糸を形成させ、
(2)当該菌糸を破砕して上清を得、
(3)この上清を80%飽和の塩析にかけ、そして
(4)上記の塩析により生ずる沈澱物から回収する、
ことを含んで成る方法を提供する。好ましくは、前記の回収工程は、前記沈殿物を緩衝液に溶解し、得られる溶液を緩衝液に対して透析して硫安を除去することにより行う。
The present invention further relates to a method for producing the 80% ammonium sulfate salting out extract of the matsutake,
(1) cultivating matsutake fungi to form mycelia,
(2) The supernatant is obtained by crushing the mycelium,
(3) subjecting the supernatant to salting out to 80% saturation; and (4) recovering from the precipitate resulting from the salting out.
A method comprising the above is provided. Preferably, the recovery step is performed by dissolving the precipitate in a buffer solution and dialyzing the resulting solution against the buffer solution to remove ammonium sulfate.
I.活性成分
1.マツタケの80%硫安塩析抽出物
マツタケの80%硫安塩析抽出物は、下記のようにして調製することが出来る。(1)マツタケ菌を培養して菌糸を形成させ、(2)当該菌糸を破砕して上清を得、(3)この上清を80%飽和の塩析にかけ、そして(4)上記の塩析により生ずる沈澱物から回収する。
I. Active ingredient 80% ammonium sulfate salting out extract of matsutake The 80% ammonium sulfate salting out extract of matsutake can be prepared as follows. (1) cultivating matsutake fungi to form mycelia; (2) crushing the mycelium to obtain a supernatant; (3) subjecting the supernatant to salting out to 80% saturation; and (4) the salt described above. It is recovered from the precipitate produced by the analysis.
マツタケ(Tricholoma matsutake)の菌糸は、当該菌糸が増殖しうる任意の培地で培養することが出来る。具体例として、後に述べる浜田氏培地が挙げられる。培養は、菌糸株の保存と増殖のためには、固体培地(例えば、寒天培地)を用い、表面培養を行う。活性成分の生産のための培地としては液培地が好ましく、例えば下記の浜田氏液体培地が上げられる。培養は好気的条件下で行う必要があり、液体培地上での表面静置培養が好ましい。また、液体培地中で、振とう、通気、撹拌などの常用の手段による好気的条件下で培養を行うことも出来る。 The mycelium of Tricholoma matsutake can be cultured in any medium in which the mycelium can grow. A specific example is Mr. Hamada's medium described later. The culture is performed by surface culture using a solid medium (for example, an agar medium) for storage and growth of the mycelium strain. As the medium for producing the active ingredient, a liquid medium is preferable, and for example, the following Mr. Hamada liquid medium can be raised. Culture must be performed under aerobic conditions, and surface static culture on a liquid medium is preferable. Moreover, it can also culture | cultivate in a liquid medium on aerobic conditions by usual means, such as shaking, aeration, and stirring.
マツタケの菌糸を液体表面培養により培養する場合、本発明の抗ウイルス活性成分は、主とした菌体内に蓄積される。このため、活性成分を含むマツタケの80%硫安塩析抽出物を得るには、先ず、培養液から菌糸を回収する。菌糸の回収方法は、濾過、遠心分離など、液体培地から菌糸(菌体)を回収するために常法を用いることが出来る。
次に、回収した菌糸を破砕する。この方法も、ミキサーや、その他の常用の菌体破砕手段を用いて行うことが出来る。
When cultivating mycelia of matsutake by liquid surface culture, the antiviral active ingredient of the present invention accumulates mainly in the fungus body. For this reason, in order to obtain an 80% ammonium sulfate salting out extract of matsutake containing active ingredients, first, the mycelium is recovered from the culture solution. As a method for collecting mycelia, conventional methods can be used for collecting mycelia (mycelium) from a liquid medium, such as filtration and centrifugation.
Next, the collected mycelium is crushed. This method can also be carried out using a mixer or other conventional cell disruption means.
次に、菌体破砕物から菌体端破片を除去して上清、即ち菌体破砕物から菌体破片等の不溶物を除去した液体を得る。この場合も、濾過、遠心分離など、常用の固液分離手段を用いることが出来る。 Next, the cell end debris is removed from the crushed cell body to obtain a supernatant, that is, a liquid from which insoluble matter such as the cell crumb is removed from the cell crushed material. In this case as well, conventional solid-liquid separation means such as filtration and centrifugation can be used.
次に、この上清に硫安を加えて硫安濃度を80%飽和にすることにより塩析を行い、これにより週ずる沈澱を得る。次に、この沈澱を水又は適当な緩衝液に溶解し、更に水又は適当な緩衝液に対して透析することにより硫安を除去する。この場合の緩衝液のpHは6〜7である。これにより本発明の「マツタケの80%硫安塩析抽出物」が得られる。この抽出物は、そのまま低温保存、例えば凍結保存することが出来、また凍結乾燥などの手段により固体にして保存することも出来る。 Next, salting out is performed by adding ammonium sulfate to this supernatant to saturate the ammonium sulfate concentration to 80%, thereby obtaining a weekly precipitate. Next, the precipitate is dissolved in water or a suitable buffer, and ammonium sulfate is removed by dialysis against water or a suitable buffer. In this case, the pH of the buffer solution is 6-7. As a result, the “80% ammonium sulfate salting out extract of matsutake” of the present invention is obtained. This extract can be stored as it is at a low temperature, for example, frozen, or it can be stored as a solid by means of lyophilization or the like.
本発明のマツタケの80%硫安塩析抽出物は100℃で10分間加熱しても、抗ウイルス活性を殆ど失わない。従って、抗ウイルス活性の本体は低分子物質であると予想される。更に、活性成分が低分子物質であることは、ゲル濾過法によっても確認される。 The 80% ammonium sulfate salting out extract of the matsutake of the present invention loses almost no antiviral activity even when heated at 100 ° C. for 10 minutes. Therefore, the main body of antiviral activity is expected to be a low molecular weight substance. Furthermore, it is also confirmed by gel filtration that the active ingredient is a low molecular weight substance.
次に、実施例により、本発明を更に具体的に説明する。
II.材料と方法
1.マツタケの80%硫安塩析抽出物の調製
マツタケ菌糸を浜田氏平板培地(水道水 1000 mL;ブドウ糖 20 g;エビオス粉末 5 g;pH=5.1〜5.2;寒天 20 g)上で30日間24℃で培養し菌糸を増殖させた。この菌糸を、200 mL 容の三角フラスコ中の20 mLの浜田氏液体培地(水道水 1000 mL;ブドウ糖 20 g;エビオス粉末 5 g;pH=5.1〜5.2)に接種し、24℃にて60日間静置培養し、液体培地表面に菌糸を増殖させた。
Next, the present invention will be described more specifically with reference to examples.
II. Materials and methods Preparation of 80% ammonium sulfate salted out extract of matsutake mushroom mycelia on Hamada plate medium (tap water 1000 mL; glucose 20 g; shrimp powder 5 g; pH = 5.1 to 5.2; agar 20 g) at 30 ° C. for 30 days The mycelium was grown by culturing. This mycelium is inoculated into 20 mL of Mr. Hamada's liquid medium (1000 mL of tap water; glucose 20 g; shrimp powder 5 g; pH = 5.1 to 5.2) in a 200 mL Erlenmeyer flask at 24 ° C. for 60 days. After static culture, the mycelium was grown on the surface of the liquid medium.
