JP2023008859A - ハダニを防除するためのタンパク質bvp10及びその応用 - Google Patents
ハダニを防除するためのタンパク質bvp10及びその応用 Download PDFInfo
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Abstract
Description
ダニを殺滅するためのBVP10タンパク質の取得:
1)配列番号1に示される配列(配列番号2に示されるタンパク質を符号化したもの)で、BVP10タンパク質遺伝子断片(Sangon Bioengineering(Shanghai)Co.Ltd.)を人工合成し、且つ大腸菌発現ベクターpet28aプラスミドに結合し、組換え発現プラスミドpet28a-BVP10を構築した。
上記の符号化配列を持つ組換え発現プラスミドpet28a-BVP10を大腸菌BL21に形質転換し、組換え菌BL21/pet28a-BVP10を得た。該組換え菌を5mL LB液体培地に接種し、37℃の振動台に置いてOD600が0.6になるまで培養した後に、1.0mmol/Lのイソプロピル-B-Dチオガラクトシド(即ちIPTGであり、Sigma会社から購入されるもの)を添加して30℃で3時間誘導培養する。上記50mL組換え菌BL21/pet28a-BVP10の誘導培養を3時間行ったものを12000rpmで30秒間遠心分離して菌体を収集し、超音波(技術パラメータ:300W、粉砕30秒、間欠時間30秒)で、細胞を粉砕した後、12000rpmで15分間遠心分離して上澄みを取り、続いて0.4μm孔径の濾過膜で濾過して異物を除去し、GST融合タンパク質のアフィニティークロマトグラフィー法で様々なタンパク質を精製した。最終的な精製生成物に対してSDS-PAGE電気泳動検出を行い、結果を図1に示すが、精製されたタンパク質とタンパク質分子量標準を比較すると、分子量が10kDaであると推定され、予想されるタンパク質BVP10の分子量10.07kDaにほぼ一致し、本発明のBVP10タンパク質の大腸菌BL21での発現が成功したことが証明された。
BVP10タンパク質のハダニ殺虫剤の調製への応用:
1)BVP10タンパク質によるナミハダニ(Tetranychus urticae)殺滅
FAO(国連食糧農業機関)で推薦する有害ダニ測定標準方法であるスライドグラス浸漬法を参照する。両面テープを2-3cmの長さに切り、顕微鏡のスライドガラスの一端に貼り付け、ピンセットでテープ上の紙を剥がし、0号毛筆で、大きさが一致し、色が鮮やかで、活発な雌成体を選択し、その背部を両面テープに貼り付け(ダニの足、髭及び口器に貼り付けないようにすることを注意する)、各シートに4行、各行に10匹貼り付けた。温度25℃、相対湿度85%の左右の生化学インキュベータに4時間放置した後、双眼鏡で観察し、死亡した又は活発でない生体を除去する。予備試験をベースとして水で薬剤を5-7種の濃度に希釈し、ダニ付きスライドグラスの一端を薬液に浸漬させ、軽く5秒間振った後に取り出し、吸水紙でダニ及びその周辺の余計な薬液を迅速に吸い取った。上記生化学インキュベータに置き、24時間後に双眼鏡で結果を検査する。毛筆でダニを軽く触れ、ダニの足が動かないものを死亡したものとした。各濃度のものを3回繰り返し、清水に浸漬したものを対照とした。上記実験ステップに従い、BVP10タンパク質懸濁液によってナミハダニをバイオアッセイすると、その結果は、以下の表1に示すように、19.07μg/mLであった。LC50値は、SPASS 19.0データ処理ソフトウェアで算出された。
FAO(国連食糧農業機関)で推薦する有害ダニ測定標準方法であるスライドグラス浸漬法を参照する。両面テープを2-3cmの長さに切り、顕微鏡のスライドガラスの一端に貼り付け、ピンセットでテープ上の紙を剥がし、0号毛筆で、大きさが一致し、色が鮮やかで、活発な雌成体を選択し、その背部を両面テープに貼り付け(ダニの足、髭及び口器に貼り付けないようにすることを注意する)、各シートに4行、各行に10匹貼り付けた。温度25℃、相対湿度85%の左右の生化学インキュベータで4時間放置した後、双眼鏡で観察し、死亡した又は活発でない生体を除去する。予備試験をベースとして水で薬剤を5-7種の濃度に希釈し、ダニ付きスライドグラスの一端を薬液に浸漬させ、軽く5秒間振った後に取り出し、吸水紙でダニ及びその周辺の余計な薬液を迅速に吸い取る。上記生化学インキュベータに置き、24時間後に双眼鏡で結果を検査する。毛筆でダニを軽く触れ、ダニの足が動かないものを死亡したものとした。各濃度のものに対して3回繰り返して行い、清水に浸漬したものを対照とした。上記実験ステップに従い、BVP10タンパク質懸濁液によってミカンハダニをバイオアッセイすると、その結果は、以下の表2に示すように、58.05μg/mLであった。LC50値は、SPASS 19.0データ処理ソフトウェアで算出された。
