JP3922869B2 - Human interferon transgenic plant - Google Patents

Description

【００７０】
５．２ 形質転換矮性イネにおける目的遺伝子の確認
５．２．１ ＰＣＲによる確認
（１）ＰＣＲ
得られた形質転換個体の各々（α−１，α−２、α−３、α−４及びα−５系統）につき確認を行った。ゲノムＤＮＡを４ｎｇ／２μｌに調製し、鋳型ＤＮＡとしてＰＣＲに用いた。次の組成に従い、鋳型ＤＮＡ以外の混合物を0.5ｍｌエッペンチューブに分注し、これに鋳型ＤＮＡを加えた。陰性対照としては、野生型矮性イネのゲノムＤＮＡと蒸留水とを用いた。
鋳型ＤＮＡ・・・・・・・・２μｌ
10×緩衝液・・・・・・・・２μｌ
25ｍＭ ＭｇＣｌ₂・・・・・２μｌ
10×ｄＮＴＰ・・・・・・・２μｌ
前方プライマー^*1・・・・・１μｌ
後方プライマー^*2・・・・・１μｌ
Ｔａｑポリメラーゼ・・・ 0.1μｌ
蒸留水 全量 20 μｌ
^*1 GUS snaFを使用
^*2 GUS sacRを使用
【００７１】
GeneAmp PCR system 2400に0.5ｍｌエッペンチューブをセットし、下記のプログラムを実行した。
〔標準プログラム〕
（ａ） 94℃ １分
（ｂ） 94℃ 0.5分
（ｃ） 60℃ １分
（ｄ） 上記（ａ）〜（ｃ）を35サイクル反復する
（ｅ） 72℃ 1.5分
（ｆ） 72℃ ５分
（ｇ） ４℃ 保存
【００７２】
（２） アガロースゲル電気泳動
上記の反応操作の後、反応液20μｌを次の手順でアガロースゲル電気泳動し、バンドの確認を行った。すなわち、アガロース0.4ｇを三角フラスコに入れ、ＴＡＥ緩衝液（0.4Ｍトリス酢酸、0.62Ｍ ＥＤＴＡ）を40ｍｌ加えた。電子レンジで溶解後、蒸留水で補正し、型に流して固化させた（ゲル濃度0.8％）。固化後、ＴＡＥ緩衝液を満たした泳動装置にゲルを入れ、ＰＣＲ産物にＰＢＰを少量加えて、ウェルに適用した。両端には、λHind IIIマーカーを適用した。約30分間泳動させた後、エチジウムブロマイド染色液に10分間浸して染色した。染色後、ゲルをImageMaster（Pharmacia Biotech）にかけてバンドの位置を確認し、併せて写真撮影した。
【００７３】
（３） 結果
ＰＣＲの結果を図４に示す。全ての形質転換系統において、目的の位置（500ｂｐ付近）にＤＮＡ断片を確認することができた。また陰性対照においてはＤＮＡの増幅は見られなかった。この結果、形質転換体において目的遺伝子が導入されていることが確認された。
【００７４】
５．２．２ ゲノミックサザンブロットによる確認
（１） イネゲノムの単離
得られた形質転換個体のうち、α−２、α−３及びα−４系統について確認を行った。約10ｇのイネ緑葉を鋏でできるだけ小さく切ってから乳鉢に入れ、これに液体窒素を加えて乳棒でパウダー状になるまですりつぶした。細かくなった組織をスパチュラで50ｍｌコーニングチューブに入れ、予め沸騰寸前まで加熱しておいた２％ＣＴＡＢ溶液〔100ｍＭトリス塩酸、20ｍＭ ＥＤＴＡ、1.4Ｍ ＮａＣｌ、２％ＣＴＡＢ（セチルトリメチルアンモニウムブロマイド）、ｐＨ8.0〕20ｍｌを加えた。56℃にて20分間震盪した後、等量のクロロホルム：イソアミルアルコール（24:1）を加え、室温にて20分間転倒混和した。遠心して上層を分離しこれに、再び等量のクロロホルム：イソアミルアルコール（24:1）を加え、室温にて20分間転倒混和した。遠心して上層を分離し、これに１〜1.5倍量の１％ＣＴＡＢ溶液（50ｍＭトリス塩酸、10ｍＭ ＥＤＴＡ、1.4Ｍ ＮａＣｌ、１％ＣＴＡＢ、ｐＨ8.0）を加えて、ゲノムＤＮＡを析出させた。3,000ｒｍｐにて20分間遠心し、沈殿を回収した。沈殿に１Ｍ ＮａＣｌを５ｍｌ加え、更に10ｍｇ／ｍｌ ＲＮａｓｅ Ａを５μｌ加えて、56℃にて沈殿が溶けるまで震盪した。クリーンベンチ内で、冷却した100％エタノール10ｍｌを静かに重層し、先を熱で融かしたパスツールピペットに巻き付けるようにしてゲノムＤＮＡを回収した。このとき、目視し得る程のゲノムＤＮＡが得られた。80％エタノールに２回、100％エタノールに１回、この順にそれぞれ約５分浸漬した後、巻き付けたＤＮＡが透明になるまで風乾した。ＴＥ２ｍｌの入った15ｍｌのコーニングチューブにゲノムＤＮＡを入れ、必要な場合は56℃に加熱して、完全に溶解させた。吸光度（ＯＤ₂₆₀）を測定してＤＮＡ濃度を求めた。
【００７５】
（２）ＤＮＡトランスファーメンブランの調製
ゲノムＤＮＡ10μgを、Hind III、Xba I、Sac I及びEco RI〔導入遺伝子（Ｔ−ＤＮＡ）中に１ヶ所しかない制限酵素部位に対する制限酵素である〕の各制限酵素で37℃にて一晩消化した。
ゲノムＤＮＡ・・・・・・・10μg
10×緩衝液・・・・・・・・10μｌ
制限酵素・・・・・・・・・ 8μｌ
蒸留水・・・・・・・全量 100 μｌ
【００７６】
反応後、エタノール沈殿を行い、全体量の1/10量の３Ｍ酢酸ナトリウムと２〜2.5倍量の100％エタノールを加え、混合した後、-80℃にて15分以上放置した。
その後卓上遠心機で15,000ｒｐｍ、室温にて20分間遠心し、上清を捨て、80％エタノール200μｌを加え、15,000ｒｐｍで室温にて10分間遠心し、上清を捨て、沈殿を減圧乾燥させた。回収されたＤＮＡを蒸留水20μｌに溶解させ、0.8％濃度のゲル、50ｍＡの条件で電気泳動を行った。泳動後のゲルをエチジウムブロマイド染色液に10分間以上浸漬した。ゲルをＢＰＢが黄変するまで0.25Ｎ塩酸中で震盪した後、0.4Ｎ水酸化ナトリウム中で20分間処理して、ＤＮＡを一本差に変性した。ゲルと同じ大きさのHybond-N⁺（Amersham）１枚と３ＭＭ濾紙２枚を切り、ブロット台を組み立て８時間〜一晩、ＤＮＡをHybond-N⁺（Amersham）にブロットした。Hybond-N⁺（Amersham）のゲルのコームに位置するところに印をつけてから、２×ＳＳＣ〔塩化ナトリウム17.53ｇ／ｌ、クエン酸ナトリウム8.82ｇ／ｌ〕で５分間洗浄した。Hybond-N⁺（Amersham）をペーパタオルに挟んで80℃にて２時間ベーキングした後、デシケーターに保存した。
【００７７】
（３） プローブＤＮＡの調製
下記の系で制限酵素処理を２時間行った。
pIG-A11 ・・・・・・・・・８μg
10×緩衝液・・・・・・・・４μｌ
SnaB I ・・・・・・・・・２μｌ
Sac I ・・・・・・・・・・２μｌ
10×BSA ・・・・・・・・・４μｌ
蒸留水 全量 40 μｌ
【００７８】
制限酵素で消化したＤＮＡを100ｍＡで電気泳動した（手順は上記参照）。泳動後、ゲルの写真を撮り、ＵＶ照射下に必要なバンドをかみそりでカットした。溶出装置を水平に合わせ、１×電気溶出緩衝液（20ｍＭトリス塩酸、0.2ｍＭ ＥＤＴＡ、５ｍＭ ＮａＣｌ、ｐＨ8.0）を注いだ。１ｍｌピペットマンで緩衝液を出し入れすることにより各チャンネルから空気を抜いた。３Ｍ 酢酸ナトリウム125μｌを、溢れないよう注意しつつ全てのチャンネルに加えた。切り出したゲルを台に載せ、100Ｖ・400Ａで１時間溶出させた後、ゲルをＵＶ照射してＤＮＡが全て回収されたことを確認した。緩衝液を抜き、キャピラリーチップをつけた１ｍｌピペットマンでチャンネル内の液を回収した。100％エタノール１ｍｌを加えて振り混ぜた後、-80℃に15分以上おいた。卓上遠心機で15,000ｒｐｍで20分間遠心した後、上清を捨てた。濯ぎのため、80％エタノール200μｌを加えて15,000ｒｐｍにて10分間遠心し、ピペットマンを用いて上清を注意深く捨てた。減圧乾燥機で沈殿を乾燥させ、蒸留水12μｌに溶解させた。
【００７９】
（４） ＤＮＡの標識化
上記プローブを25〜100ｎｇ／３μｌの濃度に調整し、３μｌのＤＮＡ溶液とBcaBEST^TMキット（宝酒造）〔ランダムプライマー（９マー）、10×緩衝液、ｄＮＴＰ混液（0.2ｍＭ ｄＡＴＰ、ｄＴＴＰ、ｄＧＴＰ）、BcaBEST^TM ＤＮＡポリメラーゼ〕付属のランダムプライマー２μｌを混ぜて、95℃にて３分間、水浴中で熱変性させた。次いで、これを氷上で５分間冷却することによりアニーリングさせた。これに10×緩衝液2.5μｌ、ｄＮＴＰ混液2.5μｌ、蒸留水9.0μｌを加えた。混合し、遠心した後、［α−³²Ｐ（3,000Ｃｉ／ｍｍｏｌ）］ｄＣＴＰを５μｌ加え、更にBcaBEST^TM ＤＮＡポリメラーゼを１μｌ加えた。55℃にて10分間反応させた。スピンカラムで3,400ｒｐｍで２分間遠心し、未反応のｄＮＴＰを除去し、［α−³²Ｐ］ｄＣＴＰの取り込み効率をチェックした後、エッペンチューブのまま４℃にて保存した。
【００８０】
（５） プレハイブリダイゼーション
サケ精子ＤＮＡを、95℃にて３分間熱変性させた後、氷上に５分間置いた。シャーレにハイブリダイゼーション緩衝液〔６×SSPE、５×Denharts 試薬、0.5％ＳＤＳ、〕〔20×SSPE：3.6Ｍ ＮａＣｌ、200ｍＭ リン酸二水素カリウム、20ｍＭ ＥＤＴＡ（ｐＨ7.4）； 50×Denharts 試薬：1.0％フィコール、1.0％ポリビニルピロリドン、1.0％ＢＳＡ〕を10ｍｌ入れ、１／100量の熱変性サケ精子ＤＮＡを加えた。気泡が入らないようにメンブランをシャーレに入れ、ビニールテープでシールした後、65℃にて３時間以上インキュベートした。
