JP2013514768A - 昆虫抵抗性管理のためのCRY1DaおよびCRY1Faタンパク質の併用 - Google Patents
昆虫抵抗性管理のためのCRY1DaおよびCRY1Faタンパク質の併用 Download PDFInfo
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Classifications
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- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
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Abstract
Description
「コーンボーラー保護Bt(Cry1AbまたはCry1F)トウモロコシ製品の特定の構造化要件は、以下の通りである:
構造化緩衝帯:
コーンベルトで20%の非鱗翅目Btトウモロコシ緩衝帯;
コットンベルトで50%の非鱗翅目Bt緩衝帯
ブロック
内部(すなわち、Bt圃場内)
外部(すなわち、任意交配を最大にするためにBt圃場から1/2マイル(可能であれば1/4マイル)以内に別個の圃場)
圃場内の帯状地
幼虫の移動の影響を低減するために、帯状地は少なくとも4列(rows)(好ましくは6列)の幅でなければならない」
緩衝帯要件に関して類似した指針を提供する。例えば:
「コーンボーラーIRMの要件:
・トウモロコシ畑地の少なくとも20%に緩衝帯雑種を植える
・綿花生産地では、緩衝帯は50%でなければならない
・緩衝帯雑種の1/2マイル以内に植えなければならない
・緩衝帯は、Bt圃場内に帯状地として植えることができる;緩衝帯の帯状地は少なくとも4列の幅でなければならない
・標的昆虫の経済的許容限界に到達する場合だけ、緩衝帯を従来の殺虫剤で処理することができる
・Btベースの噴霧可能な殺虫剤を緩衝帯トウモロコシで用いることはできない
・Btトウモロコシのあらゆる農場に適当な緩衝帯を設けなければならない」
トランスジェニックトウモロコシで発現されるCry1Da(pDAS5163)は、フォールアーミーワーム(FAW)、スポドプテラ・フルジペルダ(Spodoptera frugiperda)(J.E.Smith)による食害からの保護を提供する。同じ事象はCry1Faへの抵抗性を発達させたFAWを防除することにおいてより有効であり、おそらくFAW防除のための業界をリードする昆虫抵抗性形質であるTC1507イベントを含むトウモロコシ植物より明らかに優れる。
FAWから単離されたブラシ縁膜小胞(BBMV)を用いて125I標識Cry1Daで実施された競合結合実験を下に記載する。これらの実験からの結果は、Cry1Daがその受容体にタイトに結合し、Cry1FaはCry1Daと結合部位をめぐって競合しないことを実証する。Cry1Daへの抵抗性がこれらの試験で観察される受容体への突然変異に基づくことができるならば、Cry1Faがそのような抵抗性集団を管理するか、そのような抵抗性の発達を軽減するための優れたIRM手段となるであろうことを、これらのデータは示唆する。Cry1Fa抵抗性のFAW(rFAW)によるバイオアッセイからの結果は、Cry1Daがこの集団で活性であることを実証する。総合すると、Cry1FaおよびCry1Daがいずれかの殺虫性タンパク質への抵抗性の発達を効果的に軽減するIRMスタックである可能性を、これらのデータは示唆する。
キメラ毒素。別のCry毒素のプロトキシン部分と融合している1つのCry毒素のコア毒素ドメインを利用するキメラタンパク質は、例えば、米国特許第5593881号および米国特許第5932209号で以前に報告されている。
完全長Cry1Daキメラタンパク質を生成するように遺伝子操作された蛍光菌(Pseudomonas fluorescens)(Pf)発現構築物pDOW2848の構築では、標準のクローニング法[例えばSambrookら, (1989)およびAusubelら, (1995)、およびその最新版に記載]を用いた。タンパク質生成は、米国特許第5169760号に開示される改変ラックオペロンの挿入を有する蛍光菌(Pseudomonas fluorescens)株MB214(株MB101の誘導体;蛍光菌(P. fluorescens)生物型I)で実施された。基本的なクローニング戦略は、プラスミドベクターにCry1Daタンパク質をコードするDNA断片をサブクローニングすることを伴い、それにより、DNA断片はプラスミドpKK223−3(PL Pharmacia、Milwaukee、WI)からのPtacプロモーターおよびrrnBT1T2ターミネーターの発現制御下に置かれる。そのようなプラスミドの1つはpDOW2848と名付けられ、このプラスミドを抱えるMB214分離株はDpf150と名付けられる。
