JP3584924B2 - Transgenic plants created by new vectors - Google Patents

Description

【００５８】
表１から明らかなように、Ｎｏ．８のシュートでは、これより得られた葉がカナマイシン耐性及びＧＵＳ活性を有しているにもかかわらず、これ自体を１か月培養しても多芽体を形成しない。これは、ここで用いたベクターｐＮＰＩ１０６において、多芽体の形成に寄与するｉｐｔ遺伝子はトランスポゾンＡｃの内部に挿入された形で存在しているため、このベクターを保持するＡ．ツメファシエンスをタバコ葉片に感染させてその培養を始めた当初は、タバコ染色体中に導入され、発現していたｉｐｔ遺伝子が、その後の培養中に、このＡｃの働きにより、これと共に脱離してその機能を消失したためであると考えられる。一方、同じベクター上でも、ＮＰＴＩＩ遺伝子とＧＵＳ遺伝子は、Ａｃと挙動を一つにすることのない位置に挿入されているので、これらはＮｏ．８のシュートにおいてもなお、その染色体中に残留して発現しているのである。
【００５９】
また、表１中、Ｎｏ．３、５及び６のシュートでは、カナマイシン耐性、多芽体形成能が認められるにもかかわらず、ＧＵＳ活性のみが陰性を示している。すなわち、これらのシュートではｐＮＰＩ１０６を用いて導入した遺伝子のうち、ＧＵＳ遺伝子のみが発現していないことになるが、これは、これらの遺伝子が植物染色体に導入される時に起こった組込みミスによるものと考えられる。つまり、Ａ．ツメファシエンスを介して遺伝子導入を行った場合、ｐＮＰＩ１０６のような構造を有するプラスミドでは、本来、Ｔ−ＤＮＡ領域、すなわちＲＢサイトとＬＢサイトの内側領域全体が植物染色体に組み込まれるはずであるが、時としてこの組込みが完全には行われず、これがＬＢ末端側のある部分で千切れたような状態で組み込まれることがある。ここで用いたｐＮＰＩ１０６では、そのＴ−ＤＮＡ領域に挿入した遺伝子の中でＧＵＳ遺伝子が、最もＬＢサイトに近い位置に存在していることから、このような遺伝子導入時の組込みミスにより、これが千切れてその機能を失った状態で染色体に組み込まれたか、あるいは全く組み込まれなかったため、これらのシュートではＧＵＳ遺伝子の発現がなく、その活性も認められないものと考えられる。
ここで、Ｎｏ．２及びＮｏ．８のシュートの１か月培養後の形態を図１１、１２に示す。
【００６０】
なお、この時Ｎｏ．８のシュートから得られ、カナマイシン耐性試験に供した葉について、試験後も培養を続けたところ、５個の不定芽がこの葉から得られたが、これらはすべて正常体であった。
【００６１】
Ｂ．ＰＣＲ分析
表１におけるＮｏ．１〜９のシュートにつき、その多芽体形成能を観察した後、参考例１のＩＶ−Ｂと同様にしてＰＣＲ分析を行い、染色体中のｉｐｔ遺伝子の存在等についてさらに検討を加えた。ただしここでは、参考例１のＩＶ−Ｂで用いたプライマーに加えて、ＮＰＴＩＩ遺伝子とＧＵＳ遺伝子上に結合すべく設計されたプライマーも使用したが、これを用いてＰＣＲを行うと、ｐＮＰＩ１０６のＴ−ＤＮＡ領域から、Ａｃ、及びその内部に挿入されたｉｐｔ遺伝子の脱離が起こった場合、約３ｋｂのＤＮＡ断片が増幅されるため、この増幅を指標として、Ａｃ及びｉｐｔ遺伝子の染色体ＤＮＡからの脱離を検出できることとなる。
ここで行ったＰＣＲ分析のうち、Ｎｏ．８のシュートについての結果を図１３に示す。なお図中、左に示した数値については図６と同様である。
【００６２】
これより明らかなように、Ｎｏ．８のシュートより抽出した染色体ＤＮＡにおいては、ｉｐｔ遺伝子とＡｃの脱離を示す約３ｋｂのＤＮＡ断片の増幅が認められる一方、ｉｐｔ遺伝子の存在を示す約８００ｂｐのＤＮＡ断片の増幅は認められない。これは、ｉｐｔ遺伝子がＡｃと共に、このシュートの染色体ＤＮＡから脱離・消失したことを意味するものである。
【００６３】
なお、これに対してＮｏ．１〜７、及び９のシュートでは、このとき、その染色体ＤＮＡ試料のいずれからも約３ｋｂのＤＮＡ増幅は検出されず、その一方で、約８００ｂｐの増幅はすべてにおいて検出された。従って、これらのシュートにおいてはｉｐｔ遺伝子がなお、Ａｃと共にその染色体ＤＮＡ中に存在しているものと考えられる。
【００６４】
［参考比較例２］
参考例２のＩＩ−ＡのＡ．ツメファシエンス感染葉の培養において、多芽体と共に分化してきた正常体のうち３個を分離し、これらについて参考例２のＩＩと同様に、カナマイシン耐性試験、ＧＵＳ活性試験、及び培養１か月後の形態観察、さらにＰＣＲ分析を行った。
【００６５】
結果を表１に示すが、これらはいずれもカナマイシン耐性、ＧＵＳ活性、及び多芽体形成能を有さず、さらにＰＣＲ分析においても、約８００ｂｐ及び約３ｋｂのＤＮＡ断片は、いずれもその増幅が検出されなかった。
【００６６】
［参考例３］
参考例２で分離した多芽体６３系統のそれぞれ一部を、さらにホルモンフリーＭＳ寒天培地に植え継いで培養を継続し、約２か月後に、２系統の多芽体から肉眼で識別できる正常な形態、すなわち頂芽優勢を示すシュート、Ｎｏ．１３−１〜３、１４−１〜４の計７個を得た。このシュートを分離して同組成の培地に移植すると正常な形態の伸長発根個体が得られ、そのうち、シュートＮｏ．１３−１及び１４−１より得られた個体について、参考例２のＩＩ−Ｂと同様にＰＣＲ分析を行ったところ、約８００ｂｐのＤＮＡ断片の増幅はどちらからも検出されず、その一方、約３ｋｂのＤＮＡ断片の増幅は両者で共に検出され、ｉｐｔ遺伝子がＡｃと共に、これらの個体の染色体ＤＮＡから脱離・消失していることが確認された。結果を図１４に示す。図中、左に示した数値については図６と同様である。またＧＵＳ遺伝子の発現は、７個のシュートより得られたすべての個体において検出された。
なお、ここで多芽体から正常なシュートが分化してきた状態を図１５に示す。
【００６７】
［参考例４］
参考例２で選抜した９個のシュートのうち、表１においてＮｏ．７としたシュートより得られた葉を、ホルモンフリーＭＳ寒天培地で約１か月培養し、そこから分化してきた６個の不定芽より、正常体１個を肉眼により選抜・分離した。これを同組成の培地に移植すると正常な形態の伸長発根個体が得られ、さらに、これについて参考例２のＩＩ−Ｂと同様にＰＣＲ分析を行ったところ、約８００ｂｐのＤＮＡ断片の消失と約３ｋｂの同断片の増幅により、その染色体ＤＮＡからｉｐｔ遺伝子がＡｃと共に脱離・消失したことが確認された。結果を図１６に示す。図中、左に示した数値については図６と同様である。また同じ個体について、ＧＵＳ遺伝子の発現も確認された。
【００６８】
［実施例１］
Ｉ．酵母からの部位特異的組換え系（ｐＳＲ１系）の分離
酵母（Zygosaccharomyces rouxii、（財）発酵研究所より購入）を５ｍｌのＹＰＡＤ液体培地（酵母エキス１０ｇ／ｌ、ポリペプトン２０ｇ／ｌ、アデニン０．４ｇ／ｌ、グルコース２０ｇ／ｌ）に接種し、３０℃で一昼夜培養した。培養液を、６９００×ｇ、２０℃、３分間遠心して集菌（以下、菌体の回収はすべて同条件で行なった。）し、得られた菌体を２ｍｌの０．２Ｍ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．０）−５ｖ／ｖ％ β−メルカプトエタノールに懸濁して、時折軽く撹拌しつつ２５℃で３０分間放置した後、回収した。この菌体に、ザイモリエイス−２０Ｔ（生化学工業（株）より購入）２．５ｍｇ／ｍｌを含む１０ｗ／ｖ％ ソルビトール−５ｗ／ｖ％ ＫＰＯ4（ｐＨ６．８）１ｍｌを加えて懸濁し、３０℃、９０分間放置し、再び遠心分離により集菌した後、これを１ｍｌの溶解液（０．２Ｍ ＮａＣｌ、０．１Ｍ ＥＤＴＡ、５ｗ／ｖ％ ＳＤＳ、５０ｍＭ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．５）に懸濁し、さらにＰｒｏｔｅｉｎａｓｅＫ（２０ｍｇ／ｍｌ）を加え混合して、６０℃で１時間放置した。次いでこの混合液を室温に戻し、フェノール：クロロホルム、クロロホルム抽出により順次精製した後、得られた上清にイソプロパノールを等量加え、染色体ＤＮＡ及びプラスミドｐＳＲ１を析出させ、６９００×ｇ、４℃、１０分間遠心してこれを沈殿させた。沈殿させたＤＮＡは、７０ｖ／ｖ％エタノールで洗浄した後、真空乾燥し、１００μｌのＴＥに溶解した。
【００６９】
このようにして抽出したＤＮＡ（染色体ＤＮＡ及びプラスミドｐＳＲ１の両方を含む）より、ＰＣＲ法を用い、プラスミドｐＳＲ１に存在している部位特異的組換え系（これをｐＳＲ１系という。）のみを増幅した。ｐＳＲ１系は、組換え酵素遺伝子であるＲ遺伝子と、組換え配列Ｒｓとから構成され、その塩基配列もすでに明らかとなっている（Ｈ．Ａｒａｋｉ ｅｔ ａｌ．、Ｊ．Ｍｏｌ．Ｂｉｏｌ．、１８２：１９１、１９８５）。本発明においては、Ｒ遺伝子の増幅のため、プラスミドｐＳＲ１の配列中、５５９６〜５６１７番目に当たる２２塩基の５′位に、ＸｂａＩ制限酵素部位を付加したプライマー（５′−ＣＣＴＣＴＡＧＡＡＴＧＣＡＡＴＴＧＡＣＣＡＡＧＧＡＴＡＣＴＧ−３′）と、４１２６〜４１４７番目に当たる２２塩基の５′位に、ＳａｃＩ制限酵素部位を付加したプライマー（５′−ＣＣＧＡＧＣＴＣＴＴＡＡＴＣＴＴＧＴＣＡＧＧＡＧＧＴＧＴＣＡ−３′）を合成して使用し、一方、Ｒｓの増幅には、２セット各３０塩基（計４種類）のプライマーを合成・使用した。すなわち１セットは、プラスミドｐＳＲ１の塩基配列中、２８７〜３１６番目に当たる配列のうち３塩基を置換して、ＳｓｅＩ制限酵素部位を導入した塩基配列を有するプライマー（５′−ＡＧＧＡＴＴＧＡＧＣＴＡＣＴＧＧＡＣＧＧＧＡＡＴＣＣＴＧＣＡ−３′）と、６９０〜７１９番目に当たる配列のうち４塩基を置換して、ＨｉｎｄＩＩＩ制限酵素部位とＸｈｏＩ制限酵素部位を導入したものであるプライマー（５′−ＣＡＡＣＴＣＧＡＧＣＡＡＴＣＡＡＡＧＣＴＴＣＴＣＧＴＡＧＴＣ−３′）とからなり（このプライマーセットで増幅されるＲｓをＲｓ１と呼ぶ。）、もう１セットは、同じくプラスミドｐＳＲ１の塩基配列中、２８７〜３１６番目に当たる配列のうち３塩基を置換して、ＸｈｏＩ制限酵素部位とＥｃｏＲＩ制限酵素部位を導入した塩基配列を有するプライマー（５′−ＡＧＧＡＴＴＧＡＧＣＴＡＣＴＣＧＡＧＧＧＧＡＡＴＴＣＴＧＧＡ−３′）と、６８８〜７１７番目に当たる配列のうち５塩基を置換して、ＳｓｅＩ制限酵素部位を導入したものであるプライマー（５′−ＡＣＴＧＧＡＣＣＡＡＴＣＣＣＴＧＣＡＧＧＴＣＧＴＡＧＴＣＡＡ−３′）とからなっている（このプライマーセットで増幅されるＲｓをＲｓ２と呼ぶ。）。
【００７０】
Ｒ遺伝子とＲｓの増幅のため、ＴＥ溶液とした抽出ＤＮＡ１００μｌの内１μｌを、それぞれのプライマーセット０．２μＭを含む、参考例１のＩＶ−Ｂで使用した混合液各５０μｌ中に混合し、これらを、９５℃ ３０秒、５５℃ ３０秒、７２℃ １分３０秒の加温サイクル３０回で反応させた。得られた反応混合物をアガロースゲル電気泳動を用いて分析し、Ｒ遺伝子とＲｓの増幅を確認した。
【００７１】
ＩＩ．ベクターの作成
ＰＣＲ法により増幅したＲｓ１を、制限酵素ｐｓｔＩ及びＸｈｏＩで切断し、これをｐＳＬ１１８０（ファルマシア バイオテク（株）より購入）のＰｓｔＩ及びＸｈｏＩ制限酵素部位に挿入し、プラスミドｐＮＰＩ１２６を得た。
【００７２】
次に、ｐＨＳＧ３９８のＥｃｏＲＩ制限酵素部位、及びＨｉｎｄＩＩＩ制限酵素部位を消失させるため、これらの制限酵素による切断、Ｔ４ポリメラーゼＩ（大サブユニット）による切断末端の平滑化、さらにその平滑末端の再連結を順次繰り返し、これらの制限酵素部位が消失したプラスミドｐＮＰＩ１２１を得、そのＳａｌＩ及びＰｓｔＩ制限酵素部位に、ＰＣＲ法により増幅したＲｓ２を制限酵素ＸｈｏＩ及びＰｓｔＩで切断して挿入し、プラスミドｐＮＰＩ１２７を作成した。
【００７３】
そしてこのｐＮＰＩ１２７のＳｍａＩ及びＸｂａＩ制限酵素部位に、ｐＮＰＩ１２６から制限酵素ＳｍａＩ及びＳｐｅＩで切り出されたＲｓ１を挿入して、プラスミドｐＮＰＩ１２８を得た。
【００７４】
Ｒ遺伝子は、ＰＣＲ法により増幅したその断片を、制限酵素ＸｂａＩ及びＳａｃＩで切断して、ｐＨＳＧ３９８のＸｂａＩ及びＳａｃＩ制限酵素部位に挿入した。これにより得られたプラスミドをｐＮＰＩ１２４とした。
【００７５】
次に、ｐＢＩ２２１（ＣＬＯＮＴＥＣＨ社より購入）を制限酵素ｐｓｔＩで切断し、常法によりその切断末端を平滑化・連結して、そのＳｓｅＩ及びＰｓｔＩ制限酵素部位の消失したプラスミドｐＮＰＩ１１１を得た。続いて、このｐＮＰＩ１１１のＸｂａＩ及びＳａｃＩ制限酵素部位に、そのＧＵＳ遺伝子と置換する形で、ｐＮＰＩ１２４より制限酵素ＸｂａＩ及びＳａｃＩで切り出されたＲ遺伝子を挿入して、プラスミドｐＮＰＩ１２５を作成し、さらに、これから、カリフラワーモザイクウイルス３５Ｓプロモーターとそれに連結されたＲ遺伝子、及びノパリンシンターゼのポリアデニル化シグナルを制限酵素ＨｉｎｄＩＩＩ及びＥｃｏＲＩで切り出して、これをｐＮＰＩ１２８のＨｉｎｄＩＩＩ及びＥｃｏＲＩ制限酵素部位に挿入することにより、プラスミドｐＮＰＩ１２９を得た。
