JP2004147511A - Ethylene receptor gene-like nucleic acid of petunia hybrida and primer for detecting pcr - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a means for regulating the flower keeping quality of a plant belonging to the genus Petunia and to provide a method for detecting a transgenic plant comprising an ethylene receptor gene-like nucleic acid or its antisense DNA transferred thereinto. <P>SOLUTION: A primer set comprises a nucleic acid containing a DNA composed of a specific base sequence derived from Petunia hybrida and a combination of a primer 1 containing bases forming one boundary region selected from the group consisting of a boundary region 1 between exon 1 and exon 2, a boundary region 2 between the exon 2 and exon 3, a boundary region 3 between the exon 3 and exon 4 and a boundary region 4 between exon the 4 and exon 5 constituting a cDNA encoding the ethylene receptor gene-like nucleic acid sequence derived from the plant belonging to the genus Petunia, respectively or a complementary sequence thereof and a primer 2 containing bases forming other boundary regions or a complementary sequence thereto. The primer set is prepared by using one of the primers 1 and 2 as a forward primer and the other as a reverse primer, respectively. <P>COPYRIGHT: (C)2004,JPO

Description

本発明において「境界領域」（境界部位ともいう）とは、ゲノム上で２つのエキソンがイントロンを挟んで離れて存在している場合、イントロンがスプライシングされた後に、２つのエキソンが結合したときの結合点（境界点）、すなわちスプライス部位を指す（図２）。
【０１１０】
図２に示すＰｈｖｃ−ｅｒｓ１のｃＤＮＡにおいて、エキソン１とエキソン２との境界を境界領域１、エキソン２とエキソン３との境界を境界領域２、エキソン３とエキソン４との境界を境界領域３、エキソン４とエキソン５との境界を境界領域４という。従って、境界領域１は、エキソン１の最も３´側の塩基とエキソン２の最も５´側の塩基により形成される。具体的には、エキソン１の最も３´側の塩基は、配列番号１に示す塩基配列の第９０８番目のチミンであり、エキソン２の最も５´側の塩基は、配列番号１に示す塩基配列の第９０９番目のチミンである。また、境界領域２は、エキソン２の最も３´側の塩基とエキソン３の最も５´側の塩基により形成され、具体的には、エキソン２の最も３´側の塩基は、配列番号１に示す塩基配列の第１２７３番目のグアニンであり、エキソン３の最も５´側の塩基は、配列番号１に示す塩基配列の第１２７４番目のアデニンである。また境界領域３は、エキソン３の最も３´側の塩基とエキソン４の最も５´側の塩基により形成され、具体的には、エキソン３の最も３´側の塩基は、配列番号１に示す塩基配列の第１５４５番目のチミンであり、エキソン４の最も５´側の塩基は、配列番号１に示す塩基配列の第１５４６番目のアデニンである。さらに境界領域４は、エキソン４の最も３´側の塩基とエキソン５の最も５´側の塩基により形成され、具体的には、エキソン４の最も３´側の塩基は、配列番号１に示す塩基配列の第１６６９番目のシトシンであり、エキソン５の最も５´側の塩基は、配列番号１に示す塩基配列の第１６７０番目のグアニンである。
【０１１１】
（２）オリゴヌクレオチド
本発明のオリゴヌクレオチドは、上記のような１つの境界領域を挟む両側の領域（例えばエキソン１とエキソン２）の配列又はその相補配列を含むものである。従って、図２に示すように、オリゴヌクレオチドＤ〜Ｇ及びＨ〜Ｋは、それらの相補性に基づいて、境界領域に対しハイブリダイズするものである。本発明において、オリゴヌクレオチドに関して用いる「ハイブリダイズ」又は「ハイブリダイゼーション」とは、検出対象のＰｈｖｃ−ｅｒｓ１のｃＤＮＡに対し、その相補性に基づいて特異的に結合することを指す。オリゴヌクレオチドＤ〜Ｇは、検出対象のＰｈｖｃ−ｅｒｓ１ ｃＤＮＡに対し相補的な配列を有するものであり、導入されたＰｈｖｃ−ｅｒｓ１又はその部分配列とハイブリダイズするものである。一方、オリゴヌクレオチドＨ〜Ｋは、検出対象のＰｈｖｃ−ｅｒｓ１ ｃＤＮＡに対し相同的な配列を有するものであり、導入されたＰｈｖｃ−ｅｒｓ１のアンチセンス核酸又はその部分配列とハイブリダイズするものである。
【０１１２】
境界領域（境界部位）を挟む両側の配列、すなわち、境界領域を形成する配列（特に境界部の両隣りの配列）又はその相補配列を、以下「境界領域の塩基配列」という。
【０１１３】
本発明のオリゴヌクレオチドは、上記のような境界領域の塩基配列を含むものであり、外来的に導入されたＰｈｖｃ−ｅｒｓ１ ｃＤＮＡ又はそのアンチセンス核酸（ｃＤＮＡ）のみとハイブリダイズすることができる。
【０１１４】
従って、このような境界領域の塩基配列を含むオリゴヌクレオチドプローブを用いてハイブリダイゼーションを行った場合には、野生型植物（非形質転換体）のゲノムＤＮＡ由来の内在遺伝子のハイブリダイゼーションは起こらず、Ｐｈｖｃ−ｅｒｓ１ ｃＤＮＡ又はそのアンチセンス核酸が導入された形質転換植物の場合にのみハイブリダイゼーションが起こり、それが検出される。従って、本発明のオリゴヌクレオチドを用いてＰｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入された形質転換植物を検出する場合には、ハイブリダイゼーションが起こるかどうかの検出操作のみを行えばよい。そのため本発明のオリゴヌクレオチドを用いることにより、Ｐｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入された形質転換植物の検出を簡便かつ明確に行うことができる。
【０１１５】
（３）プライマーセット
また、本発明のオリゴヌクレオチドをプライマーとして使用する場合、本発明のプライマーセットは、上記「（１）概要」の項に記載の１つの境界領域を挟む両側の領域（例えばエキソン１とエキソン２）の塩基配列を含むプライマーと他の境界領域を挟む両側の領域（例えばエキソン３とエキソン４）の塩基配列を含むプライマーとからなる。従って、図３に示すように、フォーワードプライマーＬ、Ｍ又はＮのいずれかと、リバースプライマーＯ、Ｐ又はＱのいずれかとが組み合わされたものが１つのプライマーセットとなる。フォーワードプライマーＬとリバースプライマーＯとを組み合わせた場合には増幅産物Ｌ−Ｏが得られ、フォーワードプライマーＬとリバースプライマーＰとを組み合わせた場合には増幅産物Ｌ−Ｐが得られ、フォーワードプライマーＭとリバースプライマーＰとを組み合わせた場合には増幅産物Ｍ−Ｐが得られ、フォーワードプライマーＭとリバースプライマーＱとを組み合わせた場合には増幅産物Ｍ−Ｑが得られ、フォーワードプライマーＮとリバースプライマーＱとを組み合わせた場合には増幅産物Ｎ−Ｑが得られ、そしてフォーワードプライマーＬとリバースプライマーＱとを組み合わせた場合には増幅産物Ｌ−Ｑが得られる（図３）。
【０１１６】
本発明の境界領域の塩基配列を含むプライマーは、図４Ａに示すように、３´から５´方向のＤＮＡを鋳型としてプライマーを作製する場合には、このプライマー配列がスプライス部位から５´側の部分（以下、５´側配列という）と３´側の部分（以下、３´側配列という）とからなるものである。プライマーの設計は、最初に３´側配列を決定する。３´側配列は、増幅対象であるＰｈｖｃ−ｅｒｓ１ ｃＤＮＡの境界領域から約２〜８塩基の配列からなるように設計する。プライマーは、鋳型ＤＮＡに対し相同的な場合と相補的な場合があり、一般にフォワードプライマーの場合は鋳型ＤＮＡに対し相同配列となり、リバースプライマーの場合は鋳型ＤＮＡに対し相補配列となる。一方、５´側配列は、オリゴヌクレオチドのＴｍからプライマーの長さを調整することを優先して、５´側配列の長さを決めてから設計する。従って、５´側配列は、増幅対象であるＰｈｖｃ−ｅｒｓ１ ｃＤＮＡの境界領域と対応する約１０〜４０塩基の配列からなるように設計する。このように設計した、境界領域の塩基配列を含むプライマーは、Ｐｈｖｃ−ｅｒｓ１ ｃＤＮＡにアニーリングして、それを増幅することができる（図４Ａ）。
【０１１７】
続いて、Ｐｈｖｃ−ｅｒｓ１ゲノムＤＮＡを鋳型とした場合について説明する（図４Ｂ）。ゲノムＤＮＡは、エキソンとイントロンを含むため、境界領域はイントロンによって分断されている。従って、本発明のプライマーの５´側配列は、ゲノムＤＮＡの分断された境界領域の一部とアニーリングすることができる。しかし３´側配列は、イントロン配列に対応する配列を含まないため、ゲノムＤＮＡのイントロンにアニーリングすることができない（図４Ｂ▲１▼）。ＤＮＡポリメラーゼによる増幅には、３´側配列のアニーリングが重要であるため、３´側配列がアニーリングできない場合には、ゲノムＤＮＡが増幅されることはない。また、本発明のプライマーの３´側配列は、約２〜８塩基と非常に短い配列であるため、３´側配列のみではゲノムＤＮＡにアニーリングすることはできない（図４Ｂ▲２▼）。従って、本発明の境界領域の塩基配列を含むプライマーは、Ｐｈｖｃ−ｅｒｓ１ゲノムＤＮＡを増幅することができない。
【０１１８】
以上より、本発明の境界領域の塩基配列を含むプライマーは、外来的に導入されたＰｈｖｃ−ｅｒｓ１ ｃＤＮＡ又はそのアンチセンス核酸（ｃＤＮＡ）のみを特異的に増幅することができる。
【０１１９】
従って、このような境界領域の塩基配列を含むプライマーセットを用いて増幅反応を行った場合には、野生型植物（非形質転換体）のゲノムＤＮＡ由来の内在遺伝子の増幅は起こらず、Ｐｈｖｃ−ｅｒｓ１ ｃＤＮＡ又はそのアンチセンス核酸が導入された形質転換植物の場合にのみ増幅が起こり、増幅産物が検出される。従って、本発明のプライマーセットを用いてＰｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入された形質転換植物を検出する場合には、増幅産物が生成されるかどうかの検出操作のみを行えばよく、従来のように電気泳動によるゲノムＤＮＡとｃＤＮＡの増幅断片の長さの違いを判別する操作は不要となる。そのため本発明のプライマーセットを用いることにより、Ｐｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入された形質転換植物の検出を簡便かつ明確に行うことができる。
【０１２０】
（４）ゲノムＤＮＡ及びスプライス部位の決定
境界領域の塩基配列を含むオリゴヌクレオチド（プライマー又はプローブ）を設計するため、まず、Ｐｈｖｃ−ｅｒｓ１のゲノムＤＮＡ配列及びスプライス部位（イントロン／エキソン境界領域）を決定する。
【０１２１】
Ｐｈｖｃ−ｅｒｓ１のゲノムＤＮＡは、ペチュニア由来の全ゲノムＤＮＡを鋳型として、Ｐｈｖｃ−ｅｒｓ１の塩基配列に基づいて設計したプライマーを用いて増幅することにより調製することができる。続いて、このように増幅したＰｈｖｃ−ｅｒｓ１ゲノムＤＮＡの塩基配列を決定する。このゲノムＤＮＡの塩基配列と、Ｐｈｖｃ−ｅｒｓ１のｃＤＮＡ配列（例えば配列番号１）とを比較することによって、イントロン配列及びスプライス部位（イントロン／エキソン境界領域）を特定することができる。上記塩基配列の比較は、当技術分野で公知の配列比較プログラムなどを用いて行うことができ、そのようなプログラムとしては、例えばＧＥＮＥＴＩＸ−ＭＡＣ１０．１（ソフトウエア開発株式会社）などが挙げられる。
【０１２２】
イントロン配列は、Ｐｈｖｃ−ｅｒｓ１のセンス鎖及びアンチセンス鎖の両方の配列を解読して決定することが好ましい。ゲノムＤＮＡが長すぎるためにイントロン配列の全長を決定することができない場合には、プライマーウォーキング法を利用して全長配列を決定してもよい。プライマーウォーキング法は、当技術分野で公知の塩基配列決定法であり、決定しようとする全塩基配列の端から１回目の配列決定を行って約５００〜６５０ｂｐの領域（ただし、使用する塩基配列決定用の試薬、機器などの能力に応じて変化する）を解析し、続いて１回目の解析情報を利用してプライマーを設計し、そのプライマーを用いて２回目の配列決定を同様に行うというものである。この部分的な配列決定操作を繰り返すことによって全長の配列を決定することができる。
【０１２３】
上述した方法に従って決定したＰｈｖｃ−ｅｒｓ１のゲノムＤＮＡを配列番号３に示す。ＯＲＦ領域は３７８２塩基であり、そのうちｃＤＮＡ配列は１９１１塩基である（配列番号１）。また、Ｐｈｖｃ−ｅｒｓ１（図１Ａ）と、公開されている２１８５塩基のシロイヌナズナＥＲＳ１遺伝子（図１Ｂ；ＧｅｎＢａｎｋアクセッション・ナンバーＵ２１９５２）とのスプライス部位の比較を図１に示す。図１に示すように、Ｐｈｖｃ−ｅｒｓ１のスプライス部位（境界領域）は、Ｐｈｖｃ−ｅｒｓ１のｃＤＮＡ配列（配列番号１）の９０８番（チミン）と９０９番（チミン）の間、１２７３番（グアニン）と１２７４番（アデニン）との間、１５４５番（チミン）と１５４６番（アデニン）との間、及び１６６９番（シトシン）と１６７０番（グアニン）との間の４箇所である。これら境界領域は、それぞれ図２及び３に示す境界領域１、境界領域２、境界領域３及び境界領域４に対応している。
【０１２４】
（５）オリゴヌクレオチドの設計
上記（４）に従って決定された境界領域（境界領域１〜４）の塩基配列を含むオリゴヌクレオチドを設計する。設計するオリゴヌクレオチドとしては、図２に示すように、８種類のオリゴヌクレオチド、すなわち境界領域１（Ｄ及びＨ）、境界領域２（Ｅ及びＩ）、境界領域３（Ｆ及びＪ）、並びに境界領域４（Ｇ及びＫ）の塩基配列を含むオリゴヌクレオチドが挙げられる。
【０１２５】
オリゴヌクレオチドを設計する際の条件は、当業者であれば容易に決定することができる。例えば、プローブを設計する場合には、これに限定するものではないが、ロシュ・ダイアグノスティックス製のＤＩＧ Ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅ ３´Ｅｎｄ Ｌａｂｅｌｉｎｇ Ｋｉｔのようなキットに添付の説明書に従ってプローブとして使用可能なオリゴヌクレオチドを設計することができる。なお、プライマーとして使用可能なオリゴヌクレオチドの設計手法に関しては、以下（７）において詳細に説明する。
【０１２６】
プローブとして設計するオリゴヌクレオチドの長さは、合計約１２〜４０塩基であり、好ましくは１５〜３０塩基である。プローブとして用いるオリゴヌクレオチドは、相補性に基づいてＰｈｖｃ−ｅｒｓ１のｃＤＮＡとハイブリダイズ可能であれば、ＤＮＡプローブであってもＲＮＡプローブであってもよい。
【０１２７】
またオリゴヌクレオチドの合成法は当技術分野で周知である。例えば、ホスホロアミダイト法などの一般的なオリゴヌクレオチド合成法を用いることができる。
【０１２８】
本発明のオリゴヌクレオチドとしては、例えば以下のものが挙げられる：
（ａ）配列番号１に示す塩基配列のうち少なくとも９０８〜９０９番の塩基を含み、かつ連続した１５塩基以上の塩基配列又はその相補配列からなるオリゴヌクレオチド（境界領域１に対応する）
（ｂ）配列番号１に示す塩基配列のうち少なくとも１２７３〜１２７４番の塩基を含み、かつ連続した１５塩基以上の塩基配列又はその相補配列からなるオリゴヌクレオチド（境界領域２に対応する）
（ｃ）配列番号１に示す塩基配列のうち少なくとも１５４５〜１５４６番の塩基を含み、かつ連続した１５塩基以上の塩基配列又はその相補配列からなるオリゴヌクレオチド（境界領域３に対応する）
（ｄ）配列番号１に示す塩基配列のうち少なくとも１６６９〜１６７０番の塩基を含み、かつ連続した１５塩基以上の塩基配列又はその相補配列からなるオリゴヌクレオチド（境界領域４に対応する）
【０１２９】
さらに具体的には、例えば以下の（ａ）〜（ｇ）のオリゴヌクレオチドが挙げられる。
（ａ）配列番号４に示す塩基配列のうち少なくとも１９〜２０番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域１に対応する）
（ｂ）配列番号５に示す塩基配列のうち少なくとも１６〜１７番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域２に対応する）
（ｃ）配列番号６に示す塩基配列のうち少なくとも１９〜２０番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域２に対応する）
（ｄ）配列番号７に示す塩基配列のうち少なくとも１４〜１５番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域３に対応する）
（ｅ）配列番号８に示す塩基配列のうち少なくとも１９〜２０番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域１に対応する）
（ｆ）配列番号９に示す塩基配列のうち少なくとも１６〜１７番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域２に対応する）
（ｇ）配列番号１０に示す塩基配列のうち少なくとも１８〜１９番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるオリゴヌクレオチド（境界領域３に対応する）
【０１３０】
続いて、上述のようにして設計したオリゴヌクレオチドが、Ｐｈｖｃ−ｅｒｓ１のゲノムＤＮＡとハイブリダイズすることなく、Ｐｈｖｃ−ｅｒｓ１のｃＤＮＡと特異的にハイブリダイズ可能であるかどうかを試験する。本発明においては、上記設計したオリゴヌクレオチドが、（Ａ）ｃＤＮＡとハイブリダイズすること、及び（Ｂ）ゲノムＤＮＡとハイブリダイズしないこと、の２つを確認する。このような試験は、当技術分野で周知であり、例えばＡｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製のＴａｑＭａｎ（Ｒ）プローブのようなキットを利用することにより簡便に行い、オリゴヌクレオチドの機能を確認することができる。
【０１３１】
（６）ハイブリダイゼーション
本発明のハイブリダイゼーション方法は、エチレン受容体遺伝子様核酸配列（Ｐｈｖｃ−ｅｒｓ１）若しくはそのアンチセンス核酸、又はその部分配列が導入された形質転換体を検出するため、被検植物のＤＮＡを鋳型として、上記（５）で設計したオリゴヌクレオチドを用いてハイブリダイゼーションを行い、ハイブリダイゼーションが検出された場合に、被検植物がＰｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入された形質転換植物であると判定するものである。
【０１３２】
本方法においては、Ｐｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸（ｃＤＮＡ）が存在する場合にのみハイブリダイゼーションが起こる。従って、本方法により形質転換植物を検出しようとする場合には、ハイブリダイゼーションの有無を検出すればよい。本方法で用いられる被検植物としては、Ｐｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入されているか否かを検出しようとする植物であればいずれのものでもよく、特にペチュニア属植物が好ましい。上記被検植物から、当技術分野で周知の方法を使用してＤＮＡを抽出することができる。
【０１３３】
本発明において利用可能なハイブリダイゼーション反応の手法としては、上述したように、オリゴヌクレオチドプローブが検出対象のＤＮＡのみとハイブリダイズして、検出可能であればどのような手法でもよい。例えば、タックマン法は、ＤＮＡポリメラーゼ（例えばＡＭＰＬＩＴＡＱ ＤＮＡポリメラーゼ）の５´−３´エキソヌクレアーゼ活性を利用するものであり、５´レポーター色素（例えば蛍光色素）と３´クエンチャー色素を有するオリゴヌクレオチドプローブをＰＣＲ反応に導入し、そのＰＣＲ過程において、プローブが標的配列とハイブリダイズした場合には、ＡＭＰＬＩＴＡＱ ＤＮＡポリメラーゼの５´−３´ポリヌクレオチド分解活性によって、プローブがレポーター色素とクエンチャー色素との間で切断され、その結果、レポーター色素がクエンチャー色素と分離することによって蛍光が増大するため、そのシグナルを蛍光検出器を用いて検出するものである。
【０１３４】
例えば、上記ハイブリダイゼーション反応は、市販のキットを用いて簡便に行うことができる。そのようなキットとしては、Ｔａｑ Ｍａｎ（Ｒ）プローブ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）などが挙げられる。
【０１３５】
上記ハイブリダイゼーションの検出には、ハイブリダイゼーションを特異的に認識することができる公知の手段を用いることができる。例えば、オリゴヌクレオチドプローブを標識して用いることにより、ハイブリダイゼーションを検出することができる。
【０１３６】
以上のようにしてハイブリダイゼーションが検出された場合には、被検植物が、Ｐｈｖｃ−ｅｒｓ１遺伝子若しくはそのアンチセンス核酸、又はその部分配列が導入された形質転換植物であると判定する。
【０１３７】
（７）プライマーの設計
上記（４）に従って決定された境界領域（境界領域１〜４）の塩基配列を含むプライマーを設計する。設計するプライマーとしては、図３に示すように、３種類のフォーワードプライマー、すなわち境界領域１（Ｌ）、境界領域２（Ｍ）及び境界領域３（Ｎ）の塩基配列を含むプライマーと、３種類のリバースプライマー、すなわち境界領域２（Ｏ）、境界領域３（Ｐ）及び境界領域４（Ｑ）の塩基配列を含むプライマーが挙げられる。
【０１３８】
プライマーを設計する際の条件は、（Ａ）スプライス部位から３´末端までの鎖長（３´側配列の長さ）、及び（Ｂ）プライマーの融解温度（Ｔｍ）である。
【０１３９】
（Ａ）スプライス部位から３´末端までの鎖長
プライマーの配列が、スプライス部位から３´末端までの鎖長が長い場合には、ゲノムＤＮＡに対してもプライマーの３´側配列がアニーリングする可能性が高まると考えられる。そのためスプライス部位から３´末端までの鎖長が２〜８塩基となるように設計することが好ましい。
【０１４０】
（Ｂ）Ｔｍ（融解温度）
また設計の際には、プライマーのＴｍを確認することが好ましい。Ｔｍとは、任意のオリゴヌクレオチドの５０％がその相補鎖とハイブリッドを形成する温度を意味し、鋳型ＤＮＡとプライマーとが二本鎖を形成してプライミング反応を効果的に起こすためには、アニーリングの温度を最適化する必要がある。一方、アニーリング温度を下げすぎると非特異的増幅が起こるため、アニーリング温度は可能な限り高いことが望ましい。従って、プライマーのＴｍは、増幅反応を行う上で重要な因子である。Ｔｍは、例えば以下に示す式に従って求めることができる：
Ｔｍ（℃）＝８１．５＋１６．６×ｌｏｇ_１０［Ｓ］ ＋ ０．４１×（％ＧＣ）−（５００／ｎ）
〔式中、［Ｓ］は塩のモル濃度（Ｍ）を表し、（％ＧＣ）はプライマー配列中のＧＣ含量（％）を表し、ｎはプライマーの長さ（ｂｐ）を表す。〕
また本発明においては、プライマー配列中のＧとＣの総数に４を掛け、ＡとＴの総数に２を掛けて、その和の値をＴｍとする簡便法も利用可能である。
【０１４１】
Ｔｍの確認には、公知のプライマー設計用ソフトウエアを利用することができ、本発明で利用可能なプライマー設計用ソフトウエアとしては、例えばＯｌｉｇｏ ５．０（Ｎａｔｉｏｎａｌ Ｂｉｏｓｃｉｅｎｃｅｓ社）、ＨＹＢｓｉｍｕｌａｔｏｒ ｖｅｒｓｉｏｎ４（Ａｄｖａｎｃｅｄ Ｇｅｎｅ Ｃｏｍｐｕｔｉｎｇ Ｔｅｃｈｎｏｌｏｇｉｅｓ， Ｉｎｃ．）などが挙げられる。本発明では、平均Ｔｍが約５０〜６０℃であることが好ましい。
【０１４２】
設計するプライマーの長さは、５´側配列と３´側配列とを合わせて合計約１２〜４０塩基であり、好ましくは１５〜３０塩基である。またプライマーの合成法は当技術分野で周知である。例えば、ホスホロアミダイト法などの一般的なオリゴヌクレオチド合成法を用いることができる。
【０１４３】
本発明のプライマーとしては、例えば以下のものが挙げられる：
（ａ）配列番号４に示す塩基配列のうち少なくとも１９〜２０番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるフォワードプライマー（境界領域１に対応する）
（ｂ）配列番号５に示す塩基配列のうち少なくとも１６〜１７番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるフォワードプライマー（境界領域２に対応する）
（ｃ）配列番号６に示す塩基配列のうち少なくとも１９〜２０番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるフォワードプライマー（境界領域２に対応する）
（ｄ）配列番号７に示す塩基配列のうち少なくとも１４〜１５番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるフォワードプライマー（境界領域３に対応する）
（ｅ）配列番号８に示す塩基配列のうち少なくとも１９〜２０番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるリバースプライマー（境界領域１に対応する）
（ｆ）配列番号９に示す塩基配列のうち少なくとも１６〜１７番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるリバースプライマー（境界領域２に対応する）
（ｇ）配列番号１０に示す塩基配列のうち少なくとも１８〜１９番の塩基を含み、かつ連続した１５塩基以上の塩基配列からなるリバースプライマー（境界領域３に対応する）。
【０１４４】
上記プライマーのうち、本発明において特に好ましい組み合わせのプライマーセットは以下の通りである：
・プライマー（ａ）とプライマー（ｅ） 境界領域１〜２間が増幅される
・プライマー（ａ）とプライマー（ｆ） 境界領域１〜３間が増幅される
・プライマー（ｂ）とプライマー（ｆ） 境界領域２〜３間が増幅される
・プライマー（ｃ）とプライマー（ｇ） 境界領域２〜４間が増幅される
・プライマー（ｄ）とプライマー（ｇ） 境界領域３〜４間が増幅される
【０１４５】
（８）設計したプライマーの確認
続いて、上記（７）で設計したプライマーの確認試験を行う。本発明のプライマーは、形質転換体に導入されたＰｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸（すなわちｃＤＮＡ）のみを特異的に増幅するものでなければならない。従って、上記（７）で設計したプライマーを用いて、Ｐｈｖｃ−ｅｒｓ１のゲノムＤＮＡを増幅することなく、Ｐｈｖｃ−ｅｒｓ１のｃＤＮＡを特異的に増幅可能であるかどうかを試験する。
【０１４６】
本発明においては、上記設計したプライマーが、（Ａ）ｃＤＮＡを鋳型として増幅すること、及び（Ｂ）ゲノムＤＮＡを鋳型として増幅しないこと、の２つを確認する。
【０１４７】
（Ａ）ｃＤＮＡを鋳型とした増幅試験
本発明者は、Ｐｈｖｃ−ｅｒｓ１のｍＲＮＡを鋳型としてＲＴ−ＰＣＲ増幅反応を行って特異的な増幅産物が得られる場合には、該プライマーがＰｈｖｃ−ｅｒｓ１ ｃＤＮＡを特異的に増幅できると判断した。
【０１４８】
従って、確認試験（Ａ）においては、ペチュニア属植物由来のｍＲＮＡを鋳型として上記（７）で設計したプライマーを用いてＲＴ−ＰＣＲを行う。ｍＲＮＡの調製方法は、上記「１．Ｐｈｖｃ−ｅｒｓ１の単離・同定」の項に記載の通りである。ＲＴ−ＰＣＲは、市販のキットを用いて簡便に行うことができる。そのようなキットとしては、ＱＩＡＧＥＮ製 Ｏｎｅ ｓｔｅｐ ＲＴ−ＰＣＲ ｋｉｔ、ＳＩＧＭＡ製 Ｅｎｈａｎｃｅｄ Ａｖｉａｎ ＨＳ ＲＴ−ＰＣＲ Ｋｉｔなどが挙げられる。
【０１４９】
またＲＴ−ＰＣＲの条件は、５０℃で３０分、９４℃で１５分の後、９４℃で１分、５０．０〜７０．５℃で１分及び７２℃で１分３０秒の増幅工程を３５サイクル行い、７２℃で１５分の伸長工程後、４℃ホールドである。ここで、５０．０℃〜７０．５℃として示す温度は、例えば５℃毎に数段階に設定し、反応の最適条件を検討することにより、最適温度を決定することができる。
ＲＴ−ＰＣＲ反応の結果、Ｐｈｖｃ−ｅｒｓ１のｃＤＮＡのサイズのバンドが１本だけ検出された場合に、特異的増幅反応が起こったと判定する。
【０１５０】
（Ｂ）ゲノムＤＮＡを鋳型とした増幅試験
上記（７）で設計したプライマーを用いて、Ｐｈｖｃ−ｅｒｓ１ゲノムＤＮＡを鋳型としてＰＣＲ増幅反応を行った場合に増幅産物が検出されないことを確認する。
【０１５１】
従って、確認試験（Ｂ）においては、ペチュニア属植物由来のゲノムＤＮＡを鋳型として上記（７）で設計したプライマーを用いてＰＣＲを行う。ＤＮＡの調製方法は、当技術分野で公知であり、フェノール抽出及びエタノール沈殿を行う方法、ガラスビーズを用いる方法などが挙げられる。ＰＣＲは、市販のキットを用いて簡便に行うことができる。そのようなキットとしては、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ製 ＡｍｐｌｉＴａｑ Ｇｏｌｄ（登録商標） ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ、サワディーテクノロジー製 Ｌｅｔ’ｓ Ｇｏ ＰＣＲ Ｋｉｔなどが挙げられる。
【０１５２】
またＰＣＲの条件は、９４℃で１５分の後、９４℃で１分、５０．０〜７０．５℃で１分及び７２℃で１分３０秒の増幅工程を３０サイクル行い、７２℃で１５分の伸長工程後、４℃ホールドである。ここで、５０．０℃〜７０．５℃として示す温度は、例えば５℃毎に数段階に設定し、反応の最適条件を検討することにより、最適温度を決定することができる。
【０１５３】
ＰＣＲ反応の結果、増幅産物が検出されなかった場合に、ゲノムＤＮＡを鋳型とした増幅反応は行われなかったと判定する。
以上の確認試験（Ａ）及び（Ｂ）の両者を満たしたプライマーセットを本発明のプライマーセットとして用いることができる。
また本発明のプライマーセットは、ポリメラーゼ、緩衝液などと共にキットとしても使用可能である。
【０１５４】
（９）増幅反応
本発明の検出方法は、エチレン受容体遺伝子様核酸配列（Ｐｈｖｃ−ｅｒｓ１）若しくはそのアンチセンス核酸、又はその部分配列が導入された形質転換体を検出するため、被検植物のＤＮＡを鋳型として、上記（７）で設計したプライマーセットを用いて増幅反応を行い、増幅産物が検出された場合に、被検植物がＰｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入された形質転換植物であると判定するものである。
【０１５５】
本方法においては、Ｐｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸（ｃＤＮＡ）に特異的な断片のみを増幅する。従って、本方法により形質転換植物を検出しようとする場合には、増幅産物の有無のみを検出すればよい。プライマーとしては、上記（７）及び（８）で設計し、その機能を確認したプライマーセットを用いる。
【０１５６】
本方法で用いられる被検植物としては、Ｐｈｖｃ−ｅｒｓ１又はそのアンチセンス核酸が導入されているか否かを検出しようとする植物であればいずれのものでもよく、特にペチュニア属植物が好ましい。上記被検植物から、当技術分野で周知の方法を使用してＤＮＡを抽出することができる。
【０１５７】
増幅手法としては、特に限定されないが、ポリメラーゼ連鎖反応（ＰＣＲ）法の原理を利用した公知の方法を挙げることができる。例えば、ＰＣＲ法、ＬＡＭＰ（Ｌｏｏｐ−ｍｅｄｉａｔｅｄ ｉｓｏｔｈｅｒｍａｌ ＡＭＰｌｉｆｉｃａｔｉｏｎ）法、ＩＣＡＮ（Ｉｓｏｔｈｅｒｍａｌ ａｎｄＣｈｉｍｅｒｉｃ ｐｒｉｍｅｒ−ｉｎｉｔｉａｔｅｄ Ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ Ｎｕｃｌｅｉｃ ａｃｉｄｓ）法、ＲＣＡ（Ｒｏｌｌｉｎｇ Ｃｉｒｃｌｅ Ａｍｐｌｉｆｉｃａｔｉｏｎ）法、ＬＣＲ（Ｌｉｇａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）法、ＳＤＡ（Ｓｔｒａｎｄ Ｄｉｓｐｌａｃｅｍｅｎｔ Ａｍｐｌｉｆｉｃａｔｉｏｎ）法等を挙げることができる。増幅は、増幅産物が検出可能なレベルになるまで行う。
【０１５８】
ＰＣＲ法は、被検植物に含まれるＤＮＡを鋳型として、ＤＮＡポリメラーゼにより、プライマーセットに含まれる一対のプライマー間の塩基配列を合成するものである。ＰＣＲ法によれば、変性、アニーリング及び合成からなるサイクルを繰り返すことによって、増幅断片を指数関数的に増幅させることができる。ＰＣＲの最適条件は、当業者であれば容易に決定することができる。例えば、上記（７）において設計したプライマー（ａ）〜（ｇ）を用いてＰＣＲを行う場合には以下の通りである。
９０〜９７℃、好ましくは９３〜９５℃の変性工程を３０秒〜３分、好ましくは３０秒〜１分；３７〜７０℃、好ましくは５０〜７０℃のアニーリング工程を２０秒〜１分、好ましくは３０秒〜１分；及び５０〜８０℃、好ましくは７０〜７５℃の伸長工程を１〜５分、好ましくは１〜２分、からなるサイクルを４〜４０サイクル。
【０１５９】
但し、鋳型ＤＮＡ及びプライマーを十分変性させるために、上記増幅サイクルの前に９４℃で１〜３分の変性工程を加えてもよく、また、増幅されたＤＮＡを完全に伸長するために、増幅サイクルの後に７２℃で２〜２０分の伸長工程を加えてもよい。さらに、増幅産物の検出を直ちに行わない場合は、非特異的な増幅が起こらないようにするために、増幅産物を４℃で保存する工程を加えることが好ましい。
【０１６０】
（１０）増幅産物の検出
上記増幅反応後の増幅産物の検出には、このような産物を特異的に認識することができる公知の手段を用いることができる。例えば、増幅反応の過程で取り込まれるｄＮＴＰに、放射性同位体、蛍光物質、発光物質などの標識体を作用させ、この標識体を検出することができる。放射性同位体としては、^３２Ｐ、^１２５Ｉ、^３５Ｓなどを用いることができる。また蛍光物質としては、例えば、フルオレセン（ＦＩＴＣ）、スルホローダミン（ＴＲ）、テトラメチルローダミン（ＴＲＩＴＣ）などを用いることができる。また発光物質としてはルシフェリンなどを用いることができる。
【０１６１】
これら標識体の種類や標識体の導入方法等に関しては、特に制限されることはなく、従来公知の各種手段を用いることができる。例えば標識体の導入方法としては、放射性同位体を用いるランダムプライム法が挙げられる。
【０１６２】
本発明において、標識したｄＮＴＰを取り込んだ増幅産物を観察する方法としては、上述した標識体を検出するための当技術分野で公知の方法であればいずれの方法でもよい。例えば、標識体として放射性同位体を用いた場合には、放射活性を、例えば液体シンチレーションカウンター、γ−カウンターなどにより計測することができる。また標識体として蛍光を用いた場合には、その蛍光を蛍光顕微鏡、蛍光プレートリーダーなどを用いて検出することができる。
【０１６３】
以上のようにして増幅産物が検出された場合には、被検植物が、Ｐｈｖｃ−ｅｒｓ１若しくはそのアンチセンス核酸、又はその部分配列が導入された形質転換植物であると判定する。
【０１６４】
【実施例】
以下の実施例によって本発明をさらに詳細に説明するが、本発明はこれらの実施例に限定されるものではない。
【０１６５】
〔実施例１〕ペチュニアのエチレン受容体遺伝子様核酸（Ｐｈｖｃ−ｅｒｓ１）のクローニング
ペチュニアのエチレン受容体遺伝子様核酸をクローニングするため、以下の実験を行った。
【０１６６】
（１）植物試料
市販のペチュニア種子（ｃｖ． ｖｅｌｖｅｔ ｃａｒｐｅｔ、福花園種苗）を人工気象器（コイトトロン；小糸製作所）中で２５℃、日照１６時間、湿度５０％で栽培した植物体を試料とした。
【０１６７】
（２）ペチュニアＲＮＡの調製
ペチュニア植物体（葉、蕾、花など）を１００ｍｇ程度採取し、液体窒素凍結下で粉砕した。ＱＩＡＧＥＮ製ＲＮＡ調製キット（ＲＮｅａｓｙ Ｐｌａｎｔ Ｍｉｎｉ Ｋｉｔ）を用いてキット添付の標準プロトコールに従ってＲＮＡを調製した。
【０１６８】
（３）クローニング用ＰＣＲプライマー
ＧｅｎＢａｎｋデータベース（ＧｅｎＢａｎｋホームページ、ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ＧｅｎＢａｎｋ／ｉｎｄｅｘ．ｈｔｍｌ）に登録されている既知のＥＲＳ１遺伝子について、遺伝子解析ソフトのＧｅｎｅｔｙｘ−Ｍａｃ１０．１（ソフトウエア開発株式会社）を使用して分子系統解析を行った。作成した分子系統樹のナス科クラスターより分類学的近縁と判断されるタバコ、トマト及びジャガイモの同遺伝子の配列を参考にしてＯＲＦの両末端部分の配列を持つ下記のプライマーを設計し合成した。
フォーワードプライマー
ＥＲＳ−Ｆ：ａｔｇｇａａｔｃｃｔｇｔｇａｔｔｇｃａｔｔｇａｇ （配列番号１１）
リバースプライマー
ＥＲＳ−Ｒ：ｔｔａｃａｒａｃｔｙｃｔｔｔｇａｔａｇｃｇｔｔｇａ （配列番号１２）
上記プライマーは、ミックスプライマーとして作成され、ｒはＡ又はＧ、ｙはＣ又はＴを表す。
【０１６９】
（４）ＲＴ−ＰＣＲ増幅
上記プライマーを用いてＱＩＡＧＥＮ製 Ｏｎｅ−Ｓｔｅｐ ＲＴ−ＰＣＲ Ｋｉｔを使用してＲＴ−ＰＣＲ増幅を行った。反応液組成はキット添付の標準プロトコールに従い、Ｔａｋａｒａ製サーマルサイクラー（ＭＰ）を用いて実施した。ＰＣＲ条件は以下のとおりである。
５０℃ ３０分
９４℃ １５分
（９４℃ １分／５５℃ １分／７２℃ １分３０秒）×４０サイクル
７２℃ １５分
４℃
【０１７０】
（５）再ＰＣＲ増幅による制限酵素サイトの導入
クローニング用ベクターｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）（ＴＯＹＯＢＯ）及び植物用バイナリーベクターであるｐＢＩ１２１へのクローニングを目的として、ＲＴ−ＰＣＲ増幅断片に制限酵素サイト配列を導入するため、制限酵素サイト配列（ＸｂａＩ、ＳａｃＩ、ＳｍａＩ）を付加した以下のプライマーを合成した。
フォーワードプライマー
Ｘｂａ−ＥＲＳ−Ｆ：ｇｃｔｃｔａｇａａｔｇｇａａｔｃｃｔｇｔｇａｔｔｇｃａｔｔｇａｇ（配列番号１３）
Ｓａｃ−ＥＲＳ−Ｆ：ｃｇａｇｃｔｃａｔｇｇａａｔｃｃｔｇｔｇａｔｔｇｃａｔｔｇａｇ（配列番号１４）
リバースプライマー
Ｓａｃ−ＥＲＳ−Ｒ：ｃｇａｇｃｔｃｔｔａｃａｒａｃｔｙｃｔｔｔｇａｔａｇｃｇｔｔｇａ（配列番号１５）
Ｓｍａ−ＥＲＳ−Ｒ：ｔｃｃｃｃｃｇｇｇｔｔａｃａｒａｃｔｙｃｔｔｔｇａｔａｇｃｇｔｔｇａ（配列番号１６）
Ｘｂａ−ＥＲＳ−Ｒ：ｇｃｔｃｔａｇａｔｔａｃａｒａｃｔｙｃｔｔｔｇａｔａｇｃｇｔｔｇａ（配列番号１７）
上記プライマーは、ミックスプライマーとして作成され、ｒはＡ又はＧ、ｙはＣ又はＴを表す。
【０１７１】
これらを用いて、ＲＴ−ＰＣＲ増幅断片の再ＰＣＲ増幅を行った。ＰＣＲ増幅時の増幅エラー（間違った塩基が取り込まれて増幅される）を防止するため、３´→５´エキソヌクレアーゼ活性を有する Ｐｆｕ ＤＮＡ ｐｏｌｙｍｅｒａｓｅ（Ｐｆｕ Ｔｕｒｂｏ、ＴＯＹＯＢＯ）を使用した。反応液組成はキット添付の標準プロトコールに従い、Ｔａｋａｒａ製サーマルサイクラー（ＭＰ）を用いて実施した。ＰＣＲ条件は以下のとおりである。
９４℃ ２分
（９４℃ １分／５５℃ １分／７２℃ １分３０秒）×３０サイクル
７２℃ １５分
４℃
【０１７２】
（６）増幅断片の精製と制限酵素処理
得られたＰＣＲ増幅産物断片について、対応する制限酵素の組み合わせ（ＸｂａＩ−ＳａｃＩ、ＸｂａＩ−ＳｍａＩ）を用いて、ＰＲＯＭＥＧＡ製ＭＵＬＴＩＣＯＲＥバッファー中で３７℃で同時制限酵素消化を行った。また、クローニング・ベクターｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）（ＴＯＹＯＢＯ）についても同様の処理を行った。０．８％アガロース・ゲルを用いてＴＡＥバッファーで電気泳動後、エチジウムブロマイド（ＥｔＢｒ）染色を行い、目的断片を確認した。目的断片部分をゲルごとメスで切出し、ＱＩＡＧＥＮ製 ＱＩＡｑｕｉｃｋ Ｇｅｌ Ｅｘｔｒａｃｔｉｏｎ Ｋｉｔを用いてゲルより溶出・精製した。
【０１７３】
（７）クローニングベクターへの増幅断片の導入と増幅
Ｔａｋａｒａ製 ＤＮＡ Ｌｉｇａｔｉｏｎ ｋｉｔ ｖｅｒ．２を用いて、上記のＰＣＲ増幅断片と制限酵素消化したベクターのライゲーションを行った。ＴＯＹＯＢＯ製コンピテントセル（大腸菌ＤＨ５α）を付属プロトコールに従って上記ＤＮＡを用いて形質転換し、ＩＰＴＧ、Ｘ−ｇａｌ、アンピシリン（５０μｇ／ｍｌ）添加したＬＢ培地で培養し、形質転換体を選抜した。出現したコロニーを釣菌してアンピシリン（５０μｇ／ｍｌ）添加したＬＢ培地で液体培養した。得られた菌体からＱＩＡＧＥＮ製 Ｐｌａｓｍｉｄ Ｍｉｎｉ Ｋｉｔを用いてプラスミドを調製した。制限酵素処理後の断片の長さ比較及びＰＣＲ増幅により、インサートの確認を行い、目的断片がサブクローニングされたプラスミドを得た。
【０１７４】
（８）ＤＮＡシークエンサーによる塩基配列の確認
選られたプラスミドについてＡＢＩ製 ＤＮＡシークエンサー（３１０ Ｇｅｎｅｔｉｃ Ａｎａｌｙｚｅｒ）による塩基配列の確認を行った。シークエンス反応には ＢｉｇＤｙｅ Ｔｅｒｍｉｎａｔｏｒ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ，ＦＳキット（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いた。詳細な実験手順はＡＢＩ製のマニュアル（操作ガイド、２０００年８月版Ｒｅｖ．４．０）にしたがって実施した。
【０１７５】
（９）塩基配列の解析
得られた塩基配列は、Ｇｅｎｅｔｙｘ−Ｍａｃ／ ＡＴＳＱ ３．０（ソフトウエア開発株式会社）を用いて解析・編集した。クローニングした全長配列を決定後、インターネット上のデータベースに照会し（ＧｅｎＢａｎｋ；ｈｔｔｐ：／／ｗｗｗ．ｎｃｂｉ．ｎｌｍ．ｎｉｈ．ｇｏｖ／ＧｅｎＢａｎｋ／ｉｎｄｅｘ．ｈｔｍｌ）、類似度の高い登録配列を探索した。これら（既知のＥＲＳ１遺伝子）の配列を用いて分子系統解析（遺伝子解析ソフト；Ｇｅｎｅｔｙｘ−Ｍａｃ１０．１を使用）を行った。
【０１７６】
（１０）ペチュニアの各組識におけるＰｈｖｃ−ｅｒｓ１の配列の確認
４つの組織（葉・未熟な蕾・成熟した蕾・開花した花）より上記の方法でＲＮＡを抽出した。このＲＮＡより同様にＲＴ−ＰＣＲ増幅した断片を電気泳動後にゲルより切出し精製した。この断片をテンプレートとしてシークエンス反応を行い、ダイレクト・シークエンシングを行った。この方法で各組識由来のｃＤＮＡの全長配列をそれぞれ決定した（ただし「未熟な蕾」のみ部分配列を確認）。
【０１７７】
４つの組織（葉・未熟な蕾・成熟した蕾・開花した花）よりＲＮＡを抽出・精製し、ＲＴ−ＰＣＲ増幅によって得た断片のｃＤＮＡの全長配列をそれぞれ決定した（ただし「未熟な蕾」のみ部分配列を確認）。
【０１７８】
実験データの解析の結果、この核酸配列は１９１１塩基対（６３７アミノ酸をコードする）のＯＲＦを有すると推定された。得られた配列をデーターベースに照会した結果、エチレン受容体ＥＲＳ１様遺伝子と相同性が高い遺伝子断片であることが判明した。特に、系統分類学的に近縁なタバコ及びトマトの同遺伝子と相同性が高いという結果が得られた。データーベース上で相同性がみられた植物由来の同遺伝子配列についてＮＪ法（近隣結合法）で分子系統樹を作成した。その結果、本実施例で得たエチレン受容体ＥＲＳ１様遺伝子の核酸配列（Ｐｈｖｃ−ｅｒｓ１）は、双子葉植物・ナス科植物クラスターに属し、分類学的位置関係とも一致した。このクラスターに属する植物種の遺伝子とのホモロジー（配列相同性）は、核酸レベルでタバコ、トマト及びジャガイモに対して、それぞれ９３．６％、９１．２％及び９１．１％であった。またアミノ酸レベル（核酸配列からの推定アミノ酸配列）で比較した場合にはそれぞれ９５．３％、９２．６％及び９２．１％であった。一方、メロン果実の日持ち性改良を目指した遺伝子導入に一般的に使用されたメロンＥＲＳ１に対しては、核酸レベルで７０．７％、アミノ酸レベルで７１．０％であることが判明した。この結果から、植物体に導入したときの効果において、ペチュニア由来とその他の植物由来とで差が生じる可能性がある（Ｐｌａｎｔ Ｐｈｙｓｉｏｌｏｇｙ （１９９９） ｖｏｌ．１１９， ３２１−３２９）。
【０１７９】
（１１）Ｐｈｖｃ−ｅｒｓ１のクローニング
上記（１０）に記載の方法で得た核酸断片をクローニングベクター（ｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋））にクローニングし、ＤＮＡシークエンサーにより挿入位置及び挿入断片の核酸配列を確認した。この結果、ｐＢｌｕｅｓｃｒｉｐｔ ＩＩ ＳＫ（＋）のマルチクローニングサイト（ＭＣＳ）中のＳｍａＩ−ＸｂａＩ間に３´→５´方向に挿入されたクローン（ｐＰｈｖｃ−ｅｒｓ１）を得た。この時点で、得られたペチュニアＥＲＳ１様核酸配列をＰｈｖｃ−ｅｒｓ１と命名した。
【０１８０】
〔実施例２〕Ｐｈｖｃ−ｅｒｓ１のＯＲＦ両末端領域の決定
（１）植物試料及びペチュニアＲＮＡの調製
植物試料及びペチュニアＲＮＡの調製（葉組織由来）は、実施例１の（１）及び（２）に記載のように行った。
【０１８１】
（２）ＲＡＣＥ法
ＲＡＣＥ法（Ｒａｐｉｄ ａｍｐｌｉｆｉｃａｔｉｏｎ ｏｆ ｃＤＮＡ ｅｎｄｓ）は、市販のキット（ロシュ・ダイアグノスティク製５´／３´ ＲＡＣＥ Ｋｉｔ）を用いて行い、目的の断片を得た。
【０１８２】
（３）ＰＣＲ増幅
ＰＣＲ条件は、後述するように複数のアニーリング条件で同時検討し、良好な増幅産物が得られたものについて次の段階に使用した。３´ＲＡＣＥでは、ｎｅｓｔｅｄ−ＰＣＲを実施した。
【０１８３】
最初に、実施例１で得られたＰｈｖｃ−ｅｒｓ１の推定配列に基づいて、そのＯＲＦ部分の両末端領域近傍を標的としたＰＣＲプライマーを設計した。ｎｅｓｔｅｄ−ＰＣＲのために両末端用にそれぞれ複数個のプライマーを作製したが、目的領域の増幅に成功した際に使用したもののみを下記表１に記載した。
【０１８４】
【表１】