上記の培養液を、ブフナー漏斗(No.2濾紙;Advantec製)により濾過してマツタケ菌糸を回収し、20 mM Tris−HCl(pH 7.0)緩衝液に懸濁し、ミキサーを用いて粉砕した。粉砕した菌体を遠心分離(12,000 rpm, 20分間)にかけ、上清を回収した。この上清液に80%飽和になるように硫安を加えて塩析を行った。塩析後、沈殿物を遠心分離(12,000 rpm, 20分間)により回収し、20 mM Tris−HCl(pH=7.0)緩衝液に溶解し、この溶液を20 mM Tris−HCl(pH=7.0)緩衝液を用いて透析し、これを「マツタケの80%硫安塩析抽出物」とした。 The culture broth was filtered through a Buchner funnel (No. 2 filter paper; manufactured by Advantec) to collect matsutake mycelia, suspended in 20 mM Tris-HCl (pH 7.0) buffer, and pulverized using a mixer. The crushed cells were centrifuged (12,000 rpm, 20 minutes), and the supernatant was collected. The supernatant was salted out by adding ammonium sulfate to 80% saturation. After salting out, the precipitate is collected by centrifugation (12,000 rpm, 20 minutes), dissolved in 20 mM Tris-HCl (pH = 7.0) buffer, and this solution is buffered with 20 mM Tris-HCl (pH = 7.0). The resulting solution was dialyzed to obtain an “80% ammonium sulfate salting out extract of matsutake”.
前記マツタケの80%硫安塩析抽出物の熱に対する性質を調べた。非加熱の「マツタケの80%硫安塩析抽出物」と、100℃にて10分間加熱した「マツタケの80%硫安塩析抽出物」とについて、ササゲを用いるアッセイ系において、ウイルス感染阻害率を調べた。その結果、加熱しても90%以上(92%)のウイルス感染阻害率が維持された。このことから、「マツタケの80%硫安塩析抽出物」中の活性成分は熱に安定な低分子性物質であることが示唆された。
更に、Sephadex G-75を用いたゲル濾過によっても、阻害物質が低分子の物質であることが確認された。
The heat resistance of the 80% ammonium sulfate salting out extract of matsutake was examined. In the non-heated "Matsutake 80% ammonium sulfate salting out extract" and the "Matsutake 80% ammonium sulfate salting out extract" heated at 100 ° C for 10 minutes, the virus infection inhibition rate in the assay system using cowpea Examined. As a result, the virus infection inhibition rate of 90% or more (92%) was maintained even when heated. From this, it was suggested that the active ingredient in “80% ammonium sulfate salting out extract of matsutake” is a heat-stable low molecular weight substance.
Furthermore, gel filtration using Sephadex G-75 also confirmed that the inhibitor was a low-molecular substance.
2.供試植物
ササゲ(Vigna sesquipedalis cv. kurodane sanjaku)
バーミキュライトを満たした平鉢に播種し、温室内に置いて発芽した苗をセルトレイに移植して育苗した。実験には初生葉が十分に展開したものを、直径9 cmのプラスチックポットに移植し用いた。
タバコ(Nicotiana tabacum cx. Xanthi nc)
バーミキュライトを満たした平鉢に播種し、温室内に置いて発芽した苗をセルトレイに移植して育苗した。2~3週間後に、鉢に1個体ずつ定植し、7~8葉期のものを実験に使用した。
2. Test plant
Cowpea (Vigna sesquipedalis cv. Kurodane sanjaku)
The seedlings sown in a flat pot filled with vermiculite and placed in a greenhouse were germinated by transplanting to a cell tray. In the experiment, primary leaves that were sufficiently developed were transplanted into a 9 cm diameter plastic pot.
Tobacco (Nicotiana tabacum cx. Xanthi nc)
The seedlings sown in a flat pot filled with vermiculite and placed in a greenhouse were germinated by transplanting to a cell tray. Two to three weeks later, one individual plant was planted in a pot, and one in the 7-8 leaf stage was used for the experiment.
3.供試ウイルス
CMVのpepo系統
1973年に尾崎らが和歌山県で採種した、モザイク症状を示すペポカボチャより分離した。サテライトRNAを含まない病原力の強い分離株である。ラグナリア系統群、サブグループIに属する。
3. Test virus
CMV pepo system
It was isolated from a peppo pumpkin that showed mosaic symptoms in 1973 by Ozaki et al. It is a highly virulent isolate that does not contain satellite RNA. It belongs to Lagunaria group, subgroup I.
4.CMV純化法
CMV接種後7日目のタバコ接種葉を−80℃下で凍結し、ミキサーで磨砕した。凍結感染葉5 gに対し100 mlの抽出バッファー〔0.5Mクエン酸、0.01Mエチレンジアミン-4-酢酸-2-ナトリウム(EDTA);pH6.5,0.1%チオグリコール酸ナトリウム〕を加え、撹拌した。これを氷冷下で5分間静置した後、10,000 rpmで15分間の遠心分離を行い、上清を得た。この上清に8%(w/v)のポリエチレングリコール6000(PEG)を加え、4℃下で完全に融解させ、10,000 rpmで15分間の遠心分離を行い、ウイルス沈殿を得た。この沈殿をBEバッファー(0.005Mホウ酸;pH9.0,0.01M EDTA)で懸濁し、10,000 rpmで10分間の遠心分離をおこない、夾雑物を除去した。この操作を2、3回おこない、上清を得た。
4). CMV purification method
Tobacco-inoculated leaves on day 7 after CMV inoculation were frozen at −80 ° C. and ground with a mixer. To 5 g of frozen infected leaves, 100 ml of extraction buffer [0.5 M citric acid, 0.01 M ethylenediamine-4-acetic acid-2-sodium (EDTA); pH 6.5, 0.1% sodium thioglycolate] was added and stirred. This was allowed to stand for 5 minutes under ice cooling, and then centrifuged at 10,000 rpm for 15 minutes to obtain a supernatant. To this supernatant, 8% (w / v) polyethylene glycol 6000 (PEG) was added, completely thawed at 4 ° C., and centrifuged at 10,000 rpm for 15 minutes to obtain a virus precipitate. This precipitate was suspended in BE buffer (0.005M boric acid; pH 9.0, 0.01M EDTA), and centrifuged at 10,000 rpm for 10 minutes to remove impurities. This operation was performed 2 or 3 times to obtain a supernatant.