【００７６】
また、ｐＮＰＩ１０１は制限酵素ＳｍａＩで切断し、その切断部位に５′リン酸化ＨｉｎｄＩＩＩリンカー（宝酒造（株）より購入）を挿入し、プラスミドｐＮＰＩ１２２とした。すなわちこのｐＮＰＩ１２２は、ｐＮＰＩ１０１のＳｍａＩ制限酵素部位をＨｉｎｄＩＩＩ制限酵素部位に置換したものである。さらに、このｐＮＰＩ１２２を制限酵素ＰｓｔＩで切断し、常法によりその切断末端を平滑化・連結して、そのＳｓｅＩ及びＰｓｔＩ制限酵素部位が消失した、プラスミドｐＮＰＩ１２３を作成し、次いでこれから、カリフラワーモザイクウイルス３５Ｓプロモーターとそれに連結されたｉｐｔ遺伝子を、制限酵素ＨｉｎｄＩＩＩで切り出して、ｐＮＰＩ１２９のＨｉｎｄＩＩＩ制限酵素部位に挿入し、プラスミドｐＮＰＩ１３０を得た。
【００７７】
目的とするベクターは、このｐＮＰＩ１３０から、カリフラワーモザイクウイルス３５Ｓプロモーターにそれぞれ連結されたｉｐｔ遺伝子とＲ遺伝子、及びこれらの両端にあるＲｓを、制限酵素ＰｓｔＩで切り出して、ｐＢＩ１２１のＳｓｅＩ制限酵素部位に挿入することにより得られ、これをプラスミドｐＮＰＩ１３２と命名した。
【００７８】
なお、このプラスミドｐＮＰＩ１３２もまた、大腸菌ＪＭ１０９株に導入し、この大腸菌をＥ．ｃｏｌｉ ＪＭ１０９（ｐＮＰＩ１３２）（受託番号：ＦＥＲＭＢＰ−５０６５号）として、国際寄託に付した。
【００７９】
ｐＮＰＩ１３２の作成スキムを図１７〜１９に、また、そのＴ−ＤＮＡ領域の制限酵素地図を図２０に示す。図１７〜１９、及び２０中、網かけした三角形は酵母のプラスミドｐＳＲ１に由来する組換え配列Ｒｓと、その配列方向を表す。その他は図２〜５と同様である。
【００８０】
図２０より明らかなように、このプラスミドは、Ｔ−ＤＮＡ領域にマーカー遺伝子としてｉｐｔ遺伝子を、目的遺伝子のモデルとしてＮＰＴＩＩ遺伝子及びＧＵＳ遺伝子を有している点では、ｐＮＰＩ１０６と同様である。しかしこの場合、脱離能を有するＤＮＡ因子として機能するのは、酵母の部位特異的組換え系であるｐＳＲ１系の組換え配列、Ｒｓ間の領域であり、従って、ｉｐｔ遺伝子は、同一方向を向いたこの二つの組換え配列Ｒｓに挟まれた形で挿入されている。
【００８１】
ＩＩＩ．タバコへのｐＮＰＩ１３２の導入及び遺伝子導入を行ったタバコの解析
参考例１のＩＩ、ＩＩＩと同様に、ｐＮＰＩ１３２をＡ．ツメファシエンスＬＢＡ４４０４株に導入して、このＡ．ツメファシエンスをタバコ葉片（ここでは、Nicotiana tabacum cv. SR1を材料として用いた。）に感染させた後、感染葉を、アセトシリンゴン５０ｍｇ／ｌ添加ホルモンフリーＭＳ寒天培地にて培養し、次いでカルベニシリンのみ５００ｍｇ／ｌを添加した同培地で培養した。これを１か月後に同組成の培地に植え継いでさらに１か月培養を続け、多芽体４８系統を分離した。
【００８２】
これらを再び同組成の培地に移植してなお培養を続けたところ、約１か月後（すなわちＡ．ツメファシエンス感染後、約３か月）に、その４８系統のうち７系統から、肉眼で見て正常な形態を示すシュートが生じ、これを分離してやはり同組成の培地に移植すると、結局１０株の正常個体を得ることができた。
【００８３】
これらについて、参考例２のＩＩ−Ｂと同様にＰＣＲ分析を行った結果を図２１〜２３、及び表２に示す。但しここでは、参考例２のＩＩ−Ｂで用いたプライマーの他に、ＧＵＳ遺伝子の存在を確認できるプライマーも併せて使用した。これらを用いてＰＣＲ分析を行うことにより、ｉｐｔ遺伝子が存在する場合には約８００ｂｐ、ｉｐｔ遺伝子がｐＮＰＩ１３２のＴ−ＤＮＡ領域から、Ｒｓに挟まれた部分もろとも脱離した場合には約３ｋｂ（ここまでは、参考例２のＩＩ−Ｂで分析を行った場合と同様である。）、そしてＧＵＳ遺伝子が存在している場合には約１．７ｋｂのＤＮＡ断片が増幅されることとなる。なお図２１〜２３中、左に示した数値については図６と同様である。
【００８４】
【表２】

【００８５】
表２より明らかなように、ここで、肉眼によりその形態を観察することのみで選抜された個体の染色体からはいずれも、マーカー遺伝子であるｉｐｔ遺伝子の存在は検出されず、かわってその脱離を示すＤＮＡ断片の増幅が検出された。一方、目的遺伝子として用いたＧＵＳ遺伝子の存在は、すべての個体において検出された。
【００８６】
一方、ここで多芽体４８系統とほぼ同時に生じた正常体（正常な芽の形態をしたもの）から得られ、正常な伸長・発根を示した個体についても、その頂芽を用いてカナマイシン耐性を検定したところ（カナマイシン２００ｍｇ／ｌ添加ホルモンフリーＭＳ寒天培地使用）、その１６個体のうち２個体がカナマイシン耐性を有することが判明した。
【００８７】
そこで、このカナマイシン耐性個体についてさらに検討を加えるため、他のカナマイシン非耐性１４個体のうちの３個体と共に、多芽体より得られたものの場合と同様にしてＰＣＲ分析を行った。この結果を図２４に示す。図中の左に示した数値については、図６と同様である。
【００８８】
図２４より明らかなように、カナマイシン耐性を示す２個体では、それぞれ、ｉｐｔ遺伝子を含むＲｓに挟まれた領域の脱離、及びＧＵＳ遺伝子の存在を示すＤＮＡ断片の増幅が検出され、これらの染色体には、ｐＮＰＩ１３２に由来すると考えられる遺伝子が組込まれていることを確認した。なお、カナマイシン非耐性の３個体ではかかる増幅は全く検出されず、また、ｉｐｔ遺伝子の存在を示すＤＮＡ断片の増幅は、いずれの個体からも検出されなかった。
【００８９】
本来、多芽体形成能のない系統より得られたこれらの個体は、そもそもＡ．ツメファシエンス感染時に、ｐＮＰＩ１３２が染色体中に導入されなかった細胞を起源とするはずであるが、そう仮定する以上、このベクターに由来する遺伝子がその染色体に存在することは考えられない。ましてや、その染色体中に、これに由来する遺伝子のうち、ｉｐｔ遺伝子のみが存在せず、ＮＰＴＩＩ遺伝子（これらの個体がカナマイシン耐性を示すことより、その存在は明らかである。）、ＧＵＳ遺伝子は完全な形で存在する個体が、かかる頻度で出現することはまず有り得ないことである。
【００９０】
従って、これらの個体では、ｐＮＰＩ１３２が染色体に導入されなかったのではなく、一旦は導入されたと考えるのが妥当である。つまり、ｐＮＰＩ１３２のＴ−ＤＮＡ領域は、Ａ．ツメファシエンスの感染によってこれらの染色体に導入されたが、ここでマーカー遺伝子の脱離に利用したｐＳＲ１系の性能が良すぎたため、そのＡ．ツメファシエンスの感染後、多芽体を形成する前に、その働きによってｉｐｔ遺伝子が染色体から脱離し、結局、ＮＰＴＩＩ遺伝子、ＧＵＳ遺伝子だけが、そのままそこに残留し続けることになったのであろう。ＰＣＲ分析により、かかるカナマイシン耐性個体において、ＧＵＳ遺伝子の存在と同時に認められたｉｐｔ遺伝子を含むＲｓに挟まれた領域の脱離も、これを裏付けるものである。
【００９１】
［実施例２］
Ｉ．ベクターの作成
ｐＢｌｕｅｓｃｒｉｐｔＩＩ ＳＫ＋（東洋紡績（株）製）のＥｃｏＲＩ制限酵素部位に挿入された、ｒｏｌＡ、ｒｏｌＢ、及びｒｏｌＣを含む、全長７．６ｋｂのｒｏｌ遺伝子群（S.Kiyokawa、Plant Physiol.、104:801、1994）を制限酵素ＥｃｏＲＩで切り出し、これをｐＮＰＩ１２９のＥｃｏＲＩ制限酵素部位に挿入して、プラスミドｐＮＰＩ７００を作成した。
【００９２】
次いでこのｐＮＰＩ７００から、ｒｏｌ遺伝子群、及びカリフラワーモザイクウイルス３５Ｓプロモーターとそれに連結されたＲ遺伝子、及びこれらの両端にあるＲｓを、制限酵素ＳｓｅＩで切り出し、これをｐＢＩ１２１のＳｓｅＩ制限酵素部位に挿入して、目的とするプラスミドｐＮＰＩ７０２を得た。
【００９３】
なお、このプラスミドｐＮＰＩ７０２もまた、大腸菌ＪＭ１０９株に導入し、この大腸菌をＥ．ｃｏｌｉ ＪＭ１０９（ｐＮＰＩ７０２）（受託番号：ＦＥＲＭＢＰ−５０６６号）として、国際寄託に付した。
【００９４】
ｐＮＰＩ７０２の作成スキムを図２５に、また、そのＴ−ＤＮＡ領域の制限酵素地図を図２６に示す。なおこれらの図中、用いた記号は図２〜５と同様である。
【００９５】
図２６より明らかなように、このプラスミドはマーカー遺伝子をｐＮＰＩ１３２のｉｐｔ遺伝子から、ｒｏｌ遺伝子群に置き換えたものである。なお、本実施例で用いたｒｏｌ遺伝子群は、天然にはＡ．リゾジェネスのＴ−ＤＮＡ上に存在し、これが植物細胞に導入されると、その植物組織に毛状根を発生させ、またこれから再生した植物体においても矮化等の形態異常を引き起こすことが知られている。
【００９６】
ＩＩ．タバコへのｐＮＰＩ７０２の導入及び遺伝子導入を行ったタバコの解析
参考例１のＩＩ、ＩＩＩ と同様に、ｐＮＰＩ７０２をＡ．ツメファシエンスＬＢＡ４４０４株に導入して、このＡ．ツメファシエンスをタバコ葉片に感染させ、感染葉をアセトシリンゴン５０ｍｇ／ｌ添加ホルモンフリーＭＳ寒天培地にて３日間暗所で培養した。次いでチカルシリンのみ４００ｍｇ／ｌを添加した同培地で培養したところ、毛状根の分化が培養開始後１５日過ぎ頃より認められたので、この毛状根を順次分離し、シュート誘導培地（α−ナフタレン酢酸０．１ｍｇ／ｌ、ベンジルアデニン２．０ｍｇ／ｌ、チカルシリン４００ｍｇ／ｌ添加ＭＳ寒天培地）に置床・培養して不定芽を分化させ、そのうち正常な形態をしていると思われるもの１８個体を肉眼で選抜した。これらについて参考例２のＩＩ−Ｂと同様にＰＣＲ分析を行い、そのうちの９個体について、その染色体から、ｒｏｌ遺伝子を含むＲｓで挟まれた領域が脱離していることを確認した。なお、ここではプライマーとして、かかる脱離が検出されるプライマー（実施例１、参考例２〜４及び参考比較例２で用いたものと同じ。すなわち、約３ｋｂのＤＮＡ断片の増幅により検出。）と、ｒｏｌ遺伝子の存在を検出するためのプライマー（約１．１ｋｂのＤＮＡ断片の増幅により検出）を用いた。
【００９７】
［参考例５］
参考例２で作成したベクター、ｐＮＰＩ１０６を用いて、木本植物である交雑ヤマナラシ（ポプルス・シーボルディ×ポプルス・グランディデンタータ：Populus Sieboldii×Populus grandidentata）への遺伝子導入を行った。
【００９８】
交雑ヤマナラシＹ６３株（秋田十條化成（株）内実験林より採取）の無菌フラスコ苗の茎を、節を含まないように長さ５ｍｍに切断し、これをさらに縦に二つ割りにして材料として用い、ｐＮＰＩ１０６を導入したＡ．ツメファシエンスを参考例１のＩＩＩと同様にして感染させた。感染後、この茎切片を、アセトシリンゴン４０ｍｇ／ｌを添加したホルモンフリー修正ＭＳ寒天培地（シュークロース２ｗ／ｖ％、寒天０．８ｗ／ｖ％）に置床して３日間培養し、次いでカルベニシリンのみ５００ｍｇ／ｌを添加した同培地に移植して培養を続けた。なお、ここで用いた修正ＭＳ培地とは、通常のＭＳ培地の組成のうち、アンモニア態窒素、及び硝酸態窒素の濃度を、それぞれ１０ｍＭ、３０ｍＭに変更したものである。
【００９９】
約２か月後、この切片から生じた不定芽を分離し、さらに２か月間培養を行うことにより多芽体６系統が得られたが、これらについて引き続き継代培養を行ったところ、約４か月経過後（Ａ．ツメファシエンス感染より約８か月後）から、この多芽体より伸長する、正常な形態のシュートが認められ始めた。これらのシュートは、ＩＢＡ０．０５ｍｇ／ｌを添加した２／３倍稀釈ＭＳジェランガム培地（シュークロース２ｗ／ｖ％、ジェランガム０．３ｗ／ｖ％）に移植して培養することにより、正常な発根個体となすことができ、結局、Ａ．ツメファシエンス感染１０か月目までには、２系統の多芽体より、合わせて７個の正常個体を得ることができた。
【０１００】
これらについて、実施例１のＩＩＩと同様にＰＣＲ分析を行ったところ、ｉｐｔ遺伝子はその全ての個体において検出されず、またＧＵＳ遺伝子に関しては、このうち２個体においてその存在が検出され、本発明のベクターが、木本植物においても有効に機能することが確認された。なお、残りの５個の正常個体については、ＧＵＳ遺伝子はその一部分の存在のみしか検出されなかったが、かかる個体は、ここで用いたベクター、ｐＮＰＩ１０６により導入されたトランスポゾンＡｃが、ｉｐｔ遺伝子と共にその染色体より脱離する際に、その近辺にあったＧＵＳ遺伝子をも巻き込み、これを引き千切るようにして脱離してしまった結果、生じたものであると考えられる。
ここで、多芽体から正常なシュートが分化してきた状態を図２７に示す。
【０１０１】
［実施例３］
実施例１において、ｐＮＰＩ１３２により遺伝子を導入された正常個体（多芽体を経由したもの）に対し、さらに本発明のベクターを用いて遺伝子導入を行った。