【０１８５】
表１に示すプライマーを用いて、上記（１）で調製したペチュニア葉由来ＲＮＡから合成されたｃＤＮＡを鋳型として、Ｔａｋａｒａ製 Ｅｘ Ｔａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅを使用してＰＣＲ増幅を行った。反応液組成はキット添付の標準プロトコールに従い、Ｅｐｐｅｎｄｏｒｆ製サーマルサイクラー（マスターサイクラーグラディエント）を用いて実施した。この装置はヒートブロック上に任意の（最大１２段階の）温度勾配を設定して反応を行う事ができるので、増幅効率と特異性に重要なアニーリング温度を５０．０℃から７０．５℃（約５℃毎に５段階；すなわち、５０．０℃、５５．５℃、６０．８℃、６６．０℃、及び７０．５℃）に設定し、その最適条件を検討した。ＰＣＲ条件を以下に示す。
９４℃ １５分
（９４℃ １分／５０．０〜７０．５℃ １分／７２℃ １分３０秒）×３０サイクル
７２℃ １５分
４℃
【０１８６】
（４）増幅断片の精製
得られたＰＣＲ増幅産物断片について、２％アガロース・ゲル（０．８％は不適切）を用いてＴＡＥバッファーで電気泳動後、エチジウムブロマイド（ＥｔＢｒ）染色を行い、目的断片を確認した。目的断片部分をゲルごとメスで切出し、ＱＩＡＧＥＮ製ＱＩＡｑｕｉｃｋ Ｇｅｌ ｅｘｔｒａｃｔｉｏｎ ｋｉｔを用いてゲルより溶出・精製した。
【０１８７】
（５）クローニングベクターへの増幅断片の導入と増幅
Ｔａｋａｒａ製 ＤＮＡ Ｌｉｇａｔｉｏｎ ｋｉｔ ｖｅｒ．２を用いて、上記（４）で精製したＰＣＲ増幅断片とＴＡクローニング用（ＰＣＲ産物のクローニング用）ベクター（Ｎｏｖａｇｅｎ製 ｐＴ７Ｂｌｕｅ−２）のライゲーション（１６℃、一晩反応）を行った。ＴＯＹＯＢＯ製コンピテントセル（大腸菌ＤＨ５α）を付属プロトコールに従って形質転換し、ＩＰＴＧ、Ｘ−Ｇａｌ及びアンピシリン（５０μｇ／ｍｌ）添加したＬＢ培地で培養し、形質転換体を選抜した。出現したコロニーを釣菌してアンピシリン（５０μｇ／ｍｌ）添加したＬＢ培地で液体培養して得た菌体から、ＱＩＡＧＥＮ製 Ｐｌａｓｍｉｄ Ｍｉｎｉ Ｋｉｔを用いてプラスミドを調製した。ゲル電気泳動によるインサートの確認を行い、目的断片がサブクローニングされたと予想されるプラスミドを得た。
【０１８８】
（６）ＤＮＡシークエンサーによる塩基配列の確認
使用したシークエンサー及び実験手順は実施例１の（８）に記載と同様にした。またシークエンシング用プライマーは、ｐＴ７Ｂｌｕｅ−２ベクターのクローニングサイトの両端に存在するＴ７配列及びＭ１３配列を標的としたプライマー（Ｔａｋａｒａ製 ＢｃａＢＥＳＴ Ｓｅｑｕｅｎｃｉｎｇ Ｐｒｉｍｅｒ Ｔ７；ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧ（配列番号２３）及びＭ１３ Ｐｒｉｍｅｒ Ｍ４；ＧＴＴＴＴＣＣＣＡＧＴＣＡＣＧＡＣ（配列番号２４））を使用した。
【０１８９】
（７）塩基配列の解析
得られた塩基配列は、Ｇｅｎｅｔｙｘ−Ｍａｃ／ ＡＴＳＱ ３．０を用いて解析・編集した。実施例１においてクローニングしたｃＤＮＡ配列（１９１１塩基対）と比較してＯＲＦの両末端領域配列を再決定した。
【０１９０】
配列解析の結果得られたペチュニア由来Ｐｈｖｃ−ｅｒｓ１のｃＤＮＡ（ＯＲＦ領域）の塩基配列を配列番号１に、Ｐｈｖｃ−ｅｒｓ１タンパク質のアミノ酸配列を配列番号２に示す。
【０１９１】
〔実施例３〕スプライス部位及びイントロン配列の決定
（１）植物試料及びペチュニアＤＮＡの調製
植物試料は、実施例１の（１）に記載のように行った。
またペチュニアＤＮＡ（葉組織由来）は、ペチュニア植物体（葉）を１００ｍｇ程度採取し、液体窒素凍結下で粉砕した後、ＱＩＡＧＥＮ製ＤＮＡ調製キット（ＤＮｅａｓｙ ｐｌａｎｔ ｍｉｎｉ ｋｉｔ）を用いてキット添付の標準プロトコールに従って調製した。
【０１９２】
（２）ＰＣＲ増幅
実施例１の（３）で設計したプライマーを用いて、上記（１）で調製したペチュニア葉由来ＤＮＡを鋳型として、Ｔａｋａｒａ製 Ｅｘ Ｔａｑ ＤＮＡ ｐｏｌｙｍｅｒａｓｅを使用してＰＣＲ増幅を行った。反応液組成はキット添付の標準プロトコールに従い、Ｅｐｐｅｎｄｏｒｆ製サーマルサイクラー（マスターサイクラーグラディエント）を用いて実施した。アニーリング温度及びＰＣＲ条件は実施例２の（３）と同様にした。
【０１９３】
（３）増幅断片の精製
ＰＣＲ増幅産物断片の精製は、０．８％アガロース・ゲルを用いた以外は実施例２の（４）と同様に行った。
【０１９４】
（４）ＤＮＡシークエンサーによる塩基配列の確認と解析
得られたＰＣＲ増幅産物断片について、実施例１の（８）と同様に塩基配列を確認した。
得られた塩基配列は、Ｇｅｎｅｔｙｘ−Ｍａｃ／ ＡＴＳＱ ３．０を用いて解析・編集した。これまでにクローニングしたｃＤＮＡ配列（１９１１塩基対）と比較してイントロン配列及びスプライス部位を特定した。
【０１９５】
（５）プライマーウォーキングによる未知配列の解析
イントロン部分の配列は、隣接するエキソン領域に設計したプライマーによって両方の鎖（センス鎖／アンチセンス鎖）の配列を解読する事により決定した。イントロン部分の全域の解読ができない場合は、解析結果に基づきスプライシング領域近傍に新たにシークエンシング用プライマーを設計して解読を進めるプライマーウォーキング法により全長配列の決定を行った。この実験に使用したシークエンシング用プライマーを表２にまとめた。
【０１９６】
【表２】

【０１９７】
配列解析の結果得られた塩基配列の情報に基づいて作成したＰｈｖｃ−ｅｒｓ１遺伝子のゲノムＤＮＡの塩基配列を配列番号３に示した。染色体上にコードされる該遺伝子のＯＲＦ領域の長さは３７８２塩基（ｃＤＮＡ配列は１９１１塩基）と判明した。これはシロイヌナズナＥＲＳ１（ＧｅｎＢａｎｋアクセッション・ナンバーＵ２１９５２）の２１８５塩基（ｃＤＮＡ配列は１８４２塩基）と比較すると著しく長い。また、ＥＲＳ１タイプの遺伝子の中で唯一イントロン配列の情報がデータベース上に公開されているシロイヌナズナＥＲＳ１とのイントロン領域の比較を図１に示す。
【０１９８】
〔実施例４〕内在／外来Ｐｈｖｃ−ｅｒｓ１を識別して検出可能なＰＣＲプライマーの設計
（１）植物試料及びペチュニアＲＮＡの調製
植物試料及びペチュニアＲＮＡの調製（葉組織由来）は、実施例１の（１）及び（２）に記載のように行った。
【０１９９】
（２）プライマーの設計
実施例３から、ペチュニアのゲノムＤＮＡにコードされるＰｈｖｃ−ｅｒｓ１の配列を決定しており、該遺伝子には４箇所のイントロンが存在することが明らかになった（図１）。
【０２００】
また、ゲノムＤＮＡから転写後にスプライシングを受けたｍＲＮＡには、切り出されたイントロンを挟んだ２つのエキソン間の境界領域（スプライス部位）が４箇所存在する（以下、それぞれ境界領域１、境界領域２、境界領域３、及び境界領域４と呼ぶ）。この４箇所の境界領域を標的としてプライマーを設計した。プライマーは、境界領域１においては、フォーワードプライマーのみ、境界領域２及び境界領域３においては、フォーワードプライマーとリバースプライマーの両方、境界領域４においては、リバースプライマーのみを設計した（図３）。また、プライマーの設計時には、スプライス部位から３´末端までの鎖長（３´側配列の長さ）と、プライマーのＴｍについて考慮し、合計３１種類のプライマーを設計及び合成し、そのうち表３に示す１１種類のプライマーを選択した。表３中、方向の欄に示す「Ｆ」はフォーワードプライマーを表し、「Ｒ」はリバースプライマーを表す。また、標的部位の欄に示す数字は、配列番号１に示すＰｈｖｃ−ｅｒｓ１遺伝子の塩基配列の番号を表し、鎖長の欄に示す数字は、スプライス部位からプライマーの３´末端まで（３´側配列）の鎖長を表す。
【０２０１】
【表３】

【０２０２】
（３）ＲＴ−ＰＣＲ増幅
上記（２）で設計したプライマーを用いて、上記（１）で調製したペチュニア葉由来ＲＮＡを鋳型として、ＱＩＡＧＥＮ製 Ｏｎｅ Ｓｔｅｐ ＲＴ−ＰＣＲ Ｋｉｔを使用してＲＴ−ＰＣＲ増幅を行った。反応液組成はキット添付の標準プロトコールに従い調製した。

【０２０３】
Ｅｐｐｅｎｄｏｒｆ製 マスターサイクラー・グラディエントを用いて、５０．０℃〜７０．５℃の範囲で約５℃毎に５段階のアニーリング温度を設定し、ＲＴ−ＰＣＲを実施した。ＲＴ−ＰＣＲ増幅条件を以下に示す。
▲１▼５０℃ ３０分
▲２▼９４℃ １５分
▲３▼（９４℃ １分／５０．０〜７０．５℃ １分／７２℃ １分３０秒）×３５サイクル
▲４▼７２℃ １５分
▲５▼４℃
【０２０４】
抽出したペチュニアのＲＮＡを鋳型にして、表３に示すプライマーを用いてＲＴ−ＰＣＲ増幅を行った。４箇所の境界領域を標的として設計したプライマーを用いて増幅されるＰｈｖｃ−ｅｒｓ１遺伝子のｃＤＮＡ断片は、境界領域１〜２間、境界領域１〜３間、境界領域１〜４間、境界領域２〜３間、境界領域２〜４間、及び境界領域３〜４間の６種類である（図３）。
【０２０５】
それぞれの境界領域間のＲＴ−ＰＣＲ増幅後のゲル電気泳動の結果、目的のサイズのバンドを１本のみ検出可能であったプライマーの組み合わせを表４に示す。表４中、Ｆの欄に示すアルファベットはフォーワードプライマーの名称を表し、Ｒの欄に示すアルファベットはリバースプライマーの名称を表す。また鎖長の欄に示す数字は、スプライス部位から３´末端まで（３´側配列）の鎖長を表す。
【０２０６】
【表４】

【０２０７】
Ｔｍが６０℃程度となるように設計したプライマー（表３及び４参照）を用いてＲＴ−ＰＣＲ増幅を行った結果、境界領域１〜３間（図５Ａ）、境界領域２〜４間（図５Ｂ）、及び境界領域３〜４間（図５Ｃ）において目的のサイズの増幅産物のみが増幅され、特異的検出が可能となった。図５Ａ〜Ｃにおいて、各レーンは、レーン１：アニーリング温度５０．０℃、レーン２：アニーリング温度５５．５℃、レーン３：アニーリング温度６０．８℃、、レーン４：アニーリング温度６６．０℃、及びレーン５：アニーリング温度７０．５℃で得られた結果を示す。
【０２０８】
さらにＴｍを６０℃として設計したプライマーを用いてＲＴ−ＰＣＲ増幅を行った場合には、アニーリング温度５０．０〜７０．５℃の全範囲において非特異的な増副産物が見られる傾向にあった。そこで、プライマーのアニーリングが低い温度で起こるようにするため、Ｔｍの設定温度を６０℃以下に設定する方法を考察した。境界領域１〜２間、境界領域２〜３間において、Ｔｍの設定温度が６０℃以下になるように設計したプライマー（表３及び４参照）を用いて、上述したのと同様にＲＴ−ＰＣＲ増幅を行った。その結果、アニーリング温度７０．５℃では非特異的な増副産物が見られなくなり、境界領域１〜２間（図６Ａ）、及び境界領域２〜３間（図６Ｂ）においても、目的のサイズの増幅産物のみが検出され、特異的検出が可能となった。図６Ａ及びＢにおいて、各レーンは、レーン１：アニーリング温度５０．０℃、レーン２：アニーリング温度５５．５℃、レーン３：アニーリング温度６０．８℃、、レーン４：アニーリング温度６６．０℃、及びレーン５：アニーリング温度７０．５℃で得られた結果を示す。
【０２０９】
これらのことから、４箇所のそれぞれの境界領域を標的として設計したプライマーを用いたＲＴ−ＰＣＲ増幅により、外来遺伝子に相当するＰｈｖｃ−ｅｒｓ１遺伝子ｃＤＮＡの特異的検出が可能になった。
【０２１０】
〔実施例５〕ゲノムＤＮＡの存在下におけるＰＣＲ増幅条件の検討
ペチュニア葉由来のＤＮＡは、実施例３（１）に記載と同様にして調製した。
（１）ＰＣＲ条件の検討
Ｐｈｖｃ−ｅｒｓ１ ｃＤＮＡのＲＴ−ＰＣＲ増幅による特異的検出が可能となった至適アニーリング温度において（表３及び４参照）、ゲノムＤＮＡを鋳型としたＰＣＲ増幅を行う場合に、増幅産物が得られるか否かを検討した。そのために、実施例４で行ったＲＴ−ＰＣＲ増幅反応と反応溶液組成及びＰＣＲ反応条件などを同一条件にした比較実験を実施した。ＲＴ−ＰＣＲ増幅で用いた酵素溶液（ＱＩＡＧＥＮ製）の中には、２種類の逆転写酵素 （Ｏｍｎｉｓｃｒｉｐｔ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ及びＳｅｎｓｉｓｃｒｉｐｔ Ｒｅｖｅｒｓｅ Ｔｒａｎｓｃｒｉｐｔａｓｅ）と Ｈｏｔ Ｓｔａｒ Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ が含まれている。そこで、ＰＣＲ増幅にはｏｎｅ ｓｔｅｐ ＲＴ−ＰＣＲ増幅で用いられている酵素（ＱＩＡＧＥＮ製 Ｈｏｔ Ｓｔａｒ Ｔａｑ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ）を同じ濃度で用いた。
【０２１１】
条件の検討には、実施例１の（３）で設計したプライマーを用いた。
フォーワードプライマー
ＥＲＳ−Ｆ：ａｔｇｇａａｔｃｃｔｇｔｇａｔｔｇｃａｔｔｇａｇ（配列番号１１）
リバースプライマー
ＥＲＳ−Ｒ：ｔｔａｃａｒａｃｔｙｃｔｔｔｇａｔａｇｃｇｔｔｇａ（配列番号１２）
プライマーの添加量は、上記実施例４のＲＴ−ＰＣＲ反応液と同じ２．０μＭと、ＱＩＡＧＥＮ Ｈｏｔ Ｓｔａｒ Ｔａｑ ＰＣＲで推奨している量０．５μＭの２つの条件で比較した。
ＰＣＲ反応液中のマグネシウム濃度については、実施例４のＲＴ−ＰＣＲ反応液と同じ２．５ｍＭ（添加量１．０μｌ）と１．５ｍＭ（添加量０μｌ）の２つの条件で比較した。
以上をふまえ、以下の表５に示す４条件でＰＣＲ増幅を行った。
【０２１２】
【表５】

【０２１３】
Ｅｐｐｅｎｄｏｒｆ製マスターサイクラー・グラディエントを用いて、５０．０℃〜７０．５℃の範囲で約５℃毎に５段階のアニーリング温度を設定し、増幅反応を実施した。ＰＣＲ条件を以下に記載する。
▲１▼９４℃ １５分
▲２▼（９４℃ １分／５０．０〜７０．５℃ １分／７２℃ １分３０秒）×３０サイクル
▲３▼７２℃ １５分
▲４▼ ４℃
【０２１４】
実施例４においてプライマーＥＲＳ−Ｆ及びＥＲＳ−Ｒ（それぞれ配列番号１１及び１２）を用いたＲＴ−ＰＣＲ増幅の至適アニーリング温度は約５５〜６０℃であった。表５に示す４条件のＰＣＲ反応液を用いてアニーリング温度５５．５℃及び６０．８℃でＰＣＲ増幅行った結果、どの条件でもアニーリング温度６０．８℃での特異的検出が可能であった。これにより、今回設定したプライマーの添加量及びマグネシウム濃度では、特異的増幅に影響はないことが判明し、ＲＴ−ＰＣＲ増幅時とほぼ同じ条件を設定できたと考えられる。以上の結果に基づき、ＰＣＲ増幅の反応液組成については、実施例４のＲＴ−ＰＣＲ増幅と同じ、条件３を採用した。
【０２１５】
（２）Ｐｈｖｃ−ｅｒｓ１ ゲノムＤＮＡのＰＣＲ増幅試験
ゲノムＤＮＡの存在下で、選択した７組のプライマーセット（表４参照）を用いて、それぞれの至適アニーリング温度（表４参照）で条件３を用いてＰＣＲ増幅を行った結果、増副産物は検出されなかった（図７参照）。このことから、これらのプライマーでは内在遺伝子は増幅されないことが確認できた。
【０２１６】
各レーンは、以下を表す：
レーン１：分子量マーカー（１ｋｂｐ）
レーン２：分子量マーカー（１００ｂｐ）
レーン３：プライマー１Ｆ−Ｓ１及び２Ｒ−Ｓ４を用いて境界領域１〜２間のｃＤＮＡを増幅した
レーン４：プライマー１Ｆ−Ｓ１及び２Ｒ−Ｓ４を用いて境界領域１〜２間のゲノムＤＮＡを増幅した
レーン５：プライマー１Ｆ−Ｓ１及び２Ｒ−Ｓ５を用いて境界領域１〜２間のｃＤＮＡを増幅した
レーン６：プライマー１Ｆ−Ｓ１及び２Ｒ−Ｓ５を用いて境界領域１〜２間のゲノムＤＮＡを増幅した
レーン７：プライマー１Ｆ−Ｓ１及び２Ｒ−Ｓ６を用いて境界領域１〜２間のｃＤＮＡを増幅した
レーン８：プライマー１Ｆ−Ｓ１及び２Ｒ−Ｓ６を用いて境界領域１〜２間のゲノムＤＮＡを増幅した
レーン９：分子量マーカー（１ｋｂｐ）
レーン１０：分子量マーカー（１００ｂｐ）
レーン１１：プライマー１Ｆ０及び３Ｒ０を用いて境界領域１〜３間のｃＤＮＡを増幅した
レーン１２：プライマー１Ｆ０及び３Ｒ０を用いて境界領域１〜３間のゲノムＤＮＡを増幅した
レーン１３：分子量マーカー（１ｋｂｐ）
レーン１４：分子量マーカー（１００ｂｐ）
レーン１５：プライマー２Ｆ−Ｓ１及び３Ｒ−Ｓ１を用いて境界領域２〜３間のｃＤＮＡを増幅した
レーン１６：プライマー２Ｆ−Ｓ１及び３Ｒ−Ｓ１を用いて境界領域２〜３間のゲノムＤＮＡを増幅した
レーン１７：分子量マーカー（１ｋｂｐ）
レーン１８：分子量マーカー（１００ｂｐ）
レーン１９：プライマー２Ｆ２及び４Ｒ０を用いて境界領域２〜４間のｃＤＮＡを増幅した
レーン２０：プライマー２Ｆ２及び４Ｒ０を用いて境界領域２〜４間のゲノムＤＮＡを増幅した
レーン２１：分子量マーカー（１ｋｂｐ）
レーン２２：分子量マーカー（１００ｂｐ）
レーン２３：プライマー３Ｆ０及び４Ｒ０を用いて境界領域３〜４間のｃＤＮＡを増幅した
レーン２４：プライマー３Ｆ０及び４Ｒ０を用いて境界領域３〜４間のゲノムＤＮＡを増幅した
【０２１７】
以上より、ペチュニアのＰｈｖｃ−ｅｒｓ１ ｃＤＮＡの境界領域の塩基配列を含むプライマーを用いたＲＴ−ＰＣＲ増幅により、外来遺伝子に相当するＰｈｖｃ−ｅｒｓ１ ｃＤＮＡの特異的検出が可能になった。また、このプライマーを用いてＰＣＲ増幅を行った場合、内在遺伝子として染色体にコードされるＰｈｖｃ−ｅｒｓ１（ゲノムＤＮＡ）の特異的検出はされないという結果となった。これらのことから、ペチュニアの内在／外来Ｐｈｖｃ−ｅｒｓ１を識別して特異的に検出可能なＰＣＲプライマーを設計することができた。
【０２１８】
【発明の効果】
本発明により、ペチュニア属に属する植物の花持ち性を調節することが可能となり、商業的に有用な植物が得られる。また本発明により、そのような形質転換植物を迅速かつ簡便に検出することが可能となる。
【０２１９】
【配列表】

