JP4903051B2 - トウモロコシ植物mon88017および組成物ならびにその検出方法 - Google Patents
トウモロコシ植物mon88017および組成物ならびにその検出方法 Download PDFInfo
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- JP4903051B2 JP4903051B2 JP2006545781A JP2006545781A JP4903051B2 JP 4903051 B2 JP4903051 B2 JP 4903051B2 JP 2006545781 A JP2006545781 A JP 2006545781A JP 2006545781 A JP2006545781 A JP 2006545781A JP 4903051 B2 JP4903051 B2 JP 4903051B2
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Description
本発明は、植物分子生物学の分野に関する。より詳細には、本発明は、グリホサート耐性および昆虫抵抗性のトウモロコシ植物MON88017、ならびに植物試料および組成物中でのトウモロコシ植物MON88017 DNAのアッセイ方法およびその存在を検出する方法に関する。
トウモロコシは、世界の多くの領域において重要な作物であって、主食料源である。バイオテクノロジー手法は、その生成物の農業形質および質の改良のためにトウモロコシに適用されている。かかる1つの農業形質は、除草剤耐性、特に、グリホサート除草剤に対する耐性である。トウモロコシにおけるこの形質は、グリホサート抵抗性の5−エノールピルビル−3−シキミ酸リン酸合成酵素(CP4 EPSPSおよび米国特許第5,633,435号)を発現するトウモロコシ植物における挿入遺伝子の発現により与えられた。もう一つの農業形質は、昆虫抵抗性、例えば、遺伝子操作されたトウモロコシ植物のコーンボーラーおよびコーンルートワームに対する抵抗性である(米国特許第6,489,542号および米国特許第6,620,988号)。有***雑の子孫が、注目する導入遺伝子/ゲノムDNAを含むかを決定するために特定の植物の導入遺伝子/ゲノムDNAの存在を検出できることは有利であろう。加えて、組換えの作物植物から由来した食品の市販前承認および標識を必要とする規制に従う場合に、特定の植物の検出方法は有用であろう。
本発明は、受入番号PTA−5582を持つ米国培養菌保存施設(ATCC)に寄託された種子を有するMON88017と指定されたトランスジェニックトウモロコシ植物に関する。本発明のもう一つの態様は、植物MON88017の植物および種子の子孫植物もしくは種子または再生可能な器官を含む。また、本発明は、限定されるものではないが、花粉、胚珠、種子、根および葉を含むトウモロコシ植物MON88017の植物器官を含む。本発明は、グリホサート耐性の表現型およびコーンルートワーム抵抗性の表現型を有するトウモロコシ植物MON88017、およびMON88017のゲノムに含まれた新規な遺伝子組成物に関する。
本明細書において「植物MON88017」または「MON88017」というトランスジェニックトウモロコシ植物は、鞘翅類害虫(Coleopteran pest)コーンルートワーム(Diabrotica spp.)による食害に抵抗性であり、かつグリホサートを含有する農業用除草剤の植物毒素作用に耐性である。この二重形質のトウモロコシ植物は、コーンルートワーム幼虫による食害に対する抵抗性、およびグリホサートに対する植物耐性を与えるAgrobacterium sp.株CP4からのグリホサート抵抗性5−エノールピルビルシキミ酸−3−リン酸合成酵素(CP4 EPSPS)蛋白質(米国特許第5,633,435号)を提供するBacillus thuringiensis(亜種 kumamotoensis)からのCRY3Bb1蛋白質(米国特許第6,501,009号)の改変変異体を発現する。二重形質トウモロコシの使用は、トウモロコシ生産者に主要な利点:a)世界の多数のトウモロコシ栽培領域における生育関連における主な害虫のコーンルートワーム幼虫による経済的な損失からの保護、ならびにb)広域スペクトルの雑草制御のためのトウモロコシ作物に対する農業用除草剤を含有するグリホサートを適用する能力を提供するであろう。加えて、コーンルートワームおよびグリホサート耐性形質をコードする導入遺伝子を、同一DNAセグメントに連結し、MON88017のゲノム中の単一座にて生じさせ、これは、育種効率の増強を提供し、育種集団およびその子孫における導入遺伝子挿入断片を追跡するための分子マーカーの使用を可能にする。
実施例1
