JP5767585B2 - 大豆遺伝子組換え事象mon87705およびその検出方法 - Google Patents
大豆遺伝子組換え事象mon87705およびその検出方法 Download PDFInfo
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Description
本願は、2008年9月29日付けで出願した米国仮出願シリアル番号第61/100,859号の利益を主張する。
サイズが50,995バイト(MS−DOSにおける測定)であって、2009年9月22日に作成されたファイル名「56047−0000WO_seqlist.pdf」を含む、添付書類C/ST.25テキスト形式の配列表のコピーをEFSウェブを介してここに電子的に提出し、ここに出典明示して本明細書の一部とみなす。
本発明の前記の態様および他の態様は、以下の詳細な記載からより明らかになるであろう。
配列番号:1− 大豆ゲノムDNAと組込みT−DNAとの間の5’左境界結合部を表す20個のヌクレオチド配列。この配列は、配列番号:6の第3413〜3432位に対応する。加えて、配列番号:1(図2の[A])は、配列番号:3の第3432〜3422位に対応するヌクレオチド配列であり([C]、図2参照)、配列番号:5の第1〜10位に対応する組込みT−DNAの5’左境界である([E]、図2参照)。
配列番号:4− T−DNA挿入までのMON87705の挿入DNAに隣接する3’配列。
配列番号:5− 組込み後の残りの境界配列を含めた、挿入T−DNAの配列。
プライマーSQ20129およびSQ20130の組合せを用いて生成されたPCR アンプリコンは、事象MON87705の存在に陽性である。
以下の定義および方法は、本発明をより良好に定義し、本発明の実施において当業者を導くために提供される。特記しない限りは、用語は、関連技術分野における当業者による通常の使用に従い理解されるものである。また、分子生物学における通常の用語の定義は、Riegerら, Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; およびLewin, Genes V, Oxford University Press: New York, 1994に見出すことができる。
遺伝子組換え大豆植物MON87705をpMON95829(図1)に由来するDNA断片での大豆細胞のアグロバクテリウム媒介形質転換により生成した。バイナリー植物形質転換ベクターpMON95829は2つの植物形質転換T−DNAを含む。各T−DNAは、T−DNAの末端にて、右境界(RB)および左境界(LB)配列によって隣接してはさまれる。MON87705の形質転換後スクリーニングは、2つのカセット挿入を生成する2つのT−DNAの右境界ないし右境界共同組込みを同定し、一方のT−DNAは、グリホサート耐性を与えるアグロバクテリウムチュメファシエンスからのaroA−CP4遺伝子を発現するように設計され、他方は、内因性のFAD2およびFATB遺伝子(配列番号:7)のRNAiベースの抑制を引き起こすような逆方向反復配列で設計されている。逆方向反復配列構造はpMON95829で見出されていないが、2つのT−DNAのRBないしRBの共同組込みで形成される。この組込み配置は、発現および抑制カセットの双方を含む単一の遺伝子組換えの遺伝子座を生じ、残余の左境界配列(図2)によって隣接してはさまれる。RB:RB組込み部位にて生成されたユニークな配列は、配列番号:18下でリストされる。
MON87705におけるT−DNA挿入のフランキング配列を、Ochmanら, 1990 (PCR Protocols: A guide to Methods and Applications, Academic Press, Inc.) に記載された逆PCRを用いて、およびTAIL (Thermal Asymmetric InterLaced)PCRにより決定した。植物ゲノムDNAを野生型A3525および温室条件下で生育された組織からの遺伝子組換え系の双方から単離した。凍結した葉組織を液体窒素または機械的粉砕で乳鉢および乳棒によって粉砕した。22mlの容積の抽出緩衝剤を約1gの粉砕した葉組織に添加し、65℃にて1時間インキュベートした。CTAB抽出緩衝剤は、1.4M NaCl、2% CTAB、20mM EDTAおよび100mMトリス−HCl pH8.0よりなった。使用の直前に、0.02%ベータ−メルカプトエタノールおよび0.5mgのRNアーゼAを抽出緩衝剤に添加した。試料を12mLのフェノール/クロロホルム/イソアミルアルコール(25:24:1)溶液で抽出し、次いで、4℃にて10分間、4000×Gで遠心した。上清を新しいチューブに移し、DNAを15mLのイソプロパノールで沈殿させた。4000×Gにて10分間の遠心分離後に、ペレットを5mLの70%エタノールで洗浄した。4000×Gでの5分間の最終遠心を行い;ペレットを空気乾燥させ、次いで、300μLの水に再懸濁させた。
