JP6024901B2 - 植物の形質転換個体の取得方法 - Google Patents
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Description
また、請求項2記載の方法は、請求項1記載の方法において、TGSを発動させるためのsiRNAを穂木において産生せしめる方法として、標的遺伝子のプロモーター領域に相同な配列を有するsiRNAを産生することができる、CoYMVプロモーターを用いたベクターを導入したアグロバクテリウムを穂木に感染させる方法を用いることを特徴とする。
CaMV35Sプロモーターの−32〜−342bpの領域(Okano Y.ら、Plant Journal 53:65−77.2008)とそのアンチセンス鎖配列との逆位方向反復塩基配列構造の間にCAT1(カタラーゼ)遺伝子由来のイントロン(配列長:201bp、Ohta S.ら、Plant and Cell Physiology 31:805−813.1990)をスペーサーとして連結して組み込んだ。このユニットをバイナリーベクターpE2113−GUS(Mitsuhara I.ら、Plant Cell Physiology 37:49−59.1996.)のBamHI/SacI部位のGUS(beta−glucuronidase)遺伝子と入れ換えて35S:35S−IRを構築した。次に、伴細胞で特異的に機能するプロモーターであるCoYMVpをpCOI(Matsuda、Y.ら、Protoplasma 220:51−58.2002)によりPCR増幅し、これを35S:35S−IRのSalI/BamIH部位と入れ換えることで、目的のsiRNA産生ベクター(CoYMV:35S−IR)を得た(図1のsilencerを参照)。
アグロバクテリウムとしてAgrobacterium tumefacience EHA105株を用い、その単一コロニーをLB培地(組成は表1参照)に抗生物質(50mg/LのRifampicin)を添加した培地に植え付け、28℃で24時間振盪培養し、継代してさらに12時間振盪培養した。その後、4℃にて6000rpmで10分間遠心し、回収した菌を滅菌水および10%グリセロールで洗浄した。この菌のペレットを10%グリセロール1mLで懸濁し、そのうちの40μLを(1)で作製したsiRNA産生ベクター0.5〜1.0μgと混合し、混合液をキュベットに移し、20kV/cm,6msの条件でエレクトロポレーションすることで、siRNA産生ベクターをアグロバクテリウムに導入した。電圧をかけたキュベット内の反応液にLB培地1mLを加え、1.5mLチューブに回収し、28℃で24時間培養した。抗生物質(50mg/LのRifampicinおよび50mg/LのKanamycin)を含むLB寒天培地上に培養液を塗布し、28℃で3日間培養した。得られたコロニーを新しいLB培地で培養し、アグロバクテリウム感染に用いた。
LB培地5mLに抗生物質(50mg/LのRifampicinおよび50mg/LのKanamycin)を添加し、siRNA産生ベクターを保持するアグロバクテリウムを28℃で一晩培養し、継代してさらに12時間振盪培養した。その後、室温にて3000rpmで20分間遠心し、回収した菌をOD600=1.0になるように懸濁液培地(組成は表2参照)に懸濁した。こうして調製したsiRNA産生ベクターを保持するアグロバクテリウムの懸濁液に、明所条件下で無菌的に栽培した発芽後15日目のNicotiana benthamianaの個体の葉片を浸漬することでアグロバクテリウム感染を行った後、常法に従って目的とする形質転換が行われた細胞から再分化個体を取得した。
明所条件下の温室にてMS agar(0.7%)で栽培した発芽後7日目のNicotiana benthamiana 16C(図1のtargetに示されるこの実施例における標的遺伝子産生ベクターである35S:mGFPが導入された緑色蛍光タンパク質産生形質転換体。Jones L.ら、Plant Cell 11:2291−2301.1999)の個体の胚軸部位(子葉より約5mm下)を水平に剃刀で切断し、この根側を台木とした。一方、(3)でsiRNA産生ベクターを保持するアグロバクテリウムを感染させた発芽後7日目のNicotiana benthamianaの個体にも同様の処置を行い、この子葉側を穂木とした。両者の胚軸部位をシリコンチューブ(長さ:2mm×外径:0.5mm×内径0.4mm)内にて密着させて接ぎ木した。全ての操作は無菌的に顕微鏡下で行った。接ぎ木した個体は、無菌シャーレ内のアガロース(3mmキューブ)を利用して正立させた。7日後にチューブを外してロックウール(Nitto Boseki Co.)にて液体肥料(Otsuka House Nos.1 and 2,Otsuka Chemical Co.)を用いて栽培した。