この上清に1% Triton Xを加え、40,000 rpmで60分間の遠心分離をおこない、ウイルス沈殿を得た。沈殿をBEバッファーで懸濁し、10,000 rpmで10分間の遠心分離をおこない、夾雑物を除去した。この操作を2、3回おこない、上清を得た。この上清を42,000 rpmで90分間遠心分離し、得られたウイルス沈殿を0.005Mホウ酸バッファー(pH9.0)で懸濁した。この懸濁液に対し、10,000 rpmで10分間の遠心分離をおこない、夾雑物を除去した。この操作を2、3回おこない、上清を得た。
以上の操作により得られたウイルス懸濁液は、分光光度計(Ultrospec 3000;Amersham Pharmacia Biotech社)で定量後、-20℃で凍結保存した。CMVの濃度はE260 =5を1mg/mlとした。
1% Triton X was added to the supernatant, and centrifugation was performed at 40,000 rpm for 60 minutes to obtain a virus precipitate. The precipitate was suspended in BE buffer and centrifuged at 10,000 rpm for 10 minutes to remove contaminants. This operation was performed 2 or 3 times to obtain a supernatant. The supernatant was centrifuged at 42,000 rpm for 90 minutes, and the resulting virus precipitate was suspended in 0.005 M borate buffer (pH 9.0). The suspension was centrifuged at 10,000 rpm for 10 minutes to remove impurities. This operation was performed 2 or 3 times to obtain a supernatant.
The virus suspension obtained by the above operation was quantified with a spectrophotometer (Ultrospec 3000; Amersham Pharmacia Biotech) and stored frozen at -20 ° C. The CMV concentration was set to 1 mg / ml with E 260 = 5.
5.粒子接種
純化ウイルス液を0.1Mリン酸バッファー(pH7.0)で希釈し、3%カーボランダムを加えて懸濁し、綿棒を用いてこすりつけた後、水道水で洗浄した。
5. Particle inoculation The purified virus solution was diluted with 0.1 M phosphate buffer (pH 7.0), suspended by adding 3% carborundum, rubbed with a cotton swab, and washed with tap water.
6.局部病斑宿主を用いた対葉法
検定にはCMVと、局部病斑宿主であるササゲを用いた。ササゲの初生葉2枚のうち、片方には活性物質を、他方には活性物質の溶媒(20mM Tris-Cl 0.1%Triton X)を塗布した。葉を15分ほど乾燥させた後、CMVを接種し、2〜3日後に出現した壊死病斑数を比較して抗ウイルス性を検討した。ここでは、
感染阻害率(%)=(1−候補物質処理区局部病斑数/対照区局部病斑数)×100
とした。
6). The CMV and cowpea, a local lesion host, were used for the anti-leaf test using local lesion hosts. Of the two primary cowpea leaves, the active substance was applied to one, and the active substance solvent (20 mM Tris-Cl 0.1% Triton X) was applied to the other. After the leaves were dried for about 15 minutes, CMV was inoculated, and the number of necrotic lesions that appeared after 2 to 3 days was compared to examine antiviral properties. here,
Infection inhibition rate (%) = (1-candidate substance treatment district local lesion count / control district local lesion count) × 100
It was.
7.DAS-ELISA法
カーボネイト緩衝液〔64mM Na2CO3,136mM NaHCO3,0.8% NaN3:pH9.6〕で抗CMV CP IgGを1/1000に希釈し、ELISAプレートに100 μlずつ分注した。その後、37℃で2〜3時間、または4℃で一CP晩静置した後、PBSTでウェルを3回洗浄した。感染葉を10倍量のカーボネイト緩衝液で磨砕し、磨砕液をウェルに100 μlずつ分注した。37℃で2〜3時間、または4℃で一晩静置した。その後、PBSTでウェルを4回洗浄した後、PBSTでコンジュゲート(AP標識抗ウサギ抗体)を1/1000に希釈し、100μlずつ分注した.37℃で2〜3時間静置した後、PBSTでウェルを3回洗浄した。アルカリフォスフォターゼ基質を10%ジエタノールアミン液で溶かし、100 μlずつ分注し、発色後、microplate reader(autoreader;三光純薬)により吸光度を計測した。
7). DAS-ELISA method Anti-CMV CP IgG was diluted 1/1000 with carbonate buffer [64 mM Na 2 CO 3 , 136 mM NaHCO 3 , 0.8% NaN 3 : pH 9.6], and 100 μl was dispensed on an ELISA plate. Thereafter, the plate was allowed to stand at 37 ° C. for 2 to 3 hours, or at 4 ° C. for 1 CP overnight, and then the wells were washed 3 times with PBST. The infected leaves were ground with 10 times the amount of carbonate buffer, and 100 μl of the ground liquid was dispensed into each well. Allowed to stand at 37 ° C. for 2-3 hours or at 4 ° C. overnight. Thereafter, the wells were washed 4 times with PBST, and the conjugate (AP-labeled anti-rabbit antibody) was diluted 1/1000 with PBST and dispensed in 100 μl aliquots. After allowing to stand at 37 ° C. for 2 to 3 hours, the wells were washed 3 times with PBST. The alkaline phosphatase substrate was dissolved in 10% diethanolamine solution, dispensed in 100 μl aliquots, and after color development, the absorbance was measured with a microplate reader (autoreader; Sanko Junyaku).
8.純化ウイルスからのRNA抽出
試料に800 μlのグリシンバッファー〔100mMグリシン、100mM NaCl,10mM EDTA:pH9.5〕を加えて1.5mlマイクロチューブに移した。70 μlの10%SDS,70 μlの4%ベントナイトと500 μlフェノール/クロロフォルム(フェノール:クロロフォルム:イソアミルアルコール=25:24:1)を加えて、12,000 rpmで4分間の遠心分離を行った後、上清を別のマイクロチューブに移した。フェノール/クロロフォルムをさらに500 μl加え、12,000 rpmで4分間の遠心分離を行った。上清450 μlに45 μlの3 M酢酸ナトリウム溶液と1 mlのエタノールを加えて-80℃で15分間静置した後、15,000 rpmで15分間の遠心分離を行った。上澄みを除去し内壁に残っているエタノールをマイクロピペットで完全に除去した後、デジケーター中で15分間減圧乾燥した。得られた沈殿はDEPC処理水に融解させ、分光光度計で濃度を計測した。CMV RNAの濃度はE260=40を1 mg/mlとした。
8). RNA extraction from purified virus 800 μl of glycine buffer [100 mM glycine, 100 mM NaCl, 10 mM EDTA: pH 9.5] was added to the sample and transferred to a 1.5 ml microtube. Add 70 μl 10% SDS, 70 μl 4% bentonite and 500 μl phenol / chloroform (phenol: chloroform: isoamyl alcohol = 25: 24: 1), and centrifuge at 12,000 rpm for 4 minutes. The supernatant was transferred to another microtube. An additional 500 μl of phenol / chloroform was added and centrifuged at 12,000 rpm for 4 minutes. 45 μl of 3 M sodium acetate solution and 1 ml of ethanol were added to 450 μl of the supernatant, and the mixture was allowed to stand at −80 ° C. for 15 minutes, followed by centrifugation at 15,000 rpm for 15 minutes. The supernatant was removed, and ethanol remaining on the inner wall was completely removed with a micropipette, followed by drying under reduced pressure for 15 minutes in a desiccator. The obtained precipitate was melted in DEPC-treated water and the concentration was measured with a spectrophotometer. The CMV RNA concentration was 1 mg / ml with E 260 = 40.