試料中の事象MON87705を同定するために用いた方法は、事象特異的なエンドポイントTaqMan PCRアッセイであり、それについての条件の例を表2および表3に記載する。エンドポイントアッセイに用い得る第1のセットのDNAプライマーは、プライマーSQ20129(配列番号:8)、SQ20130(配列番号:9)および6FAM(商標)標識プライマーPB10043(配列番号:10)である。エンドポイントアッセイに用い得る第2のセットのDNAプライマーは、プライマーSQ21928(配列番号:11)、SQ20901(配列番号:12)および6FAM(商標)標識プライマーPB10164(配列番号:13)である。6FAM(商標)は、そのDNAプライマーに結合したApplied Biosystems (Foster City, CA)の蛍光色素生成物である。TaqMan MGB(Minor Groove Binding)プローブについて、Taq DNAポリメラーゼの5’エキソヌクレアーゼ活性は、フルオロフォアと消光剤との間で5’−終端からプローブを切断する。標的DNA鎖にハイブリダイズした場合、消光剤およびフルオロフォアが、蛍光シグナルを生成するのに十分に分離する。
以下の実施例は、所与の大豆試料中のMON87705事象の存在または不存在を同定し得る方法を記載する。
DNA事象プライマー対を用いて、大豆事象MON87705に特徴的なアンプリコンを生成する。MON87705に特徴的なアンプリコンは、少なくとも1つの結合配列:配列番号:1、配列番号:2または配列番号:18を含む。配列番号:1(図2)は、5’フランキング配列(配列番号:6の第3413〜3422位、図2参照)および挿入の組込み境界(配列番号:6の第3423〜3433位、図2参照)の結合部に対応するヌクレオチド配列である。配列番号:2(図2を参照)は、挿入の組込み境界(配列番号:6の第10664〜10673位、図2参照)および3’フランキング配列(配列番号:6の第10674〜10683位、図2参照)の結合部に対応するヌクレオチド配列である。配列番号:18は、事象MON87705を得るために2つのpMON95829 T−DNAの共組込みに際して生成された右境界から右境界結合部(配列番号:6の第9230〜9335位)に対応するヌクレオチド配列である。
Claims (25)
- 事象MON87705を含む大豆植物であって、該大豆植物が配列番号:1、2および18で示される配列、またはそれらに相補的である配列を有するポリヌクレオチドを含む異種DNA分子を含み、該配列番号:1および2は、該異種DNAが該大豆植物のゲノムに挿入されたポイントにわたる連結配列に相当する、大豆植物。
- 植物器官が、配列番号:1、2および18で示される配列、またはそれらに相補的である配列を有するポリヌクレオチドを含む、請求項1記載の大豆植物の植物器官。
- 後代が、配列番号:1、2および18で示される配列、またはそれらに相補的である配列を有するポリヌクレオチドを含む、請求項1記載の大豆植物の後代。
- 55〜80%のオレイン酸および8%未満の飽和脂肪酸を含む油組成物を有する種子を生成できる大豆植物であって、該大豆植物が事象MON87705、ここで、事象MON87705を含む大豆系統の種子の代表的試料は、アメリカン・タイプ・カルチャー・コレクション(ATCC)にATCC受入番号PTA−9241で寄託されている、を含み、該事象が、配列番号:1、2および18で示される配列、またはそれらに相補的である配列を有するポリヌクレオチドとのハイブリダイゼーションによって同定できる、大豆植物。
- 大豆事象MON87705を含む植物と、大豆事象MON87705を欠く大豆植物とを交配して、該大豆事象MON87705および改変された脂肪酸レベルを含む植物を得ることを含む、改変された脂肪酸レベルを含む大豆植物を生成する方法であって、事象MON87705を含む大豆系統の種子の代表的試料が、アメリカン・タイプ・カルチャー・コレクション(ATCC)にATCC受入番号PTA−9241で寄託されたものである、方法。
- 大豆事象MON87705を含む大豆変種を生成する方法であって、大豆事象MON87705を該変種に戻し交配することを含み、ここで、事象MON87705を含む大豆系統の種子の代表的試料は、アメリカン・タイプ・カルチャー・コレクション(ATCC)にATCC受入番号PTA−9241で寄託されたものである、方法。
- 事象MON87705を含む、請求項1に記載の大豆植物の種子から得た油組成物。