【００８１】
（６） ハイブリダイゼーション
³²Ｐ標識プローブＤＮＡを95℃にて３分間熱変性させた後、氷上に５分間放置して冷却させた。プレハイブリダイゼーションを終えたメンブランに³²Ｐ標識プローブＤＮＡを加え、55℃にて６時間以上インキュベートした。
【００８２】
（７） 洗浄
ハイブリダイゼーション緩衝液を捨て、洗浄緩衝液〔６×ＳＳＣ、0.1％ＳＤＳ〕30ｍｌで、42℃、20分間の洗浄を２回行った。サーベイメーターでメンブラン上の数カ所をカウントし、目的のバンドがある位置以外でカウントされないことを確認した。メンブランが乾燥しないうちにHybribagに入れて周囲をシールし、Molecular imager（BioRAD）スクリーンに露光させた。露光後、Molecular imager（BioRAD）で目的のバンドにハイブリダイゼーションが起こっていることを確認し、Ｘ線フィルム（Fujifilm: XR-1）に露光させ、フィルムを現像した。
【００８３】
（８） 結果
結果を図５に示す。形質転換系統であるα−２、α−３及びα−４系統のみで目的遺伝子のバンドが検出され、陰性対照である野生型のゲノムＤＮＡではバンドは検出されなかった。形質転換系統につき、４種の制限酵素で消化したゲノムＤＮＡについて、１〜３個のバンドが検出され、全ての制限酵素で単一バンドしか与えないような形質転換系統はなかった。このことは、これらの形質転換系統において、目的遺伝子が複数コピー導入されていることを示唆している。
【００８４】
５．３ 形質転換イネにおける組換えタンパク質の発現解析
５．３．１ ＥＬＩＳＡ法による解析
（１） タンパク質の抽出
カルス、根、葉については、組織300ｍｇに対して細胞溶解緩衝液1.0ｍｌを加えてホモジナイズした後、遠心分離し、上清をサンプルとして用いた。また種子（登熟期種子及び完熟種子）については、組織50ｍｇに対して細胞溶解緩衝液500μｌを加えて上記と同様に処理し、上清をサンプルとして用いた。また、上記で得られたカルスについても、液体倍法を行った後ヒトインターフェロンαタンパク質の発現につき更に調べた。
【００８５】
（２） ＥＬＩＺＡ
ELIZA用96ウェルプレートに、１μｌ／ｍｌの濃度の非標識抗体を100μｌ／ウェルづつ入れ、37℃にて１時間インキュベートした。抗体を除いた後、水分を拭い、１％牛血清アルブミンを含むＰＢＳを350μｌ／ウェル加え、37℃にて１時間インキュベートした。ブロック液を捨てた後、水分を拭い、ＴＴＢＳで１回洗浄した。ウェルにインターフェロンの標準品と１％牛血清アルブミン含有ＢＰＳで希釈したサンプルを100μｌ／ウェル添加し、37℃にて１時間インキュベートした。サンプルを捨て、ＴＴＢＳで３回洗浄した。１μg／ｍｌのＨＲＰ標識抗インターフェロンα抗体を100μｌ／ウェル添加し、37℃にて１時間インキュベートした。二次抗体を捨てた後、ＴＴＢＳで３回洗浄した。ＨＲＰの基質（ＴＭブルー）を50μｌ／ウェル加え、約５分間室温で発色させる。その後、0.1Ｎ硫酸を50μｌ／ウェル加えて反応を停止させた。450ｎｍにおける吸光度をプレートリーダーにより測定し、標準品について予め作成しておいた検量線からサンプル中の濃度を算出した。
【００８６】
（２） 結果
各組織についての結果を図６及び表２にまとめる。
【００８７】
形質転換個体におけるヒトインターフェロンαタンパク質量（ＥＬＩＺＡ）
【表２】

【００８８】
結果は、イネ組織50ｍｇ中におけるヒトインターフェロンαタンパク質量として示されている。図及び表が示すように、カルス及び登熟期の種子おいて高い発現量が得られた。完熟種子においては僅かな発現しか認められなかったが、操作に用いた細胞溶解緩衝液がプロテインボディを溶解させる物質を含有していないことから、ヒトインターフェロンαが完熟種子のプロテインボディ中に集積されている可能性がある。免疫電子顕微鏡観察の所見は、種子中において、ヒトインターフェロンαタンパク質がPB-Iに集積していることを示唆している（データは示さず）。
【００８９】
液体培養したカルスについての結果を次の表３に示す。上記表との対より、液体培養によっても個体培地で培養したカルスとほぼ同定のヒトインターフェロンαタンパク質が産生されていることが分かる。
【００９０】
【表３】

【００９１】
５．３．２ 抗ウイルス活性分析
発現されたヒトインターフェロンαタンパク質の抗ウイルス活性を評価した。
（１） 方法
用いたタンパク質サンプルは、上記ＥＬＩＺＡと同様の手順で作成した。但しカルスは使用しなかった。水疱性口内炎ウイルス（ＶＳＶ）、ヒト白血球インターフェロンα標準品、及びＦＬ５−１細胞（ヒト羊膜由来細胞）を用い、50％ＣＰＥ減少法（色素取り込み法）により、通常の手順に従って操作を行った。
【００９２】
（２） 結果
結果を図４及び表４にまとめる。
【００９３】
【表４】

【００９４】
図及び表は、形質転換イネより抽出されたタンパク質が抗ウイルス活性を有していることを明らかにしている。また、抗ウイルス活性が、ＥＬＩＺＡにおいて確認されたヒトインターフェロンαタンパク質の発現量と相関していることは、この抗ウイルス活性が形質転換イネ中に発現されたヒトインターフェロンαによるものであることを示している。
【００９５】
６．Camellia sinensisへのヒトインターフェロンα遺伝子の導入
６．１ Camellia sinensisのカルス培養及び不定胚形成
Camellia sinensisの未熟種子を次亜塩素酸ナトリウム（有効塩素濃度１％）溶液で10分間滅菌した後、滅菌水で３回洗浄した。滅菌した未熟種子から子葉を切除し２分割して1.0ｍｇ／ｌのＢＡを添加した１／２ＭＳ培地に置床し、4,000ｌｕｘの蛍光灯下で16時間日長、25℃の条件で培養した。培養６週間後からカルス形成が認められた。得られたカルスは、同組成の培地に継代培養することにより維持できた。ホルモンフリーの１／２ＭＳ培地に移植することにより、カルスから不定胚を経由して幼植物体が得られた。
【００９６】
６．２ 形質転換カルスの作出
大腸菌へのpNPI132の導入を、前記「２．１ 大腸菌へのプラスミドの導入」と同様にして行い、前記「２．２ プラスミドの大量調製」と同様の手順によりプラスミドの大量調製を行った。プラスミドを前記「２．３ アグロバクテリウムへのプラスミドの導入」と同様にしてアグロバクテリウム（Agrobacterium tumefaciens LBA4404）へ導入した。次いで、上記で作成したCamellia sinensisの子葉由来カルスを超音波処理（38ｋＨｚ、５秒間）した後、pNPI132（日本製紙株式会社）を有するAgrobacterium tumefaciensと、前記「３．３ アグロバクテリウムの感染による遺伝子の導入」に準じて３日間共存培養を行った。共存培養終了の１ヶ月後にＧＵＳ活性について検討した。その結果、約30％のカルスにおいてＧＵＳ活性が認められた。pNPI132の構造については図１０を参照のこと。図において、Ｒｓは、組換え酵素が認識し作用するＤＮＡ配列、Ｒは、組換え酵素をコードする遺伝子、iptは、サイトカイニンの合成に関与するイソペンテニルトランスフェラーゼをコードする遺伝子、Ｔはターミネーターを表す。
【００９７】
イネの場合の実験手順において行ったのと同様にして、Sna BIとSac IとでpNPI132を消化し、pNPI132中のＧＵＳ遺伝子の一部を残して除去し、代わりにインターフェロンをコードするＤＮＡをライゲートすることにより、インターフェロン遺伝子を組み込んだプラスミドＤＮＡが得られる。これをイネの場合に行ったのと同様にして、大腸菌中で増幅し、これを用いてアグロバクテリウムを形質転換し、形質転換されたアグロバクテリウムをCamellia sinensisのカルスに感染させることにより、インターフェロン遺伝子を導入したCamellia sinensisを得ることができる。
【００９８】
７．チャへのヒトインターフェロンα遺伝子の導入
チャ（Thea）は、Camelliaと分類学上隣接した位置にあるため、チャについても上記Camellia sinensisについて行ったのと同様の手順により、ＧＵＳ遺伝子又はヒトインターフェロンα遺伝子を導入することが可能となる。例えば、チャの未熟種子を次亜塩素酸ナトリウム（有効塩素濃度１％）溶液滅菌し、滅菌水で洗浄し、子葉を切除し２分割して1.0ｍｇ／ｌのＢＡを添加した１／２ＭＳ培地に置床し、4,000ｌｕｘの蛍光灯下で16時間日長、25℃の条件で培養する。得られるカルスを上記と同様に超音波処理し、pNPI132（日本製紙株式会社）を有するAgrobacterium tumefaciensと共存培養を行うことにより、ＧＵＳ遺伝子を組み込んだチャを得ることが可能であろう。従ってまた、pNPI132中のＧＵＳ遺伝子の一部を残して除去し、代わりにインターフェロンをコードするＤＮＡをライゲートすれば、インターフェロン遺伝子を組み込んだプラスミドＤＮＡが得られる。これをイネの場合に行ったのと同様にして、大腸菌中で増幅し、これを用いてアグロバクテリウムを形質転換し、形質転換されたアグロバクテリウムをチャのカルスに感染させればインターフェロン遺伝子を導入したチャが得られることが、合理的に推定される。