トウモロコシ植物MON88017を、グリホサートの栄養性および生殖性障害に対する耐性につき多数のトランスジェニックトウモロコシ植物から選択した。商業的に高品質のトランスジェニック植物の成功した生成は、現在、多数のトランスジェニック植物の生成を必要とする。本発明において、MON88017は、pMON53616を含む異なるDNA構築体で形質転換した約472R0事象のうちの1つの植物であった。そのMON88017事象を、一連の分子分析、グリホサート耐性スクリーニング、昆虫抵抗性スクリーニングおよび発現分析スクリーニングにより多数の事象から選択した。
MON88017および対照物質からのゲノムDNAを、最初に細粉に粒を処理することによりトウモロコシ粒から抽出した。約6グラムの処理された粒は、50ml(ミリリッター)の円錐管に移し、次いで、約16mlのCTAB抽出緩衝液の[1.5%(w/v)CTAB、75mMトリス−HCl pH8.0、100mM EDTA pH8.0、1.05M NaClおよび0.75%(w/v)PVP(MW 40,000)]および8マイクロリットルのRNアーゼ(10mg/ml、Roche)をその処理した粒に加えた。試料を間欠性の混合を用いて65℃にて30〜60分間インキュベートし、次いで、室温まで冷却させた。約15mlクロロホルム:イソアミルアルコール(24:1(v/v))を試料に加えた。その懸濁液を5分間混合し、次いでその2相を室温にて約16,000×gで5分間の遠心分離により分離した。水性の(上部)層をきれいな50mlの円錐管に移した。10%CTAB緩衝液[10%(w/v)CTABおよび0.7M NaCl]の約1/10容(約1.5ml)および当量のクロロホルム:イソアミルアルコール[24:1(v/v)]を水相に加え、次いで、それを5分間混合した。試料を約16,000×gで5分間遠心してそれらの相を分離した。水性(上部)層を取り出し、等容量(約15ml)のCTAB沈殿緩衝液[1%(w/v)CTAB、50mMトリス pH8.0および10mM EDTA pH8.0]と混合し、室温にて1〜2時間静置させた。その試料を室温にて10分間約10,000×gで遠心して、DNAをペレットとした。上清を捨て、ペレットを約2mlの高い塩TE緩衝液(10mMトリス−HCl pH8.0、10mM EDTA pH8.0および1M NaCl)に溶解した。37℃での穏やかな混合をペレットの溶解を援助するために行なった。要すれば、試料を室温にて約23,000×gで2分間遠心して、残骸をペレットとし、除去した。約1/10容(約150μl)の3M NaOAc(pH5.2)、および2容(上清に対して約4ml)の冷却した100%エタノールを加えて、DNAを沈殿させた。沈殿したDNAを70%エタノールを含む微量遠心試験管にスプールした。そのDNAを最高速度(約14,000rpm)にて約5分間の微量遠心機にてペレットとし、真空乾燥させ、TE緩衝液(pH8.0)に再溶解した。次いで、DNAを4℃冷蔵庫に貯蔵した。
25秒間の94℃、3分間の67℃の37サイクル;7分間の67℃の1サイクルを含む条件で行なうことができる。全ての引き続いての増幅は、以下の条件で行った:2秒間の94℃、4分間の72℃の7サイクル;2秒間の94℃、4分間の67℃の37サイクル;7分間の67℃の1サイクル。全てのアンプリコンは、臭化エチジウムで染色された0.8%のアガロースゲル上で視覚化される。そのDNAは、QIAquick PCR Purificationキット(カタログ番号 28104, Qiagen Inc., Valencia, CA)でPCR試料を直接的に精製することにより、またはそのゲルから適当な断片を抽出し、QIAquick Gel Extractionキット(カタログ番号28704、Qiagen Inc.)を用いるいずれかにより配列決定するために調製する。MON88017導入遺伝子/ゲノムの挿入断片のフランキング領域からのDNA断片をTOPO TA CloningRキット(Invitrogen, Carlsbad, CA)を用いて、サブクローニングした。5’導入遺伝子/ゲノム領域のDNA配列を図3に示し、3’導入遺伝子/ゲノム領域のDNA配列を図4に示し、完全な導入遺伝子挿入断片および連結したフランキングゲノムDNAを図5に示す。
DNA事象プライマー対を用いて、トウモロコシ事象MON88017に特徴的なアンプリコンを生成する。MON88017に特徴的な1つのアンプリコンは、少なくとも1つの結合配列、配列番号:1または配列番号:2を含む。MON88017に特徴的なアンプリコンを生成する事象プライマー対は、ここに、そのプライマー対は、限定されるものではないが、表2に記載された5’アンプリコン配列についての配列番号:6および配列番号:7を含む。プライマー配列番号:6の位置は、ヌクレオチド952位にて始まる図3に示すトウモロコシゲノム中にある。プライマー配列番号:7の位置は、ヌクレオチド450位で始まる図5に示す導入遺伝子挿入断片中にある。MON88017 DNAでのDNA増幅方法における配列番号:6および配列番号:7を用いる予期されたアンプリコンサイズは、約550bpである。これらのプライマー対に加えて、DNA増幅反応がそのMON88017または子孫には特徴的なアンプリコンを生成する配列番号:3または配列番号:4から誘導されたいずれのプライマー対も、本発明の1つの態様である。