【０２２０】
【配列表フリーテキスト】
配列番号４〜６３：合成オリゴヌクレオチド
【図面の簡単な説明】
【図１】ペチュニア（Ｐｅｔｕｎｉａ×ｈｙｂｒｉｄａ）Ｐｈｖｃ−ｅｒｓ１（Ａ）とシロイヌナズナ（Ａｒａｂｉｄｏｐｓｉｓ ｔｈａｌｉａｎａ）ＥＲＳ１（Ｂ）のゲノムＤＮＡの構造を表す図である。
【図２】ペチュニアのエチレン受容体遺伝子様核酸（Ｐｈｖｃ−ｅｒｓ１）のｃＤＮＡにおける境界領域と、それに対応する本発明のオリゴヌクレオチドプローブの設計を示す図である。
【図３】ペチュニアのエチレン受容体遺伝子様核酸（Ｐｈｖｃ−ｅｒｓ１）のｃＤＮＡにおける境界領域と、それに対応する本発明のプライマーの設計を示す図である。
【図４】本発明のプライマーセットを用いたＰｈｖｃ−ｅｒｓ１のｃＤＮＡ（Ａ）及びＰｈｖｃ−ｅｒｓ１ゲノムＤＮＡの増幅を示す図である。
【図５】ＲＴ−ＰＣＲ増幅反応によるＰｈｖｃ−ｅｒｓ１ ｃＤＮＡの特異的検出を示す電気泳動写真を示す。プライマーは、Ｔｍを６０℃に設定したものを使用した。
【図６】ＲＴ−ＰＣＲ増幅反応によるＰｈｖｃ−ｅｒｓ１ ｃＤＮＡの特異的検出を示す電気泳動写真を示す。プライマーは、Ｔｍを５０〜５６℃に設定したものを使用した。
【図７】境界領域の塩基配列を含むプライマーを用いたＰｈｖｃ−ｅｒｓ１ ｃＤＮＡ及びＰｈｖｃ−ｅｒｓ１ゲノムＤＮＡの検出結果を示す電気泳動写真である。写真は、ｃＤＮＡが特異的に検出され、ゲノムＤＮＡが検出されないことを示している。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a novel and novel ethylene receptor gene-like nucleic acid useful for improving the flower durability of a Petunia plant. The present invention also relates to a DNA encoding the nucleic acid or an expression vector containing the antisense nucleic acid of the nucleic acid, a transformant containing the nucleic acid or the antisense nucleic acid, and a seed obtained from the transformant. Furthermore, the present invention relates to a method for regulating the flower durability of a plant, and a method for producing a plant with enhanced flower durability.
[0002]
The present invention also relates to a method for detecting a transformed plant into which an ethylene receptor gene-like nucleic acid or an antisense nucleic acid thereof has been introduced, and a probe and primer set for detection.
[0003]
[Prior art]
In previous studies, several ethylene receptor genes and downstream genes of the signaling system have been identified. In Arabidopsis, which has been studied in the most detailed manner, five types of ethylene receptor genes ETR1, ERS1, ETR2, ERS2 and EIN4 have been isolated, and the primary structure characteristics and nucleotide sequence homology of proteins predicted from the genes have been identified. Are classified into two subfamilies. ETR1 and ERS1 are classified into the ETR1 subfamily and are characterized by having three hydrophobic domains at the N-terminus of the protein. On the other hand, ETR2, ERS2 and EIN4 are classified into the ETR2 subfamily, and these proteins have four hydrophobic domains on the N-terminal side. In addition, the homology of the nucleotide sequences within the subfamily is high, and the homology of the nucleotide sequences between the subfamilies is low.
[0004]
In the study of the ethylene receptor gene, for example, a transformant was prepared by introducing antisense DNA of the ethylene receptor gene or a mutated ethylene receptor gene into Arabidopsis thaliana or tomato, and it was found that the ethylene sensitivity of the transformant was reduced. It has been reported (referred to as Report 1).
[0005]
On the other hand, a model of an ethylene signaling system has been proposed in Arabidopsis. Plant ethylene receptors that respond to ethylene are thought to basically have a common function with Arabidopsis thaliana. According to this model, in a state before ethylene is bound, expression of a downstream ethylene-responsive gene does not occur, and ripening does not proceed. However, when ethylene binds to the receptor, downstream genes are activated and various reactions occur due to the binding of ethylene. According to this model, methods for reducing ethylene sensitivity include a method of expressing a receptor lacking ethylene binding ability in a plant by gene transfer and a method of overexpressing a normal receptor in a plant (Emen Hiro, Biotechnology, Vol. 78, No. 8, pp. 317-321).
[0006]
When the antisense method (or cosuppression method) used in the above report 1 is examined by applying the above-described method for reducing ethylene sensitivity to the Arabidopsis thaliana model, the result is that ethylene sensitivity is increased. is expected. Therefore, it is considered that the result of Report 1 has a mechanism different from that of the model of the signal transduction system of ethylene. As described above, the plurality of ethylene receptor genes that have been isolated to date have many unclear points such as the interaction of the proteins encoded by each, the mode of existence, and the expression control mechanism.
[0007]
Furthermore, in solanaceous plants to which tobacco, tomato, potato, etc. belong, ethylene and ethylene receptors have been studied to delay fruit ripening, and the ethylene receptor genes of the above plants have been isolated. However, although studies have been made on the ethylene receptor gene and flower senescence in solanaceous plants (Wilkinson, JQ, et al, Nature Biotechnology, vol. 15, 444-447, 1997), the flowering durability The ethylene receptor gene aimed at improvement has not been isolated from Solanaceae plants.
[0008]
On the other hand, when a transgenic plant into which a foreign gene has been introduced is put into practical use and put on the market, it is necessary to confirm the safety as well as the gene in accordance with the “Food Sanitation Law” by the Ministry of Health, Labor and Welfare and the “Revised JAS Law” by the Ministry of Agriculture, Forestry and Fisheries. It is necessary to clearly indicate that it is a recombinant. At present (end of 2001), since this law is applied only to foods, it is considered that this law does not apply to genetic modification of petunia, a flower variety. However, since petunia belongs to solanaceous plants including major vegetables such as eggplant, potato, capsicum, pepper, and tomato, the possibility that a transgene from transgenic petunia is transmitted to vegetables cannot be denied. Therefore, it is desirable to establish a technique for detecting a transgenic product and a recombined gene as a tool for ensuring safety in practical use of the transgenic petunia. Ministry of Health, Labor and Welfare's "Testing Methods for Recombinant DNA Technology-Applied Foods" (partially revised in April and September 2002) and JAS Analysis Test Handbook "Genetically Modified Food Testing and Analysis Manual" prepared by the Agriculture, Forestry and Fisheries Consumer Technology Center According to (April 2002), detection of a gene is described as "by qualitative analysis by PCR". Taking the "test method for foods applied with recombinant DNA technology" as an example, it is necessary to simultaneously prepare a blank reaction solution without a primer and a DNA solution without a DNA sample solution at the same time as a PCR reaction. In addition, to confirm that DNA has been extracted from the sample, it is necessary to similarly perform PCR amplification using a positive control primer pair instead of the recombinant gene detection primer for each DNA sample solution. Furthermore, when a PCR amplification band is detected by the positive control primer pair and the target recombinant gene detection primer pair, a PCR reaction solution is newly prepared using the same DNA sample solution, and the confirmation primer pair (recombination) is prepared. PCR amplification must be performed using a different primer pair than for gene detection). When performing such a test, it is essential that the presence or absence of the PCR amplified fragment can be clearly determined. In addition, when it becomes necessary to test for the presence or absence of a recombinant gene, it is necessary to quickly and reliably determine a large amount of samples.
[0009]
However, since the ethylene receptor gene is also present in a natural plant, in the case of a recombinant plant in which the gene has been re-introduced from the outside, a PCR primer designed to amplify the gene generally does not have a natural non-combination. Amplification products derived from endogenous genes are also detected in transgenic plants. This problem can be circumvented by distinguishing the foreign gene (cDNA) and the endogenous gene (genomic DNA) based on the difference in the length of the amplification products, since the gene introduced from the outside is usually introduced in the form of cDNA. it is conceivable that. However, in this case, since the operation of comparing and determining the length of the amplified fragment by electrophoresis or the like is essential, it is difficult to simply and objectively determine only the operation of determining “the presence or absence of an amplification product”. This also means that mass automated analysis is difficult. Therefore, a method for simply and clearly determining a transformant into which a gene having an endogenous gene has been introduced has been desired.
[0010]
[Patent Document 1] Japanese Patent No. 3183407
[Patent Document 2] Japanese Patent No. 3030015
[0011]
[Problems to be solved by the invention]
The present invention provides a method and means for regulating the flower durability of a Petunia plant, and a method for detecting a transformed plant into which an ethylene receptor gene-like nucleic acid or its antisense nucleic acid, or a partial sequence thereof has been introduced. The purpose is to do.
[0012]
[Means for Solving the Problems]
The present inventors have conducted intensive studies to achieve the above object, and as a result, succeeded in isolating an ethylene receptor gene-like nucleic acid involved in flower persistence from petunia, and prepared a vector containing the nucleic acid or its antisense nucleic acid. It has been found that it is possible to obtain a transformed plant in which flower durability is regulated by transforming the plant using the plant. The present inventors also revealed the exon-exon boundary region of the gene, designed primers and probes containing the nucleotide sequence of this region, and by using the primer or probe, the nucleic acid or the antisense nucleic acid was introduced. It has been found that the transformed plant can be easily and clearly detected.
[0013]
That is, the present invention relates to the following protein (a) or (b).
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
(B) a protein in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and which has an ability to bind to ethylene
[0014]
The present invention is also a nucleic acid encoding the following protein (a) or (b).
(A) a protein consisting of the amino acid sequence shown in SEQ ID NO: 2
(B) a protein in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and which has an ability to bind to ethylene
[0015]
The present invention also relates to the following nucleic acid (a) or (b).
(A) a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1
(B) a nucleic acid consisting of a base sequence having a homology of 96% or more with the DNA consisting of the base sequence shown in SEQ ID NO: 1 and having a function related to ethylene sensitivity
[0016]
The present invention also relates to a nucleic acid containing all or a part of the DNA having the nucleotide sequence shown in SEQ ID NO: 3.
Further, the present invention is an expression vector containing all or a part of the above nucleic acid.
[0017]
The present invention is also an antisense nucleic acid comprising a nucleotide sequence complementary to a DNA comprising the whole or a part of the above nucleic acid. Further, the present invention is an expression vector containing all or a part of the antisense nucleic acid.
[0018]
Further, the present invention is a transformant containing the above nucleic acid or the above antisense nucleic acid. Here, the transformant is not particularly limited, but includes a plant, preferably a flower plant, particularly preferably a plant belonging to the genus Petunia. In addition, the transformant has an enhanced flower durability compared to the wild type.
The present invention is also a seed obtained from the above transformant.
[0019]
Furthermore, the present invention is a method for regulating flower durability of a plant, comprising introducing the nucleic acid or the antisense nucleic acid into a plant.
[0020]
Furthermore, the present invention is a method for producing a transformed plant, which comprises culturing or cultivating the transformant or the seed and regenerating a plant. Here, the plant includes a flower plant, preferably a plant belonging to the genus Petunia.
[0021]
The present invention is also an oligonucleotide comprising a partial nucleotide sequence of the above nucleic acid or antisense nucleic acid.
[0022]
The present invention also provides a boundary region 1 between exon 1 and exon 2, a boundary region 2 between exon 2 and exon 3, and an exon 3 which constitute a cDNA encoding an ethylene receptor gene-like nucleic acid sequence derived from a plant belonging to the genus Petunia. An oligonucleotide comprising a base sequence forming a boundary region selected from the group consisting of boundary region 3 between exon 4 and exon 4 and boundary region 4 between exon 4 and exon 5, or a complementary sequence thereof.
[0023]
In the above oligonucleotide, for example, the bases forming boundary region 1 are thymine 908 and 909 in base sequence shown in SEQ ID NO: 1, and the bases forming boundary region 2 are guanine 1273 and 1274 The bases forming boundary region 3 are thymine 1545 and adenine 1546, and the bases forming boundary region 4 are cytosine 1669 and guanine 1670.
[0024]
Examples of the above-mentioned oligonucleotide include those selected from the group consisting of the following (a) to (d).
(A) an oligonucleotide comprising at least 908 to 909 of the base sequence shown in SEQ ID NO: 1 and comprising a continuous base sequence of 15 or more bases or a complementary sequence thereof
(B) an oligonucleotide comprising at least nucleotides 1273 to 1274 of the nucleotide sequence shown in SEQ ID NO: 1 and comprising a continuous nucleotide sequence of 15 or more nucleotides or a complementary sequence thereof
(C) an oligonucleotide comprising at least 1545 to 1546 bases in the base sequence shown in SEQ ID NO: 1 and comprising a continuous base sequence of 15 or more bases or a complementary sequence thereof
(D) an oligonucleotide comprising at least nucleotides 1669 to 1670 of the nucleotide sequence shown in SEQ ID NO: 1 and comprising a continuous nucleotide sequence of 15 or more nucleotides or a complementary sequence thereof
[0025]
Examples of the oligonucleotide include those selected from the group consisting of the following (a) to (g).
(A) an oligonucleotide comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 4 and consisting of a continuous base sequence of 15 or more bases
(B) an oligonucleotide comprising at least the 16th to 17th nucleotides of the nucleotide sequence shown in SEQ ID NO: 5 and comprising a continuous nucleotide sequence of 15 or more nucleotides
(C) an oligonucleotide comprising at least the 19th to 20th nucleotides of the nucleotide sequence shown in SEQ ID NO: 6 and comprising a continuous nucleotide sequence of 15 or more nucleotides
(D) an oligonucleotide comprising at least the 14th to 15th bases in the base sequence shown in SEQ ID NO: 7 and consisting of a continuous base sequence of 15 or more bases
(E) an oligonucleotide comprising at least the 19th to 20th bases in the base sequence shown in SEQ ID NO: 8 and consisting of a continuous base sequence of 15 or more bases
(F) an oligonucleotide comprising at least the 16th to 17th bases in the base sequence shown in SEQ ID NO: 9 and comprising a continuous base sequence of 15 bases or more
(G) an oligonucleotide comprising at least the 18th to 19th bases of the base sequence shown in SEQ ID NO: 10 and comprising a continuous base sequence of 15 or more bases
[0026]
Furthermore, the present invention is a probe for detecting a plant into which the above-mentioned nucleic acid or the above-mentioned antisense nucleic acid containing the above-mentioned oligonucleotide is introduced.
[0027]
The present invention further provides a method for detecting a plant into which the nucleic acid or the antisense nucleic acid has been introduced, wherein the oligonucleotide probe is reacted with DNA of a test plant to detect a reaction product thereof.
[0028]
Further, the present invention provides a boundary region 1 between exon 1 and exon 2, a boundary region 2 between exon 2 and exon 3, and an exon 3 which constitute a cDNA encoding an ethylene receptor gene-like nucleic acid sequence derived from a plant belonging to the genus Petunia. A primer 1 comprising a base forming one boundary region selected from the group consisting of boundary region 3 between exon 4 and exon 4 and boundary region 4 between exon 4 and exon 5, or another boundary region This is a primer set comprising a combination with a primer 2 containing a base or a complementary sequence thereof, wherein one of primers 1 and 2 is used as a forward primer and the other is used as a reverse primer.
[0029]
In the above primer set, for example, the bases forming boundary region 1 are thymine 908 and 909 in the base sequence shown in SEQ ID NO: 1, and the bases forming boundary region 2 are guanine 1273 and 1274 The bases forming boundary region 3 are thymine 1545 and adenine 1546, and the bases forming boundary region 4 are cytosine 1669 and guanine 1670.
[0030]
As the primer, for example, at least one primer selected from the group consisting of the following (a) to (d) and at least one primer selected from the group consisting of the following (e) to (g): And the like.
(A) a forward primer comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 4 and consisting of a continuous base sequence of 15 bases or more
(B) a forward primer comprising at least the 16th to 17th nucleotides of the nucleotide sequence shown in SEQ ID NO: 5 and consisting of a continuous nucleotide sequence of 15 or more nucleotides
(C) a forward primer comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 6 and consisting of a continuous base sequence of 15 bases or more
(D) a forward primer comprising at least the 14th to 15th nucleotides of the nucleotide sequence shown in SEQ ID NO: 7 and comprising a continuous nucleotide sequence of 15 or more nucleotides
(E) a reverse primer comprising at least the 19th to 20th nucleotides of the nucleotide sequence shown in SEQ ID NO: 8 and consisting of a continuous nucleotide sequence of 15 or more nucleotides
(F) a reverse primer comprising at least the 16th to 17th nucleotides of the nucleotide sequence shown in SEQ ID NO: 9 and comprising a continuous nucleotide sequence of 15 or more nucleotides
(G) a reverse primer comprising at least the 18th to 19th bases of the base sequence shown in SEQ ID NO: 10 and comprising a continuous base sequence of 15 or more bases
[0031]
Furthermore, the present invention is a method for detecting a plant into which the above-described nucleic acid or its antisense nucleic acid has been introduced, comprising amplifying the DNA of a test plant using a primer set and detecting the resulting amplification product.
[0032]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail.
[0033]
The present inventors have focused on ethylene, which is one of the factors involved in flower life, in order to regulate the flower life of plants (for example, plants belonging to the genus Petunia). Ethylene is involved as a plant hormone in promoting flower aging and fruit ripening. Therefore, when the activity of ethylene increases in the plant body, the aging of the flower progresses.
[0034]
The present invention promotes or inhibits the expression of an ethylene receptor gene-like nucleic acid (referred to as Phvc-ers1) in a plant belonging to the genus Petunia, thereby promoting or inhibiting the action of ethylene in the plant, and It is intended to control flower durability.
[0035]
As described above, Phvc-ers1 has a function related to ethylene sensitivity and is involved in senescence of petunia plants. Therefore, a plant transformed so that the expression of endogenous Phvc-ers1 is suppressed has an enhanced flowering life as a result of the inhibition of the action of ethylene.
[0036]
As a technique for suppressing gene expression, a technique of introducing a gene antisense nucleic acid (antisense method), a technique using RNA interference (RNAi), and the like are known. In the antisense method, the expression of the target gene is suppressed by specifically binding the antisense sequence to the sequence of the target gene. The antisense sequence inhibits the expression of the target gene by binding to cellular mRNA or genomic DNA and blocking translation or transcription. As another method, there is a method using cosuppression. Cosuppression refers to the expression of both an introduced gene and an endogenous gene in a plant, for example, when a gene is linked in the sense direction to a downstream of a promoter that causes constitutive and strong expression and introduced into the plant. It is a phenomenon that is suppressed (Montgomery, MK and Fire, A (1998), Trends Genet., 14, 255-8). Accordingly, in the present invention, the endogenous Phvc-ers1 is introduced into a plant by introducing a sense DNA of Phvc-ers1 controlled by a strong promoter, an antisense DNA of its nucleic acid sequence, or a partial sequence thereof into a plant. It suppresses the expression of ers1 and inhibits the action of ethylene to produce plants with enhanced flower durability. In these antisense and cosuppression methods, the higher the homology of the base sequence of the DNA or antisense DNA used to the base sequence of the endogenous ethylene receptor gene, the lower the probability that the expression of that gene is suppressed. Will be higher. Therefore, in order to efficiently enhance the flower durability of a plant, it is desirable to prepare DNA or antisense DNA to be used based on the nucleotide sequence of the endogenous ethylene receptor gene.
[0037]
In addition, RNA interference means that when double-stranded RNA (hereinafter, also referred to as double-stranded RNA) is present in a cell, endogenous mRNA homologous to the base sequence of the RNA is degraded. As a result, this is a phenomenon in which gene expression in the cell is specifically suppressed, and is also referred to as RNA interference or RNAi. This RNA interference was first reported in C. elegans, and subsequently confirmed in Drosophila, plants, mammalian cells, and the like (for example, Hannon, GJ., Nature, 2002, Vol. 418, pp. 244-251). (Review); Elbashir, SM et al., Nature, 2001, vol. 411, p. 494-498; and McCaffrey, AP. Et al., Nature, 2002, vol. 418, p. 38-39. reference). For example, in Drosophila, a method is known in which a hairpin-type double-stranded RNA is produced, and this is recognized as a double-stranded RNA in cells, and endogenous mRNA is degraded and destroyed. Therefore, in the present invention, by introducing a double-stranded RNA (antisense nucleic acid) homologous to Phvc-ers1 into a plant, the expression of endogenous Phvc-ers1 can be specifically regulated using RNA interference. It can also be suppressed.
[0038]
On the other hand, normally, when Phvc-ers1 is introduced into a plant in the sense direction, the expression of Phvc-ers1 is expected to be enhanced. In such a case, the aging of the flower is promoted, and the flower durability is reduced. Plants having reduced flower durability are useful when it is desired to obtain seeds early or when it is desired to accelerate the plant life cycle.
[0039]
Hereinafter, isolation of Phvc-ers1, production of Phvc-ers1 protein, production of an expression vector, and production of a transformant will be described.
[0040]
1. Isolation and identification of Phvc-ers1
(1) Preparation of RNA and cDNA libraries
Phvc-ers1 can be obtained by a known method, for example, RACE (rapid amplification of cDNA ends) -PCR, RT-PCR, screening from a cDNA library, or the like, using total RNA or purified mRNA extracted from plant tissues. be able to. Since Phvc-ers1 is mainly expressed in plant leaves, buds, flowers, and the like, such a tissue can be used as a source of RNA. Although the expression level is low, other tissues can also be used. For example, plant tissues or tissue culture cells aseptically cultured in an MS medium or the like can be used. Since the main object of the present invention is to control the flower life of a petunia plant, which is a flora plant, the plant serving as a source for isolating nucleic acids is particularly a plant that is closely related to petunia. Preferably, but not limited to, plants belonging to the genus Petunia.
[0041]
Examples of plants belonging to the genus Petunia include, for example, P. hybrida species, P. axillaris sp. inflata, P. inflata. violacea species; integrifolia.
[0042]
In the present invention, the term "floral plant" refers to cut flowers, cut leaves or cuttings, pots, flowerbed seedlings, bulbs, flowers and trees, lawns, and the like cultivated for ornamental use.
[0043]
The preparation of RNA can be performed, for example, by using the above-mentioned plant tissue as a material by a commonly used technique. For example, after a plant tissue frozen with liquid nitrogen is ground in a mortar or the like, a crude RNA fraction is extracted and prepared from the obtained ground material by a guanidine thiocyanate method, a cesium chloride method, a cetyltrimethylacetylbromide (CTAB) method, or the like. Then, if necessary, the crude RNA fraction is subjected to poly (A) by an affinity column method using oligo dT-cellulose as a carrier or a method using oligo dT-immobilized latex particles. ⁺ RNA (mRNA) can be obtained. Commercially available kits for preparing total RNA from plant tissues include, for example, RNeasy Plant Mini Kit (manufactured by QIAGEN).
[0044]
Using the thus obtained total RNA or mRNA as a template, oligo-dT primers or primers designed for cloning, and reverse transcriptase, a single-strand cDNA was synthesized by RT-PCR. A double-stranded cDNA is synthesized from the strand cDNA. Next, the resulting double-stranded cDNA is incorporated into an appropriate cloning vector to prepare an expression vector. Then, Escherichia coli or the like is transformed using this expression vector, and a transformant is selected using tetracycline resistance, ampicillin resistance, etc. as an index, whereby a cDNA library can be obtained. When a plasmid is used as a vector, it is necessary to contain a drug resistance gene such as tetracycline or ampicillin. In addition, a cloning vector other than a plasmid, for example, λ phage (λgt11 or the like) can also be used.
[0045]
Alternatively, using the mRNA prepared as described above as a template, a single-stranded cDNA is synthesized using oligo dT20 and reverse transcriptase using a commercially available kit (for example, cDNA Synthesis Kit manufactured by STRATAGENE), and then the single-stranded cDNA is synthesized. A double-stranded cDNA is synthesized from the cDNA. Then, by adding an appropriate adapter or cassette (for example, EcoRI cassette (manufactured by TAKARA)) to the obtained double-stranded cDNA, for example, a cDNA library serving as a template for RACE-PCR can be prepared. As a kit for performing the RACE-PCR method, for example, 5 '/ 3' RACE Kit (manufactured by Roche Diagnostics), 3'-Full RACE Core Set (manufactured by Takara), 5'-Full RACE Core Set (Manufactured by Takara) or the like can be used.
[0046]
(2) Cloning of Phvc-ers1
As a method of cloning Phvc-ers1, a fragment of the nucleic acid is used to clarify an unknown DNA region by RACE-PCR, RT-PCR, PCR, or the like, and finally, full-length cDNA is amplified by PCR, A method of cloning an amplification product containing -ers1 into an appropriate vector is exemplified.
[0047]
A fragment of Phvc-ers1 can be obtained by performing RT-PCR or PCR using a primer (for example, a degenerate primer) designed from the nucleotide sequence of the ethylene receptor gene of another Solanaceae plant. Examples of the ethylene receptor gene of solanaceous plants include tobacco (Nicotiana tabacum; Accession No. AF039921), tomato (Lycopersicon esculentum; Accession No. Z54099), potato (Solonum lycopersicum; Accession No. U38666), and the like. Have been. Here, examples of the template RNA that can be used in RT-PCR or RACE-PCR include the plant-derived total RNA or mRNA prepared in (1) above. Examples of the template DNA that can be used in PCR include the cDNA library prepared in the above (1) and genomic DNA (which can be prepared using DNeasy Plant Mini Kit manufactured by QIAGEN).
[0048]
Examples of the primer include atgggaatcctgtgattgcattgag (SEQ ID NO: 11) as a sense primer for performing RT-PCR, and ttacaractyctttttatagcgttga (SEQ ID NO: 12) as a reverse primer. The above primer is a mixed primer, and r represents A or G, and y represents C or T. However, the present invention is not limited to these primers, and those skilled in the art can design appropriate primers based on the nucleotide sequence of the ethylene receptor gene of the Solanaceae plant.
[0049]
The nucleic acid can be cloned by linking the thus-obtained amplified fragment of Phvc-ers1 to a plasmid vector such as plasmid pBluescript and transforming Escherichia coli.
[0050]
Specifically, vectors that can be used include plasmids pBluescript, pUC18, pUC119, pBR322, and the like. Since the amplified fragment usually has a tendency to add adenine (A) to the end, the vector is easily cloned by cutting with a restriction enzyme that generates blunt ends such as EcoRV and adding a thymine (T) end. Yes (TA cloning method). Alternatively, the amplified fragment of Phvc-ers1 may be re-amplified so that an appropriate restriction enzyme site is introduced, and then the amplified fragment may be inserted into a vector using the restriction enzyme site and cloned.
[0051]
Transformation of Escherichia coli can be performed using a known method. When pBluescript is used as a vector, the resulting transformant becomes ampicillin-resistant, and at the same time, a β-galactosidase activity is deleted by inserting a foreign gene into a β-galactosidase-encoding region. Therefore, a white colony that is ampicillin-resistant and cannot degrade X-gal can be selected as a transformant (blue-white selection). In this manner, a transformant having a plasmid in which Phvc-ers1 is integrated into the vector plasmid pBluescript can be obtained.
[0052]
Further, by culturing the transformant and using a well-known means such as an alkali-SDS method, it is possible to prepare a large amount of a plasmid incorporating Phvc-ers1.
[0053]
(3) Analysis of Phvc-ers1