【００９９】
【配列表】

【図面の簡単な説明】
【図１】 pINT-RP10sGUSの調製プロセスを示す図。
【図２】 pIG121Hmの調製プロセスを示す図。
【図３】 pUC19へのヒトインターフェロンα遺伝子配列の組み込みを表す図。
【図４】 ヒトインターフェロンα遺伝子配列を組み込んだpUC19断片のpIG121Hmへの組み込みを表す図。
【図５】 構築されたpIG-A11の、ヒトインターフェロンα遺伝子配列を含んだ領域を表す図。
【図６】 形質転換植物における目的遺伝子の導入を示すＰＣＲによる増幅像。
【図７】 形質転換植物における目的遺伝子の導入を示すゲノミックサザン分析。
【図８】 形質転換植物組織中のヒトインターフェロンαタンパク質量を示すグラフ。
【図９】 形質転換植物組織中のタンパク質の抗ウイルス活性を示すグラフ。
【図１０】 pNPI132の構造を示す図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a plant in which an interferon protein is incorporated, and particularly relates to a gramineous plant and a tea in which a base sequence encoding an interferon α or ω protein is incorporated into genomic DNA.
[0002]
[Prior art]
Establishing a method for introducing foreign genes into plant cells is possible with other evolutionary distant plants that were impossible with conventional hybridization methods, as well as the potential for useful characteristics of microorganisms and animals. And is expected as an innovative technology for plant breeding. There are two methods of actually introducing an isolated gene into a plant cell, one using a physical method and the other using a living organism.
[0003]
Physical methods include a polyethylene glycol method (PEG method), an electroporation method, a microinjection method, and a particle gun method. These methods are highly useful in that they can be applied to both monocotyledonous and dicotyledonous plants. However, in the polyethylene glycol method and the electroporation method, since the cell wall becomes an obstacle, protoplasts must be used, and the frequency of integration of the introduced gene into the chromosomal DNA of plant cells is a problem. In addition, in the microinjection method using callus or tissue without using protoplasts, there are many difficulties in terms of needle thickness, tissue fixation, and the like. Even in the particle gun method using tissue, there are problems such as the appearance of mutations in the form of chimeras. Moreover, in these physical methods, the introduced foreign gene is generally easily incorporated into the nuclear genome as a multi-copy gene in an incomplete state. It is known that when multiple copies of a foreign gene are introduced, the gene is easily inactivated.
[0004]
On the other hand, methods for introducing an isolated gene using a living organism include an Agrobacterium method, a virus vector method, and a method using pollen as a vector, which has been developed recently. These methods have the advantage that the culture does not last for a long time and is not susceptible to damage such as somaclonal mutation because gene transfer is performed using plant callus, tissue or plant body without using protoplasts. ing. Among them, the method using pollen as a vector has few experimental examples yet, and there are many unknown parts as plant transformation methods. Although the viral vector method has the advantage that the gene to be introduced to the entire plant infected with the virus spreads, it is only amplified and expressed in each cell, and there is no guarantee that it will be transmitted to the next generation, In addition, there is a problem in that long DNA fragments cannot be introduced. The Agrobacterium method has many advantages such as the ability to introduce DNA of 20 kbp or more into chromosomes without major rearrangement, the small number of copies of the introduced gene, and the high reproducibility. On the other hand, it has been difficult to apply to monocotyledonous plants.
[0005]
Since Agrobacterium is outside the host range for monocotyledonous plants such as gramineous plants, introduction of foreign genes into gramineous plants has been conventionally performed by the physical methods described above. However, in recent years, the Agrobacterium method has been applied to monocotyledonous plants such as rice and other plants with established culture systems.