以下の実施例は、昆虫腸組織での推定上の受容体へのCry1コア毒素タンパク質の競合結合を評価する。スポドプテラ・フルジペルダ(Spodoptera frugiperda)(フォールアーミーワーム)から調製されるブラシ縁膜小胞(BBMV)に125I標識Cry1Daコア毒素タンパク質が高い親和性で結合すること、およびCry1Faコア毒素タンパク質がこの結合で競合しないことが示される。
Cry1DaおよびCry1Faコア毒素タンパク質による結合アッセイで用いるためにBBMVタンパク質の最適量を決定するために、飽和曲線を生成した。0.5nMの125I放射性標識Cry1コア毒素タンパク質を、0μg/mLから500μg/mLのBBMVタンパク質の量と一緒に、結合緩衝液(8mM NaHPO4、2mM KH2PO4、150mM NaCl、0.1%BSA、pH7.4)中で28度で1時間インキュベートした(0.5mLの全容量)。反応混合液の150μLを3反復で別々の1.5mL遠心管に採取し、室温で8分間の14,000×gで試料を遠心分離することによって、BBMVタンパク質に結合した125I標識Cry1コア毒素タンパク質を未結合の分画から分離した。上清を静かに除去し、氷冷結合緩衝液でペレットを3回洗浄した。ペレットを含む遠心管の底を切り離し、13×75mmガラス培養管に入れ、試料を各々ガンマ計数器で5分間数えた。得られたCPM(1分あたりのカウント数)からバックグラウンドCPM(BBMVタンパク質のない反応)を引いたものを、BBMVタンパク質濃度に対してプロットした。他の者(Luoら, 1999)によって報告された結果と一致して、結合アッセイで用いるBBMVタンパク質の最適濃度は、150μg/mLであると決定された。
相同的および非相同的競合結合アッセイは、150μg/mLのS.フルジペルダ(S. frugiperda)BBMVタンパク質および0.5nMの125I放射性標識Cry1Daコア毒素タンパク質を用いて実施した。真の結合競合を保証するために、反応混合液に加えられた競合的非放射性標識Cry1Faコア毒素タンパク質の濃度は0.045nMから1000nMであって、放射性Cry1Daコア毒素タンパク質と同時に加えられた。インキュベーションを28度で1時間実施し、BBMVに結合した(特異的結合)125I標識Cry1Daコア毒素タンパク質の量を前記のように測定した。1,000nMの非放射性標識Cry1Daコア毒素タンパク質の存在下で得られたカウント数によって、非特異的結合を表した。100パーセントの全結合は、いかなる競合者Cry1Faコア毒素タンパク質もない場合の結合の量であるとみなされた。
Claims (36)
- Cry1D殺虫性タンパク質をコードするDNAおよびCry1F殺虫性タンパク質をコードするDNAを含むトランスジェニック植物。
- 請求項1に記載の植物の種子。
- Cry1D殺虫性タンパク質をコードするDNAおよびCry1F殺虫性タンパク質をコードするDNAが遺伝子移入されている、請求項1に記載の植物。
- 請求項3に記載の植物の種子。
- 非Bt緩衝帯植物および請求項1に記載の複数の植物を含む植物の圃場であって、前記緩衝帯植物は前記圃場の全ての作物植物の40%未満を構成する、圃場。
- 前記緩衝帯植物が前記圃場の全ての作物植物の30%未満を構成する、請求項5に記載の植物の圃場。
- 前記緩衝帯植物が前記圃場の全ての作物植物の20%未満を構成する、請求項5に記載の植物の圃場。
- 前記緩衝帯植物が前記圃場の全ての作物植物の10%未満を構成する、請求項5に記載の植物の圃場。
- 前記緩衝帯植物が前記圃場の全ての作物植物の5%未満を構成する、請求項5に記載の植物の圃場。
- 前記緩衝帯植物がブロックまたは帯状地にある、請求項5に記載の植物の圃場。
- 非Bt緩衝帯植物からの緩衝帯種子および請求項4に記載の複数の種子を含む種子混合物であって、前記緩衝帯種子は混合物の全ての種子の40%未満を構成する、種子混合物。
- 前記緩衝帯種子が混合物の全ての種子の30%未満を構成する、請求項11に記載の種子混合物。
- 前記緩衝帯種子が混合物の全ての種子の20%未満を構成する、請求項11に記載の種子混合物。
- 前記緩衝帯種子が混合物の全ての種子の10%未満を構成する、請求項11に記載の種子混合物。
- 前記緩衝帯種子が混合物の全ての種子の5%未満を構成する、請求項11に記載の種子混合物。
- 種子を播いて請求項5に記載の植物の圃場を作製することを含む、昆虫によるCry毒素への抵抗性の発達を管理する方法。
- Cry1Abコア毒素を含むタンパク質をコードするDNAをさらに含む、請求項1に記載のトランスジェニック植物。
- 非Bt緩衝帯植物および請求項17の複数のトランスジェニック植物を含む植物の圃場であって、前記緩衝帯植物は前記圃場の全ての作物植物の約20%未満を構成する、圃場。
- 約10%未満の緩衝帯植物を含む、請求項17に記載の複数の植物を含む植物の圃場。
- 種子を播いて請求項19に記載の植物の圃場を作製することを含む、昆虫によるCry毒素への抵抗性の発達を管理する方法。
- Cry1Fコア毒素含有タンパク質およびCry1Dコア毒素含有タンパク質の両方の有効量を発現する細胞を含む、鱗翅類の有害生物を防除するための組成物。