The nucleotide sequence of Phvc-ers1 is determined using the plasmid vector obtained in the above (2). The nucleotide sequence can be determined by a well-known method such as a die terminator method. Usually, the sequence is determined using an automatic nucleotide sequencer (for example, 310 Genetic Analyzer manufactured by ABI). As a kit for performing a base sequence determination reaction, for example, BigDye ^TM Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by Applied Biosystems) and the like are commercially available.
[0054]
SEQ ID NO: 1 shows the base sequence of Phvc-ers1 of the present invention, and SEQ ID NO: 2 shows the amino acid sequence of a protein encoding Phvc-ers1 (hereinafter, referred to as “Phvc-ers1 protein”). However, there may be some differences in amino acid sequence between plants depending on the cultivar and the like. Further, even in the same plant variety, amino acids may change due to mutation or the like. Therefore, in the present invention, the amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which a plurality (1 to several tens, for example, 1 to 30) of amino acids have been substituted, deleted or added, and Also included in the present invention are proteins having the binding ability.
[0055]
For example, 1 to 10, preferably 1 to 5 amino acids of the amino acid sequence shown in SEQ ID NO: 2 may be deleted, and 1 to 10, preferably 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO: 2 Five amino acids may be added, or the amino acid sequence shown in SEQ ID NO: 2 in which 1 to 10, preferably 1 to 5 amino acids have been substituted with other amino acids may also be used as the Phvc- ers1 protein.
[0056]
In the present invention, the “binding ability with ethylene” refers to the binding ability between ethylene receptor protein and ethylene. This function allows the expression of the ethylene receptor gene in host cells, ¹⁴ It can be confirmed by measuring the bond with C-ethylene.
[0057]
Furthermore, it hybridizes under stringent conditions to a sequence complementary to the DNA consisting of all or part of the base sequence of the above-mentioned Phvc-ers1 DNA, or has a homology with the DNA of 96% or more. And a nucleic acid sequence having a function related to ethylene sensitivity is also included in Phvc-ers1 of the present invention. Stringent conditions refer to conditions under which a specific hybrid is formed and a non-specific hybrid is not formed. In the present invention, the stringent condition relating to Phvc-ers1 refers to a condition under which only DNA having high homology (homology is 90% or more, preferably 95% or more) strongly hybridizes. In the present invention, “homology” refers to homology calculated using a sequence comparison program known in the art. For example, such a sequence comparison program includes a program (http://www.accelrys.com/products/gcg_wisconsin_package/) included in a sequence analysis package of the Genetics Computer Group (GCG). In addition, in the present invention, "a function involved in ethylene sensitivity" refers to an interaction between a nucleic acid (DNA or mRNA) of an endogenous ethylene receptor gene and a nucleic acid. Therefore, the nucleic acid of the present invention causes an interaction between the nucleic acid of the endogenous ethylene receptor gene of interest and the nucleic acid by the antisense method, cosuppression method, RNA interference method and the like, and as a result, the function of the gene product (Ie, ethylene binding ability). More specifically, as the nucleic acid of the present invention, for example, when an antisense DNA is introduced, an antisense RNA capable of specifically binding to mRNA transcribed from an endogenous ethylene receptor gene is produced. Nucleic acid sequences that can be included.
[0058]
The “partial sequence” is a nucleotide sequence of a DNA containing a part of the nucleotide sequence of Phvc-ers1, wherein the DNA encodes a protein capable of binding to ethylene or is involved in ethylene sensitivity. Refers to those having functions.
[0059]
Further, Phvc-ers1 of the present invention is a protein having the amino acid sequence shown in SEQ ID NO: 2, or one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and Is a nucleic acid encoding a protein having the binding ability of
[0060]
Once the nucleotide sequence of the Phvc-ers1 of the present invention is determined, it is subsequently hybridized by chemical synthesis, by PCR using the cDNA or genomic DNA of Phvc-ers1 as a template, or by using a DNA fragment having the nucleotide sequence as a probe. By soybean, Phvc-ers1 of the present invention can be obtained.
[0061]
Phvc-ers1 thus isolated encodes an ethylene receptor gene in a Petunia plant. Therefore, when it is intended to enhance plant flowering using the antisense method or cosuppression method as described above, the DNA or antisense DNA to be introduced is prepared based on the base sequence of Phvc-ers1. Since the sequence homology between the introduced DNA or the antisense DNA and the endogenous ethylene receptor gene is increased, the flower life can be more efficiently and reliably improved as compared with the case where other genetic information is used. Can be enhanced.
[0062]
2. Vector construction, transformation and production of Phvc-ers1 protein
The Phvc-ers1 protein of the present invention can be produced from cells or tissues of the above-mentioned plants (for example, plants of the genus Petunia) by protein purification methods known in the art. Further, it can also be produced by a chemical peptide synthesis method known in the art, for example, a solid phase synthesis method. Alternatively, it can be produced by culturing a transformant transformed with a DNA encoding the Phvc-ers1 protein, and collecting the protein from the resulting culture. A method for producing a protein using such a genetic engineering technique is well known in the art, and will be specifically described below.
[0063]
(1) Preparation of vector
An expression vector for transformation can be obtained by ligating Phvc-ers1 or a part thereof to an appropriate vector. Moreover, a transformant that produces a Phvc-ers1 protein can be obtained by introducing this expression vector into a host so that Phvc-ers1 can be expressed. "Part" refers to a portion of Phvc-ers1 that can be introduced into a host to express Phvc-ers1.
[0064]
A phage or plasmid capable of autonomous propagation in a host is used as the vector. Examples of the plasmid DNA include Escherichia coli-derived plasmids (eg, pBR322, pUC18, pUC19, etc.), Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, etc.), and yeast-derived plasmids (eg, YEp13, YEp24, YCp50, etc.). Examples of the phage DNA include λ phage (λgt10, λgt11, λZAP, etc.). Furthermore, animal viruses such as retrovirus or vaccinia virus, and insect virus vectors such as baculovirus can also be used.
[0065]
In order to insert Phvc-ers1 into a vector, first, a method in which purified DNA is cut with an appropriate restriction enzyme, inserted into an appropriate vector DNA restriction enzyme site or a multiple cloning site, and ligated to a vector is employed. Is done.
[0066]
Phvc-ers1 needs to be incorporated into a vector so that the function of the nucleic acid sequence is exerted. Thus, in addition to the promoter and Phvc-ers1, cis elements such as enhancers, splicing signals, polyA addition signals, selection markers, ribosome binding sequences (SD sequences), and the like can be ligated to the expression vector, if desired. In addition, examples of the selection marker include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, and the like. Furthermore, in addition to vectors capable of autonomous propagation in two or more host microorganisms such as Escherichia coli and yeast, various shuttle vectors can be used. Such a vector can also be cut with the restriction enzyme to obtain a fragment thereof.
[0067]
A known DNA ligase is used to link the DNA fragment and the vector fragment. Then, the DNA fragment and the vector fragment are annealed and then ligated to prepare an expression vector.
[0068]
(2) Transformation
The host used for the transformation is not particularly limited as long as it can express Phvc-ers1. Examples include bacteria (such as Escherichia coli and Bacillus subtilis), yeast, plant cells, animal cells (such as COS cells and CHO cells), and insect cells.
[0069]
When a bacterium is used as a host, it is preferable that the expression vector be capable of autonomous replication in the bacterium and be composed of a promoter, a ribosome binding sequence, Phvc-ers1, and a transcription termination sequence. Further, a gene controlling a promoter may be included. Escherichia coli includes, for example, Escherichia coli DH5α, and Bacillus subtilis includes, for example, Bacillus subtilis. Any promoter can be used as long as it can be expressed in a host such as Escherichia coli. The method for introducing the expression vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. For example, a method using calcium ions, an electroporation method and the like can be mentioned.
[0070]
When yeast is used as a host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, or the like is used. In this case, the promoter is not particularly limited as long as it can be expressed in yeast. The method for introducing the expression vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.
[0071]
When an animal cell is used as a host, monkey cells COS-7, Vero, Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3, human FL cells, and the like are used. As the promoter, SRα promoter, SV40 promoter, CMV promoter and the like are used, and an early gene promoter of human cytomegalovirus may be used. Examples of a method for introducing an expression vector into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.
[0072]
When an insect cell is used as a host, an Sf9 cell or the like is used. Examples of a method for introducing an expression vector into insect cells include a calcium phosphate method, a lipofection method, and an electroporation method.
[0073]
The case where a plant cell is used as a host will be described in the section “3. Production of transformed plant and regulation of flower life” below.
[0074]
(3) Production of Phvc-ers1 protein
In the present invention, the Phvc-ers1 protein can be obtained by culturing the transformant having Phvc-ers1 and collecting from the culture. The term “culture” means any of a culture supernatant, a cultured cell, a cultured cell, and a crushed cell or cell. The method for culturing the transformant of the present invention in a medium is performed according to a usual method used for culturing a host.
[0075]
The culture medium for culturing the transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, and inorganic salts that can be used by the microorganism to efficiently culture the transformant. As long as the medium can be used, either a natural medium or a synthetic medium may be used.
[0076]
As the carbon source, carbohydrates, organic acids, and alcohols are used. As the nitrogen source, ammonium salts of inorganic or organic acids, peptone, meat extract, corn steep liquor, or other nitrogen-containing compounds are used. As the inorganic substance, potassium (I) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like are used.
[0077]
The cultivation is usually performed at 37 ° C. for 6 to 50 hours under aerobic conditions such as shaking culture or aeration and stirring culture. During the culture period, the pH is kept at around 5-7. Adjustment of the pH is performed using an inorganic or organic acid, an alkaline solution or the like. During the culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as needed.
[0078]
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a microorganism transformed with an expression vector using the Lac promoter, isopropyl-β-D-thiogalactoside (IPTG) or the like is used. When culturing a microorganism transformed with an expression vector using the trp promoter, indoleacetic acid is used. (IAA) and the like may be added to the medium.
[0079]
As a medium for culturing a transformant obtained by using an animal cell as a host, a commonly used RPMI1640 medium, a DMEM medium, or a medium obtained by adding fetal calf serum or the like to such a medium is used. Culture is usually performed with 5% CO ₂ Perform in the presence at 37 ° C. for 1-30 days. During the culture, antibiotics such as kanamycin and penicillin may be added to the medium as needed.
[0080]
When the Phvc-ers1 protein is produced in the cells or cells after the culture, the proteins are extracted by disrupting the cells or cells. When the Phvc-ers1 protein is produced outside the cells or outside the cells, the culture solution is used as it is, or the cells or cells are removed by centrifugation or the like. Thereafter, by using a general biochemical method used for isolation and purification of proteins, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, or the like, alone or in appropriate combination, the above-mentioned culture is performed. Can be isolated and purified from Phvc-ers1 protein.
Whether or not the Phvc-ers1 protein has been obtained can be confirmed by SDS-polyacrylamide gel electrophoresis or the like.
[0081]
3. Creation of transgenic plants and regulation of flower life
The whole or part of Phvc-ers1 or its antisense nucleic acid obtained as described in the above section “1. Isolation and identification of Phvc-ers1” is introduced into a plant host to obtain a transformed plant. In the present invention, the “plant or transformant into which Phvc-ers1 or its antisense has been introduced” includes those into which Phvc-ers1 or its antisense nucleic acid, or a partial sequence thereof has been introduced. The partial sequence is a partial sequence of Phvc-ers1 or its antisense nucleic acid, and refers to a sequence that can be introduced into a plant to enhance or suppress the expression of endogenous Phvc-ers1. By enhancing or suppressing the expression of endogenous Phvc-ers1, flower durability can be regulated. Here, “adjustment” refers to increasing or decreasing flower durability. For example, by suppressing the expression of Phvc-ers1 in a plant host, it is possible to produce a transgenic plant with improved flower durability. On the other hand, by enhancing the expression of Phvc-ers1, it is possible to produce a transgenic plant with reduced flower durability.
[0082]
In the present invention, “flower persistence” means the flowering period, that is, the life of the flower. In addition, the expression “increased” or “decreased” in flowering life means that the flowering period is “extended” or “shortened”, respectively, as compared with the flowering life of a natural (wild type) plant.
[0083]
(1) Plant host for transformation
In the present invention, a plant host refers to a plant cultured cell, the whole plant of a cultivated plant, a plant organ (eg, leaf, petal, stem, root, rhizome, seed, etc.), or a plant tissue (eg, epidermis, phloem, soft tissue, Xylem, vascular bundle, etc.). The plant that can be used as a plant host is not particularly limited, but the present invention is based on the present invention that regulates flower durability using an ethylene receptor gene-like nucleic acid derived from a Petunia plant or an antisense nucleic acid thereof. In consideration of the purpose, a plant belonging to the genus Petunia is preferable. Examples of the petunia plants include those described in the section of “Isolation and Identification of Phvc-ers1”.
[0084]
(2) Expression vector
An expression vector containing all or a part of Phvc-ers1 or its antisense nucleic acid can be obtained by ligating (inserting) all or a part of this nucleic acid or its antisense nucleic acid to an appropriate vector. The vector for inserting Phvc-ers1 or its antisense nucleic acid is not particularly limited as long as it can be replicated in a plant host, and examples thereof include plasmid DNA, phage DNA, RNA / DNA virus, and binary vector system. No.
[0085]
Examples of the plasmid DNA include Escherichia coli-derived plasmids (eg, pBR322, pUC18, pUC19, etc.) and Bacillus subtilis-derived plasmids (eg, pBU110, pTP5, etc.). Phage DNA includes λ phage (λgt10, λgt11, λZAP, etc.). Is mentioned. When the transformation is performed using the Agrobacterium method (see below), a binary vector system (pBI121, pGA482, pSMAK251, etc.) can be used.
Phvc-ers1 can be obtained as described in the above section “1. Isolation and identification of Phvc-ers1”.
[0086]
Examples of the antisense nucleic acid of Phvc-ers1 include a nucleic acid having a base sequence complementary to the DNA having the base sequence shown in SEQ ID NO: 1. However, as long as the antisense nucleic acid used in the present invention can be introduced into a plant host to suppress the expression of endogenous Phvc-ers1, a sequence completely complementary to the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 Need not be. Therefore, a nucleic acid which hybridizes with a nucleic acid having a complementary nucleotide sequence to a DNA comprising the entire or partial nucleotide sequence of the nucleotide sequence shown in SEQ ID NO: 1 and suppresses the expression of Phvc-ers1 in the present invention. It can be used as a nucleic acid. Furthermore, as long as the antisense nucleic acid can be introduced into a plant host and suppress or inhibit the expression (translation or transcription) of endogenous Phvc-ers1, a base complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 1 It may be a part of the sequence (partial sequence). In the present invention, when an antisense nucleic acid is formed to form a double strand with mRNA in a living body to suppress the expression in a base sequence-specific manner, the hybridization condition is set to a high homology (a homology of 60 to the sense sequence). %, Preferably 80% or more). In the present invention, a double-stranded RNA that causes RNA interference may be used as the antisense nucleic acid. Methods for making such double-stranded RNA are known in the art along with RNA interference.
The method for synthesizing DNA or RNA is as described in the section “1. Isolation and identification of Phvc-ers1”.
[0087]
In order to insert Phvc-ers1 or its antisense nucleic acid (all or part) into an expression vector, first, purified DNA is cut with an appropriate restriction enzyme, and then an appropriate linker is ligated as necessary. And a method of inserting the vector into a restriction enzyme site or a multiple cloning site of an appropriate vector DNA and ligating the vector to the vector.
[0088]
Phvc-ers1 or its antisense nucleic acid needs to be incorporated into a vector so that its function is exhibited. Therefore, the vector contains, as expression cassettes, (i) a promoter sequence capable of being transcribed in plant cells, (ii) Phvc-ers1 or its antisense nucleic acid downstream of the promoter sequence, and (iii) Phvc-ers1 or its antisense. A terminator sequence containing sequences necessary for termination of transcription and polyadenylation, linked downstream of the nucleic acid, is ligated. To the expression cassette, besides a promoter, a DNA sequence for further promoting transcription, for example, a cis element such as an enhancer sequence, a splicing signal, a polyA addition signal, a selection marker, a ribosome binding sequence (SD sequence) and the like are linked. Can be.
[0089]
The promoter used is not particularly limited as long as it functions in a plant. For example, a promoter for constitutive expression such as the 35S promoter or an inducible promoter can be used.
[0090]
Further, if necessary, a terminator for instructing the termination of transcription can be linked to the downstream of Phvc-ers1 or its antisense nucleic acid. Examples of the terminator include a cauliflower mosaic virus-derived terminator and a nopaline synthase gene terminator. However, the terminator is not limited to this as long as it is known to function in plants.
[0091]
Furthermore, it is desirable to link an effective selectable marker gene to an expression vector in order to efficiently select a target transformed plant. The selection markers used at this time include a kanamycin resistance gene (NTPII), a hygromycin phosphinothricin acetyltransferase (http) gene that confers resistance to the antibiotic hygromycin to plants, and resistance to bialaphos (bialaphos). And the phosphinothricin acetyltransferase (bar) gene to be conferred.
[0092]
(3) Production of transgenic plants
The transformed plant of the present invention can be obtained by introducing the expression vector described in the above (2) into a plant host so that Phvc-ers1 or its antisense nucleic acid linked to the vector can function. .
[0093]
The above-described expression vector can be introduced into a plant host using various known methods. For example, an indirect introduction method using Agrobacterium or a direct introduction method such as an electroporation method, a polyethylene glycol method, or a particle gun method can be used. Among them, the method using Agrobacterium is extremely effective for dicotyledonous plants belonging to the genus Petunia because it can surely perform stable transformation. In this method, a T-DNA region which is a part of the Ti plasmid carried by Agrobacterium is transferred and integrated into a plant genome. For transfer and integration of T-DNA, imperfect repeats of 25 base pairs at both ends of T-DNA are essential. Therefore, the target DNA can be incorporated into the plant genome by inserting the foreign DNA between the boundary sequences. In addition, transfer of the T-DNA region requires a gene product of the Vir region on the Ti plasmid (plant gene manipulation manual, edited by Hirofumi Uchimiya et al., Kodansha Scientific, 1984).
[0094]
For example, when the Agrobacterium method is used, the constructed expression vector (for example, a binary vector such as pBI121 having a T-DNA region) is transferred to a suitable Agrobacterium, for example, Agrobacterium tumefaciens. C58, LBA4404, EHA101, C58C1Rif ^R , EHA105, etc., into Agrobacterium rhizogenes LBA9402, etc., using a freeze-thaw method, an electroporation method, or the like. Subsequently, the Agrobacterium strain is infected to a sterile culture of a plant host according to a floral dip method, a leaf disk method, or the like to obtain a transformed plant. The Agrobacterium infection method includes an intermediate vector method, a binary vector method, and the like. In the present invention, gene transfer can be performed using any of the infection methods.
[0095]
When the particle gun method is used, plants, plant organs or plant tissues, or sections thereof, or various samples such as callus and protoplasts can be used. The sample thus prepared is treated with a gene introduction device (for example, PDS-1000 (BIO-RAD)) to introduce DNA into a plant host. The treatment conditions vary depending on the plant or the sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.
[0096]
When a plant culture cell is used as a host, the transformation is performed by introducing an expression vector into the culture cell by a particle gun method, an electroporation method, or the like.
[0097]
From the viewpoint of recombination efficiency and ease of handling, as a preferred example of the present invention, production of a transformed plant by the Agrobacterium infection method using a binary vector system will be specifically described below.
[0098]
When DNA is introduced by the Agrobacterium infection method, a step of infecting a plant host with Agrobacterium having a plasmid containing Phvc-ers1 is required.
[0099]
When this step is performed by an in planta transformation method such as a floral dipping method, a plant host to be transformed is grown, and a solution in which Agrobacterium containing a plasmid having Phvc-ers1 or a plasmid having the antisense nucleic acid is suspended. The flower buds of the plant host are directly immersed and then the plants are grown to harvest the seeds. Then, in order to select an individual having the introduced DNA, the obtained seed is sown on an MS agar medium supplemented with an appropriate antibiotic. Transplanted plants into which Phvc-ers1 of the present invention or its antisense nucleic acid has been introduced can be obtained by transplanting the plants grown in this medium into pots and growing them (Plant Genomics Research Protocol, Takuji Sasaki et al. Supervision, Shujunsha, 2001).
[0100]
On the other hand, when the step of infecting a plant host with Agrobacterium is performed by the leaf disk method, a leaf disk collected from aseptic leaves of a plant host aseptically cultivated is used to carry a plasmid containing Phvc-ers1 or its antisense nucleic acid. After immersion in a culture solution of Agrobacterium to be grown, the cells are cultured in a foliage differentiation medium to form callus and proliferate. As a foliage differentiation medium, for example, a medium obtained by adding a plant hormone to a known medium such as an MS medium can be used. Thereafter, callus is selected using a foliage differentiation medium for selection. As a medium for selection, a medium obtained by further adding a bactericide such as kanamycin for selection of a transformant and a bactericide such as cefotaxime for killing Agrobacterium may be used in addition to the foliage differentiation medium described above. .
[0101]
Calli, shoots, hairy roots, and the like obtained as a result of the transformation can be directly used for cell culture, tissue culture, or organ culture. In addition, a plant can be regenerated by administering a plant hormone (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.) at an appropriate concentration using a conventionally known plant tissue culture method.
[0102]
Whether or not Phvc-ers1 or its antisense nucleic acid has been introduced into a plant can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, or the like. For example, DNA is prepared from a transformed plant, a DNA-specific primer is designed, and PCR is performed. Thereafter, the amplification product was subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, or capillary electrophoresis, stained with ethidium bromide, SYBR Green solution, etc., and transformed by detecting the amplification product. Can be confirmed. Alternatively, amplification products can be detected by performing PCR using primers that have been labeled with a fluorescent dye or the like in advance. Furthermore, a method of binding the amplification product to a solid phase such as a microplate and confirming the amplification product by fluorescence or an enzyme reaction can also be adopted.
[0103]
The transformed plant in which the expression of Phvc-ers1 has been enhanced can be used when it is desired to obtain seeds quickly or to accelerate the life cycle of the plant. In addition, a transformed plant in which the expression of Phvc-ers1 has been suppressed has high flower durability, and is therefore of high commercial value as an ornamental floral plant. In addition, Phvc-ers1 or its antisense nucleic acid to be introduced into the transformed plant of the present invention is prepared from information on an ethylene receptor gene-like nucleic acid endogenously present in a Petunia plant. When using Phvc-ers1 or its antisense nucleic acid, it is possible to more efficiently and surely increase flower life than when conventional ethylene receptor gene information is used. .
[0104]
4. Plants with controlled flower durability
The transformed plant in which the expression of Phvc-ers1 is suppressed, which has been produced according to the section “3. Production of transformed plant and regulation of flower durability”, has enhanced flower persistence, and expression of the nucleic acid. The transgenic plant in which has been enhanced has reduced flower durability.
[0105]
The evaluation of the flower durability of the transformed plant was performed by cultivating a transformed plant into which Phvc-ers1 or its antisense nucleic acid was introduced and a non-transformed plant (wild-type plant) under the same conditions, and from flowering to flowering. This can be done by comparing the periods up to. For example, the petunia flower durability test can be performed by the following method. That is, a flower is used for a test up to two days after flowering, a stem is cut off at about 3 cm below the flower neck, and the flower part is placed in a flask containing water (for example, a 100 ml Erlenmeyer flask). Is placed in a 25 ° C. artificial air chamber (16-hour light period, 8-hour dark period), and changes in petals are observed thereafter. In addition, detailed description of the method of evaluating flower durability can be found in, for example, a manual for maintaining freshness of cut flowers (supervised by the Japan Flower Spread Center, Distribution System Research Center, 1997), maintaining freshness of cut flowers (city) See Kazuo Mura, Tsukuba Shobo, 2000).
[0106]
In addition, using the procedures for differentiation / induction of plants from Agrobacterium-infected cells described above, tissues (eg, roots, stems, leaves) or organs (eg, growth points, pollen) of the transformed plants. Can be used to obtain a further transformed plant without going through the reproductive process (seed). Such techniques and procedures are known to those skilled in the art, and general methods for tissue culture are described in various laboratory manuals.
[0107]
Seeds obtained from the thus-produced transformed plant of the present invention also have normal germination and growth, and their flowering properties are regulated. This indicates that the introduced Phvc-ers1 or its antisense nucleic acid is conserved in the next generation, thus indicating that this regulated flower persistence is stably passed on to progeny. Therefore, according to the present invention, a practical and useful plant having improved (or reduced) flower durability can be obtained.
[0108]
5. Oligonucleotide
(1) Overview
The ethylene receptor gene-like nucleic acid (Phvc-ers1) is a nucleic acid endogenously present in a non-transformant (wild-type plant). The present inventor designed an oligonucleotide containing a base sequence corresponding to the following boundary region in SEQ ID NO: 1 to detect a transformed plant into which Phvc-ers1 or its antisense nucleic acid was introduced, and used it as a primer. Alternatively, it was used as a probe.
[0109]
Embedded image