[0006]
In the introduction of foreign genes by the Agrobacterium method, when a low molecular weight phenolic compound such as acetosyringone synthesized by plants acts on the Ti plasmid Vir region, the T-DNA region is excised from the Ti plasmid and undergoes several processes. It is integrated into the nuclear chromosomal DNA of plant cells. In the dicotyledonous plant, since the plant itself has such a phenol compound synthesis mechanism, a foreign gene can be easily introduced by the leaf disk method or the like, and reproducibility is also high. On the other hand, in monocotyledonous plants, since such plants do not synthesize such phenolic compounds, it was difficult to produce transformed plants with Agrobacterium. However, it is now possible to introduce foreign genes into monocotyledons by adding acetosyringone during Agrobacterium infection.
[0007]
Regarding the production of useful plants, herbicide-tolerant plants, virus-resistant plants, insect-resistant plants such as insect-resistant plants, and environmental stress (pollution, cold damage, drought, strong light) resistant plants have been reported so far. ing. On the other hand, research has also begun on the possibility of using plants in terms of pharmaceutical production.
[0008]
One of the current attentions is the production of bioactive peptides using a seed protein storage system. Proteins stored in seeds are relatively easy to separate. Moreover, since the original role of the storage protein is to be used as a nitrogen source, it is possible to change its amino acid sequence without impairing its physiological role.
[0009]
For example, prolamin and glutelin, storage proteins in rice seed endosperm cells, are synthesized in endosperm cells almost simultaneously with the ripening period, and accumulate in different types of intracellular granules called protein bodies (PB). Is done. Glutelin, which is most abundant in endosperm cells, is accumulated in protein body type II (PB-II), and the other major storage protein, prolamin, is accumulated in protein body type I (PB-I). This selection is thought to be based on the distinctive signal sequences present in each protein being distinguished by different endoplasmic reticulum regions.
[0010]
If these signal sequences can be used appropriately to introduce foreign genes into rice, the expressed foreign proteins can be accumulated in the protein body, and stable accumulation and easy isolation and recovery of foreign proteins can be achieved. May be easier. Similar storage proteins are also contained in the endosperm of seeds of gramineous plants other than rice, such as wheat, barley, rye, and corn, and their signal sequences are also useful.
[0011]
When human proteins are expressed using plants originally edible, such as rice, wheat, corn, etc., components harmful to humans are mixed in human proteins isolated and purified from recombinant plants. In addition to the advantage that the possibility is low, it is possible to use the protein in a method in which a plant or a callus is taken orally as it is. The same applies to the tea used as a drink in terms of ingestion by humans. If foreign useful genes can be introduced into the tea, a new application can be opened.
[0012]
On the other hand, human interferon (INF) used for the treatment of hepatitis and the like needs to be administered to a patient over a long period of time, but the supply amount is limited, and the period of administration to a patient is usually Limited to a relatively short time. For this reason, there is a need for the production of plants that can make interferon production more efficient and can promote its use in the treatment of hepatitis and the like.
[0013]
[Problems to be solved by the invention]
In the above background, the present invention aims to produce rice and tea capable of producing human interferon protein.
[0014]
[Means for Solving the Problems]
The present inventor constructed an artificial gene in which a base sequence encoding a signal sequence of a prolamin of rice, which is a grass family plant, is fused upstream of the human interferon gene (INF), and this artificial gene is ablated using the Agrobacterium method. Introduced to Ginger Rice. As a result, the expression of human interferon protein was confirmed in various tissues including callus, and it was confirmed that the protein maintained the physiological activity of human interferon. Furthermore, as a result of observation with an immunoelectron microscope, it was suggested that human interferon protein was accumulated in PB-I due to the function of the signal sequence. On the other hand, Camellia sinensis was also transformed with callus, and the introduction and expression of the foreign gene GUS was confirmed. The base sequence encoding the human interferon protein can be inserted in the form replaced with a part of the base sequence of the reporter gene GUS, as shown in the examples for fertile rice, and thereby the Camellia sinensis tissue Can also be expressed by introducing a human interferon gene. Since Camellia is adjacent to Thea in taxonomics, human interferon gene can be introduced and expressed in tea in the same manner as Camellia sinensis.
[0015]
That is, the present invention incorporates a base sequence encoding a human interferon protein, in which a base sequence encoding a signal amino acid sequence that causes accumulation of endosperm storage protein into a protein body is linked upstream, so that it can be expressed in genomic DNA. A grass plant is provided. Preferable examples of the grass family include rice, wheat, barley and oats or corn. The gramineous plant can be provided in any form of individual somatic cells, calli and plants.
[0016]
Furthermore, the present invention also provides a tea characterized in that a base sequence encoding human interferon protein is incorporated into genomic DNA so that it can be expressed. The tea can be provided in any form of individual somatic cells, calli and plants.
[0017]
Furthermore, the present invention constructs a recombinant plasmid DNA having a base sequence encoding a human interferon protein, wherein a base sequence encoding a signal amino acid sequence that causes accumulation of the endosperm storage protein into the protein body is linked upstream. Agrobacterium is transformed with the plasmid DNA, the transformed Agrobacterium is added to the callus of the Gramineae plant, and the callus expressing the human interferon protein is selected. The present invention also provides a method for producing a grass family, wherein a nucleotide sequence encoding a human interferon protein is incorporated into a genomic DNA such that it can be expressed. Preferable examples of the grass family include rice, wheat, barley and oats or corn.
[0018]
Furthermore, the present invention constructs a recombinant plasmid DNA having a base sequence encoding human interferon protein, transforms Agrobacterium with the plasmid DNA, and adds the transformed Agrobacterium to tea callus. The present invention also provides a method for producing a tea comprising a base sequence encoding a human interferon protein, which can be expressed in a genomic DNA, wherein the callus expressing the human interferon protein is selected by infection.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
The Gramineae plant or tea expressing human interferon protein produced according to the present invention can be used for extracting and recovering human interferon protein from callus, for example, and the callus itself can be used as it is or after being subjected to a treatment such as drying. Can be used for oral supplementation of human interferon.
[0020]
【Example】
Hereinafter, the present invention will be described more specifically with reference to representative examples. However, the present invention is not intended to be limited to these specific examples.
[0021]
1. Construction of plasmid vector containing human interferon gene
A plasmid vector containing the human interferon gene was constructed by the following procedures A to C (see FIGS. 1 to 5). As the interferons, interferons α and ω were used as examples.
[0022]
A. Preparation of pINT-RP10sGUS
1) A genomic DNA clone containing rice superoxide dismutase (rice SOD) was digested with BamHI (Takara Shuzo), and the DNA fragments were separated by agarose gel electrophoresis and purified using the GENE CLEAN kit (Funakoshi). Subcloning was performed on pBSK (+) (Toyobo) digested with BamHI using ligation kit ver2 (Takara Shuzo).
[0023]
2) Forward primer (5-aatcagcaatggcagcatac-3) (shown in SEQ ID NO: 5) and reverse primer (5-catggctaatgttggtgggaa-3) (shown in SEQ ID NO: 6) using rice genomic DNA as a template And amplified by PCR to obtain a DNA fragment containing a rice 10 kDa prolamin signal. Further, after treatment with a blunting kit (Takara Shuzo) and polynucleotide kinase, it was inserted into pBI221 (Clontech) digested with SmaI.
[0024]
3) The plasmid of 1) above is digested with BamHI and NheI (Takara Shuzo), the DNA fragment containing the SOD first intron is separated by agarose gel electrophoresis, and then purified using the GENE CLEAN kit. BamHI and XbaI (Takara Shuzo) PINT-RP10sGUS was obtained by subcloning using the ligation kit ver2 (Takara Shuzo).
[0025]
B. Preparation of pIG121Hm
1) Forward primer INT-f (5-ggatccactagttctagagtcatcttc-3) (shown as SEQ ID NO: 7 in the sequence listing) and reverse primer INF-r (5-cgtcctaggaagtgataacagata-3) (in the sequence listing) using rice genomic DNA as a template The DNA containing the rice SOD first intron was amplified by PCR using SEQ ID NO: 8) and digested with XbaI and BamHI. The XbaI and BamHI fragments were separated by agarose gel electrophoresis, purified using the GENE CLEAN kit, and subcloned into pBI121 (Clontech) digested with XbaI and BamHI using the ligation kit ver2.
[0026]
2) The plasmid obtained in 1) above was digested with HindIII (Takara Shuzo) and EcoRI (Takara Shuzo), and the HindIII and EcoRI fragments were separated by agarose gel electrophoresis and purified using the GENE CLEAN kit. This was then subcloned into pUC19 (Takara Shuzo) digested with HindIII and EcoRI using the ligation kit ver2.