【０１０２】
ｐＮＰＩ１３２のＧＵＳ遺伝子のみをＨＰＴ遺伝子（ハイグロマイシン抵抗性遺伝子）に置き換え、これ（ｐＮＰＩ１４０、図２８）を用いて上記正常個体に対し、参考例１のＩＩ、ＩＩＩと同様に遺伝子導入操作を行った。多芽体は、このベクターを導入したＡ．ツメファシエンスの感染から４０日後に１０系統を得ることができ、これを分離し、同組成の培地（カルベニシリン５００ｍｇ／ｌ添加ホルモンフリーＭＳ寒天培地）に移植してなお培養を続けたところ、２０日後（すなわち、Ａ．ツメファシエンスの感染から約２か月後）には、そのうち１系統で、肉眼で見て正常な形態を示すシュートの分化が認められた。
【０１０３】
この分化してきた正常なシュートのうちの１個について、参考例１のＩＶ−Ｂと同様にＰＣＲ分析を行った結果を図２９に示す。但しここでは、参考例１のＩＶ−Ｂで用いたプライマーの他に、ｉｐｔ遺伝子を含むＲｓに挟まれた領域の染色体ＤＮＡからの脱離を検出するため、ＮＰＴＩＩ遺伝子とＨＰＴ遺伝子上に結合すべく設計されたプライマー、及びＨＰＴ遺伝子の存在を検出するためのプライマーを併せて使用した（それぞれ、約４ｋｂ及び約１ｋｂのＤＮＡ断片の増幅により検出。）。なお図中、左に示した数値については図６と同様である。
【０１０４】
図２９より明らかなように、このシュートから抽出した染色体ＤＮＡのＰＣＲ分析においては、Ｒｓに挟まれた領域と共にｉｐｔ遺伝子が脱離したことを示す約４ｋｂのＤＮＡ断片、及びＨＰＴ遺伝子の存在を示す約１ｋｂのＤＮＡ断片の増幅が認められる一方、ｉｐｔ遺伝子の存在を示す約８００ｂｐのＤＮＡ断片の増幅は認められない。これは、このシュートの染色体ＤＮＡ中に、一旦は導入されたｉｐｔ遺伝子が、その後、Ｒｓに挟まれた領域と共にそこから脱離・消失した一方、ＨＰＴ遺伝子はそのＤＮＡ中に残留していること、つまり、先にｐＮＰ１１３２により目的遺伝子（ＮＰＴＩＩ遺伝子及びＧＵＳ遺伝子）を導入された個体に対し、その構成のうち目的遺伝子に関する構成のみを変えたベクターにより、ｉｐｔ遺伝子という同じ遺伝子をマーカーとして、新たな目的遺伝子（ＨＰＴ遺伝子）が重ねて導入されたこと、さらには再び同じマーカー遺伝子を用いて、第３、第４、あるいはそれ以上の目的遺伝子の導入が可能であることを示すものである。
【０１０５】
以上の実施例及び参考例より明らかなように、ここで得られた多芽体の系統は、必ずｉｐｔ遺伝子をその染色体中に有し（図６）、また、この多芽体という、肉眼で識別可能な顕著な形態異常を示す組織はすべて、ｉｐｔ遺伝子と共に導入したモデル的な目的遺伝子である、ＮＰＴＩＩ遺伝子の発現によるカナマイシン耐性を例外なく示した。これは、かかる形態異常誘導遺伝子が、植物へ遺伝子導入を行う際のマーカー遺伝子として十分に適用可能であり、これをマーカー遺伝子とする本発明のベクターもまた、植物への遺伝子導入用ベクターとして有用であることを証明するものである。
【０１０６】
また、このｉｐｔ遺伝子をトランスポゾンＡｃの内部に組み込んだベクター、ｐＮＰＩ１０６を用いて植物への遺伝子導入を行うと、遺伝子導入操作を行った直後の培養当初には多芽体を形成した組織から、目的遺伝子（ＮＰＴＩＩ遺伝子及び／またはＧＵＳ遺伝子）により付与される特性は保持したまま、ｉｐｔ遺伝子が染色体から消失して多芽体形成能を失ったシュート等が得られ（表１、図１３、１４、１６）、しかも、かかるシュート等の形態は肉眼で識別可能であり（図１５、２７）、これらを選抜・分離して培養することにより、正常な形態をした伸長発根個体が得られた。また、このシュートより得られた組織からさらに再分化した組織も、すべて多芽体形成能はなく正常な形態を示し、このようなシュート等が均一な細胞からなることを明示した。
【０１０７】
さらに同様の結果は、脱離能を有するＤＮＡ因子として部位特異的組換え系に由来するものを用いた場合、形態異常誘導遺伝子としてｒｏｌ遺伝子を用いた場合にも観察された。すなわち、かかるベクターを用いて植物への遺伝子導入を行った場合（実施例１、２）でも、遺伝子導入操作後ある時期までは異常な形態を形成した組織から、目的遺伝子は保持したまま、形態異常誘導遺伝子がその染色体から消失し、正常な形態を示す組織、そして個体が得られ（図２１〜２３、表２）、しかもこの同じ個体に対し、目的遺伝子に関する構成のみを変えたベクターにより、同じ形態異常誘導遺伝子をマーカーとして、遺伝子導入操作・培養・肉眼による選抜を繰り返すことにより、目的遺伝子を多重に導入することも可能であった（実施例３、図２９）。
【０１０８】
従って、マーカー遺伝子として形態異常誘導遺伝子、脱離能を有するＤＮＡ因子として部位特異的組換え系に由来するＤＮＡ因子を用い、この形態異常誘導遺伝子が脱離能を有するＤＮＡ因子と挙動を一つにする位置に組込んだ、かかるベクターを用いれば、まず、遺伝子導入操作後の細胞を培養して生じてくる異常な形態を示す組織を肉眼で選抜し、これを分離してさらに培養を続け、次いで生じてくる今度は正常な形態を示す組織をやはり肉眼で選抜するだけで、目的遺伝子のみが染色体等に残留してその機能を保持している細胞のみからなる組織、さらには植物体が得られることとなる。
【０１０９】
しかも脱離能を有するＤＮＡ因子として、部位特異的組換え系を用いると、かなり高い確率で形態異常組織から正常個体が得られる。加えて、その脱離も速やかに起こるため、正常な形態をした組織もまた、より速やかに効率よく検出可能となるのである。
【０１１０】
脱離能を有するＤＮＡ因子として、トランスポゾンを用いた場合、及び部位特異的組換え系に由来するものを用いた場合に、生じた形態異常組織から目的遺伝子を保持した正常個体が得られる効率を、表３に比較して示す。
【０１１１】
【表３】

【０１１２】
なお、実施例１及び参考例１〜４においてはいずれの場合にも、植物ホルモン無添加の条件で、遺伝子導入細胞を含む組織は増殖し、不定芽を分化し、さらには植物体を再生したが、これは、マーカー遺伝子として遺伝子導入細胞の染色体に組込まれた、ｉｐｔ遺伝子の働きによるものと考えられる。つまり、これらの遺伝子の発現により、その連結されたプロモーターの働きによる多少はあるものの、植物ホルモンがその細胞内で過剰に生産されるため、その細胞自体が多芽体等の組織を分化するだけではなく、これが隣接する組織にもその産生する植物ホルモンがある程度作用し、結局、培地中に植物ホルモンを人為的に添加したのと同様な状態になったのである。
【０１１３】
【発明の効果】
本発明で使用するベクターは、マーカー遺伝子として形態異常誘導遺伝子を使用したものである。このため、これを用いて植物への遺伝子導入を行った場合、目的遺伝子導入組織の選抜にあたっては、遺伝子導入操作後の細胞を通常の培地を用い、通常の培養条件で培養して、生じてくる異常な形態をした組織を肉眼により識別するだけでよく、選抜のため化学物質等を培地中へ添加する必要がないため、作業が簡略化され、またその選抜中に植物細胞の活性低下を招くおそれもない。
【０１１４】
また、この形態異常誘導遺伝子としてｉｐｔ遺伝子を用いた場合には、その働きにより、遺伝子導入細胞を含む組織は増殖し、不定芽等を分化するので、通常は植物細胞の培養において、その増殖・分化のために必須とされる、培地中への植物ホルモン添加も不要とすることができる。
【０１１５】
加えて、このベクターは、形態異常誘導遺伝子を、部位特異的組換え系に由来する脱離能を有するＤＮＡ因子と挙動を一つにする位置に組み込んで使用する。すると、このマーカー遺伝子は、植物細胞への遺伝子導入後に一定の確率で、かかるＤＮＡ因子と共に、これが存在し機能するＤＮＡ上から脱離してその機能を失い、同時に導入されたこれとは挙動を一つにしない位置に存在する目的遺伝子のみが、同じＤＮＡ上に発現可能な状態で残留することとなる。このため、このような構成をとった場合、このベクターは、導入しようとする目的遺伝子に関する部分を変更するのみで、マーカー遺伝子を始めとする他の構成に何らの変更をも加えることなく、ある一つの植物体へ遺伝子の多重導入を行うために、何度でも無制限に繰り返して用いることができる。
【０１１６】
しかもこの場合も、マーカー遺伝子である形態異常誘導遺伝子の機能の消失は、遺伝子導入の際と同様に、遺伝子導入組織の形態の変化として肉眼で検出できるので、目的遺伝子のみが染色体等に残留してその機能を保持している細胞だけからなる組織を、確実・容易に選抜できることとなる。従って、遺伝子の多重導入も効率良く行え、また、かかる細胞だけからなる遺伝子導入個体、すなわちマーカー遺伝子の影響が排除され、その遺伝子産物がもたらす危惧から完全に解放された個体も、交配過程を経ることなく得ることができる。
【０１１７】
つまり、マーカー遺伝子の影響が排除され、その遺伝子産物がもたらす危惧から完全に解放され、かつ、遺伝子導入に用いたベクターに由来する構成以外の部分が、遺伝子導入の対象となった親植物と同一の遺伝子構成を有する個体が作成されるのである。
【図面の簡単な説明】
【図１】Ｔｉプラスミドの一般的構造、及びＡ．ツメファシエンスＰＯ２２株Ｔ−ＤＮＡ領域のＰｓｔＩ断片制限酵素地図を含む概念図である。
【図２】ｐＩＰＴ４作成スキムのうち、ｐＩＰＴ２の作成までを示す図である。
【図３】ｐＩＰＴ４作成スキムのうち、ｐＩＰＴ２からｐＩＰＴ３の作成までを示す図である。
【図４】ｐＩＰＴ４作成スキムのうち、ｐＩＰＴ３からｐＩＰＴ４の作成までを示す図である。
【図５】ｐＩＰＴ４の構造のうち、Ｔ−ＤＮＡ領域の制限酵素地図である。
【図６】ｐＩＰＴ４を用いて遺伝子導入を行ったタバコより生じた、多芽体のＰＣＲ分析結果を示す図である。
【図７】ｐＮＰＩ１０６作成スキムのうち、ｐＮＰＩ１０２の作成までを示す図である。
【図８】ｐＮＰＩ１０６作成スキムのうち、ｐＩＰＴ４及びｐＮＰＩ１０２からｐＮＰＩ１０３の作成までを示す図である。
【図９】ｐＮＰＩ１０６作成スキムのうち、ｐＮＰＩ１０３からｐＮＰＩ１０６の作成までを示す図である。
【図１０】ｐＮＰＩ１０６の構造のうち、Ｔ−ＤＮＡ領域の制限酵素地図である。
【図１１】生物の形態を示す写真であり、参考例２におけるＮｏ．２のシュートの１か月培養後の状態を示している。
【図１２】生物の形態を示す写真であり、参考例２におけるＮｏ．８のシュートの１か月培養後の状態を示している。
【図１３】参考例２におけるＮｏ．８のシュートのＰＣＲ分析結果を示す図である。
【図１４】参考例３におけるＮｏ．１３−１、１４−１のシュートより得られた正常個体のＰＣＲ分析結果を示す図である
【図１５】生物の形態を示す写真であり、参考例３においてタバコの多芽体から正常なシュートが分化してきた状態を示している。
【図１６】参考例４において、参考例２におけるＮｏ．７のシュートより生じた葉から得られた、正常個体のＰＣＲ分析結果を示す図である。
【図１７】ｐＮＰＩ１３２作成スキムのうち、ｐＮＰＩ１２８の作成までを示す図である。
【図１８】ｐＮＰＩ１３２作成スキムのうち、ｐＮＰＩ１２８からｐＮＰＩ１２９の作成までを示す図である。
【図１９】ｐＮＰＩ１３２作成スキムのうち、ｐＮＰＩ１０１及びｐＮＰＩ１２９からｐＮＰＩ１３２の作成までを示す図である。
【図２０】ｐＮＰＩ１３２の構造のうち、Ｔ−ＤＮＡ領域の制限酵素地図である。
【図２１】実施例１において、系統Ｎｏ．１５〜２１のシュートより得られた正常個体のＰＣＲ分析結果を示す図のうち、ｉｐｔ遺伝子の存在が検出されるプライマーを使用して行った結果を示すものである。
【図２２】実施例１において、系統Ｎｏ．１５〜２１のシュートより得られた正常個体のＰＣＲ分析結果を示す図のうち、ｉｐｔ遺伝子を含むＲｓに挟まれた領域の脱離が検出されるプライマーを使用して行った結果を示すものである。
【図２３】実施例１において、系統Ｎｏ．１５〜２１のシュートより得られた正常個体のＰＣＲ分析結果を示す図のうち、ＧＵＳ遺伝子の存在が検出されるプライマーを使用して行った結果を示すものである。
【図２４】実施例１において、多芽体形成能のない系統より生じた正常個体のＰＣＲ分析結果を示す図である。
【図２５】ｐＮＰＩ７０２の作成スキムを示す図である。
【図２６】ｐＮＰＩ７０２の構造のうち、Ｔ−ＤＮＡ領域の制限酵素地図である。
【図２７】生物の形態を示す写真であり、参考例５において交雑ヤマナラシの多芽体から正常なシュートが分化してきた状態を示している。
【図２８】ｐＮＰＩ１４０の構造のうち、Ｔ−ＤＮＡ領域の制限酵素地図である。
【図２９】実施例３において、遺伝子の多重導入後、多芽体より分化してきた正常なシュートのＰＣＲ分析結果を示す図である。[0001]
[Industrial applications]
The present invention relates to a plant into which a target gene has been introduced using a novel vector.