9.RNA接種
ウイルス粒子から抽出したRNAを0.1Mリン酸バッファー(pH7.0)/ベントナイトで希釈し、粒子接種と同様にして供試植物に接種した。RNA濃度はササゲの場合は0.4 μg/ml、タバコの場合は2 μg/mlに調節した。
9. RNA inoculation RNA extracted from virus particles was diluted with 0.1 M phosphate buffer (pH 7.0) / bentonite and inoculated into test plants in the same manner as particle inoculation. The RNA concentration was adjusted to 0.4 μg / ml for cowpea and 2 μg / ml for tobacco.
10.ティッシュブロット法
あらかじめ、ニトロセルロースメンブレンと濾紙4枚を0.2M CaCl2で15分間振とうしておいた。サンプル葉の主脈を除去し、蒸留水で洗浄した。葉の裏側にセルライトを散布した後、歯ブラシで研磨して表皮組織を除去した。その後、サンプル葉を0.2M CaCl2で軽く洗浄した。ゲル乾燥機の上に濾紙2枚を置き、ニトロセルロースメンブレン、サンプル葉、濾紙2枚の順に重ねた。それらにトランスファーバッファーを十分に含ませた後、真空状態で50℃15分間加熱し転写を行った。
Ten. Tissue blotting The nitrocellulose membrane and 4 filter papers were shaken in advance with 0.2 M CaCl 2 for 15 minutes. The main veins of the sample leaves were removed and washed with distilled water. After cellulite was sprayed on the back side of the leaves, the epidermal tissue was removed by polishing with a toothbrush. Thereafter, the sample leaves were gently washed with 0.2 M CaCl 2 . Two filter papers were placed on the gel dryer, and a nitrocellulose membrane, sample leaves, and two filter papers were stacked in this order. After sufficiently containing a transfer buffer, they were heated in a vacuum state at 50 ° C. for 15 minutes for transfer.
転写後、ニトロセルロースメンブレンをTTBS(1×TBS, pH7.5 (25mM Tris, 137mM NaCl, 25mM KCl), 0.05% Tween 20)に浸し、10分間振とうした。振とう後、TTBSを除去し、ブロッキング溶液に浸し30分間振とうした。ブロッキング溶液を除去し、ニトロセルロースメンブレンをTTBSで軽く洗浄した。その後、TTBSで6000倍に希釈したCMV IgG (1 mg/ml)にニトロセルロースメンブレンを浸し、90分間振とうした。反応終了後、ニトロセルロースメンブレンをTTBSで10分間振とう洗浄した。 After the transfer, the nitrocellulose membrane was immersed in TTBS (1 × TBS, pH 7.5 (25 mM Tris, 137 mM NaCl, 25 mM KCl), 0.05% Tween 20) and shaken for 10 minutes. After shaking, TTBS was removed, immersed in a blocking solution and shaken for 30 minutes. The blocking solution was removed and the nitrocellulose membrane was lightly washed with TTBS. Thereafter, the nitrocellulose membrane was immersed in CMV IgG (1 mg / ml) diluted 6000 times with TTBS, and shaken for 90 minutes. After completion of the reaction, the nitrocellulose membrane was washed with TTBS for 10 minutes with shaking.
この洗浄操作を2階繰り返した後、TTBSで10000倍に希釈したアルカリフォスフォターゼ標識抗ウサギIgG - ヤギIgGにニトロセルロースメンブレンを浸し、90分間振とうした.反応終了後、ニトロセルロースメンブレンをTTBSで10分間振とう洗浄した。この洗浄操作を2回繰り返した後、ニトロセルロースメンブレンを蒸留水で軽くすすいだ。ニトロセルロースメンブレンをAP 9.5(pH7.4, 100mM Tris, 100mM NaCl, 50mM MgCl2)に5分間浸した後、BCIP/NBTに浸し暗所で発色させた。 This washing operation was repeated on the second floor, and then the nitrocellulose membrane was soaked in alkaline phosphatase-labeled anti-rabbit IgG-goat IgG diluted 10,000 times with TTBS and shaken for 90 minutes. After completion of the reaction, the nitrocellulose membrane was washed with TTBS for 10 minutes with shaking. After repeating this washing operation twice, the nitrocellulose membrane was rinsed lightly with distilled water. The nitrocellulose membrane was immersed in AP 9.5 (pH 7.4, 100 mM Tris, 100 mM NaCl, 50 mM MgCl 2 ) for 5 minutes, and then immersed in BCIP / NBT for color development in the dark.
III.結果
1.候補物質のスクリーニング
抗植物ウイルス活性の評価には、CMVとCMVの局部感染宿主であるジュウロクササゲを用いた。
その結果、マツタケ菌糸磨粋物80%硫安塩析で得られた抽出物で処理したササゲでは局部病斑数が認められなくなることから、高い感染阻害効果を有していると考えられた。
III. Result 1. Screening for Candidate Substances For the evaluation of anti-plant virus activity, CMV and Dwarf cowpea, a locally infected host of CMV, were used.
As a result, the number of local lesions was not observed in the cowpeas treated with the extract obtained from 80% ammonium sulfate salting out of matsutake mycelia, so it was considered to have a high infection inhibitory effect.
2.マツタケ80%硫安塩析抽出物の抗ウイルス性
次にマツタケ80%硫安塩析抽出物の有効濃度や、CMV接種に対する予防効果または治療効果の有無、CMV局部感染宿主であるササゲとCMV全身感染宿主であるタバコにおける増殖阻害活性について調査した。
2. Antiviral properties of matsutake 80% ammonium sulfate salted out extract Next, effective concentration of matsutake 80% ammonium sulfate salted out extract, presence or absence of preventive or therapeutic effect against CMV inoculation, cowpea and CMV systemic infected host which are CMV local infection hosts The growth inhibitory activity in tobacco was investigated.
(1)マツタケ80%硫安塩析抽出物の抗ウイルス性効果
(i)局部感染宿主におけるマツタケ80%硫安塩析抽出物の抗ウイルス性
(a)マツタケ80%硫安塩析抽出物の有効濃度
選抜したマツタケ80%硫安塩析抽出物の有効濃度をササゲにおける対葉法で調査した。マツタケ80%硫安塩析抽出物は、100 μg/ml,10 μg/ml及び1 μg/mの3つの濃度で塗布した。その結果、感染阻害率を80%以上示したのは100 μg/mlであり、濃度10 μg/ml,1 μg/mlは感染阻害率を40%以下示した(図1)。
マツタケ80%硫安塩析抽出物をCMVと混合させ、100 μg/ml,10 μg/ml,1 μg/mlの3つの濃度で接種した。その結果、濃度100μg/mlは96%の感染阻害率を示した.濃度10 μg/ml,1 μg/mlは40%以下の感染阻害率を示した(図2)。
(1) Antiviral effect of matsutake 80% ammonium sulfate salting out extract (i) Antiviral property of matsutake 80% ammonium sulfate salting out extract in locally infected host (a) Effective concentration of matsutake 80% ammonium sulfate salting out extract The effective concentration of matsutake 80% ammonium sulfate salting-out extract was investigated by the counter-leaf method in cowpea. Matsutake 80% ammonium sulfate salting out extract was applied at three concentrations of 100 μg / ml, 10 μg / ml and 1 μg / m. As a result, 100 μg / ml showed an infection inhibition rate of 80% or more, and concentrations of 10 μg / ml and 1 μg / ml showed an infection inhibition rate of 40% or less (FIG. 1).