[0027]
3) pGPTV-HPT (ATCC) was digested with XbaI and SstI (Gibco), and XbaI and SstI fragments were separated by agarose gel electrophoresis and purified using the GENE CLEAN kit.
[0028]
4) The plasmid obtained in 2) above is digested with XbaI and SstI, the plasmid is purified by agarose gel electrophoresis, the GUS gene is removed, and XbaI having the HPT gene (hygromycin resistance) of 3) above. The SstI fragment was inserted using ligation kit ver2.
[0029]
5) The plasmid obtained in 4) above was digested with HindIII and EcoRI, and HindIII and EcoRI DNA fragments were separated by agarose gel electrophoresis and then purified using the GENE CLEAN kit. This was then made blunt using a blunting kit.
[0030]
6) The plasmid obtained in 1) above was digested with EcoRI and made blunt using a blunting kit. The HindIII and EcoRI DNA fragments of 5) above were inserted into this plasmid using ligation kit ver2 to obtain pIG121Hm.
[0031]
C. Preparation of pIG-A11 and pIG-O89
1) Forward primer A-GUS-SF (5-ctcctggtgctcatacgtaagtcacgcggctctgtgggctg-3) (shown as SEQ ID NO: 9 in the sequence listing) and reverse primer A-SacR (5-catttccatgttggagctcttttcattccttacttc-3) (sequence listing) DNA containing human interferon α2A (INF-α2A) was amplified by PCR using SEQ ID NO: 10). In addition, the forward primer O-Gus-SF (5-ccctagtgatgatacgtaatagccctcgtggatctctgggc-3) (shown by SEQ ID NO: 11 in the sequence table) and the reverse primer O-SacR (5-gcaaattaatcaatgagctccgtatcaagatgag-3) (SEQ ID NO: 12 in the sequence table) DNA containing human interferon ω (INF-ω) was amplified by PCR.
[0032]
2) The pINT-RP10sGUS and the PCR product from 1) above were digested with SnaBI (Takara Shuzo) and SacI (Takara Shuzo), separated by agarose gel electrophoresis, purified using the GENE CLEAN kit, and the ligation kit ver2 was obtained. And INF-α2A and INF-ω genes were cloned.
[0033]
3) The plasmid obtained in 2) above was digested with XbaI and SacI, separated by agarose gel electrophoresis, and then purified using the GENE CLEAN kit.
[0034]
4) pIG121Hm was digested with XbaI and SacI, separated by agarose gel electrophoresis, and then purified using the GENE CLEAN kit to remove the SOD intron and GUS gene.
[0035]
5) Insert the XbaI and SacI fragments of 3) above into the plasmid of 4) above using ligation kit ver2 to obtain pIG-A11 containing the INF-α2A gene and pIG-O89 containing the INF-ω gene. It was.
[0036]
SEQ ID NO: 1 in the Sequence Listing shows the base sequence constituting the characteristic part of plasmid DNA containing the base sequence encoding interferon α protein used in the present invention. In the sequence, bases 1 to 6 are XbaI sites, bases 10 to 894 are rice SOD intron base sequences, bases 916 to 987 are base sequences encoding a prolamin 10k signal consisting of 24 amino acids, base 988 ~ 1044 encodes a 19-amino acid prolamin N-terminal sequence, bases 1045 to 1437 encode a 126-amino acid GUS N-terminal region followed by a 5-amino acid junction Base sequence, bases 1438 to 1446 are base sequences encoding thrombin recognition site consisting of 3 amino acids, bases 1447 to 1947 are base sequences encoding interferon alpha protein (full length) consisting of 167 amino acids, bases 1948 to 1950 Is a stop codon and bases 1954 to 1959 are SacI sites.
[0037]
SEQ ID NO: 2 shows the amino acid sequence of the protein encoded by the DNA of SEQ ID NO: 1 and expressed in rice. In the amino acid sequence, amino acids 1 to 24 are prolamin signals, which are cleaved and removed upon passage through the membrane. Amino acids 25-43 correspond to 19 amino acids in the prolamin N-terminal region. Amino acids 44 to 169 correspond to 126 amino acids in the N-terminal region of GUS, and amino acids 170 to 174 are amino acids constituting the linking part. Amino acids 175 to 177 are amino acids (Arg-Gly-Ser) constituting the thrombin recognition site, and are cleaved between arginine and glycine by thrombin. Amino acids 178 to 344 constitute the full-length amino acid sequence of interferon α protein consisting of 167 amino acids. When the interferon α protein is cut out by thrombin, a part of the thrombin recognition sequence (Gly-Ser) remains on the N-terminal side of the interferon α protein consisting of amino acids 178 to 344, but more than 20 types are found. Since some of the natural interferon α proteins that contain this sequence Gly-Ser exist, the sequence of 169 amino acids including this sequence Gly-Ser is also that of the interferon α protein. is there.
[0038]
SEQ ID NO: 3 in the sequence listing shows the base sequence constituting the characteristic portion of the plasmid DNA containing the base sequence encoding the interferon ω protein used in the present invention. In the sequence, bases 1 to 6 are XbaI sites, bases 10 to 894 are rice SOD intron base sequences, bases 916 to 987 are base sequences encoding a prolamin 10k signal consisting of 24 amino acids, base 988 ~ 1044 encodes a 19-amino acid prolamin N-terminal sequence, bases 1045 to 1440 encode a 126-amino acid GUS N-terminal region followed by a 6-amino acid junction Base sequence, bases 1441-1449 are base sequences encoding thrombin recognition site consisting of 3 amino acids, bases 1450-1971 are base sequences encoding interferon ω protein (full length) consisting of 174 amino acids, bases 1972-1974 Is a stop codon and bases 1975 to 1980 are SacI sites.
[0039]
SEQ ID NO: 4 shows the amino acid sequence of the protein encoded by the DNA of SEQ ID NO: 3 and expressed in rice. In the amino acid sequence, amino acids 1 to 24 are prolamin signals, which are cleaved and removed upon passage through the membrane. Amino acids 25-43 correspond to 19 amino acids in the prolamin N-terminal region. Amino acids 44 to 169 correspond to 126 amino acids in the N-terminal region of GUS, and amino acids 170 to 175 are amino acids constituting the linking part. Amino acids 176 to 178 are amino acids (Arg-Gly-Ser) constituting the thrombin recognition site, and are cleaved between arginine and glycine by thrombin. Amino acids 179 to 352 constitute the full-length amino acid sequence of interferon ω protein consisting of 174 amino acids.
[0040]
Hereinafter, the experimental operation will be described based on an example using the interferon α gene. The operations using the interferon ω and other interferon genes are performed in the same manner as when interferon α is used.
[0041]
2. Agrobacterium transformation
2.1 Introduction of plasmid into E. coli
E. coli was used as the host cell. To the host cells thawed at room temperature, 1-2 μl of the recombinant plasmid vector pIG-A11 constructed above was added and allowed to stand on ice for 1 minute. The cells were then transferred to a cuvette (0.2 CM, Bio Rad Inc.) and an electrical pulse (25 μFD, 200Ω, 2.5 kV) was applied (GENE PULSER, Bio Rad Inc.).
. Heat shock by adding 1.0 ml of SOC medium (tryptophan 10 g / l, yeast extract 5.0 g / l, sodium chloride 10 mM, magnesium chloride 2.5 mM, magnesium sulfate 10 mM, glucose 20 mM) preheated to 37 ° C. to the cuvette Was transferred to a sterilized tube and incubated at 37 ° C. for 1 hour at 180-220 rpm using a rotary shaker. It was plated on LB plate (5.0 g of sodium chloride, 5.0 g of tryptophan, 10 g of yeast extract, 1.5% agar, amypicillin or kanamycin 50 μg / ml in 1 L of LB medium), and incubated at 37 ° C. overnight.