[0002]
[Prior art]
Transformation of microorganisms, cultured cells, and the like using genetic engineering technology is currently applied for the purpose of producing a physiologically active substance useful as a pharmaceutical, and has made a great contribution to the actual industry. In the field of plant breeding, the application of genetic engineering technology to the actual industry is slightly delayed due to the fact that the life cycle of plants is much longer than that of microorganisms, etc. Since the gene can be directly introduced into a plant to be bred, (a) only the trait to be modified can be introduced, and (b) traits of a species other than the plant (such as a microorganism) can also be introduced into the plant. , (C) the breeding period can be significantly shortened, and has many advantages as compared with classical breeding in which crosses are repeated, and its application is expected to bring dramatic progress in plant breeding, And this expectation is becoming a reality.
[0003]
Specifically, in order to prepare a transgenic plant by introducing a target gene into a target plant, (1) introducing the target gene into a plant cell (including a case where the target gene is introduced into a chromosome, a nucleus, etc.), and (2). This necessarily involves three steps: selection of a plant tissue consisting solely of cells into which the target gene has been introduced, and (3) regeneration of a plant from the selected plant tissue. In selecting the target gene-introduced tissue, a tissue in which the target gene is expressed (a tissue in which the target gene is expressed is, of course, a tissue composed of cells into which the gene has been introduced) is generally selected from plants. The target gene is introduced into a plant cell together with a marker gene whose expression can be easily detected at the stage of cell culture, because the gene is not regenerated and is difficult to confirm with the naked eye. The presence or absence of a marker gene) is usually used as an index for introducing a target gene. For example, such a marker gene is involved in kanamycin resistance gene (NPTII; neomycin kinase gene) or hygromycin resistance gene (HPT; hygromycin kinase gene) conferring antibiotic resistance, and amino acid synthesis. Synthase gene (NOS), octopine synthase gene (OCS), and sulfonylurea resistance gene (ALS; acetolactate synthase gene) that confer pesticide resistance.
[0004]
However, the expression of the marker gene is also a serious obstacle when such transgenic plants are intended to be used for food or the like. That is, it is extremely difficult to ensure the safety of the gene product generated by the expression of such a marker gene in the human body. Therefore, when a transgenic plant created using these marker genes as an indicator is sold as a food, a detailed investigation is required on the effect of the gene product on the human body. For example, the NPTII gene has been used extensively at the laboratory level as a marker gene since the early 1980's, but only in 1994 was its gene product identified by the U.S. Food and Drug Administration (FDA) as a food additive. This gene was used as a marker gene, and the transgenic plant thus transformed was used for edible purposes. However, at the level of the consumer who really wants to say this, such anxiety about the NPTII gene product is still hard to wipe out.
[0005]
At present, only genes contributing to the detoxification of growth inhibitors on plant cells, including this NPTII gene, have been put to practical use as marker genes. The culture is performed in a medium containing a growth inhibitor, and the presence or absence of the expression of the marker gene, that is, the resistance to the substance is evaluated, and this is used as an index. However, in this case, if plant tissue grows in the presence of resistance, i.e., in the presence of such substances, this is a matter of degree, and culturing in the presence of such inhibitors is not preferred for plant cells. Influence is inevitable, and in reality, side effects such as a reduction in the rate of regeneration and regeneration of the transgenic tissue due to a decrease in the activity of the plant cells have become a problem.
[0006]
Furthermore, the expression of a marker gene in a plant cell after selection of a transgenic tissue causes a great obstacle in plant breeding by gene transfer. That is, when another gene is to be newly introduced into a transgenic plant created using a certain marker gene, the gene cannot be introduced again using the same marker gene. Since the marker gene is already present in the plant, even if it is introduced again into the same plant together with a new target gene, the new target gene may or may not be introduced, but the plant cells and This is because this marker gene is always expressed in tissues, and it can no longer be used as an index for introducing a target gene. Therefore, such multiple gene transfer uses a different marker gene each time each transfer process is repeated. However, the effectiveness of the marker gene varies depending on the plant species, and each use of the marker gene requires a preliminary experiment for setting conditions (for example, in rice, the HPT gene is more effective than the NPTII gene). (K. Shimamoto et al., Nature (London), 338: 274, 1989) In addition, since the types of marker genes are limited in the first place, multiplexing of genes is only possible by changing the marker genes. It is impossible to repeat gene transfer indefinitely, that is, the number of times gene transfer can be repeated for a plant is naturally limited by the number of types of marker genes that can be used for that plant. Moreover, the types of marker genes that can be used at present are described above. Therefore, after the selection of the transgenic plant tissue, the marker gene used at that time is removed from the DNA of the chromosome or the like where it exists and functions, and the influence of this is removed. It can be said that the technology for eliminating cells, tissues, and plants from plants is essential for the new development of plant breeding using genetic engineering technology.
[0007]
At present, as such a technique for eliminating the influence of a marker gene, a method of using a marker gene and a plant transposon as an integral body and removing this together with the transposon from the introduced plant chromosome (International Publication WO92 / 01370). And a method using a site-specific recombination system of P1 phage instead of this transposon (WO 93/01283). According to these methods, although the probability is fairly low, cells in which the marker gene has been removed from the plant chromosome at a certain rate after gene transfer, that is, only the target gene remains expressed in the chromosome, and the marker gene Transgenic plant cells with lost function can be obtained.
[0008]
However, even if these methods result in a plant cell in which the marker gene has been removed from its chromosome, these cells are scattered in cells where it is still present and expressing, Since these two types of cells cannot be distinguished with the naked eye, they cannot be separated as they are. As in the case of introducing the target gene, when trying to select cells in which the marker gene is expressed, it is possible to perform selection using an index such as drug resistance and auxotrophy to be imparted to the plant cells. At the time of this selection, cells without expression of the marker gene cause severe growth impairment and many of them can be killed, so that the purpose here is to obtain cells from which the marker gene has been removed. Cannot be applied at all.
[0009]
Therefore, in order to obtain a plant in which the marker gene has been removed from the chromosome by these methods and only the target gene has remained, cells in which the marker gene has been removed from the chromosome and cells in which the marker gene is still present are present. Analyze plant tissues obtained by culturing and growing to some extent with the mixture remaining, and then re-differentiating the cultured tissues using techniques such as Southern hybridization or polymerase chain reaction. It is conceivable to carry out selection. The premise in this case is that such a redifferentiated individual is derived from a single cell, and therefore all the constituent cells are of equal properties, e.g. from a cell from which the marker gene has been removed. Needless to say, it is an assumption that an individual consists solely of such cells. However, in many cases, it is already well known that the cells constituting such a redifferentiated individual are not always uniform, and even if the marker gene is used, the cell from which it has been removed from the chromosome and the cell which still exists there This means that coexistence and distribution are completely irregular even within the same individual of a regenerated plant or even within the same tissue. Therefore, with these methods, it is very difficult to obtain an individual consisting only of cells whose marker gene has been removed from the chromosome at the stage of merely regenerating a cultured tissue and regenerating a plant individual. In addition, in the analysis for selecting this, no matter what method is used, all of the currently known methods, including the above-described analysis method, are used as co-test samples even for the whole individual when applied. Since a tissue having an appropriate mass, for example, a single leaf, is used instead of one cell, the result is limited to measuring and analyzing the overall tendency of the marker gene in one leaf. . Therefore, in the same individual / tissue, cells in which the marker gene has been removed and cells in which the marker gene is present are usually in a mixed state. Even if an individual consisting solely of the removed cells occurs accidentally, it must be said that it is almost impossible to select that individual based on the analysis results. Even if the presence of the marker gene was not detected in this result, there is no guarantee that the same applies to the tissues of other parts of the same individual, and the presence of the marker gene was not detected in the first place. In other words, this merely indicates that the abundance or the like is below the detection limit, and in this case, it cannot be said that the sample does not contain any cells in which the marker gene is present. Because.
[0010]
After all, in such a case, it is only by passing through germ cells such as pollen and egg cells that an individual without the effect of the marker gene is obtained. That is, since these germ cells are unicellular, for example, if this is an egg cell in which the marker gene has been removed from the chromosome of the cell, when self-pollination is performed using this, certain By chance, a fertilized egg will be obtained that does not contain the marker gene in its chromosome, and the fertilized egg will give rise to an individual consisting only of cells having the same characteristics. Selection by an analysis technique such as the Southern hybridization method may be performed on this individual. In other words, according to the method described in the report mentioned here, even if a cell in which the marker gene has been removed from the chromosome is obtained, the plant is re-differentiated from the cultured tissue in which the cell is present, and the re-differentiated plant is further regenerated. Only after obtaining the F1 or progeny progeny, an individual consisting of only such cells can be obtained, and the individual can be selected as an individual from which the influence of the marker gene has been eliminated. is there.
[0011]
In Japanese Patent Application Laid-Open No. Hei 6-276872, in order to eliminate the influence of the marker gene, the marker gene is inserted into a plasmid vector separate from the target gene during gene transfer, and only the marker gene is transferred from the cells after the transfer. Although techniques for removal are reported, even in this case, a mating process must be performed for removal, which is similar to the above two reports.
[0012]
However, such a method is difficult to apply to a slow-growing woody plant, a sterile individual, or a hybrid individual that is worth F1 itself. Even if this is not the case, it is often the case that these are present, and the probability of their removal from functional chromosomal DNA or viral vector DNA is very low, so that their removal, that is, In practice, it is required that the loss of function of a marker gene can be easily detected at least at the stage of a cultured tissue. If this cannot be reliably detected without regenerating the cultured tissue and producing a progeny by crossing the regenerated individuals, practical application of the progeny is almost impossible.
[0013]
[Problems to be solved by the invention]
An object of the present invention is to provide a transgenic plant which is prepared using a novel vector without using a plant cell growth inhibitor or the like that reduces the activity of a plant cell.
[0014]
Further, the present invention provides a transgenic plant prepared by using a novel vector, which does not undergo the process of producing F1 or progeny by crossing, in which the effects of marker genes have been eliminated, and further, the effects of marker genes have been eliminated, Another object of the present invention is to provide a plant into which multiple genes have been introduced.
[0015]
[Means for Solving the Problems]
An object of the present invention is a morphological abnormality-inducing gene as a marker gene,DNA factor derived from site-specific recombination system as DNA factor having elimination ability, The morphological abnormality-inducing gene is located at a position where the behavior is integrated with the DNA element having the elimination ability, and the target gene is not integrated with the DNA element having the elimination ability. This can be achieved by using a vector present at the position.
[0016]
Here, the morphological abnormality-inducing gene causes unusual morphological differentiation in plant tissues, for example, dwarfing, collapse of apical dominance, pigment change, apical carcinoma, hairy root, leaf waving, etc. Gene means. For example, as these morphological abnormality induction genes,iptGenes (AC Smigocki, LD Owens, Proc. Natl. Acad. Sci. USA, 85: 5131, 1988),iaaM (tryptophan monooxygenase) gene (HJ Klee et al., GENES & DEVELOPMENT, 1:86, 1987), gene5Gene (H. koerber et al., EMBO Journal, 10: 3983, 1991), gene6bGenes (PJJ Hooyas et al., Plant Mol. Biol., 11: 791, 1988), andlolA~DofrolSpecific genes such as genes (FF White et al., J. Bacteriol., 164: 33, 1985) are found in various plants to produce carcinomas and teratomas (ie, the formation of adventitious buds and adventitious roots). It has been reported that it is present in Agrobacterium, which is a bacterium that causes germplasm, and also a subspecies of Pseudomonas syringae (Ps eudomonas  syringae  subsp.savastanoi)iaaThe presence of the L (indoleactic acid-lysine synthase) gene (A. Spena et al., Mol. Gen. Genet., 227: 205, 1991), and the presence of homeobox genes and phytochrome genes from various plants. Have been reported.
[0017]
Any of these genes can be used in the present invention, but in particular causes a collapse of apical dominanceiptCauses genes and hairy root formation, dwarfing of plants regenerated from hairy roots and wavy leavesrolThe gene group is preferable as a marker gene used in the present invention because it causes a characteristic morphological abnormality among various morphological abnormality-inducing genes. Furthermore, they can also be designed to combine them to redifferentiate specific structures, such as adventitious buds and adventitious roots, into specific plants into which they have been introduced. In the present invention, such a combination of genes can be used to construct and use a morphological abnormality-inducing gene in accordance with the conditions for producing a transgenic plant, such as the type of plant to be transfected.
[0018]
Since the tissue having an abnormal morphology caused by the introduction of such a morphological abnormality-inducing gene into cells is composed only of cells having this gene, a vector is constructed together with the target gene using this as a marker gene, and As long as the cells are introduced into the plant cells and cultured, only tissues with abnormal morphology that arise from this can be selected with the naked eye to select tissues consisting only of cells into which the marker gene, that is, the target gene, has been introduced. You can do it. Here, a vector refers to a DNA sequence used for the purpose of introducing a foreign gene into a host cell and having a function necessary for expressing the foreign gene in the host cell. The gene is often introduced into the host cell in an integrated form.
[0019]
That is, if the gene is introduced using the vector of the present invention, the cells after the gene introduction are simply cultured under ordinary culture conditions using a normal medium such as an MS medium, and only the cells into which the target gene has been introduced are formed. The selection of plant tissues by the naked eye becomes possible. Therefore, in the selection, it is not necessary to use a special substance for selecting a transgenic tissue, such as a plant cell growth inhibitor, which not only simplifies the operation but also reduces the activity of the plant cell due to these effects. There is no risk of the decrease.
[0020]
On the other hand, a DNA element having an elimination ability refers to a DNA sequence capable of elimination itself from chromosomal DNA or the like in which these exist and function.Such DNA factors include site-specific recombination systems ( site-specific recombination system ) Are known.
[0023]
This site-specific recombination system comprises a recombination site having a characteristic DNA sequence (corresponding to a DNA element having an elimination ability of the present invention), and a sequence specifically binding to this DNA sequence and When present, it is composed of two elements, an enzyme that catalyzes recombination between the sequences, and this DNA sequence is present at two locations on the same DNA molecule at a certain interval in the same direction. If this sequence is present, the region flanked by it is detached from the DNA molecule (plasmid, chromosome, etc.), and if this sequence is present in two opposing directions, this region is It shows the behavior of reversing. In the present invention, the former elimination action is utilized, but such elimination / reversal inside the recombination site occurs as a result of so-called homologous recombination by a site-specific recombination system. This is the most different point from the mechanism using a transposon, which causes its detachment as a process of transposition. The gene coding for the recombinase does not necessarily need to be present on the same DNA molecule as the recombination site. It is known that reversal can occur (NLCraig, Annu. Rev. Genet., 22:77, 1988).