Matsutake 80% ammonium sulfate precipitation was mixed with CMV and inoculated at three concentrations of 100 μg / ml, 10 μg / ml, and 1 μg / ml. As a result, a concentration of 100 μg / ml showed a 96% infection inhibition rate. Concentrations of 10 μg / ml and 1 μg / ml showed an infection inhibition rate of 40% or less (FIG. 2).
(b)マツタケ80%硫安塩析抽出物処理の有効時間
マツタケ80%硫安塩析抽出物を塗布し乾燥直後にCMVを接種すると、80%以上の感染阻害率を示した。ここでは、マツタケ80%硫安塩析抽出物塗布前と後に時間をおき、その後CMVを接種し、マツタケ80%硫安塩析抽出物の効果の持続性について調査した。
濃度100 μg/mlのマツタケ80%硫安塩析抽出物をササゲの初生葉の一方に綿棒で塗布し、他方に溶媒を塗布した。6,12,24時間後に10 μg/mlのCMVを粒子接種した。その結果、マツタケ80%硫安塩析抽出物処理後6時間では84%の感染阻害率を示した。処理後12及び24時間では40%以下の感染阻害率を示した(図3)。よって、マツタケ80%硫安塩析抽出物は葉面上で6時間にわたり感染阻害効果を持続することがわかった。
(B) Effective time of treatment with matsutake 80% ammonium sulfate salting out extract When the matsutake 80% ammonium sulfate salting out extract was applied and CMV was inoculated immediately after drying, an infection inhibition rate of 80% or more was shown. Here, before and after application of matsutake 80% ammonium sulfate salting-out extract, time was taken, and then CMV was inoculated to investigate the persistence of the effect of matsutake 80% ammonium sulfate salting-out extract.
A matsutake 80% ammonium sulfate salting-out extract with a concentration of 100 μg / ml was applied to one of the primary leaves of cowpea with a cotton swab and a solvent was applied to the other. Particles were inoculated with 10 μg / ml CMV after 6, 12, and 24 hours. As a result, an infection inhibition rate of 84% was exhibited 6 hours after treatment with matsutake 80% ammonium sulfate salting out extract. At 12 and 24 hours after treatment, the infection inhibition rate was 40% or less (FIG. 3). Therefore, it was found that the matsutake 80% ammonium sulfate salting-out extract maintained the infection-inhibiting effect on the leaf surface for 6 hours.
続いてCMV接種後に濃度100 μg/mlのマツタケ80%硫安塩析抽出物を処理することにより、マツタケ80%硫安塩析抽出物の病害治療の可能性について調査した。ササゲの初生葉にCMVを接種し、6,12時間後にマツタケ80%硫安塩析抽出物を塗布した。その結果、CMV接種後6,12時間では40%以下の感染阻害率を示した(図4)。よって、CMV接種後のマツタケ80%硫安塩析抽出物塗布処理では病斑形成は抑制できず、病害治療効果はなかった。 Subsequently, after treatment with CMV, the possibility of disease treatment of matsutake 80% ammonium sulfate salting-out extract was examined by treating matsutake 80% ammonium sulfate salting-out extract at a concentration of 100 μg / ml. The primary leaves of cowpea were inoculated with CMV, and after 6 to 12 hours, matsutake 80% ammonium sulfate salting out extract was applied. As a result, the infection inhibition rate was 40% or less at 6, 12 hours after CMV inoculation (FIG. 4). Therefore, the application of matsutake 80% ammonium sulfate salting-out extract after CMV inoculation did not suppress the formation of lesions, and there was no disease treatment effect.
(ii)全身感染宿主におけるマツタケ80%硫安塩析抽出物の抗ウイルス性
マツタケ80%硫安塩析抽出物のCMV増殖阻害活性をCMV全身感染宿主であるタバコを用いて調査した。
(a)マツタケ80%硫安塩析抽出物のタバコ葉面塗布処理におけるCMV CP蓄積量
タバコに濃度100 μg/mlのマツタケ80%硫安塩析抽出物を塗布し、完全乾燥させた30分後にCMV粒子を20 μg/mlで接種した。対照区には溶媒を塗布し、完全乾燥させた30分後にCMV粒子を接種した。2日おきに、接種葉から2枚上の上位葉をサンプリングし、DAS-ELISA法でCMV CP蓄積量を測定した。
(Ii) Antiviral activity of matsutake 80% ammonium sulfate salting out extract in systemically infected host The CMV growth inhibitory activity of matsutake 80% ammonium sulfate salting out extract was investigated using tobacco, a CMV systemic infected host.
(A) Amount of CMV CP accumulated in the tobacco leaf surface application of matsutake 80% ammonium sulfate salting out extract 30 minutes after applying 100% matsutake 80% ammonium sulfate salting out extract to tobacco and completely drying it The particles were inoculated at 20 μg / ml. The control group was coated with a solvent and inoculated with CMV particles 30 minutes after complete drying. Every two days, the upper two leaves from the inoculated leaves were sampled, and the CMV CP accumulation amount was measured by the DAS-ELISA method.
その結果、接種後におけるマツタケ80%硫安塩析抽出物処理区のCMV CPの蓄積量が対照区より明らかに低いことが観察された。また、対照区では接種5日後に葉の奇形と葉脈の透明化が上位葉で観察され、12日後にはモザイク病徴が確認されたが、マツタケ80%硫安塩析抽出物処理区では病徴発見が遅延し、8日後になってはじめて葉に軽い黄化が観察され、15日後にモザイク病徴が確認された。マツタケ80%硫安塩析抽出物処理タバコの病徴はその後も進行し、対照区と差はなくなった(図4、5及び6)。 As a result, it was observed that the amount of CMV CP accumulated in the matsutake 80% ammonium sulfate salting-out treatment group after inoculation was clearly lower than that in the control group. In the control plot, leaf malformation and clear veins were observed in the upper leaves 5 days after inoculation, and mosaic symptoms were confirmed 12 days later, but in the matsutake 80% ammonium sulfate salting-out extract treatment plot, symptoms were observed. The discovery was delayed and light yellowing was observed on the leaves only after 8 days and mosaic symptoms were confirmed after 15 days. The symptom of the matsutake 80% ammonium sulfate salting-out extract treated tobacco continued to progress and was no different from the control group (FIGS. 4, 5 and 6).