[0042]
2.2 Large-scale preparation of plasmids
E. coli carrying the recombinant plasmid DNApIG-A11 was cultured overnight in LB medium containing antibiotics (LB medium 50 ml + ampicillin 50 μg / ml) by shaking. Transfer the culture to a centrifuge tube and add 7,000 rpm (desktop microcentrifuge BECKMAN Microfuge^TM R) was centrifuged at 4 ° C. for 5 minutes, and 1 ml of alkaline lysis solution I (25 mM Tris-HCl, 50 mM glucose, 10 mM EDTA) was added to the precipitate, suspended well, and left at room temperature for 5 minutes. Next, 2 ml of alkaline solution II (0.2N sodium hydroxide, 1% SDS) was added, mixed gently, and left on ice for 5 minutes. 1.5 ml of 3M potassium acetate solution (pH 5.7) was added and mixed, and left on ice for 10 minutes. 12,000 rpm (High-speed cooling centrifuge: BECKMAN Avanti^TM Centrifuge for 10 minutes with HP-25I, rotor: JA-12), transfer the supernatant to a new centrifuge tube, add 3.5 ml of isopropanol (final concentration 60%), mix and leave on ice for 10 minutes to 1 hour did. After centrifuging at 12,000 rpm for 10 minutes, the precipitate was collected and dried, and then 1 ml of distilled water was added and dissolved. After the DNA dissolved in distilled water was transferred to a Spitz tube and filled up to 1.6 ml, 1.75 g of CsCl and 100 μl of ethidium bromide were added and mixed. This is put in a quick seal tube for ultracentrifugation and sealed, 80,000 rpm (supercentrifuge: BECKMAN Optima^TM TLX, rotor: TLA 120.2) was ultracentrifuged at 20 ° C. for 16-20 hours. It collect | recovered with the 1 ml sterilization syringe (for tuberculin), and moved to the Eppendorf type | mold tube. Filled up with distilled water to 0.5 ml, added 0.5 ml of n-butanol, shaken 3-4 times, and added 1 ml of ethanol (room temperature). Distilled water was added dropwise until the precipitated CsCl disappeared. The mixture was allowed to stand at −80 ° C. for 30 minutes, precipitated with ethanol, then returned to room temperature and shaken. 15,000 rpm (BECKMAN Microfuge^TM R) was centrifuged at 4 ° C. for 5 minutes, the supernatant was discarded, 200 μl of distilled water, 20 μl of 3M sodium acetate, and 550 μl of 100% ethanol were added to the precipitate, and then left at −80 ° C. for 20 minutes. The mixture was centrifuged at 15,000 rpm for 5 minutes at 4 ° C., the supernatant was discarded, and 200 μl of 80% ethanol was added to the precipitate. The mixture was allowed to stand at −80 for 20 minutes, centrifuged at 15,000 rpm for 3 minutes at 4 ° C., the supernatant was discarded, the precipitate was dried, and then dissolved in 50 μl of distilled water. The DNA concentration was measured by the absorbance method (measured by filling up 5 μl to 1 ml, OD₂₆₀Calculated as x10 μg / ml).
[0043]
2.3 Introduction of plasmid into Agrobacterium
2.3.1 Preparation of competent cells
Agrobacterium tumefaciens wild strains (EHA101 strain and EHA105 strain) were streaked so that single colonies appeared on LB plates containing antibiotics (LB medium + kanamycin 50 μg / ml) and cultured at 28 ° C. for 2 to 3 days did. The colonies were inoculated into 3 ml of antibiotic-containing LB medium (sodium chloride 5.0 g / l, tryptone 5.0 g / l, yeast extract 10 g / l) and cultured at 28 ° C. overnight. Inoculate 0.1-0.5ml of culture solution into 50ml of LB medium containing antibiotics and OD at 28 ° C₆₀₀= Cultured to about 0.4. The flask containing the Agrobacterium tumefaciens culture solution was cooled in ice water for 15 minutes, then transferred to a 50 ml Corning tube and centrifuged at 3,500 rpm for 15 minutes at 4 ° C. to remove the medium. After adding about 10 ml of 10% glycerol at 4 ° C., the cells were suspended, and further cold 10% glycerol was added to make a total of 40 ml, followed by centrifugation at 4,000 rpm for 15 minutes. Glycerol was removed and about 10 ml of cold 10% glycerol was added again, and then the cells were suspended. Further, cold 10% glycerol was added to make a total of 40 ml and centrifuged at 4,000 rpm for 15 minutes. After sufficiently removing glycerol, 400 μl of 10% glycerol was added to suspend the Agrobacterium tumefaciens cells. 40 μl each was dispensed into 1.5 ml Eppendorf tubes, quenched with liquid nitrogen, and stored at -80.
[0044]
2.3.2 Transformation of Agrobacterium tumefaciens
To the host cells thawed at room temperature, 1-2 μl of recombinant plasmid DNApIG-A11 DNA was added and allowed to stand on ice for 1 minute. Host cells were transferred to a pre-cooled cuvette and given an electrical pulse (25 μFD, 200Ω, 2.5 kV). Add 1.0 ml of SOC medium preheated to 28 ° C. to the cuvette, apply heat shock, transfer to a sterilized test tube, and incubate at 180 ° C. to 220 rpm using a rotary shaker at 28 ° C. for 3 hours. Cells were plated on LB plates (LB medium, kanamycin 50 μg / ml, hygromycin 50 μg / ml, 1.5% agar) and cultured at 28 ° C. for 2-3 days. A single colony was inoculated into an antibiotic-containing LB medium, and then the plasmid was recovered with Flex prep (Pharmacia), and introduction of the target gene was confirmed by PCR in a conventional manner.
[0045]
3. Transformation of ripening rice
3.1 Preparation of Agrobacterium
Inside the safety cabinet, platinum ears were inserted into vials of Agrobacterium tumefaciens (EHA101 / pIG-A11 and EHA105 / pIG-A11) stocked with glycerol at -80 ° C. and applied to AB medium (see medium list). Sealed with vinyl tape and cultured in the dark at 28 ° C for 3 days.
[0046]
3.2 Induction of callus
After removing the cocoons of the raw rice seeds, they were put in a bottle with a picket, 70% ethanol was added and the mixture was stirred with a stirrer for about 1 minute. After discarding ethanol, sodium hypochlorite diluted to 50% was added and stirred with a stirrer for about 1 hour. Sodium hypochlorite was completely cut using a tea strainer in a clean bench, the seeds were transferred to a petri dish, and washed 5-6 times with distilled water. The seeds were transferred to N6CP medium (see medium list) with tweezers and cultured at 28 ° C. in the light for 2 weeks. The resulting callus was removed with tweezers and transferred to fresh N6CP medium and used within 1-2 weeks.
[0047]
3.3 Gene transfer by Agrobacterium infection
Attempts were made in two ways with and without sonication.
[0048]
3.3.1 Method without sonication
About 25 ml of AA suspension medium (see medium list) was dispensed into a 50 ml Corning tube. On the AB medium (see medium list), acetosyringone (dissolved in DMSO at a concentration of 50 mg / ml and stored at 4 ° C. protected from light) was added to the AA suspension medium to a concentration of 10 mg / ml. Agrobacterium (EHA101 strain) was scraped and suspended with a spatula several centimeters. The callus on the 31st day from the start of induction was transferred to an AA suspension medium in which Agrobacterium cells were suspended, shaken, and then infected for 5 minutes with occasional shaking. The cell suspension was discarded a little, and the suspension and callus were transferred to a petri dish together. Callus was placed on N6 co-culture medium (see medium list), sealed with surgical tape, and co-cultured at 28 ° C. in the dark for 3 days.
[0049]
3.3.2 Method of performing ultrasonic treatment
About 25 ml of AA suspension medium (see medium list) was dispensed into a 50 ml Corning tube. Callus on the 17th day of induction was placed in a Corning tube and sonicated for 30 seconds. Agrobacterium cells on the AB medium were scraped and suspended in an AA suspension medium (see medium list) with a spatula. The cells of Agrobacterium (EHA105 strain) were suspended by shaking and then infected for 5 minutes with occasional shaking. The cell suspension was discarded a little, and the suspension and callus were transferred to a petri dish together. Callus was placed on N6 co-culture medium (see medium list), sealed with surgical tape, and co-cultured in the dark at 28 ° C. for 2 days.