MON88017に特徴的なアンプリコンを生成するDNA増幅方法に有用である配列番号:3の少なくとも11個の近接するヌクレオチドまたはその相補体を含むいずれかの単一の単離されたDNAポリヌクレオチドプライマー分子は、本発明の1つの態様である。MON88017に特徴的なアンプリコンを生成する方法に有用である配列番号:4の少なくとも11個の近接するヌクレオチドまたはその相補体を含むいずれの単一の単離されたDNAポリヌクレオチドプライマー分子も、本発明の1つの態様である。この分析のための増幅条件の一例を表2および表3に示すが、MON88017に特徴的なアンプリコンを生成するMON88017の導入遺伝子挿入断片(配列番号:5)に含まれた遺伝子エレメントの配列番号:3もしくは配列番号:4またはDNA配列に相同的または相補的なDNAプライマーのこれらの方法または使用のいずれの変更も当該技術分野内ものである。特徴的なアンプリコンは、少なくとも1つの導入遺伝子/ゲノムの結合DNA(配列番号:1または配列番号:2)に相同的または相補的であるDNA分子またはその実質的な部分を含む。
事象MON88017を含む同型接合体の子孫からの異型接合を識別するために用いた方法を、接合状態アッセイに記載し、その条件の例を表4および表5に記載する。接合状態アッセイに用いたDNAプライマーは、プライマー(配列番号:30)、(配列番号:31)、(配列番号:32)、6FAMTM標識プライマー(配列番号:33)およびVICTM標識プライマー(配列番号:34)であり、6FAMおよびVIC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。
食料および商品のごとき生成物は、トウモロコシ事象MON88017から生成でき、かかる食料および商品は、かかる食料および商品において十分なレベルにて検出される場合、かかる商品および食料内のトウモロコシ事象MON88017物質の存在に特徴的になり得るヌクレオチド配列を含むと予期される。かかる食料および商品の例は、限定されるものではないが、動物による食料源として消費のための、さもなければ、バルキング剤(bulking agent)として意図された、または化粧品用途のためのメークアップ組成物における成分として意図されたトウモロコシ油、コーンミール、コーンフラワー、トウモロコシグルテン、コーンケーキ、コーンスターチおよび他の食料等を含む。配列番号:1、配列番号:2、配列番号:3、配列番号:4および配列番号:5に記載された配列よりなる群から選択されるプローブまたはプライマー対に基づいた核酸検出方法および/またはキットが開発され、それは、生物学的試料中の配列番号:1および配列番号:2のごときMON88017ヌクレオチド配列の検出を可能とし、かかる検出は、かかる試料中のトウモロコシ事象MON88017に特徴的であろう。
Claims (23)
- 配列番号:1および配列番号:2よりなる群から選択されるDNA分子である、または該DNA分子に完全に相補的である、トウモロコシ事象MON88017ゲノムDNAから単離されたDNA分子。
- 試料中のトウモロコシ事象MON88017 DNAを検出するのに有用なDNAプローブとして用いる単離されたDNA分子であって、該プローブが配列番号:1の少なくとも18個の隣接するヌクレオチド、またはその完全な相補体を含む該単離されたDNA分子。
- 試料中のトウモロコシ事象MON88017 DNAを検出するのに有用なDNAプローブとして用いる単離されたDNA分子であって、該プローブが配列番号:2の少なくとも18個の隣接するヌクレオチド、またはその完全な相補体を含む該単離されたDNA分子。
- 生物学的試料中のトウモロコシ事象MON88017ヌクレオチド配列の存在の検出方法であって:
(a)該試料とDNAプライマー対とを接触させ;
(b)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
(c)該アンプリコンを検出する
ことを含み、ここに、該アンプリコンは配列番号:1または配列番号:2を含むことを特徴とする該検出方法。 - 昆虫抵抗性およびグリホサート耐性を示す安定的に形質転換されたトウモロコシ植物であって、そのDNAが請求項4記載の検出方法により付された場合に、トウモロコシ事象MON88017に特徴的である配列番号:1または配列番号:2を含むDNAアンプリコンを生成する該植物。
- 該DNAプライマー対が、配列番号:6に記載のヌクレオチド配列を有する第1のプライマーおよび配列番号:7に記載のヌクレオチド配列を有する第2のプライマーを含むことを特徴とする請求項4記載の検出方法。
- 生物学的試料中のトウモロコシ事象MON88017DNAの存在の検出方法であって、
(a)ストリンジェントなハイブリダイゼーション条件下でMON88017DNAとハイブリダイズし、かつストリンジェントなハイブリダイゼーション条件下でMON88017DNAではないトウモロコシ植物ゲノムDNAとハイブリダイズしないプローブと、該試料とを接触させ、