(b)マツタケ80%硫安塩析抽出物のタバコ葉面混合処理におけるCMV CP蓄積量
タバコに濃度100 μg/mlのマツタケ80%硫安塩析抽出物とCMVの混合液を塗布した。対照区には溶媒とCMVの混合液を塗布した。2日おきに、接種葉から2枚上の上位葉をサンプリングし、DAS-ELISA法でCMV CP蓄積量を測定した。
その結果、接種後におけるマツタケ80%硫安塩析抽出物処理区のCMV CPの蓄積量が対照区より明らかに低いことが観察された。また、対照区では接種6日後に葉の奇形と葉脈の透明化が上位葉で観察され、11日後にはモザイク病徴が確認されたが、マツタケ80%硫安塩析抽出物処理区では病徴発見が遅延し、9日後になってはじめて葉に軽い黄化が観察され、14日後にモザイク病徴が確認された。マツタケ80%硫安塩析抽出物処理タバコの病徴はその後も進行し、対照区と差はなくなった(図7、8及び9)。
(B) Accumulated CMV CP in Tobacco Leaf Surface Mixing Process of Matsutake 80% Ammonium Sulfate Salting Extract A mixed solution of Matsutake 80% ammonium sulfate salting out extract and CMV at a concentration of 100 μg / ml was applied to tobacco. A solvent and CMV mixture was applied to the control group. Every two days, two upper leaves from the inoculated leaves were sampled, and CMV CP accumulation was measured by the DAS-ELISA method.
As a result, it was observed that the accumulated amount of CMV CP in the matsutake 80% ammonium sulfate salting-out treatment group after inoculation was clearly lower than that in the control group. In the control group, leaf deformity and vein transparency were observed in the upper leaves 6 days after inoculation, and mosaic symptoms were confirmed 11 days later, but in the matsutake 80% ammonium sulfate salting-out extract treatment group, disease symptoms were observed. The discovery was delayed and light yellowing was observed on the leaves only after 9 days, and mosaic symptoms were confirmed after 14 days. Symptoms of matsutake 80% ammonium sulfate salted-out extract treated tobacco continued to progress, and were no different from the control group (FIGS. 7, 8 and 9).
(c)ティッシュブロット法でタバコの葉のウイルス分布
タバコに濃度100 μg/mlのマツタケ80%硫安塩析抽出物とCMVの混合液を塗布した。対照区には溶媒とCMVの混合液を塗布した。2日、4日おきに、接種葉をサンプリングし、ティッシュブロット法でタバコの葉のウイルス分布を調査した。
タバコに濃度100 μg/mlのマツタケ80%硫安塩析抽出物を塗布し、完全乾燥させた30分後にCMV粒子を20 μg/mlで接種した。対照区には溶媒を塗布し、完全乾燥させた30分後にCMV粒子を接種した。2日、4日おきに、接種葉をサンプリングし、ティッシュブロット法でタバコの葉のウイルス分布を調査した。
(C) Virus distribution of tobacco leaves by tissue blotting To a tobacco, a mixed solution of matsutake 80% ammonium sulfate salting-out extract and CMV having a concentration of 100 μg / ml was applied. A solvent and CMV mixture was applied to the control group. Every 2 days and 4 days, the inoculated leaves were sampled, and the virus distribution of tobacco leaves was examined by tissue blotting.
Tobacco was coated with matsutake 80% ammonium sulfate salting-out extract at a concentration of 100 μg / ml, and after 30 minutes of complete drying, CMV particles were inoculated at 20 μg / ml. The control group was coated with a solvent and inoculated with CMV particles 30 minutes after complete drying. Every 2 days and 4 days, the inoculated leaves were sampled, and the virus distribution of tobacco leaves was examined by tissue blotting.
その結果、2日目に、混合した処理区では葉の縁側に少し分布した。対照区では葉の葉脈周囲に多量に分布した。4日目に、処理区と対照区は両方ともウイルスが葉に全面的に拡大した(図10)。
塗布した処理区では2日目に、葉の主葉脈の周囲に少し分布した。対照区では葉の葉脈の周囲に多量に分布した。4日目に、処理区と対照区は両方ともウイルスが葉に全面的に拡大した(図11及び12)。
As a result, on the second day, the mixed treatment was slightly distributed on the edge of the leaves. In the control group, it was distributed in large quantities around the leaf veins. On the 4th day, both the treated and control plots had the virus fully spread to the leaves (Figure 10).
In the applied treatment section, it was slightly distributed around the main veins of the leaves on the second day. In the control group, it was distributed in large quantities around the leaf veins. On the 4th day, both the treatment group and the control group had the virus fully expanded to the leaves (FIGS. 11 and 12).
3.マツタケ80%硫安塩析抽出物の抗ウイルス作用機構
次に、マツタケ80%硫安塩析抽出物の抗ウイルス作用機構の解明を試みた。
(1)ササゲに対する抵抗性の誘導性の有無
葉の裏面に濃度100 μg/mlのマツタケ80%硫安塩析抽出物を塗布し、さらに表面にウイルスを接種した裏面処理でササゲに対する抵抗性の誘導性を調査した。その結果、ウイルスの感染阻害率は40%以下であり、抵抗性の誘導は認められなかった。
3. Antiviral action mechanism of matsutake 80% ammonium sulfate salting out extract Next, we tried to elucidate the antiviral action mechanism of matsutake 80% ammonium sulfate precipitation.
(1) Presence or absence of induction of resistance to cowpea Induction of resistance to cowpea by applying backside treatment with 100% matsutake 80% ammonium sulfate salting out extract on the backside of the leaf and inoculating the surface with virus. The sex was investigated. As a result, the virus infection inhibition rate was 40% or less, and no induction of resistance was observed.
(2)ササゲにおける抽出物の移行性の有無
ササゲの片葉に濃度100 μg/mlのマツタケ80%硫安塩析抽出物を塗布し、もう一方の片葉にウイルスを接種し、抽出物の阻害移行性の有無を調査した。その結果、感染阻害率も30%以下であり、浸透移行性は認められなかった。
(2) The presence or absence of extract migration in cowpea Applying 100% matsutake 80% ammonium sulfate salting out extract to one leaf of cowpea, inoculating the virus on the other leaf, inhibiting the extract The presence or absence of migration was investigated. As a result, the infection inhibition rate was 30% or less, and no permeability was observed.
(3)マツタケ80%硫安塩析抽出物とCMVの混合接種
マツタケ80%硫安塩析抽出物がCMV粒子またはCMV RNAのどちらに作用しているかを調査するために、マツタケ80%硫安塩析抽出物とCMV RNAの混合接種を行った.マツタケ80%硫安塩析抽出物とCMV RNAの混合液を作製し、ササゲに接種した。混合液中のマツタケ80%硫安塩析抽出物の濃度は20%とした。
その結果、対照区の葉面に局部病斑が数十程度出た。処理区の葉面には局部病斑がまったくなく、100%の感染阻害率を示した(図13)。よって、マツタケ80%硫安塩析抽出物がウイルスRNAを阻害することが考えられた。
(3) Mixed inoculation of matsutake 80% ammonium sulfate salted out extract and CMV To investigate whether matsutake 80% ammonium sulfate salted out extract acts on CMV particles or CMV RNA, matsutake 80% ammonium sulfate salted out extract The mixture was inoculated with CMV RNA. A mixed solution of matsutake 80% ammonium sulfate salting-out extract and CMV RNA was prepared and inoculated in cowpea. The concentration of Matsutake 80% ammonium sulfate salting-out extract in the mixed solution was 20%.