[0050]
3.4 Agrobacterium removal and recovery
About 25 ml of distilled water was placed in a 50 ml Corning tube, and callus after co-culture was added. After shaking, it was allowed to stand still, and it was confirmed that the callus had settled, and the washing water was discarded. The operations of adding about 25 ml of distilled water, mixing, standing, and discarding the washing water were repeated until the distilled water did not become cloudy (2 to 3 times). Add 25 ml of distilled water, add 50 μl of carbenicillin (dissolved in sterilized water to 250 mg / ml, filter sterilize in a clean bench and store at 4 ° C.) (final concentration 500 mg / ml) It was confirmed that the callus had settled, and the washing water was discarded. Repeat the operation of adding and mixing carbenicillin-containing distilled water, allowing to stand, and discarding the wash water until the wash water does not become cloudy (3-4 times). Moved to a petri dish. Callus was placed in N6C + carbenicillin medium (see medium list) with tweezers, sealed with surgical tape, cultured at 28 ° C for 10 days for callus infected with EHA101 strain, and for 9 days for callus infected with EJHA105 strain for 9 days. .
[0051]
This recovery operation is aimed at recovery from the damage to the plant caused by Agrobacterium infection, and omission of this operation is not preferable because it reduces the possibility of obtaining a transformant. The optimal culture period in N6C + carbenicillin medium is about one week.
[0052]
3.5 Selection
All calli cultured for 1 week in N6C + carbenicillin medium were placed on N6 selective medium (see medium list). At this time, the callus that was growing was placed in small portions while avoiding excessive force. After sealing with surgical tape, the placed callus was cultured at 28 ° C. in the light. The medium was changed about every 2 weeks. The hygromycin concentration of the N6C + carbenicillin medium used was 25 mg / l for the first 15 days for the EHA101 strain-infected callus, 40 mg / l for the remaining period (17 days), and the entire period ( 27 days) 30 mg / l.
[0053]
3.6 Redifferentiation
Only calli that had been grown in N6 selective medium and grown were placed on MS regeneration medium (see medium list) with a hygromycin concentration of 25 mg / l. Callus greened in 1-2 weeks. Redifferentiated individuals were transferred to MS hormone-free medium (see medium list). The presence or absence of hygromycin resistance was confirmed, and the plant body was grown.
[0054]
3.7 Acclimatization
When the roots grew sufficiently and the shoots grew, the lid was gradually opened over 3 days. At this time, in order to prevent the generation of mold, the medium was filled with water and the water was changed once a day. After sufficiently removing the medium from the acclimatized plant body, it was replanted in soil and allowed to grow with hypox.
[0055]
4). Medium list
The composition of each medium used in the above operation is as follows.
[0056]
AA medium: sodium dihydrogen phosphate 150 mg / l, calcium chloride dihydrate 150 mg / l, magnesium sulfate heptahydrate 250 mg / l, potassium iodide 0.75 mg / l, cobalt chloride hexahydrate 0.025 mg / l, boric acid 3.0 mg / l, sodium molybdate dihydrate 0.25 mg / l, manganese sulfate tetrahydrate 10.0 mg / l, copper sulfate pentahydrate 0.025 mg / l, zinc sulfate heptahydrate 2.0 mg / l, edetic acid first Iron 40.0 mg / l, Inositol 100 mg / l, Thiamine hydrochloride 10.0 mg / l, Nicotinic acid 1.0 mg / l, Pyridoxine hydrochloride 1.0 mg / l, Glycine 7.5 mg / l, Arginine 177 mg / l, Potassium chloride 3 g / l, Glutamine 900 mg / l, aspartic acid 300 mg / l, sucrose 2 g / l
[0057]
AA suspension medium: AA salts and amino acids, B5 vitamins, sucrose 20 g / l, kinetin 0.2 mg / l, 2,4-D 2 mg / l, acetosyringone 10 mg / l, pH 5.8
[0058]
AB medium: potassium hydrogen phosphate 3 g / l, potassium dihydrogen phosphate 1 g / l, ammonium chloride 1 g / l, magnesium sulfate heptahydrate 0.3 g / l, potassium chloride 0.15 g / l, calcium chloride 0.01 g / l, Ferrous sulfate heptahydrate 2.5 mg / l, glucose 5 g / l, agar 15 g / l, pH 7.2
[0059]
N6 medium: ammonium sulfate 463 mg / l, potassium nitrate 2830 mg / l, potassium dihydrogen phosphate 400 mg / l, calcium chloride dihydrate 166 mg / l, magnesium sulfate heptahydrate 185 mg / l, potassium iodide 0.8 mg / l, cobalt chloride Hexahydrate 0.025 mg / l, boric acid 1.6 mg / l, sodium molybdate dihydrate 0.25 mg / l, manganese sulfate tetrahydrate 4.4 mg / l, copper sulfate pentahydrate 0.025 mg / l, zinc sulfate 7 water 1.5 mg / l salt, ferrous sulfate heptahydrate 27.85 mg / l, disodium edetate 37.25 mg / l, inositol 100 mg / l, thiamine hydrochloride 1.0 mg / l, nicotinic acid 0.5 mg / l, pyridoxine hydrochloride 0.5 mg / L, glycine 2.0 mg / l, sucrose 30 g / l
[0060]
N6 co-culture medium (N6co): N6 salts and vitamins, sucrose 30 g / l, glucose 10 g / l, 2,4-D 2 mg / l, acetosyringone 10 mg / l, agar 8 g / l, pH 5.2
[0061]
N6 callus induction medium (N6C): N6 salts and vitamins, sucrose 30 g / l, 2,4-D 2 mg / l, agar 8 g / l, pH 5.8
[0062]
N6 callus induction + proline medium: N6 callus induction medium, proline 2.8 g / l
[0063]
N6C + Carbenicillin medium: NC6 callus induction medium, Carbenicillin 500 mg / l, pH 5.8
[0064]
N6 selective medium: N6 salts and vitamins, sucrose 30 g / l, 2,4-D 2 mg / l, gelrite 2 g / l, carbenicillin 500 mg / l, hygromycin 30-50 mg / l, pH 5.8
[0065]
MS medium: 440 mg / l calcium chloride dihydrate, 1650 mg / l ammonium nitrate, 1900 mg / l potassium nitrate, 170 mg / l potassium dihydrogen phosphate, 370 mg / l magnesium sulfate heptahydrate, 0.83 mg / l potassium iodide, cobalt chloride Hexahydrate 0.025 mg / l, boric acid 6.2 mg / l, sodium molybdate tetrahydrate 0.25 mg / l, manganese sulfate tetrahydrate 22.3 mg / l, copper sulfate pentahydrate 0.025 mg / l, zinc sulfate 7 water Salt 8.6 mg / l, Ferrous sulfate heptahydrate 27.85 mg / l, Disodium edetate 37.25 mg / l, Inositol 100 mg / l, Thiamine hydrochloride 1.0 mg / l, Nicotinic acid 0.5 mg / l, Pyridoxine hydrochloride 0.5 mg / L, glycine 2.0 mg / l, sucrose 30 g / l
[0066]
MS regeneration medium (MSre): MS salts and vitamins, sucrose 30 g / l, sorbitol 30 g / l, NAA 1 mg / l, BAP 2 mg / l, carbenicillin 250 mg / l, hygromycin 25 mg / l, gelrite 2 g / l, pH 5.8
[0067]
MS hormone-free medium (MSh): MS salts and vitamins, sucrose 30 g / l, carbenicillin 250 mg / l, hygromycin 25 mg / l, gelrite 2 g / l, pH 5.8
[0068]
5. Examination of the transformed fertile rice obtained
5.1 Introduction efficiency of human interferon α in fertile rice
By the above method, fertile rice transformed by introduction of human interferon α gene was obtained. The number of seeds used, the number of callus, the number of transformed solids obtained, and the seed-based transformation efficiency are shown.
[0069]
[Table 1]

[0070]
5.2 Confirmation of target genes in transformed fertile rice
5.2.1 Confirmation by PCR
(1) PCR
Each of the obtained transformed individuals (α-1, α-2, α-3, α-4 and α-5 lines) was confirmed. Genomic DNA was prepared to 4 ng / 2 μl and used as PCR as template DNA. According to the following composition, a mixture other than the template DNA was dispensed into a 0.5 ml Eppendorf tube, and the template DNA was added thereto. As negative controls, genomic DNA of wild-type dwarf rice and distilled water were used.
Template DNA: 2 μl
10 x buffer solution 2 μl
25 mM MgCl₂... 2 μl
10 x dNTP ··· 2 μl
Forward primer^{* 1}... 1 μl
Back primer^{* 2}... 1 μl
Taq polymerase ... 0.1μl
All distilled water 20 μl
^{* 1}  Use GUS snaF
^{* 2}  Use GUS sacR
[0071]
A 0.5 ml Eppendorf tube was set in the GeneAmp PCR system 2400, and the following program was executed.