In the present invention, the term “boundary region” (also referred to as a boundary site) refers to the case where two exons are separated from each other across an intron on the genome and the two exons are joined after the intron is spliced. Point of attachment (boundary point), that is, a splice site (FIG. 2).
[0110]
In the cDNA of Phvc-ers1 shown in FIG. 2, the boundary between exon 1 and exon 2 is boundary region 1, the boundary between exon 2 and exon 3 is boundary region 2, the boundary between exon 3 and exon 4 is boundary region 3, The boundary between exon 4 and exon 5 is called boundary region 4. Therefore, boundary region 1 is formed by the 3'-most base of exon 1 and the 5'-base of exon 2. Specifically, the 3'-most base of exon 1 is the 908th thymine of the base sequence shown in SEQ ID NO: 1, and the 5'-most base of exon 2 is the base sequence shown in SEQ ID NO: 1. Is the 909th thymine. In addition, the boundary region 2 is formed by the most 3 ′ base of exon 2 and the most 5 ′ base of exon 3, and specifically, the most 3 ′ base of exon 2 is represented by SEQ ID NO: 1. The 1273rd guanine of the nucleotide sequence shown in SEQ ID NO: 1 is the 1273th guanine of the nucleotide sequence shown, and the 5'-most base of exon 3 is the 1274th adenine of the nucleotide sequence shown in SEQ ID NO: 1. The boundary region 3 is formed by the 3'-most nucleotide of exon 3 and the 5'-most nucleotide of exon 4. Specifically, the 3'-most nucleotide of exon 3 is represented by SEQ ID NO: 1. It is the 1545th thymine in the base sequence, and the 5'-most base of exon 4 is the 1546th adenine in the base sequence shown in SEQ ID NO: 1. Further, the boundary region 4 is formed by the most 3 ′ base of exon 4 and the most 5 ′ base of exon 5, and specifically, the most 3 ′ base of exon 4 is represented by SEQ ID NO: 1. It is the 1669th cytosine in the base sequence, and the 5'-most base of exon 5 is the 1670th guanine in the base sequence shown in SEQ ID NO: 1.
[0111]
(2) Oligonucleotide
The oligonucleotide of the present invention contains the sequence of the region on both sides (for example, exon 1 and exon 2) sandwiching one boundary region as described above, or its complementary sequence. Therefore, as shown in FIG. 2, the oligonucleotides DG and HK hybridize to the boundary region based on their complementarity. In the present invention, "hybridization" or "hybridization" used for an oligonucleotide refers to specific binding to a detection target Phvc-ers1 cDNA based on its complementarity. Oligonucleotides D to G have a sequence complementary to the Phvc-ers1 cDNA to be detected, and hybridize with the introduced Phvc-ers1 or a partial sequence thereof. On the other hand, the oligonucleotides HK have a sequence homologous to the Phvc-ers1 cDNA to be detected, and hybridize with the introduced Phvc-ers1 antisense nucleic acid or a partial sequence thereof.
[0112]
The sequence on both sides of the boundary region (boundary region), that is, the sequence forming the boundary region (particularly, the sequence on both sides of the boundary portion) or its complementary sequence is hereinafter referred to as “base sequence of the boundary region”.
[0113]
The oligonucleotide of the present invention contains the base sequence of the boundary region as described above, and can hybridize only with exogenously introduced Phvc-ers1 cDNA or its antisense nucleic acid (cDNA).
[0114]
Therefore, when hybridization is performed using an oligonucleotide probe containing the nucleotide sequence of such a boundary region, hybridization of an endogenous gene derived from genomic DNA of a wild-type plant (non-transformant) does not occur. Hybridization occurs only in the case of a transformed plant into which Phvc-ers1 cDNA or its antisense nucleic acid has been introduced, and is detected. Therefore, when detecting a transformed plant into which Phvc-ers1 or its antisense nucleic acid has been introduced using the oligonucleotide of the present invention, only the operation of detecting whether hybridization occurs may be performed. Therefore, by using the oligonucleotide of the present invention, it is possible to simply and clearly detect a transformed plant into which Phvc-ers1 or its antisense nucleic acid has been introduced.
[0115]
(3) Primer set
When the oligonucleotide of the present invention is used as a primer, the primer set of the present invention comprises a region on both sides (for example, exon 1 and exon 2) sandwiching one boundary region described in the above section “(1) Outline”. And a primer containing the nucleotide sequence of the region on both sides (for example, exon 3 and exon 4) sandwiching the other boundary region. Therefore, as shown in FIG. 3, a combination of any of the forward primers L, M or N and any of the reverse primers O, P or Q constitutes one primer set. When the forward primer L and the reverse primer O are combined, an amplification product LO is obtained, and when the forward primer L and the reverse primer P are combined, the amplification product LP is obtained. When the primer M and the reverse primer P are combined, an amplification product MP is obtained. When the forward primer M and the reverse primer Q are combined, an amplification product MQ is obtained. When N and reverse primer Q are combined, amplification product NQ is obtained, and when forward primer L and reverse primer Q are combined, amplification product LQ is obtained (FIG. 3).
[0116]
As shown in FIG. 4A, when a primer containing the base sequence of the boundary region of the present invention is prepared using DNA in the 3 ′ to 5 ′ direction as a template, this primer sequence is located 5 ′ from the splice site. (Hereinafter referred to as 5 'side arrangement) and 3' side (hereinafter referred to as 3 'side arrangement). In designing primers, the 3 'sequence is first determined. The 3'-side sequence is designed to be a sequence of about 2 to 8 bases from the boundary region of Phvc-ers1 cDNA to be amplified. The primer may be homologous or complementary to the template DNA. In general, a forward primer has a sequence homologous to the template DNA, and a reverse primer has a sequence complementary to the template DNA. On the other hand, the 5'-side sequence is designed after determining the length of the 5'-side sequence, with priority given to adjusting the length of the primer based on the Tm of the oligonucleotide. Therefore, the 5'-side sequence is designed to have a sequence of about 10 to 40 bases corresponding to the boundary region of the Phvc-ers1 cDNA to be amplified. The thus designed primer containing the nucleotide sequence of the boundary region can anneal to Phvc-ers1 cDNA and amplify it (FIG. 4A).
[0117]
Subsequently, a case where the Phvc-ers1 genomic DNA is used as a template will be described (FIG. 4B). Since genomic DNA contains exons and introns, border regions are separated by introns. Therefore, the 5′-side sequence of the primer of the present invention can anneal to a part of the divided boundary region of genomic DNA. However, since the 3 'side sequence does not contain a sequence corresponding to the intron sequence, it cannot be annealed to the intron of the genomic DNA (FIG. 4B (1)). Annealing of the 3 'side sequence is important for amplification by a DNA polymerase, and therefore, if the 3' side sequence cannot be annealed, genomic DNA will not be amplified. Further, since the 3 'side sequence of the primer of the present invention is a very short sequence of about 2 to 8 bases, it is not possible to anneal to genomic DNA only with the 3' side sequence (FIG. 4B (2)). Therefore, the primer containing the nucleotide sequence of the boundary region of the present invention cannot amplify Phvc-ers1 genomic DNA.
[0118]
As described above, the primer containing the base sequence of the boundary region of the present invention can specifically amplify only the exogenously introduced Phvc-ers1 cDNA or its antisense nucleic acid (cDNA).
[0119]
Therefore, when an amplification reaction is performed using a primer set containing the base sequence of such a boundary region, amplification of an endogenous gene derived from genomic DNA of a wild-type plant (non-transformant) does not occur, and the Phvc- Amplification occurs only in the case of a transformed plant into which ers1 cDNA or its antisense nucleic acid has been introduced, and the amplification product is detected. Therefore, when detecting a transformed plant into which Phvc-ers1 or its antisense nucleic acid has been introduced using the primer set of the present invention, it is sufficient to perform only the operation of detecting whether an amplification product is produced. As described above, the operation of discriminating the difference between the lengths of the amplified fragments of genomic DNA and cDNA by electrophoresis becomes unnecessary. Therefore, by using the primer set of the present invention, it is possible to easily and clearly detect a transformed plant into which Phvc-ers1 or its antisense nucleic acid has been introduced.
[0120]
(4) Determination of genomic DNA and splice sites
In order to design an oligonucleotide (primer or probe) containing the base sequence of the boundary region, first, the genomic DNA sequence of Phvc-ers1 and a splice site (intron / exon boundary region) are determined.
[0121]
The genomic DNA of Phvc-ers1 can be prepared by amplifying the whole genomic DNA derived from petunia as a template using primers designed based on the nucleotide sequence of Phvc-ers1. Subsequently, the base sequence of the thus amplified Phvc-ers1 genomic DNA is determined. By comparing the nucleotide sequence of this genomic DNA with the cDNA sequence of Phvc-ers1 (for example, SEQ ID NO: 1), an intron sequence and a splice site (intron / exon boundary region) can be specified. The base sequence comparison can be performed using a sequence comparison program known in the art, and such a program includes, for example, GENETIX-MAC 10.1 (Software Development Co., Ltd.).
[0122]
The intron sequence is preferably determined by decoding the sequence of both the sense strand and the antisense strand of Phvc-ers1. If the full length of the intron sequence cannot be determined because the genomic DNA is too long, the full length sequence may be determined using the primer walking method. The primer walking method is a nucleotide sequencing method known in the art, and the first sequencing is performed from the end of the entire nucleotide sequence to be determined to a region of about 500 to 650 bp (however, the nucleotide sequencing to be used). (Depending on the capacity of reagents, equipment, etc.) and then design primers using the first analysis information, and perform the second sequencing in the same manner using the primers It is. By repeating this partial sequencing operation, the full-length sequence can be determined.
[0123]
The genomic DNA of Phvc-ers1 determined according to the method described above is shown in SEQ ID NO: 3. The ORF region has 3782 bases, of which the cDNA sequence has 1911 bases (SEQ ID NO: 1). FIG. 1 shows a comparison of the splice site between Phvc-ers1 (FIG. 1A) and the published 2185 base Arabidopsis ERS1 gene (FIG. 1B; GenBank Accession Number U21952). As shown in FIG. 1, the splice site (boundary region) of Phvc-ers1 is located between 908 (thymine) and 909 (thymine) and 1273 (guanine) of the cDNA sequence of Phvc-ers1 (SEQ ID NO: 1). And No. 1274 (adenine), No. 1545 (thymine) and No. 1546 (adenine), and No. 1669 (cytosine) and No. 1670 (guanine). These boundary areas correspond to boundary area 1, boundary area 2, boundary area 3 and boundary area 4 shown in FIGS. 2 and 3, respectively.
[0124]
(5) Design of oligonucleotide
An oligonucleotide including the base sequence of the boundary region (boundary regions 1 to 4) determined according to the above (4) is designed. As shown in FIG. 2, eight kinds of oligonucleotides to be designed, namely, boundary region 1 (D and H), boundary region 2 (E and I), boundary region 3 (F and J), and boundary Oligonucleotides containing the nucleotide sequence of region 4 (G and K) are exemplified.
[0125]
Conditions for designing oligonucleotides can be easily determined by those skilled in the art. For example, when designing a probe, an oligo that can be used as a probe according to the instruction attached to a kit such as, but not limited to, DIG Oligonucleotide 3 'End Labeling Kit manufactured by Roche Diagnostics, Inc. Nucleotides can be designed. The method of designing an oligonucleotide that can be used as a primer will be described in detail in (7) below.
[0126]
The length of the oligonucleotide designed as a probe is about 12 to 40 bases in total, and preferably 15 to 30 bases. The oligonucleotide used as a probe may be a DNA probe or an RNA probe as long as it can hybridize with the cDNA of Phvc-ers1 based on complementarity.
[0127]
Oligonucleotide synthesis methods are also well known in the art. For example, a general oligonucleotide synthesis method such as a phosphoramidite method can be used.
[0128]
Oligonucleotides of the invention include, for example:
(A) an oligonucleotide comprising at least bases 908 to 909 of the base sequence shown in SEQ ID NO: 1 and consisting of a continuous base sequence of 15 or more bases or a complementary sequence thereof (corresponding to boundary region 1)
(B) an oligonucleotide comprising at least nucleotides 1273 to 1274 of the nucleotide sequence shown in SEQ ID NO: 1 and comprising a continuous nucleotide sequence of 15 or more nucleotides or a complementary sequence thereof (corresponding to boundary region 2)
(C) an oligonucleotide comprising at least 1545 to 1546 bases in the base sequence shown in SEQ ID NO: 1 and consisting of a continuous base sequence of 15 or more bases or a complementary sequence thereof (corresponding to boundary region 3)
(D) an oligonucleotide comprising at least bases 1669 to 1670 of the base sequence shown in SEQ ID NO: 1 and consisting of a continuous base sequence of 15 or more bases or a complementary sequence thereof (corresponding to boundary region 4)
[0129]
More specifically, the following oligonucleotides (a) to (g) are exemplified.
(A) an oligonucleotide containing at least the 19th to 20th bases in the base sequence shown in SEQ ID NO: 4 and consisting of a continuous base sequence of 15 bases or more (corresponding to boundary region 1)
(B) an oligonucleotide comprising at least the 16th to 17th bases of the base sequence shown in SEQ ID NO: 5 and consisting of a continuous base sequence of 15 or more bases (corresponding to boundary region 2)
(C) an oligonucleotide comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 6 and consisting of a continuous base sequence of 15 bases or more (corresponding to boundary region 2)
(D) an oligonucleotide comprising at least the 14th to 15th bases of the base sequence shown in SEQ ID NO: 7 and consisting of a continuous base sequence of 15 bases or more (corresponding to boundary region 3)
(E) an oligonucleotide comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 8 and consisting of a continuous base sequence of 15 or more bases (corresponding to boundary region 1)
(F) an oligonucleotide containing at least the 16th to 17th bases in the base sequence shown in SEQ ID NO: 9 and comprising a continuous base sequence of 15 or more bases (corresponding to boundary region 2)
(G) an oligonucleotide comprising at least the 18th to 19th bases of the base sequence shown in SEQ ID NO: 10 and comprising a continuous base sequence of 15 or more bases (corresponding to boundary region 3)
[0130]
Subsequently, it is tested whether or not the oligonucleotide designed as described above can specifically hybridize to the cDNA of Phvc-ers1 without hybridizing to the genomic DNA of Phvc-ers1. In the present invention, it is confirmed that (A) the designed oligonucleotide hybridizes with cDNA and (B) does not hybridize with genomic DNA. Such tests are well known in the art and can be conveniently performed, for example, by utilizing a kit such as a TaqMan® probe from Applied Biosystems to confirm the function of the oligonucleotide.
[0131]
(6) Hybridization
The hybridization method of the present invention uses the DNA of a test plant as a template to detect a transformant into which an ethylene receptor gene-like nucleic acid sequence (Phvc-ers1) or its antisense nucleic acid or a partial sequence thereof has been introduced. Hybridization is performed using the oligonucleotide designed in (5) above, and when hybridization is detected, it is determined that the test plant is a transformed plant into which Phvc-ers1 or its antisense nucleic acid has been introduced. Is what you do.
[0132]
In the present method, hybridization occurs only when Phvc-ers1 or its antisense nucleic acid (cDNA) is present. Therefore, when trying to detect a transformed plant by this method, the presence or absence of hybridization may be detected. As the test plant used in the present method, any plant may be used as long as it is a plant which is to detect whether Phvc-ers1 or its antisense nucleic acid has been introduced, and a plant of the genus Petunia is particularly preferable. DNA can be extracted from the test plants using methods well known in the art.
[0133]
As described above, any method of the hybridization reaction that can be used in the present invention may be any method as long as the oligonucleotide probe hybridizes with only the DNA to be detected and can be detected. For example, the TaqMan method utilizes the 5'-3 'exonuclease activity of a DNA polymerase (e.g., AMPLITAQ DNA polymerase), and comprises an oligonucleotide probe having a 5' reporter dye (e.g., a fluorescent dye) and a 3 'quencher dye. Is introduced into a PCR reaction, and in the PCR process, when the probe hybridizes to the target sequence, the probe is caused to move between the reporter dye and the quencher dye by the 5′-3 ′ polynucleotide degrading activity of AMPLITAQ DNA polymerase. And as a result, the fluorescence increases due to the separation of the reporter dye from the quencher dye, and the signal is detected using a fluorescence detector.
[0134]
For example, the above hybridization reaction can be easily performed using a commercially available kit. Such kits include Taq Man® probes (Applied Biosystems) and the like.
[0135]
A known means capable of specifically recognizing hybridization can be used for the detection of hybridization. For example, hybridization can be detected by labeling and using an oligonucleotide probe.
[0136]
When hybridization is detected as described above, the test plant is determined to be a transformed plant into which the Phvc-ers1 gene or its antisense nucleic acid, or a partial sequence thereof has been introduced.
[0137]
(7) Primer design
A primer containing the base sequence of the boundary region (boundary regions 1 to 4) determined according to the above (4) is designed. As the primers to be designed, as shown in FIG. 3, three types of forward primers, namely, a primer containing the base sequence of boundary region 1 (L), boundary region 2 (M) and boundary region 3 (N), There are various types of reverse primers, that is, primers containing the base sequences of boundary region 2 (O), boundary region 3 (P) and boundary region 4 (Q).
[0138]
The conditions for designing primers are (A) the chain length from the splice site to the 3 'end (length of the 3' side sequence), and (B) the melting temperature (Tm) of the primer.
[0139]
(A) Chain length from splice site to 3 'end
When the primer sequence has a long chain length from the splice site to the 3 'end, it is considered that the possibility that the 3' side sequence of the primer will anneal to genomic DNA is also increased. Therefore, it is preferable to design the chain length from the splice site to the 3 'end to be 2 to 8 bases.
[0140]
(B) Tm (melting temperature)
In designing, it is preferable to confirm the Tm of the primer. Tm means a temperature at which 50% of an arbitrary oligonucleotide forms a hybrid with its complementary strand. In order for a template DNA and a primer to form a double strand and to effectively cause a priming reaction, annealing is required. Temperature needs to be optimized. On the other hand, if the annealing temperature is too low, non-specific amplification occurs, so it is desirable that the annealing temperature be as high as possible. Therefore, the Tm of a primer is an important factor in performing an amplification reaction. Tm can be determined, for example, according to the following equation:
Tm (° C.) = 81.5 + 16.6 × log ₁₀ [S] + 0.41 x (% GC)-(500 / n)
[Wherein [S] represents the molar concentration (M) of the salt, (% GC) represents the GC content (%) in the primer sequence, and n represents the length (bp) of the primer. ]
In the present invention, a simple method of multiplying the total number of G and C in the primer sequence by 4 and multiplying the total number of A and T by 2 and setting the sum to Tm can also be used.
[0141]
For confirmation of Tm, known primer design software can be used. Examples of primer design software that can be used in the present invention include Oligo 5.0 (National Biosciences) and HYBsimulator version 4 (Advanced Gene). Computing Technologies, Inc.). In the present invention, the average Tm is preferably about 50 to 60 ° C.
[0142]
The length of the primer to be designed is a total of about 12 to 40 bases, preferably 15 to 30 bases, including the 5'-side sequence and the 3'-side sequence. Also, methods for synthesizing primers are well known in the art. For example, a general oligonucleotide synthesis method such as a phosphoramidite method can be used.
[0143]
The primers of the present invention include, for example, the following:
(A) A forward primer (corresponding to boundary region 1) comprising at least the 19th to 20th bases in the base sequence shown in SEQ ID NO: 4 and consisting of a continuous base sequence of 15 bases or more
(B) a forward primer (corresponding to boundary region 2) comprising at least the 16th to 17th bases of the base sequence shown in SEQ ID NO: 5 and consisting of a continuous base sequence of 15 bases or more
(C) a forward primer (corresponding to boundary region 2) comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 6 and consisting of a continuous base sequence of 15 bases or more
(D) a forward primer (corresponding to boundary region 3) comprising at least the 14th to 15th bases in the base sequence shown in SEQ ID NO: 7 and consisting of a continuous base sequence of 15 bases or more
(E) a reverse primer comprising at least the 19th to 20th nucleotides of the nucleotide sequence shown in SEQ ID NO: 8 and consisting of a continuous nucleotide sequence of 15 nucleotides or more (corresponding to boundary region 1)
(F) A reverse primer comprising at least the 16th to 17th bases of the base sequence shown in SEQ ID NO: 9 and consisting of a continuous base sequence of 15 bases or more (corresponding to boundary region 2)
(G) a reverse primer (corresponding to boundary region 3) comprising at least the 18th to 19th bases of the base sequence shown in SEQ ID NO: 10 and consisting of a continuous base sequence of 15 bases or more.
[0144]
Among the above primers, particularly preferred combinations of primer sets in the present invention are as follows:
・ Amplifier between primer (a) and primer (e) boundary region 1-2
・ Amplification between primer (a) and primer (f) boundary regions 1 to 3
-Primer (b) and primer (f) are amplified between the boundary regions 2 and 3.
・ Primer (c) and primer (g) Amplify between boundary areas 2 and 4
・ Primer (d) and primer (g) Amplify between boundary areas 3 and 4
[0145]
(8) Confirmation of designed primer
Subsequently, a confirmation test of the primer designed in the above (7) is performed. The primer of the present invention must specifically amplify only Phvc-ers1 or its antisense nucleic acid (ie, cDNA) introduced into the transformant. Therefore, using the primers designed in (7) above, it is tested whether the cDNA of Phvc-ers1 can be specifically amplified without amplifying the genomic DNA of Phvc-ers1.
[0146]
In the present invention, it is confirmed that the designed primers (A) amplify using cDNA as a template and (B) do not amplify using genomic DNA as a template.
[0147]
(A) Amplification test using cDNA as template
The present inventors have determined that when a specific amplification product is obtained by performing an RT-PCR amplification reaction using the mRNA of Phvc-ers1 as a template, the primer can specifically amplify the Phvc-ers1 cDNA.
[0148]
Therefore, in the confirmation test (A), RT-PCR is performed using mRNA derived from a plant belonging to the genus Petunia as a template and the primers designed in (7) above. The method for preparing the mRNA is as described in the above section “1. Isolation and identification of Phvc-ers1”. RT-PCR can be easily performed using a commercially available kit. Examples of such a kit include One Step RT-PCR Kit manufactured by QIAGEN and Enhanced Avian HS RT-PCR Kit manufactured by SIGMA.
[0149]
The RT-PCR conditions were as follows: 50 ° C. for 30 minutes, 94 ° C. for 15 minutes, 94 ° C. for 1 minute, 50.0 to 70.5 ° C. for 1 minute, and 72 ° C. for 1 minute 30 seconds. Are performed for 35 cycles, and the elongation step is performed at 72 ° C. for 15 minutes, followed by 4 ° C. hold. Here, the temperature indicated as 50.0 ° C. to 70.5 ° C. is set in several steps, for example, every 5 ° C., and the optimum temperature can be determined by examining the optimum conditions for the reaction.
As a result of the RT-PCR reaction, when only one band of the size of the cDNA of Phvc-ers1 is detected, it is determined that the specific amplification reaction has occurred.
[0150]
(B) Amplification test using genomic DNA as template
Using the primers designed in (7) above, confirm that no amplification product is detected when a PCR amplification reaction is performed using Phvc-ers1 genomic DNA as a template.
[0151]
Accordingly, in the confirmation test (B), PCR is performed using the genomic DNA derived from a plant belonging to the genus Petunia as a template and the primers designed in (7) above. Methods for preparing DNA are known in the art, and include methods for performing phenol extraction and ethanol precipitation, methods using glass beads, and the like. PCR can be easily performed using a commercially available kit. Examples of such a kit include AmpliTaq Gold (registered trademark) PCR Master Mix manufactured by Applied Biosystems and Let's Go PCR Kit manufactured by Sawasdee Technology.
[0152]
The PCR conditions were as follows: after 30 minutes at 94 ° C., 30 cycles of an amplification step of 94 ° C. for 1 minute, 50.0 to 70.5 ° C. for 1 minute, and 72 ° C. for 1 minute and 30 seconds. After a 15-minute extension step, hold at 4 ° C. Here, the temperature indicated as 50.0 ° C. to 70.5 ° C. is set in several steps, for example, every 5 ° C., and the optimum temperature can be determined by examining the optimum conditions for the reaction.
[0153]
When no amplification product is detected as a result of the PCR reaction, it is determined that the amplification reaction using the genomic DNA as a template has not been performed.
A primer set satisfying both of the above confirmation tests (A) and (B) can be used as the primer set of the present invention.
The primer set of the present invention can be used as a kit together with a polymerase, a buffer and the like.
[0154]
(9) Amplification reaction
The detection method of the present invention detects a transformant into which an ethylene receptor gene-like nucleic acid sequence (Phvc-ers1) or its antisense nucleic acid, or a partial sequence thereof has been introduced, An amplification reaction is performed using the primer set designed in (7) above, and when an amplification product is detected, it is determined that the test plant is a transformed plant into which Phvc-ers1 or its antisense nucleic acid has been introduced. Things.
[0155]
In this method, only a fragment specific to Phvc-ers1 or its antisense nucleic acid (cDNA) is amplified. Therefore, when trying to detect a transformed plant by this method, only the presence or absence of the amplification product need be detected. As the primer, a primer set designed in (7) and (8) above and whose function has been confirmed is used.
[0156]
As the test plant used in the present method, any plant may be used as long as it is a plant which is to detect whether Phvc-ers1 or its antisense nucleic acid has been introduced, and a plant of the genus Petunia is particularly preferable. DNA can be extracted from the test plants using methods well known in the art.
[0157]
The amplification method is not particularly limited, but may be a known method utilizing the principle of the polymerase chain reaction (PCR). For example, PCR method, LAMP (Loop-mediated isothermal AMPlification) method, ICAN (Isothermal andChimeric primer-initiated Amplification of Nucleic acids) method, RCA (Rolling Circle Amplification) method, LCR (Ligase Chain Reaction) method, SDA (Strand Displacement Amplification ) Method. Amplification is performed until the amplification product reaches a detectable level.
[0158]
The PCR method uses a DNA contained in a test plant as a template to synthesize a base sequence between a pair of primers contained in a primer set by a DNA polymerase. According to the PCR method, an amplified fragment can be amplified exponentially by repeating a cycle consisting of denaturation, annealing and synthesis. Optimal conditions for PCR can be easily determined by those skilled in the art. For example, when PCR is performed using the primers (a) to (g) designed in the above (7), the following is performed.
A denaturation step at 90-97 ° C, preferably 93-95 ° C for 30 seconds to 3 minutes, preferably 30 seconds to 1 minute; an annealing step at 37-70 ° C, preferably 50-70 ° C for 20 seconds to 1 minute; Preferably, the elongation step at 50 to 80 ° C., preferably 70 to 75 ° C. is performed for 1 to 5 minutes, preferably 1 to 2 minutes, for 4 to 40 cycles.
[0159]
However, in order to sufficiently denature the template DNA and the primer, a denaturation step at 94 ° C. for 1 to 3 minutes may be added before the amplification cycle, and the amplification is performed to completely extend the amplified DNA. An extension step at 72 ° C. for 2-20 minutes may be added after the cycle. Further, when the amplification product is not detected immediately, it is preferable to add a step of storing the amplification product at 4 ° C. in order to prevent non-specific amplification from occurring.
[0160]
(10) Detection of amplification products
For the detection of the amplification product after the amplification reaction, known means capable of specifically recognizing such a product can be used. For example, a label such as a radioisotope, a fluorescent substance, a luminescent substance, or the like is allowed to act on dNTP incorporated in the course of the amplification reaction, and the label can be detected. As radioisotopes, ³² P, ¹²⁵ I, ³⁵ S or the like can be used. As the fluorescent substance, for example, fluorescein (FITC), sulforhodamine (TR), tetramethylrhodamine (TRITC), or the like can be used. Luciferin or the like can be used as a light-emitting substance.
[0161]
There is no particular limitation on the type of the label or the method for introducing the label, and various conventionally known means can be used. For example, as a method for introducing a label, a random prime method using a radioisotope can be mentioned.
[0162]
In the present invention, as a method for observing the amplification product incorporating the labeled dNTP, any method known in the art for detecting the above-mentioned labeled substance may be used. For example, when a radioisotope is used as the label, the radioactivity can be measured by, for example, a liquid scintillation counter or a γ-counter. When fluorescence is used as the label, the fluorescence can be detected using a fluorescence microscope, a fluorescence plate reader, or the like.
[0163]
When an amplification product is detected as described above, it is determined that the test plant is a transformed plant into which Phvc-ers1 or its antisense nucleic acid, or a partial sequence thereof has been introduced.
[0164]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0165]
[Example 1] Cloning of petunia ethylene receptor gene-like nucleic acid (Phvc-ers1)
The following experiments were performed to clone a petunia ethylene receptor gene-like nucleic acid.
[0166]
(1) Plant sample
A commercially available petunia seed (cv. Velvet carpet, seedling of Fukuhanaen) was cultivated in an artificial weather device (Coittron; Koito Seisakusho) at 25 ° C., 16 hours of sunshine, and 50% humidity as a sample.
[0167]
(2) Preparation of petunia RNA
About 100 mg of a petunia plant (leaves, buds, flowers, etc.) was collected and pulverized under liquid nitrogen freezing. RNA was prepared using QIAGEN RNA preparation kit (RNeasy Plant Mini Kit) according to the standard protocol attached to the kit.
[0168]
(3) Cloning PCR primer
For the known ERS1 gene registered in the GenBank database (GenBank homepage, http://www.ncbi.nlm.nih.gov/GenBank/index.html), Genetyx-Mac 10.1 of gene analysis software (software development) Was used for molecular phylogenetic analysis. Based on the sequence of the same gene of tobacco, tomato and potato, which is judged to be taxonomically related from the Solanaceae cluster of the created molecular phylogenetic tree, the following primers having the sequence of both ends of the ORF were designed and synthesized with reference to the sequence of the same gene of tobacco, tomato and potato .
Forward primer
ERS-F: atgggaatcctgtgattgcattgag (SEQ ID NO: 11)
Reverse primer
ERS-R: ttacaractyctttgatagcgttga (SEQ ID NO: 12)
The above primer is prepared as a mixed primer, and r represents A or G, and y represents C or T.
[0169]
(4) RT-PCR amplification
RT-PCR amplification was performed using the above primers and QIAGEN One-Step RT-PCR Kit. The composition of the reaction solution was carried out using a thermal cycler (MP) manufactured by Takara according to the standard protocol attached to the kit. The PCR conditions are as follows.
50 ° C for 30 minutes
94 ° C for 15 minutes
(94 ° C 1 minute / 55 ° C 1 minute / 72 ° C 1 minute 30 seconds) x 40 cycles
72 ° C for 15 minutes
4 ℃
[0170]
(5) Introduction of restriction enzyme sites by re-PCR amplification
In order to introduce a restriction enzyme site sequence into an RT-PCR amplified fragment for the purpose of cloning into a cloning vector pBluescript II SK (+) (TOYOBO) and a plant binary vector pBI121, restriction enzyme site sequences (XbaI, SacI , SmaI) were synthesized.
Forward primer
Xba-ERS-F: gctctagaatgggaatcctgtgattgcattgag (SEQ ID NO: 13)
Sac-ERS-F: cgagctcatggaatcctgtgattgcattgag (SEQ ID NO: 14)
Reverse primer
Sac-ERS-R: cgagctcttcaractacttttgatagcgttga (SEQ ID NO: 15)
Sma-ERS-R: tcccccggggttacaractectttgatagcgttga (SEQ ID NO: 16)
Xba-ERS-R: gctctagattacataracttttgatagcgttga (SEQ ID NO: 17)
The above primer is prepared as a mixed primer, and r represents A or G, and y represents C or T.
[0171]
Using these, re-PCR amplification of the RT-PCR amplified fragment was performed. In order to prevent an amplification error during PCR amplification (incorrectly incorporated base is amplified), Pfu DNA polymerase (Pfu Turbo, TOYOBO) having 3 ′ → 5 ′ exonuclease activity was used. The composition of the reaction solution was carried out using a thermal cycler (MP) manufactured by Takara according to the standard protocol attached to the kit. The PCR conditions are as follows.
94 ° C for 2 minutes
(94 ° C 1 minute / 55 ° C 1 minute / 72 ° C 1 minute 30 seconds) x 30 cycles
72 ° C for 15 minutes
4 ℃
[0172]
(6) Purification of amplified fragments and treatment with restriction enzymes
The obtained PCR amplification product fragment was subjected to simultaneous restriction enzyme digestion at 37 ° C. in a PROMEGA MULTICORE buffer using the corresponding restriction enzyme combinations (XbaI-SacI, XbaI-SmaI). In addition, the same treatment was performed for the cloning vector pBluescript II SK (+) (TOYOBO). After electrophoresis in a TAE buffer using 0.8% agarose gel, ethidium bromide (EtBr) staining was performed to confirm the target fragment. The target fragment was excised together with the gel with a scalpel, and eluted and purified from the gel using QIAQUICK Gel Extraction Kit manufactured by QIAGEN.
[0173]
(7) Introduction and amplification of amplified fragment into cloning vector
Takara's DNA Ligation kit ver. Ligation of the PCR-amplified fragment and the vector digested with the restriction enzyme was carried out using No. 2. A competent cell manufactured by TOYOBO (Escherichia coli DH5α) was transformed with the above DNA according to the attached protocol, and cultured in an LB medium supplemented with IPTG, X-gal and ampicillin (50 μg / ml) to select transformants. The emerged colonies were picked and subjected to liquid culture in an LB medium supplemented with ampicillin (50 μg / ml). A plasmid was prepared from the obtained cells using Plasmid Mini Kit manufactured by QIAGEN. The insert was confirmed by length comparison of the fragments after restriction enzyme treatment and PCR amplification to obtain a plasmid into which the target fragment was subcloned.
[0174]
(8) Confirmation of base sequence by DNA sequencer
The nucleotide sequence of the selected plasmid was confirmed using an ABI DNA sequencer (310 Genetic Analyzer). The BigDye Terminator Cycle Sequencing, FS kit (Applied Biosystems) was used for the sequencing reaction. Detailed experimental procedures were performed according to the manual (operation guide, August 2000, Rev. 4.0) manufactured by ABI.
[0175]
(9) Analysis of base sequence
The obtained base sequence was analyzed and edited using Genetyx-Mac / ATSQ 3.0 (Software Development Co., Ltd.). After determining the cloned full-length sequence, a query on a database on the Internet (GenBank; http://www.ncbi.nlm.nih.gov/GenBank/index.html) was performed to search for a registered sequence having a high degree of similarity. Molecular phylogenetic analysis (using gene analysis software; Genetyx-Mac 10.1) was performed using the sequences of these (known ERS1 genes).
[0176]
(10) Confirmation of Phvc-ers1 sequence in each tissue of Petunia
RNA was extracted from the four tissues (leaves, immature buds, mature buds, flowering flowers) by the above method. A fragment which was similarly amplified by RT-PCR from this RNA was cut out from the gel after electrophoresis and purified. Using this fragment as a template, a sequencing reaction was performed to perform direct sequencing. By this method, the full-length sequence of cDNA derived from each tissue was determined (however, only the partial sequence of "immature buds" was confirmed).
[0177]
RNA was extracted and purified from the four tissues (leaves, immature buds, mature buds, flowering flowers), and the full-length sequence of the cDNA of the fragment obtained by RT-PCR amplification was determined (provided that “immature buds”). Check only the partial sequence).
[0178]
As a result of analyzing the experimental data, this nucleic acid sequence was estimated to have an ORF of 1911 base pairs (encoding 637 amino acids). When the obtained sequence was referred to a database, it was found to be a gene fragment having high homology to the ethylene receptor ERS1-like gene. In particular, the results showed high homology with the same gene of tobacco and tomato which are phylogenetically related. A molecular phylogenetic tree was created by the NJ method (neighborhood joining method) for the same gene sequence derived from a plant having homology in the database. As a result, the nucleic acid sequence (Phvc-ers1) of the ethylene receptor ERS1-like gene obtained in this example belongs to the dicotyledonous plant / Solanaceous plant cluster, and the taxonomic positional relationship was also consistent. The homology (sequence homology) with the gene of the plant species belonging to this cluster was 93.6%, 91.2% and 91.1% for tobacco, tomato and potato at the nucleic acid level, respectively. When compared at the amino acid level (the deduced amino acid sequence from the nucleic acid sequence), they were 95.3%, 92.6% and 92.1%, respectively. On the other hand, it was revealed that the melon ERS1 was 70.7% at the nucleic acid level and 71.0% at the amino acid level with respect to melon ERS1, which is generally used for gene transfer aimed at improving the shelf life of melon fruit. From these results, there is a possibility that a difference occurs between the petunia-derived and other plant-derived effects in the effect when introduced into a plant (Plant Physiology (1999) vol. 119, 321-329).
[0179]
(11) Cloning of Phvc-ers1
The nucleic acid fragment obtained by the method described in the above (10) was cloned into a cloning vector (pBluescript II SK (+)), and the insertion position and the nucleic acid sequence of the inserted fragment were confirmed using a DNA sequencer. As a result, a clone (pPhvc-ers1) inserted in the 3 ′ → 5 ′ direction between SmaI and XbaI in the multicloning site (MCS) of pBluescript II SK (+) was obtained. At this point, the resulting petunia ERS1-like nucleic acid sequence was named Phvc-ers1.
[0180]
[Example 2] Determination of both end regions of ORF of Phvc-ers1
(1) Preparation of plant sample and petunia RNA
Preparation of plant samples and petunia RNA (derived from leaf tissue) was performed as described in Example 1, (1) and (2).
[0181]
(2) RACE method
The RACE method (Rapid amplification of cDNA ends) was performed using a commercially available kit (5 '/ 3' RACE Kit manufactured by Roche Diagnostics) to obtain a target fragment.
[0182]
(3) PCR amplification
The PCR conditions were examined simultaneously under a plurality of annealing conditions as described later, and those obtained as good amplification products were used in the next step. In 3'RACE, nested-PCR was performed.
[0183]
First, based on the deduced sequence of Phvc-ers1 obtained in Example 1, PCR primers targeting near both ends of the ORF were designed. A plurality of primers were prepared for both ends for nested-PCR. Only those used when the target region was successfully amplified are described in Table 1 below.
[0184]
[Table 1]