As a result, several tens of local lesions appeared on the leaves of the control group. There were no local lesions on the leaves of the treated area, and the infection inhibition rate was 100% (FIG. 13). Therefore, it was considered that matsutake 80% ammonium sulfate salting out extract inhibits viral RNA.
III.考察
本発明においては、キノコ由来候補物質を材料として、抗ウイルス活性のある物質の選抜を行い、その結果得られたマツタケ80%硫安塩析抽出物の抗植物ウイルス活性と、その作用機構の解析を試みた。
マツタケ80%硫安塩析抽出物を更に精製すると、各々の分子量のタンパクの抗ウイルス活性が低く、CMVに対する抵抗性がほとんどなくなった。マツタケ80%硫安塩析抽出物自体が抗ウイルス活性を有すると考えられる.更に精製できない原因は、マツタケ80%硫安塩析抽出物の抗ウイルス活性が複数の抗ウイルス物質によると考えられる。
III. DISCUSSION In the present invention, using mushroom-derived candidate substances as materials, selection of substances having antiviral activity was performed, and the anti-plant virus activity of the resulting matsutake 80% ammonium sulfate salting-out extract and analysis of its action mechanism Tried.
When the matsutake 80% ammonium sulfate salting-out extract was further purified, the antiviral activity of each molecular weight protein was low and the resistance to CMV was almost lost. It is considered that matsutake 80% ammonium sulfate salting out extract itself has antiviral activity. The cause of further purification is thought to be due to the antiviral activity of the matsutake 80% ammonium sulfate salting out extract due to multiple antiviral substances.
マツタケ80%硫安塩析抽出物は塗布処理により、ササゲの病斑形成を抑制した。マツタケ80%硫安塩析抽出物とCMV粒子の混合処理により、ササゲの病斑形成も抑制した。CMV接種6時間前にマツタケ80%硫安塩析抽出物をササゲの塗布した場合においても高い感染阻害率を示したが、接種後のマツタケ80%硫安塩析抽出物処理では病害治療効果がなくなった。濃度100 μg/mlでマツタケ80%硫安塩析抽出物をタバコに塗布すると、病徴遅延とCMV CP蓄積量の低下が観察された。 The matsutake 80% ammonium sulfate salting-out extract suppressed the formation of lesion spots in cowpea by application treatment. By combining matsutake 80% ammonium sulfate salting-out extract and CMV particles, cowpea lesions were also suppressed. Even when application of cowpea with matsutake 80% ammonium sulfate salting out extract 6 hours before CMV inoculation, high infection inhibition rate was shown, but treatment with matsutake 80% ammonium sulfate salting out extract after inoculation lost the disease treatment effect . When matsutake 80% ammonium sulfate salting-out extract was applied to tobacco at a concentration of 100 μg / ml, symptom delay and CMV CP accumulation were reduced.
濃度100μg/mlでマツタケ80%硫安塩析抽出物とCMV粒子の混合液をタバコに塗布すると、病徴遅延とCMV CP蓄積量の低下も観察された。塗布処理と混合処理したタバコの葉をテイッシュブロット法で分析すると、CMVのタバコにおける葉面増殖が2日まで抑制したことが観察された。さらに、マツタケ80%硫安塩析抽出物とCMV RNAの混合液をササゲに接種すると、病斑が完全に観察されなかった。以上のことから、マツタケ80%硫安塩析抽出物は感染阻害活性と増殖阻害活性を持ち、CMV粒子に直接作用して阻害効果を示していることだけではなく、CMV RNAにも作用し、阻害効果を示すと考えられる。 When a mixture of matsutake 80% ammonium sulfate salting out extract and CMV particles was applied to tobacco at a concentration of 100 μg / ml, symptom delay and a decrease in CMV CP accumulation were also observed. Tobacco leaves treated with the application treatment and mixed treatment were analyzed by tissue blotting, and it was observed that leaf growth in CMV tobacco was suppressed up to 2 days. Furthermore, when the cowpea was inoculated with a mixed solution of matsutake 80% ammonium sulfate salting-out extract and CMV RNA, lesions were not completely observed. Based on the above, matsutake 80% ammonium sulfate salting out extract has infection inhibitory activity and growth inhibitory activity, not only acting directly on CMV particles but also showing inhibitory effect, also acting on CMV RNA and inhibiting It is considered to show an effect.
フィガレンは葉内に浸透してウイルスに作用、または抵抗性を誘導すると考えられている(Fujiwara et al,2001)。しかし、ササゲにおいて、CMV接種後のマツタケ80%硫安塩析抽出物処理では病害治療効果がない。ササゲにおいてマツタケ80%硫安塩析抽出物とCMVで裏面処理すると、対照区と変わらない病斑数が計測された。また、ササゲの方初生葉にマツタケ80%硫安塩析抽出物を塗布し、もう一方の葉にCMVを接種する移行性調査を行うと、対照区と変わらない病斑数が計測された。以上のことから、マツタケ80%硫安塩析抽出物は抵抗性を誘導することがなく、植物の葉から他方の葉への移行もしない、施用した葉に分布し、CMV粒子の侵入を阻害し、またCMV粒子とCMV RNAの増殖を阻害すると考えられる。 Figalene is thought to penetrate into the leaves and act on the virus or induce resistance (Fujiwara et al, 2001). However, in cowpea, treatment with matsutake 80% ammonium sulfate salting-out extract after CMV inoculation has no therapeutic effect. In the cowpea, the backside treatment with matsutake 80% ammonium sulfate salted-out extract and CMV showed the same number of lesions as the control. In addition, when a migration survey was conducted by applying matsutake 80% ammonium sulfate salting-out extract to cowpea primary leaves and inoculating CMV on the other leaves, the number of lesions was the same as in the control group. Based on the above, matsutake 80% ammonium sulfate salting-out extract does not induce resistance, does not migrate from the leaves of the plant to the other leaves, is distributed in the applied leaves and inhibits the invasion of CMV particles It is also thought to inhibit the growth of CMV particles and CMV RNA.
アルギン酸塩には高いTMVの感染阻止作用がある。また、葉組織内へ浸透し、移行性がない。さらに、アルギン酸塩はin vitroでTMVを凝集させる性質がある(Tomaruら、1965)。マツタケ80%硫安塩析抽出物も同じように植物の葉に浸透し、中に感染したウイルスを阻害する。また、先に葉組織に浸透し、ウイルスの侵入を阻害すると考えられる。 Alginate has a high anti-TMV effect. Moreover, it penetrates into the leaf tissue and has no migration. In addition, alginate has the property of aggregating TMV in vitro (Tomaru et al., 1965). The matsutake 80% ammonium sulfate salting-out extract also penetrates the leaves of the plant in the same way and inhibits the virus infected inside. Moreover, it is thought that it penetrates into the leaf tissue first and inhibits the entry of the virus.