- Cry1Fコア毒素含有タンパク質およびCry1Dコア毒素含有タンパク質の両方を発現するように形質転換された、微生物または植物細胞である宿主を含む、請求項21に記載の組成物。
- 鱗翅類の有害生物を防除する方法であって、前記有害生物に、または前記有害生物の環境に請求項21に記載の組成物の有効量を提示することを含む方法。
- 同じ標的昆虫に対して殺虫性である3つの殺虫性Cryタンパク質を生成するトランスジェニック植物であって、前記昆虫は前記Cryタンパク質のいずれか1つへの抵抗性を発達させる能力を有し、各前記Cryタンパク質は前記標的昆虫の異なる腸管内受容体に結合する植物。
- 前記昆虫がフォールアーミーワームである、請求項24に記載の植物。
- Cry1Faタンパク質と、Cry1Daタンパク質と、Vip3A、Cry1C、Cry1BeおよびCry1Eタンパク質からなる群から選択される第三のタンパク質とを生成するトランスジェニック植物。
- 種子を播いて請求項26に記載の植物の圃場を作製することを含む、昆虫によるCry毒素への抵抗性の発達を管理する方法。
- 非Bt緩衝帯植物および請求項26に記載の複数の植物を含む植物の圃場であって、前記緩衝帯植物は前記圃場の全ての作物植物の約10%未満を構成する、圃場。
- 前記圃場が約5%未満の緩衝帯植物を含む、請求項28に記載の圃場。
- 種子を播いて請求項28または29に記載の植物の圃場を作製することを含む、昆虫によるCry毒素への抵抗性の発達を管理する方法。
- 非Bt緩衝帯植物からの緩衝帯種子および請求項26に記載の植物からの複数の種子を含む種子混合物であって、前記緩衝帯種子は混合物の全ての種子の10%未満を構成する、種子混合物。
- 前記植物が10エーカーよりも多くを占める、請求項5、18および28のいずれか1項に記載の圃場。
- トウモロコシ、ダイズおよびワタからなる群から選択される、請求項1、2、17、24および26のいずれかに記載の植物。
- メイズ植物である、請求項1、2、17、24および26のいずれか1項に記載の植物。
- 前記植物細胞が、前記Cry1D殺虫性タンパク質をコードする前記DNAおよび前記Cry1F殺虫性タンパク質をコードする前記DNAを含み、前記Cry1F殺虫性タンパク質が配列番号1と少なくとも99%同一であり、前記Cry1D殺虫性タンパク質が配列番号2と少なくとも99%同一である、請求項1、2、17、24、26、33および34のいずれか1項に記載の植物の植物細胞。
- 前記Cry1F殺虫性タンパク質が配列番号1を含み、前記Cry1D殺虫性タンパク質が配列番号2を含む、請求項1、2、17、24、26、33および34のいずれか1項に記載の植物。
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CA2782546C (en) | 2023-03-21 |
UA112409C2 (uk) | 2016-09-12 |
KR20120096574A (ko) | 2012-08-30 |
CL2012001622A1 (es) | 2013-01-25 |
AU2010330916B2 (en) | 2015-07-16 |
CA2782546A1 (en) | 2011-06-23 |
CN102843903A (zh) | 2012-12-26 |
EP2512221A4 (en) | 2013-10-09 |
BR112012014575A2 (pt) | 2017-06-20 |
BR122019001711B1 (pt) | 2020-03-31 |
UA111710C2 (uk) | 2016-06-10 |
CN102843903B (zh) | 2016-02-10 |
ZA201204914B (en) | 2013-02-27 |
AU2010330916A1 (en) | 2012-07-12 |
EP2512221A1 (en) | 2012-10-24 |
RU2603257C2 (ru) | 2016-11-27 |
WO2011075587A1 (en) | 2011-06-23 |
MX358710B (es) | 2018-08-31 |
UA113273C2 (xx) | 2017-01-10 |
EP2512221B1 (en) | 2018-11-07 |
US20120331589A1 (en) | 2012-12-27 |
RU2012129906A (ru) | 2014-01-27 |
KR101841298B1 (ko) | 2018-03-22 |
BR122019001711B8 (pt) | 2022-10-11 |
CO6602146A2 (es) | 2013-01-18 |
AR079499A1 (es) | 2012-02-01 |
NZ601096A (en) | 2014-10-31 |
JP5908409B2 (ja) | 2016-04-26 |
IL220333A (en) | 2016-07-31 |
MX2012007138A (es) | 2012-10-09 |
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