[0024]
At present, site-specific recombination systems are Cre / lox systems, pSR1 systems, FLP systems isolated from microorganisms such as phages, bacteria (for example, Escherichia coli) and yeast.cersystem,fimAlthough the system is known (for review, NL Craig, Annu. Rev. Genet., 22:17, 1988), its existence is not yet known in higher organisms. However, the site-specific recombination system isolated from these microorganisms also seems to use the Cre / lox system derived from P1 phage as a vector for gene transfer into plants in the above-mentioned WO 93/01283. Furthermore, it has been clarified that even when introduced into a species (including a plant) different from the species from which it is derived, it behaves the same as the behavior in the original organism. Incidentally, in one embodiment of the present invention, yeast (Zygosaccharomyces  rouxiiPSR1 system (H. Matsuzaki et al., J. Bacteriology, 172: 610, 1990), which is a site-specific recombination system, was used by inserting a recombination enzyme between the recombination sites. The pSR1 line has also been reported to maintain its original function in higher plants (H. Onouchi et al, Nucleic Acid Res., 19: 6373, 1991).
[0025]
In the present invention, the place where the morphological abnormality-inducing gene is to be inserted may be a site at which it can be removed together with a DNA element having a removing ability. For example, as a DNA element having detachment abilityOf site-specific recombination systemsWhen the pSR1 system is used, it can be inserted anywhere as long as it does not inhibit the expression of the recombinase within the region between the recombination sites.
[0026]
If a vector is constructed by combining a morphological abnormality-inducing gene with a DNA element capable of detachment in this way, when a gene is introduced into a plant using this vector, the morphological abnormality-inducing gene used as a marker gene after introduction is used. Desorbs from the DNA into which they have been introduced and functioning, such as plant chromosomes, along with a DNA element having the detaching ability, and although its frequency varies, it loses its function with a certain probability. In this case, the target gene that does not behave as one will continue to remain on the same DNA. Therefore, this vector can be used indefinitely and repeatedly for multiple introduction of a gene into a single plant simply by changing the configuration of the target gene to be introduced. Moreover, the loss of the function of the morphological abnormality-inducing gene can be detected by the naked eye as a change in the form, which occurs during the culture of the transgenic tissue, as in the case of gene transfer, so that only the target gene remains on the chromosome and the like. A tissue consisting only of cells having the function can be reliably and easily selected only by culturing the tissue without performing any special operation. Therefore, even if the probability that such cells are actually produced is low, this vector can be used practically, and multiple transfer of a gene using this vector can not only be repeated any number of times, but also a complete plant can be obtained. Since this can be repeated at the stage of the cultured tissue before regeneration, it can be performed efficiently. In addition, in order to obtain a transgenic individual consisting of only such cells, it is only necessary to regenerate a plant from the tissue selected as described above, and it is not necessary to go through a mating process. And the transgenic individual thus obtained has just been completely freed from fears that the gene product of the marker gene may cause harm to the human body. In addition, this vector uses other characteristics derived from the use of a morphological abnormality-inducing gene as a marker gene, that is, a cell growth inhibitor or the like that may cause a decrease in cell activity in the process of selecting a transgenic tissue. Needless to say, it has the above-mentioned advantage that there is no need.
[0027]
The vector of the present invention can be used in any plant that can be transfected by genetic engineering techniques, and the target gene that can be introduced into a plant by the vector of the present invention can impart an agriculturally excellent trait. Genes and agriculturally excellent traits are not always imparted, but various types can be selected depending on the purpose, such as genes required for studying gene expression mechanisms.
[0028]
Generally, in order to produce a protein such as an enzyme from a gene, in addition to the structural gene sequence encoding the information of these polypeptides, a promoter sequence (expression start sequence) and a terminator sequence (expression termination sequence) of the structural gene are used. ) Is required. For example, as a promoter sequence that functions in plants, cauliflower mosaic virus 35S promoter (JT Odell et al., Nature (London), 313: 810, 1985), nopaline Synthetic enzyme promoter (WHR Langridge et al., Plant Cell Rep., 4: 355, 1985), ribulose diphosphate carboxylase / oxygenase small subunit promoter (R. Fluhret al., Proc. atl.Acad.Sci.USA, 83: 2358, 1986), and the terminator sequence includes a polyadenylation signal of nopaline synthase (A. Depicker et al., J. Mol. Appl. Gen., 1: 561). , 1982), and the polyadenylation signal of octopine synthase (J. Gielen et al., EMBO J., 3: 835, 1984). Therefore, when simply referred to as a gene in the present invention, it refers to a structural gene and a gene expression regulatory sequence. Incidentally, in the present invention, various gene expression regulatory sequences as described above can be used without being limited to the gene expression regulatory sequences used in the examples. Furthermore, the gene, that is, DNA, can be obtained by cloning cDNA or genomic DNA, and if its sequence is known in advance, it can also be obtained by chemical synthesis.
[0029]
The vector of the present invention can be indirectly introduced into plant cells via viruses or bacteria that infect plants (I. Potrykus, Annu. Rev. Plant Physiol. Plant Mol. Biol., 42: 205, 1991). In this case, for example, cauliflower mosaic virus, gemini virus, tobacco mosaic virus, brom mosaic virus and the like can be used as the virus, and bacteria such as Agrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens) and Agrobacteria can be used. Um rhizogenes (hereinafter abbreviated as A. rhizogenes) and the like can be used. In general, Agrobacterium does not infect monocotyledonous plants, but only dicotyledonous plants.In recent years, there have been reports of cases in which these genes are transmitted to monocotyledonous plants and transfected. (For example, International Publication WO94 / 00977).
[0030]
The vector of the present invention can also be directly introduced into plant cells by a physical / chemical method such as a microinjection method, an electroporation method, a polyethylene glycol method, a fusion method, and a high-speed ballistic penetration method ( I. Potrykus, Annu.Rev.Plant Physiol.Plant Mol.Biol., 42: 205, 1991). For many monocotyledonous plants and dicotyledonous plants that are difficult to infect with Agrobacterium, the indirect transfection method using Agrobacterium, which is widely used for gene transfer, cannot be used. It is.
[0031]
[Action]
In the present invention, the morphological abnormality-inducing gene used as a marker gene, when expressed, causes an abnormal production of plant growth hormone, etc., in the introduced plant cell, thereby causing an abnormality in the physiological state in the cell, As a result, the direction of the growth and differentiation is changed, and various morphological abnormalities are caused. In other words, the abnormally morphologically occurring tissues, such as disordered bud aggregates (polyblasts) and hairy roots in which apical dominance has collapsed, are derived from cells into which such morphological abnormality-inducing genes have been introduced. Since this is the result of abnormal growth and differentiation, it consists only of cells having this gene. Therefore, if this is introduced into a plant cell together with the target gene as a marker gene and the cells are cultured, the marker gene, that is, the target gene is introduced only by selecting the resulting tissue showing an abnormal morphology with the naked eye. This makes it possible to select a tissue consisting solely of damaged cells, so that the transfected tissue can be selected by the naked eye without any special operation such as addition of a plant cell growth inhibitor to the medium.
[0032]
Further, in the present invention, a DNA element having an elimination ability is combined with this morphological abnormality-inducing gene, and is used by incorporating the morphological abnormality-inducing gene into a position where the behavior and the DNA element having an elimination ability become one. If a gene is introduced into a plant using a vector having such a configuration, after the introduction, a morphological abnormality-inducing gene that is a marker gene, together with a DNA factor having an elimination ability, from the DNA on which they have been introduced and functioning, While it loses its function with a certain probability and loses its function, the target gene whose behavior does not become one remains on the same DNA and keeps its function. Therefore, this vector is used only for changing the configuration of the target gene to be introduced, and for carrying out the multiple introduction of a gene into a single plant without any change in other configurations including the marker gene. In addition, it can be used repeatedly indefinitely. In a transgenic tissue or the like in which a marker gene has lost its function, the expression of the marker gene can no longer occur, so the expression of the same marker gene can be repeated many times to serve as an index for introducing a new target gene. It is.
[0033]
Moreover, the loss of the function of the marker gene, that is, the morphological abnormality-inducing gene, can be detected by the naked eye as a change in the morphology of the transfected tissue, as in the case of gene transfer. Then, a tissue consisting of only cells in which only the target gene remains on its chromosome or the like and retains its function can be reliably and easily selected. That is, in order to obtain a tissue consisting of only such cells, first, the cells after the gene transfer treatment are cultured to remove abnormal morphologies such as polyblasts and hairy roots resulting from the expression of the morphological abnormality induction gene. The tissue to be shown may be selected with the naked eye, separated from the tissue, and further cultured, and then the tissue, which is generated from the tissue having the abnormal morphology and has the normal morphology, may be selected with the naked eye. In other words, there is no need to perform any special operation for selection, just repeating the culture, selection with the naked eye, and separation.BecauseIn addition, multiple gene transfer can be performed efficiently. Furthermore, a plant consisting solely of such cells can be obtained by simply regenerating the plant from the obtained transgenic tissue without going through a mating process.
[0034]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described based on examples. In the following Examples, more detailed experimental procedures were performed by Molecular Cloning, 2nd edition (Sambrook et al. Eds., Cold Spring Harbor Laboratory Press, New York, 1989) or by the manufacturer unless otherwise specified. It was performed according to the instruction manual.
[0035]
【Example】
[referenceExample 1]
I. Creating a vector
Pathogenic A. Present on the T-DNA of Tumefaciens strain PO22 (Hiroetsu Gahiko, Chemical Regulation of Plants, 24:35, 1989, (see FIG. 1)).iptRestriction enzymes for genesPstI and excised from the plasmid pUC7 (Molecular Cloning, 2nd Edition, Volume 1, 4.10).PstBy ligating with the I restriction enzyme site, a recombinant plasmid pIPT1 was obtained. From this plasmid, contains the promoter and polyadenylation signaliptRestriction enzymes for genesBamHI andPstI and excised from plasmid pUC119 (purchased from Takara Shuzo).BamHI-PstLigation between the I restriction sites yielded plasmid pIPT2, which was theniptRestriction enzymes for structural genes and polyadenylation signal among genesRsaI and excised from the plasmid pUC119.SmaBy ligating with the I restriction enzyme site, a recombinant plasmid pIPT3 was obtained. And further inserted into pIPT3iptRestriction enzymes for genesBamHI andSacI and the vector plasmid pBI121 (purchased from CLONTECH) for gene transfer into plants.BamHI-SacPlasmid pIPT4 was created by ligation between the I restriction sites. A. harboring this plasmid When a plant is infected with Tumefaciens, the inside of the structure, the LB site and the RB site called the so-called T-DNA region, here the NPTII gene,iptA region of about 5 kb containing the gene will be integrated into the plant chromosome.
[0036]
This plasmid pIPT4 is used for E. coli (Escherichia  coli) Was introduced into JM109 strain, and this E. coli was transformed into E. coli. E. coli JM109 (pIPT4) (accession number: FERM BP-5063) and deposited in an international deposit.
[0037]
FIGS. 2 to 4 show a scheme for preparing pIPT4, and FIG. 5 shows a restriction enzyme map of the T-DNA region. In FIGS. 2 to 4 and FIG. 5, P and T circled are respectively:ipt35S-P indicates the cauliflower mosaic virus 35S promoter, Nos-P indicates the promoter of the nopaline synthase gene, and T (FIG. 4) or Nos-T (FIG. 5) indicates the promoter of the gene itself and the polyadenylation signal. 1 shows the polyadenylation signal of the nopaline synthase gene.
[0038]
BookreferenceIn the example, as apparent from FIG. 5, among the morphological abnormality-inducing genes as marker genes, apical dominance predominates to be disrupted and contributes to polyblast formation.iptThe gene was used as a model, and the NPTII gene was used as a target gene as a model.iptThe gene is pathogenic A. It is a member of a tumorigenic gene maintained by Tumefaciens. In a plant cell into which this gene has been introduced, the overexpression of cytokinin, a kind of plant hormone, causes the direction of differentiation toward multibud formation.
[0039]
Also bookreferenceIn the example,iptAs a gene expression control sequence, the promoter sequence is the cauliflower mosaic virus 35S promoter, and the terminator sequence isiptThe polyadenylation signal of the gene itself was used.
[0040]
II. Introduction of pIPT4 into Agrobacterium
A. Tumefaciens strain LBA4404 (purchased from CLONTECH) was added to 10 ml of YEB liquid medium (beef extract 5 g / l, yeast extract 1 g / l, peptone 1 g / l, sucrose 5 g / l, 2 mM MgSO4).₄, PH 7.2 at 22 ° C (hereinafter referred to as pH at 22 ° C unless otherwise specified), and cultured at 28 ° C until the OD630 reaches the range of 0.4 to 0.6. did. The culture solution was centrifuged at 6900 × g at 4 ° C. for 10 minutes to collect the cells, and then the cells were suspended in 20 ml of 10 mM Tris-HCl (pH 8.0). The cells were collected by centrifugation, and then the cells were suspended in 200 μl of YEB liquid medium, which was used as a bacterial solution for plasmid introduction.
[0041]
In a 15 ml tube (Falcon), 200 μl of the bacterial solution for plasmid introduction and 6 μg of the plasmid pIPT4 prepared in I were mixed, and the mixture was added to ethanol, which had been cooled in liquid nitrogen for 30 to 40 minutes, for 5 minutes each tube. After immersion and cooling, the plate was placed in a water bath at 29 ° C. for 25 minutes, and then 750 μl of YEB liquid medium was added thereto and cultured at 29 ° C. for 1 hour with shaking. This bacterial solution was inoculated on a 50 mg / l kanamycin-added YEB agar medium (agar 1.2 w / v%, other composition is the same as described above) and cultured at 28 ° C. for 2 days. After transplanting to a liquid medium and further culturing, a plasmid was extracted from the cells by an alkaline method. Extracted plasmid into restriction enzymePstI,BamHI andEcoCleavage using RI and analysis by agarose gel electrophoresis showed that A. It was confirmed that the plasmid pIPT4 was introduced into the Tumefaciens LBA4404 strain.
[0042]
III. Introduction of pIPT4 from Agrobacterium into tobacco
Tobacco grown in a greenhouse (Nicotiana  tabacum  cv. xanthi, the same applies hereinafter unless otherwise specified. ) Was sterilized by immersion in a 1 v / v% sodium hypochlorite aqueous solution for 5 minutes, washed three times with sterile water, and then the middle vein was removed to prepare leaf pieces of about 8 mm square. The tobacco leaf pieces were transformed with A.p. The cells were immersed in a bacterial solution of Tumefaciens strain LBA4404 (OD630 = 0.25, cultured in a YEB liquid medium overnight, diluted with sterile water to adjust the bacterial cell concentration) for about 1 minute, infected with this, and sterilized. After removing the excess bacterial solution by placing it on a filter paper, a plant hormone-free (hormone-free) MS agar medium (T. Murashige and F. Skoog, Physiol. Plant., Supplemented with 50 mg / l of acetosyringone, 15: 473, 1962, provided that 0.8 ag / w% of agar was added.). This was cultured at 25 ° C. for 3 days at Zenmei (explants and plant tissues / plants were cultured under these conditions unless otherwise specified), and then hormone-free MS agar medium containing only 500 mg / l carbenicillin. As a result, 22 adventitious buds were re-differentiated, and the adventitious buds were separated and further cultured in a medium having the same composition. As a result, six lines of polyblasts were obtained. These were subcultured to the same medium every month, and from the third month, were subcultured several times on a hormone-free MS agar medium containing no carbenicillin to confirm that there was no growth of Agrobacterium. , Kanamycin resistance test and PCR analysis.