[0185]
PCR amplification was carried out using the primers shown in Table 1 and the cDNA synthesized from the RNA derived from petunia leaves prepared in the above (1) as a template and using Ex Taq DNA polymerase from Takara. The composition of the reaction solution was performed using a thermal cycler (master cycler gradient) manufactured by Eppendorf according to the standard protocol attached to the kit. Since this device can perform the reaction by setting an arbitrary (up to 12 steps) temperature gradient on the heat block, the annealing temperature, which is important for amplification efficiency and specificity, is increased from 50.0 ° C to 70.5 ° C ( The temperature was set at about 5 ° C. in five steps; namely, 50.0 ° C., 55.5 ° C., 60.8 ° C., 66.0 ° C., and 70.5 ° C.), and the optimum conditions were examined. The PCR conditions are shown below.
94 ° C for 15 minutes
(94 ° C 1 minute / 50.0-70.5 ° C 1 minute / 72 ° C 1 minute 30 seconds) x 30 cycles
72 ° C for 15 minutes
4 ℃
[0186]
(4) Purification of amplified fragment
The obtained PCR amplification product fragment was subjected to electrophoresis using a 2% agarose gel (0.8% is inappropriate) with a TAE buffer, and then stained with ethidium bromide (EtBr) to confirm the target fragment. The target fragment was cut out together with the gel with a scalpel, and eluted and purified from the gel using QIAGEN's QIAquick Gel extraction kit.
[0187]
(5) Introduction of amplified fragment into cloning vector and amplification
Takara's DNA Ligation kit ver. Ligation of the PCR-amplified fragment purified in (4) above and a vector for TA cloning (for cloning of PCR product) (pT7Blue-2 manufactured by Novagen) using (2) was performed at 16 ° C. overnight. TOYOBO competent cells (Escherichia coli DH5α) were transformed according to the attached protocol, and cultured in an LB medium supplemented with IPTG, X-Gal and ampicillin (50 μg / ml) to select transformants. Plasmids were prepared from the cells obtained by collecting the colonies, and performing liquid culture in an LB medium supplemented with ampicillin (50 μg / ml), using Plasmid Mini Kit manufactured by QIAGEN. The insert was confirmed by gel electrophoresis, and a plasmid was obtained from which the target fragment was expected to be subcloned.
[0188]
(6) Confirmation of base sequence by DNA sequencer
The sequencer and experimental procedure used were the same as described in Example 1, (8). The sequencing primers were primers targeting the T7 sequence and the M13 sequence present at both ends of the cloning site of the pT7Blue-2 vector (Takara BcaBEST Sequencing Primer T7; TAATACGACTCACTATAGGGG (SEQ ID NO: 23) and M13 Primer M4; GTCTCGATCGATCCATCTCGATCTCGATCACTCTCGATCTCGATCACTCTCGATCTCGATCACTCTCGATCGATCACTCTCTCGATCGATCACTCTCGTGA SEQ ID NO: 24)) was used.
[0189]
(7) Base sequence analysis
The obtained base sequence was analyzed and edited using Genetyx-Mac / ATSQ 3.0. Compared to the cDNA sequence (1911 base pairs) cloned in Example 1, the sequence of both terminal regions of the ORF was re-determined.
[0190]
The nucleotide sequence of the cDNA (ORF region) of Petunia-derived Phvc-ers1 obtained as a result of sequence analysis is shown in SEQ ID NO: 1, and the amino acid sequence of the Phvc-ers1 protein is shown in SEQ ID NO: 2.
[0191]
[Example 3] Determination of splice site and intron sequence
(1) Preparation of plant sample and petunia DNA
Plant samples were performed as described in Example 1, (1).
For petunia DNA (derived from leaf tissue), about 100 mg of a petunia plant (leaf) was collected and pulverized under liquid nitrogen freezing, and then using a DNA preparation kit (DNeasy plant mini kit) manufactured by QIAGEN, a standard protocol attached to the kit was used. Prepared according to
[0192]
(2) PCR amplification
Using the primers designed in (3) of Example 1, PCR amplification was carried out using the petunia leaf-derived DNA prepared in (1) above as a template and Ex Taq DNA polymerase from Takara. The composition of the reaction solution was performed using a thermal cycler (master cycler gradient) manufactured by Eppendorf according to the standard protocol attached to the kit. Annealing temperature and PCR conditions were the same as in Example 2, (3).
[0193]
(3) Purification of amplified fragment
Purification of the PCR amplification product fragment was performed in the same manner as in Example 2, (4) except that 0.8% agarose gel was used.
[0194]
(4) Confirmation and analysis of base sequence by DNA sequencer
The nucleotide sequence of the obtained PCR amplification product fragment was confirmed in the same manner as in Example 1, (8).
The obtained base sequence was analyzed and edited using Genetyx-Mac / ATSQ 3.0. The intron sequence and splice site were identified by comparison with the cDNA sequence (1911 base pairs) cloned so far.
[0195]
(5) Analysis of unknown sequence by primer walking
The sequence of the intron portion was determined by decoding the sequences of both strands (sense strand / antisense strand) using primers designed in adjacent exon regions. When the entire region of the intron portion could not be decoded, a full-length sequence was determined by a primer walking method in which a new sequencing primer was designed near the splicing region based on the analysis result and the decoding was advanced. Table 2 summarizes the sequencing primers used in this experiment.
[0196]
[Table 2]