接ぎ木した7日後に行った。可視光下とUV下において接ぎ木個体を観察した結果を図2に示す(35SIR/16c:左が可視光下で右がUV下。←は接ぎ木点)。なお、図2には、siRNA発現ユニットを含まないベクターを用いて同様の操作を行って得た接ぎ木個体の可視光下とUV下において観察した結果をあわせて示す(Empty/16c:左が可視光下で右がUV下。←は接ぎ木点)。また、それぞれの接ぎ木個体のサンプルを7%低融点アガロースブロックに包埋し、ビブラトーム(Series 1500 Leica,St.Louis,MO)を用いて100μm厚の切片を作製し、共焦点レーザー顕微鏡(Confocal laser scanning microscopy system FluoVie 1000,Olympus,Tokyo)を用いて台木の主根からの側根の分岐部分を観察した結果を図3に、側根の先端を観察した結果を図4にそれぞれ示す(図3の右側と図4の下側が可視光下で図3の左側と図4の上側がUV下)。図2から明らかなように、siRNA産生ベクターを用いて得た接ぎ木個体(35SIR/16c)は、siRNA発現ユニットを含まないベクターを用いて得た接ぎ木個体(Empty/16c)と異なり、接ぎ木点付近に若干の緑色蛍光が認められたが、この部分を除けば緑色蛍光は認められず、穂木において産生されたsiRNAが篩管を通して長距離輸送されて台木においてTGSを効果的に発動したことがわかった。また、図3と図4から明らかなように、siRNA産生ベクターを用いて得た接ぎ木個体では、台木の主根の篩管周辺でTGSが顕著に発動されること(別途の実験による主根の断面のTGS発動の観察によっても確認済み)、ここから形成される側根は全体にわたってTGSが発動していることがわかった。なお、TGSが発動している側根の切片を用いた組織培養によって得られたカルス由来の再分化個体について緑色蛍光の有無を確認したところ、TGSが後代に遺伝し、サイレンシングが維持されていることで、緑色蛍光は認められなかった。なお、比較実験として、台木において産生されたsiRNAを接ぎ木を介して穂木に輸送した場合、穂木の展開葉においてTGS発動が認められたが、TGSの発動部位は葉身全域ではなく葉脈に沿った箇所に限られていた。腋芽のシンク力を高めるための切り戻しを行っても葉身全域でのTGS発動は認められなかった。よって、穂木において産生されたsiRNAを接ぎ木を介して台木に輸送することによる台木におけるTGS発動の方が、台木において産生されたsiRNAを接ぎ木を介して穂木に輸送することによる穂木におけるTGS発動よりも効果的であり、サイレンシングが維持された形質転換個体を取得する上においても有利であることがわかった。
Claims (2)
- 植物の形質転換個体の取得方法であって、転写型遺伝子サイレンシングを発動させるためのsiRNAを穂木において産生せしめ、穂木において産生されたsiRNAを接ぎ木を介して台木に輸送し、転写型遺伝子サイレンシングを台木において発動させることによって台木の形質転換を行った後、台木の主根の側根からの再分化個体、または根系不定芽体(Root sucker)を、形質転換個体として取得することを特徴とする方法。
- 転写型遺伝子サイレンシングを発動させるためのsiRNAを穂木において産生せしめる方法として、標的遺伝子のプロモーター領域に相同な配列を有するsiRNAを産生することができる、CoYMVプロモーターを用いたベクターを導入したアグロバクテリウムを穂木に感染させる方法を用いることを特徴とする請求項1記載の方法。
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JPN6015043774; 植物ウイルス病研究会レポート vol.9, 2008, pp.1-10 * |
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JPN6016017716; 育種学研究 vol.12, suppl.2, 201009, p.216 * |
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