[0043]
IV. Analysis of transgenic tobacco
A. Kanamycin resistance test
By culturing the polyblasts of the 6 lines obtained in III as they were without subculture, the leaves that had developed were cut out, prepared in about 3 mm square, and MS agar containing kanamycin 200 mg / l. The medium was placed on a culture medium (containing 1 mg / l of BA and 0.2 mg / l of NAA) and observed one month after the culture. As a result, even in the same medium, formation of multiple shoots was observed in all leaf pieces obtained from these multiple shoots.
[0044]
B. PCR analysis
Chromosomal DNA was extracted from all of the 6 multiblasts obtained in III, and the transgene was confirmed by PCR.
[0045]
The extraction of chromosomal DNA was performed using the following improved CTAB method.
First, about 1 g of the leaves of the multi-bud was crushed in a mortar under liquid nitrogen using a mortar, which was previously kept at 60 ° C. with 2 w / v% hexadecytrimethylammonium bromide (1.4 M NaCl, 0%). The suspension was suspended in 5 ml of a buffer consisting of 0.2 v / v% β-mercaptoethanol, 20 mM EDTA, and 100 mM Tris-HCl (pH 8.0). The suspension was warmed at 60 ° C. for 30-60 minutes with gentle shaking, then cooled to room temperature, to which was added an equal volume of chloroform: isoamyl alcohol (24: 1) and gently mixed. . Subsequently, the supernatant was collected by centrifugation at 1600 × g for 5 minutes, ２ volume of isopropyl alcohol was added to the supernatant, mixed gently again, and allowed to stand on ice for 10 minutes. Chromosomal DNA was precipitated, which was precipitated by centrifugation at 1600 × g for 10 minutes. The precipitated chromosomal DNA was washed with 70 v / v% ethanol, dried under vacuum, and dissolved in 300 μl of TE (10 mM Tris-HCl, 1 mM EDTA).
[0046]
on the other hand,iptTo detect genes by PCR,iptWhen bound to the gene, the primers (oligonucleotides) used were synthesized with a DNA synthesizer (Applied Biosystems) so that the distance between the two primers was about 800 bp.iptFor gene amplification, 1 μg of the extracted chromosomal DNA was mixed with 10 μM Tris-HCl (pH 8.8 at 25 ° C.), 50 mM KCl, 1.5 mM MgCl containing 0.2 μM of this primer.₂Dissolved in 50 μl of a mixture having a composition of 1 w / v% Triton X-100, 0.1 mM dNTP, and 1.25 units of Taq polymerase (purchased from CETUS), and added at 94 ° C. for 1 minute and 30 seconds. After heating, the reaction was repeated 30 times at 94 ° C. for 1 minute, 55 ° C. for 2 minutes, and 72 ° C. for 3 minutes. The resulting reaction mixture was analyzed using agarose gel electrophoresis, and theiptThe presence of the gene was confirmed by amplification of an approximately 800 bp gene derived therefrom.
[0047]
The results are shown in FIG. 6. As is clear from the figure, the gene amplification of about 800 bp was observed in all of the six buds of the line. In the figure, the numerical values shown on the left represent the number of bases of each band component detected when the DNA size marker is electrophoresed.
[0048]
[referenceComparative Example 1]
referenceIn Example 1, III, The analysis was also performed on 16 buds having no bud formation ability obtained from adventitious buds regenerated from Tumefaciens infected leaves. That is, as a result of isolating and culturing 22 adventitious buds in the same III, when the 6 buds were selected, they were not budded but normal buds (hereinafter referred to as normal bodies). In the same manner as in the case of the multibud line in III and IV, the bacteria were removed, a kanamycin resistance test was performed, and PCR analysis was performed on 9 of them. However, in these normal strains, the leaf pieces placed on the kanamycin-supplemented medium all turned brown and died in about 3 months.iptAmplification of a DNA fragment of about 800 bp indicating the presence of the gene could not be detected in any of the nine lines analyzed.
FIG. 6 shows the results of the PCR analysis.
[0049]
[referenceExample 2]
I. Creating a vector
Plasmid pHSG398 (purchased from Takara Shuzo Co., Ltd.)BamAfter cleavage with HI, the protruding end generated by the cleavage was blunt-ended with T4 DNA polymerase I (large subunit), and this end was ligated again to obtain plasmid pNPI100. That is, this pNPI100 isBamThe HI restriction enzyme site has been eliminated. On the other hand, plasmid pCKR97 (T. Izawa et al., Mol. Gen. Genet., 227: 391, 1991) was replaced with a restriction enzyme.PstCut with corn transposonAcIs cut out, and this is used for pNPI100.PstIt was inserted into the I restriction enzyme site to obtain plasmid pNPI102.
[0050]
next,referenceFrom the plasmid pIPT4 (FIG. 5) prepared in Example 1, the cauliflower mosaic virus 35S promoter was ligated thereto.iptGenes, restriction enzymesHindIII andSacI, and the protruding end was blunt-ended with T4 DNA polymerase I.HinInsertion into the cII restriction enzyme site yielded plasmid pNPI101. From this plasmid pNPI101, the cauliflower mosaic virus 35S promoter wasiptGenes, this time with restriction enzymesPstI andEcoAfter excision with RI, the protruding end was blunted with T4 DNA polymerase I, and thenBamPlasmid pNPI103 was obtained by ligation with plasmid pNPI102, which had been cut with HI and the protruding ends were similarly blunt-ended. That is, in this plasmid pNPI103, it was ligated to the 35S promoter.iptGene is a transposonAcOld insideBamIt will be at the HI restriction enzyme site.
[0051]
A vector of interest was prepared from this plasmid pNPI103 by using a cauliflower mosaic virus 35S promoter andiptTransposons containing genesAcThe restriction enzymePstI, which was excised from the vector plasmid pBI121 for gene transfer into plants.SseIt was obtained by insertion into the I restriction enzyme site, and this was designated as plasmid pNPI106.
[0052]
This plasmid pNPI106 was also introduced into E. coli JM109 strain, and this E. coli was transformed into E. coli. E. coli JM109 (pNPI106) (accession number: FERMBP-5064) and deposited in an international deposit.
[0053]
The construction scheme of plasmid pNPI106 is shown in FIGS. 7 to 9, and the restriction enzyme map of its T-DNA region is shown in FIG. 7 to 9 and FIG.AcAre indicated by opposing black triangles. In FIG. 10, Ac-P isAcIndicates an endogenous promoter. Other symbols are the same as in FIGS.
[0054]
As is clear from FIG. 10, this plasmid contains a T-DNA region, that is, a region to be integrated into the plant chromosome, as a marker gene.iptThe gene has an NPTII gene and a GUS (β-galactosidase) gene as a model of the target gene, andiptGene is a transposonAcIt exists in the form inserted inside. The GUS gene can be used to analyze gene expression in plants, since cells containing the GUS metabolize a special substrate and produce a blue pigment. Is a gene that has been
[0055]
II. Analysis of transgenic tobacco with pNPI106 and gene transfer into tobacco
A. Introduction of pNPI106 into tobacco and expression test of transgene
referenceSimilarly to II and III of Example 1, pNPI106 was converted to A. The A. tumefaciens strain LBA4404 was introduced into this A. After infecting tobacco leaf pieces with Tumefaciens, the infected leaves were cultured on a hormone-free MS agar medium supplemented with 50 mg / l acetosyringone, and then cultured on the same medium supplemented with only 500 mg / l carbenicillin. At the month, 63 multiblasts were isolated.
[0056]
These cells were further transplanted to a medium of the same composition (hormone-free MS agar medium supplemented with 500 mg / l of carbenicillin), and continued to be cultured. One month later, shoots of slightly elongated polyblasts (hereinafter referred to as polyblasts) were generated. Each of the shoots is referred to as a shoot.), And the shoots are extended to about twice or more the size of other shoots, and the development of lateral shoots is not observed.iptNine shoots which seemed to have a weakened effect of the gene were selected with the naked eye, and the leaves attached to the shoots were used in accordance with the same kanamycin resistance test as IV-A of Reference Example 1 and the method of Jefferson et al. A GUS gene expression test (GUS activity test) was performed. Also, shoots after leaves were cut out were transplanted to a hormone-free MS agar medium and continued to be cultured. One month later, the morphology was observed.iptGene expression was assayed.
[0057]
Table 1 shows the results.
[Table 1]

[0058]
As is clear from Table 1, In the shoot of No. 8, despite the fact that the obtained leaves have kanamycin resistance and GUS activity, they do not form polyblasts even if they are cultured for one month. This contributes to the formation of multiple shoots in the vector pNPI106 used here.iptGene is a transposonAcA. harbors this vector since it is present in a form inserted inside A. When Tumefaciens was used to infect tobacco leaf pieces and started culturing, they were introduced and expressed in tobacco chromosomesiptDuring subsequent culture, the geneAcIt is probable that this function was eliminated along with this and the function was lost. On the other hand, even on the same vector, the NPTII gene and the GUS geneAcNo. and No. are inserted at positions where the behavior is not united. Even in the shoots of No. 8, they are still expressed in the chromosome.
[0059]
In Table 1, No. In shoots 3, 5, and 6, kanamycin resistance and polyblast formation were observed, but only GUS activity was negative. That is, among these shoots, only the GUS gene among the genes introduced using pNPI106 was not expressed, which was attributed to an integration error that occurred when these genes were introduced into the plant chromosome. Conceivable. That is, A. When a gene is introduced through Tumefaciens, in a plasmid having a structure such as pNPI106, the entire T-DNA region, that is, the entire inner region of the RB site and LB site should be integrated into the plant chromosome. As a result, the integration may not be completely performed, and may be incorporated in a state where a part of the LB terminal side is broken. In the pNPI106 used here, among the genes inserted into the T-DNA region, the GUS gene is located at the position closest to the LB site. These shoots did not express the GUS gene and did not show any activity since they were integrated into the chromosome in a state where they were cut and lost their functions, or were not integrated at all.
Here, No. 2 and No. The morphology of the eight shoots after one month of culture is shown in FIGS.
[0060]
At this time, No. The leaves obtained from 8 shoots and subjected to the kanamycin resistance test were continuously cultured after the test. As a result, five adventitious buds were obtained from these leaves, all of which were normal.
[0061]
B. PCR analysis
No. 1 in Table 1. After observing the multibud formation ability of 1 to 9 shoots,referencePCR analysis was performed in the same manner as in Example 1 IV-B,iptFurther studies were conducted on the existence of the gene. But here,referenceIn addition to the primers used in IV-B of Example 1, primers designed to bind to the NPTII gene and the GUS gene were also used. When PCR was performed using these primers, the T-DNA region of pNPI106 wasAc, And inserted insideiptWhen gene elimination occurs, a DNA fragment of about 3 kb is amplified.Acas well asiptThus, the detachment of the gene from the chromosomal DNA can be detected.
Among the PCR analyzes performed here, no. The results for the 8 shots are shown in FIG. In the figure, the numerical values shown on the left are the same as in FIG.
[0062]
As is clear from this, No. In the chromosomal DNA extracted from the shoot No. 8,iptGenes andAcWhile the amplification of a DNA fragment of about 3 kb indicating the elimination ofiptNo amplification of a DNA fragment of about 800 bp indicating the presence of the gene is observed. this is,iptGene isAcIn addition, it means that the shoot has been detached and disappeared from the chromosomal DNA.
[0063]
In contrast, No. For shoots 1-7 and 9 at this time, about 3 kb of DNA amplification was not detected from any of the chromosomal DNA samples, while about 800 bp of amplification was detected in all. Therefore, in these shootsiptThe gene is stillAcTogether with the chromosomal DNA.
[0064]
[referenceComparative Example 2]
referenceA. of II-A of Example 2 In the culture of Tumefaciens-infected leaves, three of the normal bodies that had differentiated along with the buds were isolated.referenceAs in II of Example 2, a kanamycin resistance test, a GUS activity test, a morphological observation one month after culture, and a PCR analysis were performed.
[0065]
The results are shown in Table 1. None of them have kanamycin resistance, GUS activity, and polyblast formation ability. Further, in PCR analysis, about 800 bp and about 3 kb of DNA fragments were all amplified. Not detected.
[0066]
[referenceExample 3]
referenceA part of each of the 63 multibud lines isolated in Example 2 was further subcultured on a hormone-free MS agar medium, and cultivation was continued. After about two months, a normal normal bud that could be visually identified from the two multibud lines was obtained. Morphology, shoots showing dominant apical buds, no. 13-1 to 3 and 14-1 to 7 were obtained in total. When this shoot was separated and transplanted to a medium having the same composition, an elongating rooted individual in a normal form was obtained. For individuals obtained from 13-1 and 14-1,referenceWhen a PCR analysis was carried out in the same manner as in II-B of Example 2, amplification of a DNA fragment of about 800 bp was not detected from either, while amplification of a DNA fragment of about 3 kb was detected in both,iptGene isAcAt the same time, it was confirmed that these individuals were detached and disappeared from chromosomal DNA. FIG. 14 shows the results. In the figure, the numerical values shown on the left are the same as in FIG. GUS gene expression was detected in all individuals obtained from seven shoots.
FIG. 15 shows a state in which normal shoots have differentiated from the multiple shoots.
[0067]
[referenceExample 4]
referenceOf the nine shoots selected in Example 2, in Table 1, No. The leaves obtained from the shoots designated as No. 7 were cultured on a hormone-free MS agar medium for about one month, and one normal body was selected and separated visually from the six adventitious buds differentiated therefrom. When this was transplanted to a medium of the same composition, an elongating rooted individual in a normal form was obtained.referenceWhen a PCR analysis was carried out in the same manner as in Example II-B, the disappearance of a DNA fragment of about 800 bp and the amplification of the same fragment of about 3 kb revealed thatiptGene isAcIt was confirmed that the compound was desorbed / dissipated. FIG. 16 shows the results. In the figure, the numerical values shown on the left are the same as in FIG. GUS gene expression was also confirmed for the same individual.
[0068]
[Example1]
I. Isolation of site-specific recombination system (pSR1 system) from yeast
yeast(Zygosaccharomyces  rouxii, 5 g of YPAD liquid medium (yeast extract 10 g / l, polypeptone 20 g / l, adenine 0.4 g / l, glucose 20 g / l) and cultured at 30 ° C. overnight. . The culture was centrifuged at 6900 × g at 20 ° C. for 3 minutes to collect cells (hereinafter, all the cells were collected under the same conditions), and the obtained cells were mixed with 2 ml of 0.2 M Tris-HCl ( (pH 8.0) -5 v / v% The suspension was suspended in β-mercaptoethanol, left at 25 ° C. for 30 minutes with occasional gentle stirring, and then collected. To the cells, 1 ml of 10 w / v% sorbitol-5 w / v% KPO4 (pH 6.8) containing 2.5 mg / ml of Zymolyase-20T (purchased from Seikagaku Corporation) was added, and suspended. , Left for 90 minutes, collected again by centrifugation, and suspended in 1 ml of a lysis solution (0.2 M NaCl, 0.1 M EDTA, 5 w / v% SDS, 50 mM Tris-HCl, pH 8.5). Further, Proteinase K (20 mg / ml) was added, mixed, and left at 60 ° C. for 1 hour. Then, the mixture was returned to room temperature and purified sequentially by extraction with phenol: chloroform and chloroform. Then, an equal amount of isopropanol was added to the obtained supernatant to precipitate chromosomal DNA and plasmid pSR1, and the precipitate was 6900 × g, 4 ° C., 10 ° C. This was spun down by centrifugation for minutes. The precipitated DNA was washed with 70 v / v% ethanol, dried under vacuum, and dissolved in 100 μl of TE.