[0197]
The nucleotide sequence of the genomic DNA of the Phvc-ers1 gene prepared based on the information on the nucleotide sequence obtained as a result of the sequence analysis is shown in SEQ ID NO: 3. The length of the ORF region of the gene encoded on the chromosome was found to be 3782 bases (cDNA sequence was 1911 bases). This is significantly longer than 2185 bases (cDNA sequence is 1842 bases) of Arabidopsis thaliana ERS1 (GenBank accession number U21952). FIG. 1 shows a comparison of the intron region with Arabidopsis thaliana ERS1, in which only the information of the intron sequence among the ERS1 type genes is disclosed on the database.
[0198]
[Example 4] Design of PCR primer capable of distinguishing and detecting endogenous / exogenous Phvc-ers1
(1) Preparation of plant sample and petunia RNA
Preparation of plant samples and petunia RNA (derived from leaf tissue) was performed as described in Example 1, (1) and (2).
[0199]
(2) Design of primer
From Example 3, the sequence of Phvc-ers1 encoded in petunia genomic DNA was determined, and it was revealed that the gene had four introns (FIG. 1).
[0200]
Also, mRNA that has been spliced after being transcribed from genomic DNA has four boundary regions (splice sites) between two exons sandwiching the cut out intron (hereinafter, boundary region 1, boundary region 2, respectively). Boundary region 3 and boundary region 4). Primers were designed using these four boundary regions as targets. As the primer, only the forward primer was designed in the boundary region 1, both the forward primer and the reverse primer were designed in the boundary region 2 and the boundary region 3, and only the reverse primer was designed in the boundary region 4 (FIG. 3). When designing primers, a total of 31 types of primers were designed and synthesized in consideration of the chain length from the splice site to the 3 'end (the length of the 3' side sequence) and the Tm of the primer. The 11 types of primers shown were selected. In Table 3, "F" shown in the direction column indicates a forward primer, and "R" indicates a reverse primer. The numbers shown in the column of the target site represent the nucleotide sequence numbers of the Phvc-ers1 gene shown in SEQ ID NO: 1, and the numbers shown in the column of the chain length are from the splice site to the 3 'end of the primer (3' side). Sequence).
[0201]
[Table 3]