- 食用油、サラダ油、ショートニング、レシチン、無毒性プラスチック、印刷用インク、滑沢剤、ワックス、作動液、電気的変圧器流体、溶剤、化粧品、ヘアケア製品およびバイオディーゼルよりなる群から選択される請求項7記載の油組成物に由来した商品。
- 配列番号:1、2および18下でリストされた配列、またはそれらに相補的である配列を有するポリヌクレオチドを含むDNA分子。
- 配列番号:1および配列番号:2で示される配列、またはそれらに相補的である配列を含む単離されたDNA分子。
- 事象MON87705を含む大豆細胞のゲノムであって、該ゲノムは配列番号:1、2および18、またはそれらの相補体を含む配列を有するポリヌクレオチドを含む異種DNA分子を含み、該配列番号:1および2は、該異種DNAが該細胞のゲノムに挿入されたポイントにわたる連結配列に相当する、ゲノム。
- 生物学的試料中の大豆事象MON87705 DNAの存在を検出する方法であって、
i.配列番号:1および配列番号:2ならびにそれらの相補体よりなる群から選択される配列とストリンジェントなハイブリダイゼーション条件下でハイブリダイズし、かつ配列番号:1および配列番号:2ならびにそれらの相補体よりなる群から選択される配列を含まない大豆植物ゲノムDNAとストリンジェントなハイブリダイゼーション条件下でハイブリダイズしないプローブと、試料とを接触させ;
ii.該試料およびプローブをストリンジェントなハイブリダイゼーション条件に付し;次いで
iii.該試料に対する該プローブの結合を検出することを含み;
結合は、該試料中の該DNAの存在に特徴的であり、該プローブがアメリカン・タイプ・カルチャー・コレクション(ATCC)にATCC受入番号PTA−9241で寄託された事象MON87705を含む大豆系統とストリンジェントなハイブリダイゼーション条件でハイブリダイズする、
ことを特徴とする方法。 - (i)該プローブが配列番号:3またはそれらの相補体の少なくとも11個の連続ヌクレオチドを含む第二のプローブをさらに含むか、または
(ii)該プローブが配列番号:4またはそれらの相補体の少なくとも11個の連続ヌクレオチドを含む第二のプローブをさらに含むか、または
(iii)該プローブが、配列5’〜配列番号:18であるかまたはそれに相補的であり、配列番号:5またはそれらの相補体の少なくとも11個の連続ヌクレオチドであり、さらに配列3’〜配列番号:18であるかまたはそれに相補的であり、配列番号:5またはそれらの相補体の少なくとも11個の連続ヌクレオチドである第二のプローブを含む、
請求項12記載の方法。 - 配列番号:1、2および18またはそれらの相補体を含むヌクレオチド配列の存在を検出することを含む、生物学的試料中の大豆事象MON87705の存在を検出する方法であって、
該生物学的試料は、大豆ミール、大豆粉末、大豆蛋白濃縮物、大豆蛋白分離物、組織状大豆蛋白濃縮物、大豆蛋白加水分解物およびホイップトッピングよりなる群から選択されることを特徴とする、方法。 - 配列番号:1、2および18下で示された少なくとも2つの配列の検出に基づいて設計されたヌクレオチド成分を含む、大豆試料中の大豆遺伝子組換え事象MON87705の存在または不存在を検出するためのキットであって、該ヌクレオチド成分が配列番号:1、2、18またはそれらの相補体の少なくとも11個の連続ヌクレオチドを含む、キット。
- 配列番号:1、2および18下で示された少なくとも2つの配列またはそれらの相補体の検出に基づいて設計されたヌクレオチド成分を含む、大豆試料中の大豆遺伝子組換え事象MON87705の存在または不存在を検出するためのキットであって、
i.該試料とDNAプライマー対とを接触させ;
ii.核酸増幅反応を行い、それにより、アンプリコンを生成し;次いで
iii.該アンプリコンを検出し、ここに、該アンプリコンは、配列番号:1、2および18下でリストされた少なくとも2つの配列を含み、
該DNAプライマー対は、
(a)配列番号:3の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドおよび配列番号:5の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドを含むか、または
(b)配列番号:5の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドおよび配列番号:4の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドを含むか、または
(c)配列番号:5の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドを含み、一方のプライマーは配列5’〜配列番号:18であるかまたはそれに相補的であり、他方のプライマーは配列3’〜配列番号:18であるかまたはそれに相補的である、