ここに、該プローブは、配列番号:1または配列番号:2よりなる群から選択される配列であるか、または選択される該配列に相補的であり;
(b)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
(c)MON88017DNAへのプローブのハイブリダイゼーションを検出することを特徴とする該検出方法。 - 試料中のトウモロコシ事象MON88017DNAを含むトウモロコシ植物のゲノムDNAの接合状態を決定する方法であって、
(a)(1)トウモロコシ事象MON88017DNAを含む核酸増幅反応に用いられた場合に、トウモロコシ事象MON88017に特徴的である第1のアンプリコンを生成する、および(2)MON88017DNA以外の天然のトウモロコシゲノムDNAを含む核酸増幅反応に用いられた場合に、天然のトウモロコシゲノムDNAに特徴的である第2のアンプリコンを生成する、配列番号:30、配列番号:31および配列番号:32を含む3つの異なるプライマーと、試料を接触させ;
(b)核酸増幅反応を行ない;次いで
(c)生成されたアンプリコンを比較し、ここに、双方のアンプリコンの存在は、該試料の接合状態に特徴的であることを特徴とする該方法。 - 少なくとも1つの親がトウモロコシ事象MON88017である、トウモロコシ事象MON88017の異種のDNAを含む雑種トウモロコシ種子。
- ATCC受入番号PTA−5582下寄託されたトウモロコシ植物の種子。
- 請求項10記載の種子を生育させることにより生成したトウモロコシ植物MON88017またはその器官。
- 請求項11記載のトウモロコシ植物MON88017の花粉、胚珠、種子、根または葉。
- 子孫が配列番号:1および配列番号:2を含む請求項11記載のトウモロコシ植物MON88017の子孫。
- 配列番号:1または配列番号:2に表されるヌクレオチド配列の18〜20の連続ヌクレオチドを含む、トウモロコシ事象MON88017のゲノム中のDNAに由来するヌクレオチド。
- 配列番号:1および配列番号:2よりなる群から選択される配列であるか、または選択される該配列に相補的であるヌクレオチド配列を含む、トウモロコシ事象MON88017のゲノム中のDNAに由来するポリヌクレオチド。
- 生物学的試料中のトウモロコシ事象MON88017ポリヌクレオチドの存在の検出方法であって:
(a)ストリンジェントなハイブリダイゼーション条件下で該試料と、プローブを接触させ、ここに、該プローブは、配列番号:1および配列番号:2よりなる群から選択される配列であるか、または選択される該配列に相補的である連続ヌクレオチド配列を含み;次いで
(b)該試料に対する該プローブのハイブリダイゼーションを検出し、ここに、該試料に対する該プローブのハイブリダイゼーションは、該試料中の該トウモロコシ事象MON88017ポリヌクレオチドの存在に特徴的である
ことを特徴とする該検出方法。 - 配列番号:1および配列番号:2を含むトウモロコシ事象MON88017のゲノム中のDNAに由来するヌクレオチドを含み、コーンミール、コーンフラワー、トウモロコシ油、コーンシルク、コーンスターチおよび加工食品よりなる群から選択される商品生産物である組成物。
- 生物学的試料中のトウモロコシ事象MON88017DNAの存在の検出に用いる長さが18〜20の連続ヌクレオチドを含むヌクレオチドプローブであって、該連続ヌクレオチドが、配列番号:1または配列番号:2に表されるヌクレオチド配列から選択される該プローブ。
- 該プローブが、デオキシリボ核酸、リボ核酸およびヌクレオチドアナログよりなる群から選択されるヌクレオチドを含む請求項18記載のヌクレオチドプローブ。
- 該プローブが、少なくとも1つのフルオロフォアで標識された請求項19記載のプローブ。
- 配列番号:1、配列番号:2、配列番号:3、配列番号:4および配列番号:5よりなる群から選択される配列を有するヌクレオチドを含むトウモロコシ事象MON88017から生成された商品または食料であって、そのヌクレオチドの検出がかかる商品または食料中のトウモロコシ事象MON88017物質の存在に特徴的である該商品または食料。
- トウモロコシ油、コーンスターチ、コーンミール、コーンフラワー、化粧品およびバルキング剤よりなる群から選択される請求項21記載の商品または食料。
- 配列番号:1および配列番号:2を含むトウモロコシ事象MON88017のゲノム中のDNAに由来する十分なレベルのヌクレオチドを含むトウモロコシ油、コーンミール、コーンフラワー、トウモロコシグルテン、コーンケーキおよびコーンスターチよりなる群から選択される生物学的試料であって、
該試料中の該ヌクレオチドの検出は、該試料中のトウモロコシ事象MON88017の存在に特徴的であることを特徴とする該生物学的試料。
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