[0069]
From the DNA thus extracted (including both chromosomal DNA and plasmid pSR1), only the site-specific recombination system (this is referred to as pSR1 system) present in plasmid pSR1 was amplified by PCR. . The pSR1 system is composed of an R gene, which is a recombinase gene, and a recombination sequence Rs, and its base sequence has already been clarified (H. Araki et al., J. Mol. Biol., 182: 191, 1985). In the present invention, in order to amplify the R gene, at the 5 ′ position of 22 bases corresponding to positions 5596 to 5617 in the sequence of plasmid pSR1,XbaIA primer to which a restriction enzyme site has been added (5'-CCTCTAGAATGCAATTGACCAAGGACTACTG-3 ') and a 5' position of 22 bases corresponding to positions 4126 to 4147,SacIPrimers (5'-CCGAGCCTCTTAATCTTGTCAGGAGGTGTCA-3 ') to which restriction enzyme sites were added were synthesized and used. On the other hand, for amplification of Rs, two sets of 30 bases each (a total of four types) of primers were synthesized and used. That is, one set replaces three bases in the sequence corresponding to positions 287 to 316 in the base sequence of plasmid pSR1,SseA primer having a base sequence into which an I restriction enzyme site has been introduced (5'-AGGGATTGAGCTTACTGGACGGGAATCCTGCA-3 '), and replacing 4 bases in the sequence corresponding to positions 690 to 719,HindIII restriction enzyme siteXhoA primer (5'-CAACTCGAGCAATCAAAGCTTCTCGTAGTC-3 ') into which an I restriction enzyme site has been introduced (Rs amplified by this primer set is referred to as Rs1). The other set also has a base sequence of plasmid pSR1. Of the sequences corresponding to positions 287 to 316, three bases are substituted,XhoI restriction enzyme siteEcoA primer having a base sequence into which an RI restriction enzyme site has been introduced (5′-AGGGATTGAGCTTACTCGAGGGGAATTCTGGA-3 ′), and replacing 5 bases in the sequence corresponding to positions 688 to 717,SseAnd a primer (5'-ACTGGACCAATCCCTGCAGGTCGTAGTCAA-3 ') into which an I restriction enzyme site has been introduced (Rs amplified by this primer set is called Rs2).
[0070]
To amplify the R gene and Rs, 1 μl of 100 μl of the extracted DNA in the form of a TE solution contains 0.2 μM of each primer set.referenceEach of the mixed solutions used in IV-B of Example 1 was mixed in 50 μl, and these were reacted in 30 heating cycles of 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute and 30 seconds. The resulting reaction mixture was analyzed using agarose gel electrophoresis to confirm the amplification of the R gene and Rs.
[0071]
II. Creating a vector
Rs1 amplified by the PCR method is replaced with a restriction enzymepstI andXhoI and cut it with pSL1180 (purchased from Pharmacia Biotech Co., Ltd.).PstI andXhoThis was inserted into the I restriction enzyme site to obtain a plasmid pNPI126.
[0072]
Next, pHSG398EcoAn RI restriction enzyme site, andHindIn order to eliminate the III restriction enzyme site, cleavage with these restriction enzymes, blunting of the cleaved end with T4 polymerase I (large subunit), and re-ligation of the blunt end were sequentially repeated, and these restriction enzyme sites were eliminated. Plasmid pNPI121 was obtained and itsSalI andPstRs2 amplified by the PCR method at the restriction site IXhoI andPstThe plasmid pNPI127 was prepared by digesting with I and inserting.
[0073]
And this pNPI127SmaI andXbaRestriction enzyme from pNPI126 to I restriction enzyme siteSmaI andSpeRs1 cut out with I was inserted to obtain plasmid pNPI128.
[0074]
The R gene is obtained by subjecting the fragment amplified by the PCR method to a restriction enzyme.XbaI andSacI and digested with pHSG398.XbaI andSacI was inserted into the restriction enzyme site. The resulting plasmid was designated as pNPI124.
[0075]
Next, pBI221 (purchased from CLONTECH) was replaced with a restriction enzyme.pstI, and the cleaved end is blunted and ligated by a conventional method.SseI andPstA plasmid pNPI111 in which the I restriction enzyme site had disappeared was obtained. Then, this pNPI111XbaI andSacIn the form of substituting the GUS gene for the I restriction enzyme site, the restriction enzymeXbaI andSacThe R gene excised with I was inserted to create plasmid pNPI125, from which the cauliflower mosaic virus 35S promoter and its linked R gene, and the polyadenylation signal of nopaline synthase wereHindIII andEcoIt is cut out by RI and this is pNPI128.HindIII andEcoPlasmid pNPI129 was obtained by insertion into the RI restriction enzyme site.
[0076]
PNPI101 is a restriction enzymeSmaAnd 5 'phosphorylation at the cleavage siteHinA dIII linker (purchased from Takara Shuzo Co., Ltd.) was inserted to give a plasmid pNPI122. That is, this pNPI 122 isSmaI restriction enzyme siteHinIt has been replaced with a dIII restriction enzyme site. Furthermore, this pNPI122 is used as a restriction enzyme.PstI, and the cleaved end is blunted and ligated by a conventional method.SseI andPstPlasmid pNPI123, in which the I restriction enzyme site had been deleted, was created and then the cauliflower mosaic virus 35S promoter was linked to it.iptGenes, restriction enzymesHinexcised with dIII and pNPI129HinInsertion into the dIII restriction enzyme site yielded plasmid pNPI130.
[0077]
The target vector was ligated from this pNPI130 to the cauliflower mosaic virus 35S promoter.iptGene and R gene, and Rs at both ends of these genes,PstI and cut out the pBI121SseIt was obtained by insertion into the I restriction enzyme site, which was named plasmid pNPI132.
[0078]
In addition, this plasmid pNPI132 was also introduced into E. coli JM109 strain, and this E. coli was transformed into E. coli. E. coli JM109 (pNPI132) (accession number: FERMBP-5065) and deposited in an international deposit.
[0079]
The construction scheme of pNPI132 is shown in FIGS. 17 to 19, and the restriction enzyme map of the T-DNA region is shown in FIG. 17 to 19 and 20, the shaded triangles indicate the recombinant sequence Rs derived from the yeast plasmid pSR1 and the sequence direction thereof. Others are the same as FIGS.
[0080]
As is clear from FIG. 20, this plasmid was used as a marker gene in the T-DNA region.iptIt is the same as pNPI106 in that the gene has the NPTII gene and the GUS gene as a model of the target gene. However, in this case, the function as a DNA element capable of detachment is a region between Rs, a recombination sequence of the pSR1 system, which is a site-specific recombination system of yeast, andiptThe gene has been inserted between the two recombination sequences Rs oriented in the same direction.
[0081]
III. Analysis of tobacco transfected with pNPI132 and transgenic tobacco
referenceSimilarly to II and III of Example 1, pNPI132 was converted to A. The A. tumefaciens strain LBA4404 was introduced into this A. Claw fascination is tobacco leaf pieces (here,Nicotiana  tabacum cv. SR1 was used as the material. ), The infected leaves were cultured on a hormone-free MS agar medium supplemented with 50 mg / l acetosyringone, and then cultured on the same medium supplemented with only 500 mg / l carbenicillin. One month later, the cells were subcultured into a medium having the same composition, and cultivation was further continued for one month. Thus, 48 multiblasts were isolated.
[0082]
When these were transplanted again to a medium having the same composition and continued to be cultured, about one month later (that is, about three months after infection with A. tumefaciens), seven of the 48 strains were visually observed. When shoots exhibiting a normal morphology were produced and isolated and transplanted to a medium having the same composition, 10 normal individuals were finally obtained.
[0083]
For these,referenceThe results of performing PCR analysis in the same manner as in Example II-B are shown in FIGS. 21 to 23 and Table 2. However, herereferenceIn addition to the primer used in II-B of Example 2, a primer capable of confirming the presence of the GUS gene was also used. By performing PCR analysis using these,iptAbout 800 bp if the gene is present,iptWhen the gene is eliminated from the T-DNA region of pNPI132, including the portion sandwiched by Rs, about 3 kb (up to this point,referenceThis is the same as the case where the analysis was performed using II-B in Example 2. ), And when the GUS gene is present, a DNA fragment of about 1.7 kb is amplified. The numerical values shown on the left in FIGS. 21 to 23 are the same as those in FIG.
[0084]
[Table 2]

[0085]
As is evident from Table 2, here, from the chromosomes of individuals selected only by observing their morphology with the naked eye, all are marker genes.iptThe presence of the gene was not detected, but instead amplification of a DNA fragment indicating its elimination was detected. On the other hand, the presence of the GUS gene used as the target gene was detected in all individuals.
[0086]
On the other hand, an individual obtained from a normal body (having a normal bud morphology) almost simultaneously with the 48 multibud lines and showing normal elongation and rooting was also used for kanamycin using the apical bud. When the resistance was assayed (using a hormone-free MS agar medium supplemented with 200 mg / l of kanamycin), it was found that two of the 16 individuals had kanamycin resistance.
[0087]
Therefore, in order to further examine this kanamycin-resistant individual, PCR analysis was performed together with three of the other 14 kanamycin-non-resistant individuals in the same manner as in the case of those obtained from multiple buds. The result is shown in FIG. Numerical values shown on the left side of the figure are the same as in FIG.
[0088]
As is clear from FIG. 24, in the two individuals showing kanamycin resistance,iptThe elimination of the region flanked by Rs containing the gene and the amplification of a DNA fragment indicating the presence of the GUS gene were detected, and it was confirmed that these chromosomes had integrated a gene considered to be derived from pNPI132. . Such amplification was not detected at all in the three kanamycin non-resistant individuals.iptNo amplification of the DNA fragment indicating the presence of the gene was detected in any of the individuals.
[0089]
Originally, these individuals obtained from a line that did not have the ability to form multiple shoots were A. cerevisiae. At the time of T. faecalis infection, pNPI132 should have originated from a cell that was not introduced into the chromosome, but assuming so, it is unlikely that the gene from this vector is present on that chromosome. Furthermore, among the genes derived from this in the chromosome,iptOnly the gene is absent, and the NPTII gene (the presence of these individuals is evident from the fact that they exhibit kanamycin resistance) and the GUS gene are present in such a frequency that individuals present in perfect form are unlikely to appear at such a frequency. It is impossible.
[0090]
Therefore, in these individuals, it is reasonable to assume that pNPI132 was not, but was, once introduced into the chromosome. That is, the T-DNA region of pNPI132 is Introduced into these chromosomes by infection with Tumefaciens. Here, the performance of the pSR1 system used for elimination of the marker gene was too good. After the infection of Tumefaciens, before its formation of multiple buds,iptThe gene would have detached from the chromosome, and eventually only the NPTII gene and GUS gene would continue to remain there. PCR analysis revealed the presence of the GUS gene in such kanamycin-resistant individuals at the same time.iptThe elimination of the region flanked by Rs containing genes also supports this.
[0091]
[Example2]
I. Creating a vector
pBluescript II SK + (manufactured by Toyobo Co., Ltd.)EcoInserted into the RI restriction enzyme site,lolA,lolB,as well asrolC7.6 kb in lengthrolRestriction enzymes for genes (S. Kiyokawa, Plant Physiol., 104: 801, 1994)EcoIt is cut out by RI, and this is pNPI129.EcoPlasmid pNPI700 was created by insertion into the RI restriction enzyme site.
[0092]
Then, from this pNPI700,rolThe genes, the cauliflower mosaic virus 35S promoter and the R gene linked thereto, and the Rs at both ends thereof wereSseI and cut it out with pBI121.SseInsertion into the I restriction enzyme site yielded the desired plasmid pNPI702.
[0093]
This plasmid pNPI702 was also introduced into E. coli JM109, and this E. coli was transformed into E. coli. E. coli JM109 (pNPI702) (accession number: FERMBP-5066) and deposited in an international depository.
[0094]
FIG. 25 shows a scheme for preparing pNPI702, and FIG. 26 shows a restriction enzyme map of the T-DNA region. The symbols used in these figures are the same as those in FIGS.
[0095]
As is clear from FIG. 26, this plasmid changed the marker gene to pNPI132.iptFrom the gene,rolIt is replaced with a group of genes. In addition, it was used in this example.rolThe gene group is naturally A.. It is known that it is present on the T-DNA of lysogenes, and when it is introduced into plant cells, it causes hairy roots in the plant tissue, and also causes morphological abnormalities such as dwarfing in the regenerated plants. ing.
[0096]
II. Analysis of tobacco transfected with pNPI702 and transgenic tobacco
referenceAs in II and III of Example 1, pNPI702 was converted to A. The A. tumefaciens strain LBA4404 was introduced into this A. Tumefaciens was infected to tobacco leaf pieces, and the infected leaves were cultured on a hormone-free MS agar medium supplemented with acetosyringone 50 mg / l for 3 days in a dark place. Then, when cultured in the same medium supplemented with 400 mg / l of ticarcillin alone, hairy root differentiation was observed around 15 days after the start of the culture. The hairy roots were sequentially separated, and a shoot induction medium (α- An adventitious bud is differentiated by placing and culturing on an MS agar medium containing 0.1 mg / l of naphthalene acetic acid, 2.0 mg / l of benzyladenine, and 400 mg / l of ticarcillin, of which the normal morphology is considered to be normal. Individuals were selected visually. About thesereferencePCR analysis was performed in the same manner as in Example II-B, and nine of the ninerolIt was confirmed that the region flanked by Rs containing the gene was eliminated. Here, as a primer, a primer from which such desorption is detected (Example 1)1, Reference Examples 2 to 4as well asreferenceSame as that used in Comparative Example 2. That is, detection was performed by amplification of a DNA fragment of about 3 kb. )When,rolPrimers for detecting the presence of the gene (detected by amplification of a DNA fragment of about 1.1 kb) were used.
[0097]
[referenceAn example5]
referenceUsing the vector created in Example 2, pNPI106, a hybrid woodpecker (Populus sieboldii x Populus grandidententa) as a woody plant:Populus  Sieboldii×Populus  grandidentata) Was introduced.
[0098]
The stem of a sterile flask seedling of the hybrid Yamanashi Y63 strain (collected from an experimental forest in Akita Jujo Kasei Co., Ltd.) was cut to a length of 5 mm so as not to include nodes, and this was further divided vertically and used as a material. A. Introduction of pNPI106 ClawsreferenceInfection was carried out as in Example 1, III. After infection, the stem section was placed on a hormone-free modified MS agar medium (sucrose 2 w / v%, agar 0.8 w / v%) supplemented with 40 mg / l acetosyringone, cultured for 3 days, and then carbenicillin. The cells were transplanted to the same medium supplemented with only 500 mg / l, and the culture was continued. The modified MS medium used here is one in which the concentrations of ammonia nitrogen and nitrate nitrogen are changed to 10 mM and 30 mM, respectively, in the composition of a normal MS medium.