ことを特徴とするキット。 - 該DNAプライマー対が、配列番号:3の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドおよび配列番号:5の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドを含む請求項16記載のキット。
- 該DNAプライマー対が、配列番号:5の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドおよび配列番号:4の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドを含む請求項16記載のキット。
- 該DNAプライマー対が、配列番号:5の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列を有するポリヌクレオチドを含み、ここに、一方のプライマーは配列5’〜配列番号:18の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列であり、他方のプライマーは、配列3’〜配列番号:18の少なくとも11個の連続ヌクレオチドである配列またはその相補的な配列であることを特徴とする請求項16記載のキット。
- 配列番号:1、2および18で示される配列、またはそれらに相補的である配列を含むDNA分子を有する組成物であって、大豆ミール、大豆粉末、大豆蛋白濃縮物、大豆蛋白分離物、組織状大豆蛋白濃縮物、大豆蛋白加水分解物およびホイップトッピングよりなる群から選択される商品である組成物。
- 油組成物が72.8%のオレイン酸を含む、請求項7記載の油組成物。
- 油組成物がさらに2.5%のパルミチン酸および3.4%のステアリン酸を含む、請求項21記載の油組成物。
- 容器に入れた大豆遺伝子組換え事象MON87705を含む大豆の種子から得られる、55〜80%のオレイン酸および8%未満の飽和脂肪酸を含む油組成物であって、事象MON87705を含む大豆系統の種子の代表的試料が、アメリカン・タイプ・カルチャー・コレクション(ATCC)にATCC受入番号PTA−9241で寄託されており、該事象が、配列番号:1、2および18の少なくとも11個の連続ヌクレオチドである配列、またはそれらに相補的である配列を有するポリヌクレオチドとのハイブリダイゼーションによって同定できる、油組成物。
- 容器に入れた大豆の種子が10,000個を超える、請求項23記載の油組成物。
- 配列番号:5の配列であるか、またはその配列に相補的である配列を有するポリヌクレオチドをさらに含む、請求項1記載の大豆植物。
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US9572311B2 (en) | 2017-02-21 |
JP2015144602A (ja) | 2015-08-13 |
JP2012503989A (ja) | 2012-02-16 |
KR20110063817A (ko) | 2011-06-14 |
CN107699545A (zh) | 2018-02-16 |
WO2010037016A1 (en) | 2010-04-01 |
UY32145A (es) | 2010-04-30 |
CA2738474C (en) | 2020-05-12 |
CA2738474A1 (en) | 2010-04-01 |
EP2328400A1 (en) | 2011-06-08 |
US20170112083A1 (en) | 2017-04-27 |
US8329989B2 (en) | 2012-12-11 |
US20130309672A1 (en) | 2013-11-21 |
EP2328400B1 (en) | 2019-05-29 |
KR101647522B1 (ko) | 2016-08-10 |
US20100080887A1 (en) | 2010-04-01 |
AR075549A1 (es) | 2011-04-20 |
MX356687B (es) | 2018-06-07 |
EP2328400A4 (en) | 2012-04-18 |
US20130074224A1 (en) | 2013-03-21 |
US8692080B2 (en) | 2014-04-08 |
US10344292B2 (en) | 2019-07-09 |
CN102164476A (zh) | 2011-08-24 |
BRPI0920827A2 (pt) | 2015-08-18 |
MX2011003297A (es) | 2011-04-21 |
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