ブラストサイジンS(BcS)はタバコ葉に感染するタバコモザイクウイルス(Tobacco mosaic virus : TMV)を抑制する効果があると報告された(Hirai et al.)。接種後と接種後12時間に抑制効果が非常に高い。ブラストサイジンS(BcS)は感染後6~24時間にタバコ葉の細胞質と葉緑体またミトコンドリアでアミノC結合を阻害する。平常のタンパクとTMVタンパクの両方とも抑制される。マツタケ80%硫安塩析抽出物はCMV RNAとの混合接種で高い感染阻害率を示したので、ウイルスRNAに阻害する作用機構を有すると考えられる。 Blasticidin S (BcS) has been reported to be effective in suppressing tobacco mosaic virus (Tobacco mosaic virus: TMV) that infects tobacco leaves (Hirai et al.). The inhibitory effect is very high after inoculation and 12 hours after inoculation. Blasticidin S (BcS) inhibits amino C binding in the cytoplasm, chloroplasts and mitochondria of tobacco leaves 6-24 hours after infection. Both normal and TMV proteins are suppressed. The matsutake 80% ammonium sulfate salted out extract showed a high infection inhibition rate when mixed with CMV RNA, so it is considered to have a mechanism of action that inhibits viral RNA.
また、Pokeweed antiviral protein : PAPは細胞間移行を通じて、下の葉から上の葉まで浸透し、ウイルス阻害効果があるという報告がある(Chenら)。マツタケ80%硫安塩析抽出物は移行性調査で調べた結果から、移行性を有しないことが考えられる。
抗ウイルス剤として使用する場合、薬害を示さずに抗ウイルス活性を示すことが重要である(平井、1972)。本実験で対象としたマツタケ80%硫安塩析抽出物については、薬害を示さずに抗ウイルス効果を示したので、抗植物ウイルスとして有望と考えられる。
In addition, Pokeweed antiviral protein: PAP has been reported to penetrate from the lower leaves to the upper leaves through cell-to-cell transition and have a virus inhibitory effect (Chen et al.). The matsutake 80% ammonium sulfate salting-out extract is considered to have no migratory properties based on the results of the migratory investigation.
When used as an antiviral agent, it is important to show antiviral activity without showing phytotoxicity (Hirai, 1972). The matsutake 80% ammonium sulfate salted-out extract, which was the subject of this experiment, was considered promising as an anti-plant virus because it showed antiviral effects without showing any phytotoxicity.
VI.引用文献
平井篤造 ( 1979). ウイルスと植物. 南江堂.
久能均・白石友紀・高橋壮・露無慎二・眞山滋志 ( 1998 ). 新編 植物病理学概論. 養賢堂.
M. Fujiwara, M. Sakaguchi, B. Ryan, S. T. Ohki and T. Osaki ( 2001). Effect of Figaren, an antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection. J. Gen. Plant Pathology. 67:159-163.
R. I. B. Francki, D.W.Mossop, T. Hatta,( 1979 ). CMI/AAB Description of Plant Virus July 1979 No,213.
VI. Cited by Atsushi Hirai (1979). Viruses and plants. Nanedou.
Hitoshi Kuno, Yuki Shiraishi, So Takahashi, Shinji Tsukumu, Shigeru Kajiyama (1998). Introduction to new plant pathology. Yokendo.
M. Fujiwara, M. Sakaguchi, B. Ryan, ST Ohki and T. Osaki (2001) .Effect of Figaren, an antiviral Protein from Cucumis figarei, on Cucumber mosaic virus Infection.J.Gen.Plant Pathology.67: 159- 163.
RIB Francki, DWMossop, T. Hatta, (1979) .CMI / AAB Description of Plant Virus July 1979 No, 213.
N. Chen, Z-J Wu, Q-Y. Lin, L-H. Xie, ( 2004 ). Purification and Partial Characterization of a Protein Inhibitor of Tobacco mosaic virus Infection From The Maitake (Grifola frndosa). Prog. Biochem. Biophys. ( 2004 ): 31(3)283-286.
Z. C. Chen, R. F. White, J. F. Antonniw and Q. Lin. ( 1991 ). Effect of pokeweed antiviral piotein ( PAP ) on the infection of plant viruses. Plant Pathology 40:612-620.
T. Hirai, A. Hiashima, T. Itoh, T.Takahashi, T. Shimomura, and Y. Hayashi. ( 1966 ). Inhibitory Effect of Blasticidin S on Tobacco Mosaic Virus Multiplication. Phytopathology 56:1236-1239.
K. Tomaru and Y. Ohkawa. ( 1975 ) The Inhibitory Activity of Alginic Acid Against Tobacco Mosaic Virus Infection. Ann. Phytopath. Soc. Japan 41:155-161.
N. Chen, ZJ Wu, QY. Lin, LH. Xie, (2004). Purification and Partial Characterization of a Protein Inhibitor of Tobacco mosaic virus Infection From The Maitake (Grifola frndosa). Prog. Biochem. Biophys. (2004): 31 (3) 283-286.
ZC Chen, RF White, JF Antonniw and Q. Lin. (1991) .Effect of pokeweed antiviral piotein (PAP) on the infection of plant viruses.Plant Pathology 40: 612-620.
T. Hirai, A. Hiashima, T. Itoh, T. Takahashi, T. Shimomura, and Y. Hayashi. (1966). Inhibitory Effect of Blasticidin S on Tobacco Mosaic Virus Multiplication. Phytopathology 56: 1236-1239.
K. Tomaru and Y. Ohkawa. (1975) The Inhibitory Activity of Alginic Acid Against Tobacco Mosaic Virus Infection. Ann. Phytopath. Soc. Japan 41: 155-161.
Claims (4)
(1)マツタケ菌を培養して菌糸を形成させ、
(2)当該菌糸を破砕して上清を得、
(3)この上清を80%飽和の塩析にかけ、そして
(4)上記の塩析により生ずる沈澱物から回収する、
ことを含んで成る方法。 In the manufacturing method of the 80% ammonium sulfate salting out extract of the matsutake of Claim 1,
(1) cultivating matsutake fungi to form mycelia,
(2) The supernatant is obtained by crushing the mycelium,
(3) subjecting the supernatant to salting out to 80% saturation; and (4) recovering from the precipitate resulting from the salting out.
A method comprising that.
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CN105176934A (en) * | 2015-10-16 | 2015-12-23 | 西南大学柑桔研究所 | Long-time in vitro preservation method for citrus yellow vein clearing virus |
JPWO2020009012A1 (en) * | 2018-07-03 | 2021-08-12 | 株式会社微生物化学研究所 | Cucumber mosaic virus attenuated strain |
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Publication number | Priority date | Publication date | Assignee | Title |
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CN105176934A (en) * | 2015-10-16 | 2015-12-23 | 西南大学柑桔研究所 | Long-time in vitro preservation method for citrus yellow vein clearing virus |
CN105176934B (en) * | 2015-10-16 | 2018-09-18 | 西南大学柑桔研究所 | The long-term in-vitro conservation method of Citrus Yellowing vein clearing virus |
JPWO2020009012A1 (en) * | 2018-07-03 | 2021-08-12 | 株式会社微生物化学研究所 | Cucumber mosaic virus attenuated strain |
JP7281126B2 (en) | 2018-07-03 | 2023-05-25 | 株式会社微生物化学研究所 | Attenuated strain of cucumber mosaic virus |
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