[0099]
After about two months, the adventitious buds generated from these sections were separated and cultured for another two months to obtain six lines of polyblasts, which were subsequently subcultured to about 4 After a lapse of months (approximately 8 months after the infection with A. tumefaciens), normal morphological shoots extending from the multiblasts began to be observed. These shoots were transplanted to a 2 / 3-fold diluted MS gellan gum medium (sucrose 2 w / v%, gellan gum 0.3 w / v%) supplemented with 0.05 mg / l of IBA, and cultured to obtain normal rooting. Individual. By the tenth month of infection with T. faecalis, a total of seven normal individuals could be obtained from the two lines of polyblasts.
[0100]
When PCR analysis was performed on these in the same manner as in III of Example 1,iptThe gene was not detected in all of the individuals, and the presence of the GUS gene was detected in two of the individuals, confirming that the vector of the present invention functions effectively also in woody plants. In the remaining five normal individuals, only the presence of a part of the GUS gene was detected, but such individuals were identified as transposons introduced by the vector used here, pNPI106.AcBut,iptIt is considered that this was caused as a result of involving the GUS gene in the vicinity when the gene was detached from the chromosome together with the gene, and detaching the GUS gene in such a manner as to cut it apart.
Here, FIG. 27 shows a state in which normal shoots have differentiated from the multiple shoots.
[0101]
[Example3]
Example1In the above, the gene was further transfected into a normal individual (through multiple buds) transfected with pNPI132 using the vector of the present invention.
[0102]
Only the GUS gene of pNPI132 was replaced with the HPT gene (hygromycin resistance gene), and using this (pNPI140, FIG. 28),referenceThe gene transfer operation was performed in the same manner as in Examples II and III. Multiple shoots were obtained from A. cerevisiae transfected with this vector. Forty days after the infection with T. faecalis, 10 strains were obtained. These strains were isolated, transplanted to a medium of the same composition (hormone-free MS agar medium supplemented with carbenicillin 500 mg / l), and continued to be cultured. That is, in about 2 months after infection with A. tumefaciens), differentiation of shoots showing a normal morphology with the naked eye was observed in one of them.
[0103]
For one of the differentiated normal shoots,referenceFIG. 29 shows the result of performing PCR analysis in the same manner as in Example 1 IV-B. However, herereferenceIn addition to the primers used in IV-B of Example 1,iptA primer designed to bind to the NPTII gene and the HPT gene, and a primer to detect the presence of the HPT gene, in order to detect detachment of the region between Rs containing the gene from chromosomal DNA, (Detected by amplification of about 4 kb and about 1 kb DNA fragments, respectively). In the figure, the numerical values shown on the left are the same as in FIG.
[0104]
As is clear from FIG. 29, in the PCR analysis of the chromosomal DNA extracted from this shoot, together with the region sandwiched by RsiptWhile amplification of a DNA fragment of about 4 kb indicating that the gene was eliminated and a DNA fragment of about 1 kb indicating the presence of the HPT gene were observed,iptNo amplification of a DNA fragment of about 800 bp indicating the presence of the gene is observed. This was once introduced into the chromosomal DNA of this shoot.iptThe gene was then detached and disappeared therefrom together with the region sandwiched by Rs, while the HPT gene remained in the DNA, that is, the target genes (NPTII gene and GUS gene) were firstly pNP1132. With respect to the introduced individual, a vector in which only the configuration related to the target gene in the configuration is changed,iptA new target gene (HPT gene) was repeatedly introduced using the same gene as a marker, and third, fourth or more target genes can be introduced again using the same marker gene. It is shown that it is.
[0105]
Example of the aboveAnd reference examplesAs is more evident, the multi-blast lines obtained here alwaysiptAll of the tissues that have the gene in their chromosome (FIG. 6), and that show prominent morphological abnormalities that are visually identifiable,iptKanamycin resistance due to expression of the NPTII gene, a model target gene introduced together with the gene, was shown without exception. This indicates that such a morphological abnormality-inducing gene can be sufficiently applied as a marker gene when introducing a gene into a plant, and the vector of the present invention using this as a marker gene is also useful as a vector for introducing a gene into a plant. It proves that
[0106]
Also thisiptTransposon geneAcWhen a gene is introduced into a plant using pNPI106, which is a vector incorporated in the inside of the target, the target gene (NPTII gene and / or GUS Gene)iptShoots and the like that lost the ability to form multiple shoots due to the loss of the gene from the chromosome were obtained (Table 1, FIGS. 13, 14, and 16), and the form of such shoots and the like was visually recognizable (FIG. 15, FIG. 27), these were selected, separated, and cultured to obtain an elongating rooted individual having a normal morphology. In addition, the tissues further regenerated from the tissues obtained from the shoots also showed normal morphology without polyblast formation ability, which clearly showed that such shoots and the like consisted of uniform cells.
[0107]
Furthermore, a similar result was obtained when a DNA element having a detachment ability derived from a site-specific recombination system was used as a morphological abnormality-inducing gene.rolIt was also observed when using the gene. That is,TakeWhen a gene is introduced into a plant using a vector (Example1,2) However, from the tissue that formed the abnormal morphology until a certain time after the gene transfer operation, the morphological abnormality-inducing gene disappeared from its chromosome while retaining the target gene, and tissues and individuals showing the normal morphology were obtained. (FIGS. 21 to 23, Table 2) Further, the same individual is repeatedly subjected to gene transfer operation, culture, and visual selection using the same morphological abnormality-inducing gene as a marker by using a vector in which only the configuration relating to the target gene is changed. In addition, it was possible to introduce multiple target genes (Example329).
[0108]
Therefore,As a marker geneA morphological abnormality-inducing gene, a DNA element capable of detachment,Using a DNA element derived from a site-specific recombination system,By using such a vector, which is integrated at a position where the behavior is united, first, a tissue exhibiting an abnormal morphology resulting from culturing the cells after the gene transfer operation is visually selected, and this is separated and separated. Further cultivation is continued, and then the resulting tissue showing a normal morphology is also selected with the naked eye again, and only the target gene remains in the chromosome etc. and consists only of cells retaining the function, Means that a plant is obtained.
[0109]
In addition, when a site-specific recombination system is used as a DNA element having an elimination ability, a normal individual can be obtained from a morphologically abnormal tissue with a considerably high probability. In addition, since the detachment also occurs promptly, a tissue having a normal morphology can be detected more quickly and efficiently.
[0110]
When a transposon is used as the DNA element having detachment ability, and when a DNA derived from a site-specific recombination system is used, the efficiency of obtaining a normal individual holding the target gene from the resulting morphologically abnormal tissue can be improved. , And Table 3.
[0111]
[Table 3]

[0112]
Example 1And Reference Example 1~4In any case, in the absence of plant hormones, the tissue containing the transgenic cells proliferated, differentiated adventitious buds, and regenerated the plant. Integrated into the chromosome ofiptThis is probably due to the function of the gene. In other words, due to the expression of these genes, plant hormones are excessively produced in the cells, albeit to some extent due to the action of the linked promoter, and the cells themselves only differentiate tissues such as polyblasts. Instead, the plant hormones produced by the plant hormones also acted on adjacent tissues to some extent, resulting in a state similar to the case where plant hormones were artificially added to the medium.
[0113]
【The invention's effect】
The vector used in the present invention uses a morphological abnormality induction gene as a marker gene. For this reason, when a gene is introduced into a plant using the same, in selecting a target gene-transfected tissue, the cells after the gene transfer operation are cultured in a normal culture medium under normal culture conditions, resulting in It is only necessary to visually identify the abnormally formed tissue, and it is not necessary to add chemicals or the like to the medium for selection, which simplifies the work and reduces the activity of plant cells during the selection. There is no possibility of inviting.
[0114]
Also, as this morphological abnormality induction geneiptWhen a gene is used, its function causes the tissue containing the transfected cell to proliferate and differentiate adventitious buds and the like, so that it is usually essential for the growth and differentiation of plant cells in culture. The addition of plant hormones to the medium can also be dispensed with.
[0115]
In addition, this vector containsDerived from a site-specific recombination systemIt is used by incorporating it into a position where its behavior is integrated with the DNA factor having detachment ability. Then, with a certain probability after introduction of the gene into the plant cell, the marker gene, together with such a DNA element, is detached from the DNA where it exists and functions, loses its function, and behaves at the same time as the introduced gene. Only the target gene existing at the unrecognized position remains in a state that can be expressed on the same DNA. For this reason, when such a configuration is adopted, this vector only changes the portion related to the target gene to be introduced, and does not make any changes to other configurations including the marker gene. In order to carry out multiple introduction of a gene into one plant, the gene can be used repeatedly indefinitely.
[0116]
Furthermore, in this case, the loss of the function of the morphological abnormality-inducing gene, which is a marker gene, can be visually detected as a change in the morphology of the transfected tissue, as in the case of gene transfer. Thus, a tissue consisting solely of cells retaining the function can be reliably and easily selected. Therefore, multiple transfection of genes can be performed efficiently, and transgenic individuals consisting only of such cells, that is, individuals in which the influence of the marker gene has been eliminated and the fear of the gene product has been completely eliminated, also undergo the mating process. You can get without.
[0117]
In other words, the effects of the marker gene are eliminated, the fear of the gene product is completely released, and the parts other than the structure derived from the vector used for gene transfer are identical to the parent plant to which the gene was transferred. Thus, an individual having the gene constitution of is created.
[Brief description of the drawings]
1 shows the general structure of the Ti plasmid, and FIG. Tumefaciens PO22 strain T-DNA regionPstIt is a conceptual diagram containing an I fragment restriction enzyme map.
FIG. 2 is a diagram showing up to the creation of pIPT2 in the pIPT4 creation scheme.
FIG. 3 is a diagram showing the steps from creation of pIPT2 to creation of pIPT3 in the pIPT4 creation scheme.
FIG. 4 is a diagram showing the steps from pIPT3 to pIPT4 creation in the pIPT4 creation scheme.
FIG. 5 is a restriction map of a T-DNA region in the structure of pIPT4.
FIG. 6 is a diagram showing the results of PCR analysis of multiple shoots generated from tobacco into which a gene has been introduced using pIPT4.
FIG. 7 is a diagram showing up to creation of a pNPI in a pNPI creation scheme;
FIG. 8 is a diagram showing the steps from the pIPT4 and pNPI102 to the creation of pNPI103 in the pNPI106 creation scheme.
FIG. 9 is a diagram showing the steps from pNPI103 to creation of pNPI106 in the pNPI106 creation scheme.
FIG. 10 is a restriction map of a T-DNA region in the structure of pNPI106.
FIG. 11 is a photograph showing a form of an organism;referenceNo. 2 in Example 2. 2 shows the state of one shoot after one month of culture.
FIG. 12 is a photograph showing a form of an organism;referenceNo. 2 in Example 2. 8 shows the state of one shoot after one month of culture.
FIG. 13referenceNo. 2 in Example 2. FIG. 9 is a view showing a PCR analysis result of 8 shoots.
FIG. 14referenceNo. 3 in Example 3. It is a figure which shows the PCR analysis result of the normal individual obtained from 13-1 and 14-1 shoots.
FIG. 15 is a photograph showing a form of an organism;referenceExample 3 shows a state in which normal shoots have differentiated from the multibud of tobacco.
FIG.referenceIn Example 4,referenceNo. 2 in Example 2. FIG. 10 is a view showing a PCR analysis result of a normal individual obtained from leaves produced from the shoots of No. 7;
FIG. 17 is a diagram showing the steps up to the creation of pNPI128 in the pNPI132 creation scheme.
FIG. 18 is a diagram showing a process from pNPI128 to creation of pNPI129 in a pNPI132 creation scheme.
FIG. 19 is a diagram showing, from the pNPI132 creation scheme, the processes from pNPI101 and pNPI129 to creation of pNPI132.
FIG. 20 is a restriction map of a T-DNA region in the structure of pNPI132.
FIG. 21 Example1In the system No. Among the figures showing the results of PCR analysis of normal individuals obtained from 15 to 21 shoots,iptFIG. 9 shows the results obtained by using a primer for detecting the presence of a gene.
FIG. 22 Example1In the system No. Among the figures showing the results of PCR analysis of normal individuals obtained from 15 to 21 shoots,iptFIG. 9 shows the results obtained by using a primer capable of detecting detachment of a region sandwiched by Rs containing a gene.
FIG. 23 Example1In the system No. Among the figures showing the results of PCR analysis of normal individuals obtained from 15 to 21 shoots, the figure shows the results obtained using primers that detect the presence of the GUS gene.
FIG. 24 Example1FIG. 3 is a diagram showing the results of PCR analysis of normal individuals generated from a line having no ability to form multiple shoots.
FIG. 25 is a diagram showing a creation scheme of pNPI702.
FIG. 26 is a restriction map of a T-DNA region in the structure of pNPI702.
FIG. 27 is a photograph showing a form of an organism;referenceAn example5Shows a state in which normal shoots have been differentiated from the multiple shoots of the hybrid porcupine.
FIG. 28 is a restriction map of a T-DNA region in the structure of pNPI140.
FIG. 29 Example3FIG. 3 is a diagram showing the results of PCR analysis of normal shoots differentiated from multiple buds after multiple gene transfer.

Claims (2)

A transgenic plant produced without the mating process by undergoing the following steps (A), (B), (C), and (D), wherein the effect of the marker gene is eliminated.
(A) a target gene, a morphological abnormality-inducing gene as a marker gene, and a DNA element having an elimination ability derived from a site-specific recombination system ; A vector for introducing a gene into a plant, which is located at a position where the behavior is unified, and the target gene is located at a position where the behavior is not integrated with the DNA element having the elimination ability, is a plant cell. (B) a step of culturing plant cells transfected with this vector, detecting abnormal morphology of plant tissue occurring during the culturing, and selecting a tissue showing this morphological abnormality (C) B) A process of culturing a tissue showing a morphological abnormality selected in B), and detecting and selecting a tissue having a normal morphology appearing in the culture (D) A tissue having a normal morphology selected in the above (C) To And nourishment, the process of regenerating plants A transgenic plant into which two or more target genes have been introduced, which is produced without the mating process by going through the following steps (A), (B), (C), and (D).
(A) a target gene, a morphological abnormality-inducing gene as a marker gene, and a DNA element having an elimination ability derived from a site-specific recombination system ; A vector for introducing a gene into a plant, which is located at a position where the behavior is unified, and the target gene is located at a position where the behavior is not integrated with the DNA element having the elimination ability, is a plant cell. (B) a step of culturing plant cells transfected with this vector, detecting abnormal morphology of plant tissue occurring during the culturing, and selecting a tissue showing this morphological abnormality (C) Culturing the tissue showing the morphological abnormality selected in B), detecting and selecting the tissue having a normal morphology which appears during the culturing (D) The process of (A) to (C) is performed once Repeated , Culturing the tissues with normal morphology were selected in (C), the process of regenerating plants

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