[0202]
(3) RT-PCR amplification
RT-PCR amplification was performed using the primers designed in (2) above and the One-Step RT-PCR Kit from QIAGEN using the petunia leaf-derived RNA prepared in (1) as a template. The composition of the reaction solution was prepared according to the standard protocol attached to the kit.

[0203]
Using a master cycler gradient manufactured by Eppendorf, RT-PCR was performed by setting the annealing temperature in 5 steps every 5 ° C. in the range of 50.0 ° C. to 70.5 ° C. RT-PCR amplification conditions are shown below.
(1) 50 ° C for 30 minutes
(2) 94 ° C for 15 minutes
(3) (94 ° C. 1 minute / 50.0-70.5 ° C. 1 minute / 72 ° C. 1 minute 30 seconds) × 35 cycles
▲ 4 ▼ 72 ℃ 15 minutes
▲ 5 ▼ 4 ℃
[0204]
RT-PCR amplification was performed using the extracted petunia RNA as a template and the primers shown in Table 3. The cDNA fragments of the Phvc-ers1 gene amplified using the primers designed to target the four boundary regions are: boundary region 1-2, boundary region 1-3, boundary region 1-4, boundary region 2 6, between boundary areas 2 and 4, and between boundary areas 3 and 4 (FIG. 3).
[0205]
As a result of gel electrophoresis after RT-PCR amplification between the respective boundary regions, combinations of primers that could detect only one band of the target size are shown in Table 4. In Table 4, the alphabet shown in the column of F represents the name of the forward primer, and the alphabet shown in the column of R represents the name of the reverse primer. The numbers shown in the column of the chain length represent the chain length from the splice site to the 3 ′ end (3 ′ side sequence).
[0206]
[Table 4]

[0207]
As a result of performing RT-PCR amplification using primers (see Tables 3 and 4) designed to have a Tm of about 60 ° C., a result was obtained between boundary regions 1 to 3 (FIG. 5A) and between boundary regions 2 to 4 (FIG. 5A). 5B) and between boundary regions 3 and 4 (FIG. 5C), only the amplification product of the target size was amplified, and specific detection became possible. In FIGS. 5A to 5C, each lane is as follows: lane 1: annealing temperature of 50.0 ° C., lane 2: annealing temperature of 55.5 ° C., lane 3: annealing temperature of 60.8 ° C., lane 4: annealing temperature of 66.0 ° C. , And lane 5 show the results obtained at an annealing temperature of 70.5 ° C.
[0208]
Furthermore, when RT-PCR amplification was performed using primers designed with Tm of 60 ° C., non-specific increasing by-products tended to be observed in the entire range of annealing temperature from 50.0 to 70.5 ° C. . Therefore, in order to make the annealing of the primer occur at a low temperature, a method of setting the set temperature of Tm to 60 ° C. or less was considered. RT-PCR was performed in the same manner as described above using primers (see Tables 3 and 4) designed so that the set temperature of Tm was 60 ° C. or lower between boundary regions 1 and 2 and between boundary regions 2 and 3. Amplification was performed. As a result, at the annealing temperature of 70.5 ° C., no non-specific increase in by-products was observed, and the target size of the desired size was also found between the boundary regions 1 and 2 (FIG. 6A) and between the boundary regions 2 and 3 (FIG. 6B). Only the amplification product was detected, allowing specific detection. In FIGS. 6A and 6B, each lane is as follows: lane 1: annealing temperature of 50.0 ° C., lane 2: annealing temperature of 55.5 ° C., lane 3: annealing temperature of 60.8 ° C., lane 4: annealing temperature of 66.0 ° C. , And Lane 5 show the results obtained at an annealing temperature of 70.5 ° C.
[0209]
From these facts, specific detection of the Phvc-ers1 gene cDNA corresponding to a foreign gene became possible by RT-PCR amplification using primers designed to target each of the four boundary regions.
[0210]
[Example 5] Examination of PCR amplification conditions in the presence of genomic DNA
Petunia leaf-derived DNA was prepared in the same manner as described in Example 3 (1).
(1) Examination of PCR conditions
At the optimal annealing temperature at which specific detection of Phvc-ers1 cDNA by RT-PCR amplification is possible (see Tables 3 and 4), is it possible to obtain an amplification product when performing PCR amplification using genomic DNA as a template? We examined whether or not. For this purpose, a comparative experiment was performed in which the RT-PCR amplification reaction performed in Example 4 was the same as the reaction solution composition, PCR reaction conditions, and the like. The enzyme solution (manufactured by QIAGEN) used for the RT-PCR amplification contains two types of reverse transcriptases (Omniscript Reverse Transcriptase and Sensiscript Reverse Transcriptase) and Hot Star Taq DNA Polymerase. Therefore, the enzyme (Hot Star Taq DNA Polymerase manufactured by QIAGEN) used in one-step RT-PCR amplification was used at the same concentration for the PCR amplification.
[0211]
For the examination of the conditions, the primers designed in (3) of Example 1 were used.
Forward primer
ERS-F: atgggaatcctgtgattgcattgag (SEQ ID NO: 11)
Reverse primer
ERS-R: ttacaractyctttgatagcgttga (SEQ ID NO: 12)
The amount of the primer added was compared under the two conditions of 2.0 μM, which is the same as the RT-PCR reaction solution in Example 4, and 0.5 μM, which is the amount recommended for QIAGEN Hot Star Taq PCR.
The magnesium concentration in the PCR reaction solution was compared under the same two conditions of the RT-PCR reaction solution of Example 4, 2.5 mM (addition amount: 1.0 μl) and 1.5 mM (addition amount: 0 μl).
Based on the above, PCR amplification was performed under the four conditions shown in Table 5 below.
[0212]
[Table 5]

[0213]
Using a master cycler gradient manufactured by Eppendorf, an annealing reaction was performed at five annealing temperatures set at about 5 ° C. in the range of 50.0 ° C. to 70.5 ° C., and the amplification reaction was performed. The PCR conditions are described below.
(1) 94 ° C for 15 minutes
(2) (94 ° C 1 minute / 50.0-70.5 ° C 1 minute / 72 ° C 1 minute 30 seconds) x 30 cycles
(3) 72 ° C for 15 minutes
▲ 4 ▼ 4 ℃
[0214]
In Example 4, the optimal annealing temperature for RT-PCR amplification using primers ERS-F and ERS-R (SEQ ID NOS: 11 and 12, respectively) was about 55-60 ° C. As a result of performing PCR amplification at the annealing temperatures of 55.5 ° C. and 60.8 ° C. using the PCR reaction solutions under the four conditions shown in Table 5, specific detection was possible at the annealing temperature of 60.8 ° C. under any conditions. . As a result, it was found that the addition amount of the primer and the magnesium concentration set this time did not affect the specific amplification, and it is considered that almost the same conditions as in the RT-PCR amplification could be set. Based on the above results, the same condition 3 as in the RT-PCR amplification of Example 4 was adopted for the reaction solution composition of the PCR amplification.
[0215]
(2) PCR amplification test of Phvc-ers1 genomic DNA
As a result of performing PCR amplification in the presence of genomic DNA using the selected seven primer sets (see Table 4) at each optimum annealing temperature (see Table 4) under condition 3, the increased by-product was Not detected (see FIG. 7). This confirmed that these primers did not amplify the endogenous gene.
[0216]
Each lane represents:
Lane 1: molecular weight marker (1 kbp)
Lane 2: molecular weight marker (100 bp)
Lane 3: cDNA between border regions 1 and 2 was amplified using primers 1F-S1 and 2R-S4
Lane 4: genomic DNA between border regions 1 and 2 was amplified using primers 1F-S1 and 2R-S4
Lane 5: cDNA between border regions 1 and 2 was amplified using primers 1F-S1 and 2R-S5
Lane 6: genomic DNA between boundary regions 1 and 2 was amplified using primers 1F-S1 and 2R-S5
Lane 7: cDNA between boundary regions 1 and 2 was amplified using primers 1F-S1 and 2R-S6
Lane 8: genomic DNA between boundary regions 1 and 2 was amplified using primers 1F-S1 and 2R-S6
Lane 9: molecular weight marker (1 kbp)
Lane 10: molecular weight marker (100 bp)
Lane 11: cDNA between border regions 1 to 3 was amplified using primers 1F0 and 3R0
Lane 12: genomic DNA between border regions 1 to 3 was amplified using primers 1F0 and 3R0
Lane 13: molecular weight marker (1 kbp)
Lane 14: molecular weight marker (100 bp)
Lane 15: cDNA between border regions 2 and 3 was amplified using primers 2F-S1 and 3R-S1
Lane 16: genomic DNA between border regions 2 and 3 was amplified using primers 2F-S1 and 3R-S1
Lane 17: molecular weight marker (1 kbp)
Lane 18: molecular weight marker (100 bp)
Lane 19: cDNA between border regions 2 to 4 was amplified using primers 2F2 and 4R0
Lane 20: genomic DNA between border regions 2 to 4 was amplified using primers 2F2 and 4R0
Lane 21: molecular weight marker (1 kbp)
Lane 22: molecular weight marker (100 bp)
Lane 23: cDNA between boundary regions 3 and 4 was amplified using primers 3F0 and 4R0
Lane 24: genomic DNA between boundary regions 3 and 4 was amplified using primers 3F0 and 4R0
[0217]
As described above, the specific detection of the Phvc-ers1 cDNA corresponding to the foreign gene became possible by RT-PCR amplification using the primer containing the base sequence of the border region of the Petunia Phvc-ers1 cDNA. Moreover, when PCR amplification was performed using these primers, the result was that Phvc-ers1 (genomic DNA) encoded on the chromosome as an endogenous gene was not specifically detected. From these facts, it was possible to design a PCR primer capable of discriminating endogenous / external Phvc-ers1 of Petunia and specifically detecting it.
[0218]
【The invention's effect】
ADVANTAGE OF THE INVENTION By this invention, it becomes possible to adjust the flower durability of the plant which belongs to the genus Petunia, and a commercially useful plant is obtained. The present invention also makes it possible to detect such a transformed plant quickly and easily.
[0219]
[Sequence list]

[0220]
[Sequence List Free Text]
SEQ ID NOs: 4 to 63: synthetic oligonucleotides
[Brief description of the drawings]
FIG. 1 is a diagram showing the structures of genomic DNA of Petunia (Petunia × hybrida) Phvc-ers1 (A) and Arabidopsis thaliana (Arabidopsis thaliana) ERS1 (B).
FIG. 2 is a diagram showing a boundary region in the cDNA of a petunia ethylene receptor gene-like nucleic acid (Phvc-ers1) and the corresponding design of the oligonucleotide probe of the present invention.
FIG. 3 is a diagram showing a boundary region in a cDNA of a petunia ethylene receptor gene-like nucleic acid (Phvc-ers1) and the design of a primer corresponding to the boundary region in the cDNA.
FIG. 4 is a diagram showing amplification of Phvc-ers1 cDNA (A) and Phvc-ers1 genomic DNA using the primer set of the present invention.
FIG. 5 shows an electrophoretic photograph showing specific detection of Phvc-ers1 cDNA by RT-PCR amplification reaction. The primer used had a Tm set at 60 ° C.
FIG. 6 shows an electrophoresis photograph showing specific detection of Phvc-ers1 cDNA by RT-PCR amplification reaction. The primer used had a Tm set at 50 to 56 ° C.
FIG. 7 is an electrophoretic photograph showing the results of detection of Phvc-ers1 cDNA and Phvc-ers1 genomic DNA using primers containing the base sequence of the boundary region. The photograph shows that the cDNA was specifically detected and no genomic DNA was detected.

Claims (29)

A protein comprising the amino acid sequence shown in SEQ ID NO: 2. A nucleic acid encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 2. The following nucleic acid (a) or (b):
(A) a nucleic acid containing all or part of the DNA consisting of the base sequence shown in SEQ ID NO: 1; (b) a nucleic acid consisting of a base sequence having a homology of 96% or more with the DNA consisting of the base sequence shown in SEQ ID NO: 1, and ethylene-sensitive Nucleic acid having a function related to A nucleic acid comprising all or part of a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3. An antisense nucleic acid comprising a nucleotide sequence complementary to a DNA comprising a whole or a part of the nucleotide sequence of the nucleic acid according to any one of claims 2 to 4. An expression vector comprising all or a part of the nucleic acid according to any one of claims 2 to 4. An expression vector comprising all or a part of the antisense nucleic acid according to claim 5. A transformant comprising the nucleic acid according to any one of claims 2 to 4 or the antisense nucleic acid according to claim 5. The transformant according to claim 8, which is a plant. The transformant according to claim 9, which is a flower plant. The transformant according to claim 10, wherein the flower plant belongs to the genus Petunia. The transformant according to any one of claims 9 to 11, wherein flowering ability is enhanced as compared with a wild-type plant. A seed obtained from the transformant according to any one of claims 9 to 12. A method for regulating flower durability of a plant, comprising introducing the nucleic acid according to any one of claims 2 to 4 or the antisense nucleic acid according to claim 5 into a plant. A method for producing a transformed plant, comprising culturing or cultivating the transformant according to any one of claims 9 to 12 or the seed according to claim 13, and regenerating a plant. The method according to claim 15, wherein the plant is a flower plant. The method according to claim 15 or 16, wherein the plant belongs to the genus Petunia. A method for producing an ethylene receptor, comprising culturing the transformant according to claim 8, and collecting an ethylene receptor from the resulting culture. An oligonucleotide comprising a partial nucleotide sequence of the nucleic acid according to any one of claims 2 to 4 or the antisense nucleic acid according to claim 5. The boundary region 1 between exon 1 and exon 2, the boundary region 2 between exon 2 and exon 3, and the boundary region between exon 3 and exon 4 which constitute the cDNA encoding the ethylene receptor gene-like nucleic acid sequence derived from a plant belonging to the genus Petunia An oligonucleotide comprising a base sequence forming a boundary region selected from the group consisting of a boundary region 3 and a boundary region 4 between exon 4 and exon 5, or a complementary sequence thereof. The bases forming the boundary region 1 are thymine 908 and 909 of the base sequence shown in SEQ ID NO: 1, the bases forming the boundary region 2 are guanine 1273 and adenine 1274, and 21. The oligonucleotide according to claim 20, wherein bases forming No. 3 are thymine No. 1545 and adenine No. 1546, and bases forming a boundary region 4 are cytosine No. 1669 and guanine No. 1670. An oligonucleotide selected from the group consisting of the following (a) to (d).
(A) an oligonucleotide comprising at least bases 908 to 909 of the base sequence shown in SEQ ID NO: 1 and consisting of a continuous base sequence of 15 or more bases or a complementary sequence thereof; (b) an oligonucleotide having a base sequence shown in SEQ ID NO: 1 Among them, the oligonucleotide comprising at least 1273 to 1274 bases, and a continuous base sequence of 15 or more bases or a complementary sequence thereof (c) contains at least 1545 to 1546 bases in the base sequence shown in SEQ ID NO: 1, And an oligonucleotide consisting of a continuous base sequence of 15 or more bases or a complementary sequence thereof (d) a base sequence of at least 1669 to 1670 of the base sequence shown in SEQ ID NO: 1 and a continuous base sequence of 15 or more bases or Oligonucleotide consisting of complementary sequence An oligonucleotide selected from the group consisting of the following (a) to (g).
(A) an oligonucleotide comprising at least the 19th to 20th bases in the base sequence shown in SEQ ID NO: 4 and consisting of a continuous base sequence of 15 or more bases (b) at least 16 to 16 bases in the base sequence shown in SEQ ID NO: 5 Oligonucleotide comprising base No. 17 and comprising a continuous base sequence of 15 or more bases (c) Consecutive bases of 15 or more bases including at least bases 19 to 20 in the base sequence shown in SEQ ID NO: 6 Oligonucleotide consisting of sequence (d) Oligonucleotide containing at least 14 to 15 bases in the base sequence shown in SEQ ID NO: 7 and consisting of a continuous base sequence of 15 or more bases (e) Nucleotide sequence shown in SEQ ID NO: 8 Oligonucleotide (f) comprising at least the 19th to 20th bases and consisting of a continuous base sequence of 15 or more bases An oligonucleotide comprising at least the 16th to 17th bases in the base sequence shown in SEQ ID NO: 9 and comprising a continuous base sequence of 15 or more bases (g) at least 18th to 19th bases in the base sequence shown in SEQ ID NO: 10 Oligonucleotides containing bases and consisting of consecutive 15 or more base sequences A probe for detecting a plant into which the nucleic acid according to claim 2 or 3 or the antisense nucleic acid according to claim 5, comprising the oligonucleotide according to any one of claims 19 to 23. The nucleic acid according to claim 2 or 3, or the antisense nucleic acid according to claim 5, wherein the probe according to claim 24 is reacted with the DNA of a test plant, and the reaction product is detected. How to detect plants. The boundary region 1 between exon 1 and exon 2, the boundary region 2 between exon 2 and exon 3, and the boundary region between exon 3 and exon 4 which constitute the cDNA encoding the ethylene receptor gene-like nucleic acid sequence derived from a plant belonging to the genus Petunia A primer 1 containing a base forming one boundary region selected from the group consisting of a boundary region 3 and a boundary region 4 between exon 4 and exon 5 or a complementary sequence thereof, and a base forming another boundary region or a complement thereof A primer set comprising a combination with a primer 2 containing a sequence, wherein one of primers 1 and 2 is prepared as a forward primer and the other is prepared as a reverse primer. The bases forming the boundary region 1 are thymine 908 and 909 of the base sequence shown in SEQ ID NO: 1, the bases forming the boundary region 2 are guanine 1273 and adenine 1274, and 27. The primer set according to claim 26, wherein the bases forming No. 3 are thymine No. 1545 and adenine No. 1546, and the bases forming boundary region 4 are cytosine No. 1669 and guanine No. 1670. A primer set comprising at least one primer selected from the group consisting of the following (a) to (d) and at least one primer selected from the group consisting of the following (e) to (g).
(A) a forward primer comprising at least the 19th to 20th bases of the base sequence shown in SEQ ID NO: 4 and consisting of a continuous base sequence of 15 or more bases; (b) at least 16 to 16 bases out of the base sequence shown in SEQ ID NO: 5 A forward primer comprising base number 17 and comprising a continuous base sequence of 15 or more bases (c) a base comprising at least bases 19 to 20 of the base sequence shown in SEQ ID NO: 6 and comprising 15 or more continuous bases (D) a forward primer comprising at least the 14th to 15th bases of the base sequence shown in SEQ ID NO: 7 and comprising a continuous base sequence of 15 or more bases (e) a base sequence shown in SEQ ID NO: 8 A reverse primer comprising at least the 19th to 20th bases and comprising a continuous base sequence of 15 or more bases (F) a reverse primer comprising at least the 16th to 17th bases in the base sequence shown in SEQ ID NO: 9 and consisting of a continuous base sequence of 15 bases or more; (g) at least 18 to 18 bases in the base sequence shown in SEQ ID NO: 10 Reverse primer containing the 19th base and consisting of a continuous base sequence of 15 or more bases The nucleic acid according to claim 2 or 3, wherein the primer set according to any one of claims 26 to 28 is used to amplify the DNA of a test plant, and the resulting amplification product is detected. Item 6. A method for detecting a plant into which the antisense nucleic acid according to Item 5 has been introduced.

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