上記実験ステップに従い、BVP10タンパク質懸濁液によってニセナミハダニをバイオアッセイすると、その結果は、以下の表3に示すように、36.08μg/mLであった。LC50値は、SPASS 19.0データ処理ソフトウェアで算出された。
BVP10タンパク質の大きい田畑でのイチゴハダニ類(Tetranychus cinnabarinus)防除への応用
試験施薬剤:20%アベルメクチンとスピロフェナクを配合したものの懸濁剤(河北興柏農業科学技術有限公司、使用時、3000倍希釈する)、BVP10タンパク質の有効な濃度が389.5μg/mLの生物殺ダニ剤。
生存ダニ減少率=(施薬前の生存ダニ数-施薬後の生存ダニ数)÷試薬前の生存ダニ数×100%
防除効果=(処理領域の生存ダニ減少率-対照領域の生存ダニ減少率)÷(100-対照領域の生存ダニ減少率)×100%
[付記1]
配列番号2に示されるタンパク質の殺ハダニ剤の調製への応用。
前記ハダニが、ナミハダニ、ミカンハダニ、ニセナミハダニ、又はイチゴハダニ類である、付記1に記載の応用。
Claims (2)
- 配列番号2に示されるタンパク質の殺ハダニ剤の調製への応用。
- 前記ハダニが、ナミハダニ、ミカンハダニ、ニセナミハダニ、又はイチゴハダニ類である、請求項1に記載の応用。
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JP2015518021A (ja) * | 2012-05-30 | 2015-06-25 | バイエル・クロップサイエンス・アーゲーBayer Cropscience Ag | 生物農薬および殺虫剤を含む組成物 |
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Patent Citations (2)
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JP2009508862A (ja) * | 2005-09-16 | 2009-03-05 | ユニバーシティ オブ コネチカット | 殺ダニ組成物およびその使用法 |
JP2015518021A (ja) * | 2012-05-30 | 2015-06-25 | バイエル・クロップサイエンス・アーゲーBayer Cropscience Ag | 生物農薬および殺虫剤を含む組成物 |
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GENBANK DATABASES, NCBI. ACCESSION NO. ABS74171, vol. [online], JPN7022004962, 24 June 2019 (2019-06-24), pages 4 - 10, ISSN: 0004905401 * |
GENBANK DATABASES, NCBI. ACCESSION NO. AJE78819, vol. [online], JPN6022044398, 24 March 2015 (2015-03-24), pages 4 - 10, ISSN: 0004905399 * |
GENBANK DATABASES, NCBI. ACCESSION NO. ATX84098, vol. [online], JPN6022044399, 27 November 2017 (2017-11-27), pages 4 - 10, ISSN: 0004905400 * |
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BR102022013219A2 (pt) | 2023-01-17 |
CN113444152B (zh) | 2022-02-01 |
US20230019438A1 (en) | 2023-01-19 |
KR20230008623A (ko) | 2023-01-16 |
US11839219B2 (en) | 2023-12-12 |
JP7263601B2 (ja) | 2023-04-24 |
EP4115736A1 (en) | 2023-01-11 |
CN113444152A (zh) | 2021-09-28 |
KR102663880B1 (ko) | 2024-06-10 |
EP4115736B1 (en) | 2024-02-28 |
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