[Standard program]
(A) 94 ° C for 1 minute
(B) 94 ° C 0.5 minutes
(C) 1 minute at 60 ° C
(D) Repeat (a) to (c) above for 35 cycles
(E) 72 ° C 1.5 minutes
(F) 72 ° C 5 minutes
(G) Storage at 4 ° C
[0072]
(2) Agarose gel electrophoresis
After the above reaction operation, 20 μl of the reaction solution was subjected to agarose gel electrophoresis according to the following procedure to confirm the band. That is, 0.4 g of agarose was placed in an Erlenmeyer flask, and 40 ml of TAE buffer (0.4 M Tris acetate, 0.62 M EDTA) was added. After dissolution in a microwave oven, the solution was corrected with distilled water and poured into a mold to be solidified (gel concentration 0.8%). After solidification, the gel was placed in an electrophoresis apparatus filled with TAE buffer, and a small amount of PBP was added to the PCR product and applied to the well. A λ Hind III marker was applied to both ends. After electrophoresis for about 30 minutes, the cells were immersed in ethidium bromide staining solution for 10 minutes for staining. After staining, the gel was subjected to ImageMaster (Pharmacia Biotech) to confirm the position of the band, and photographed together.
[0073]
(3) Results
The results of PCR are shown in FIG. In all transformed lines, a DNA fragment could be confirmed at the target position (around 500 bp). In the negative control, no DNA amplification was observed. As a result, it was confirmed that the target gene was introduced into the transformant.
[0074]
5.2.2 Confirmation by genomic Southern blot
(1) Isolation of rice genome
Among the obtained transformed individuals, the α-2, α-3, and α-4 strains were confirmed. About 10 g of green rice leaves were cut as small as possible with a boil and placed in a mortar. Liquid nitrogen was added to this and crushed until powdered with a pestle. 2% CTAB solution (100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl, 2% CTAB (cetyltrimethylammonium bromide), pH 8. 0] 20 ml was added. After shaking at 56 ° C. for 20 minutes, an equal volume of chloroform: isoamyl alcohol (24: 1) was added and mixed by inverting at room temperature for 20 minutes. Centrifugation was performed to separate the upper layer, and an equal amount of chloroform: isoamyl alcohol (24: 1) was again added thereto, followed by inverting at room temperature for 20 minutes. The upper layer was separated by centrifugation, and 1 to 1.5 volume of 1% CTAB solution (50 mM Tris-HCl, 10 mM EDTA, 1.4 M NaCl, 1% CTAB, pH 8.0) was added thereto to precipitate genomic DNA. The precipitate was recovered by centrifugation at 3,000 rpm for 20 minutes. To the precipitate was added 5 ml of 1M NaCl, and 5 μl of 10 mg / ml RNase A was further added, followed by shaking at 56 ° C. until the precipitate was dissolved. In a clean bench, 10 ml of cooled 100% ethanol was gently layered, and genomic DNA was collected by wrapping around a Pasteur pipette melted with heat. At this time, genomic DNA was obtained to such an extent that it could be visually observed. After dipping in 80% ethanol twice and 100% ethanol once in this order for about 5 minutes, each was air-dried until the wrapped DNA became transparent. Genomic DNA was placed in a 15 ml Corning tube containing 2 ml of TE and heated to 56 ° C if necessary to completely dissolve. Absorbance (OD₂₆₀) Was measured to determine the DNA concentration.
[0075]
(2) Preparation of DNA transfer membrane
Digest 10 μg of genomic DNA overnight at 37 ° C. with each restriction enzyme of Hind III, Xba I, Sac I and Eco RI (which is a restriction enzyme for a restriction enzyme site in the transgene (T-DNA)). did.
Genomic DNA ... 10μg
10 x buffer ... 10 μl
Restriction enzyme ... 8μl
Distilled water 100 μl
[0076]
After the reaction, ethanol precipitation was performed, 1/10 of the total amount of 3M sodium acetate and 2-2.5 times the amount of 100% ethanol were added and mixed, and then left at -80 ° C for 15 minutes or longer.
Then centrifuge at 15,000 rpm and room temperature for 20 minutes in a tabletop centrifuge, discard the supernatant, add 200 μl of 80% ethanol, centrifuge at 15,000 rpm for 10 minutes at room temperature, discard the supernatant, and dry the precipitate under reduced pressure . The recovered DNA was dissolved in 20 μl of distilled water, and electrophoresed under the conditions of 0.8% gel and 50 mA. The gel after electrophoresis was immersed in ethidium bromide staining solution for 10 minutes or more. The gel was shaken in 0.25N hydrochloric acid until BPB turned yellow and then treated in 0.4N sodium hydroxide for 20 minutes to denature the DNA by one difference. Hybond-N the same size as the gel⁺(Amersham) Cut one sheet and two 3MM filter papers, assemble the blot table, and place DNA into Hybond-N for 8 hours to overnight.⁺(Amersham). Hybond-N⁺(Amersham) was marked on the gel comb and washed with 2 × SSC [sodium chloride 17.53 g / l, sodium citrate 8.82 g / l] for 5 minutes. Hybond-N⁺(Amersham) was sandwiched between paper towels, baked at 80 ° C. for 2 hours, and stored in a desiccator.
[0077]
(3) Preparation of probe DNA
Restriction enzyme treatment was performed for 2 hours in the following system.
pIG-A11 ・ ・ ・ ・ ・ ・ ・ ・ ・ 8μg
10 x buffer ... 4 μl
SnaB I ... 2 μl
Sac I ... 2 μl
10 × BSA ・ ・ ・ ・ ・ ・ ・ ・ ・ 4μl
All distilled water 40 μl
[0078]
DNA digested with restriction enzymes was electrophoresed at 100 mA (see above for procedure). After electrophoresis, the gel was photographed and the necessary bands were cut with a razor under UV irradiation. The elution apparatus was horizontally aligned, and 1 × electroelution buffer (20 mM Tris-HCl, 0.2 mM EDTA, 5 mM NaCl, pH 8.0) was poured. Air was evacuated from each channel by adding and removing buffer with a 1 ml pipetman. 125 μl of 3M sodium acetate was added to all channels, taking care not to overflow. The excised gel was placed on a table and eluted at 100 V / 400 A for 1 hour, and then the gel was irradiated with UV to confirm that all DNA was recovered. The buffer solution was removed, and the liquid in the channel was collected with a 1 ml pipetman fitted with a capillary tip. After adding 1 ml of 100% ethanol and shaking, it was placed at −80 ° C. for 15 minutes or more. After centrifugation at 15,000 rpm for 20 minutes with a tabletop centrifuge, the supernatant was discarded. For rinsing, 200 μl of 80% ethanol was added and centrifuged at 15,000 rpm for 10 minutes, and the supernatant was carefully discarded using a pipetman. The precipitate was dried with a vacuum drier and dissolved in 12 μl of distilled water.
[0079]
(4) DNA labeling
Adjust the probe to a concentration of 25-100 ng / 3 μl, 3 μl of DNA solution and BcaBEST^TMKit (Takara Shuzo) [Random primer (9mer), 10x buffer, dNTP mixed solution (0.2 mM dATP, dTTP, dGTP), BcaBEST^TM DNA polymerase] 2 μl of the attached random primer was mixed and heat denatured in a water bath at 95 ° C. for 3 minutes. This was then annealed by cooling on ice for 5 minutes. To this was added 2.5 μl of 10 × buffer, 2.5 μl of dNTP mixture, and 9.0 μl of distilled water. After mixing and centrifugation, [α-³²5 μl of P (3,000 Ci / mmol)] dCTP and BcaBEST^TM 1 μl of DNA polymerase was added. The reaction was carried out at 55 ° C for 10 minutes. Centrifuge at 3,400 rpm for 2 minutes in a spin column to remove unreacted dNTPs, [α-³²After checking the efficiency of P] dCTP uptake, it was stored in an Eppendorf tube at 4 ° C.
[0080]
(5) Prehybridization
Salmon sperm DNA was heat denatured at 95 ° C. for 3 minutes and then placed on ice for 5 minutes. Petri dish with hybridization buffer [6 × SSPE, 5 × Denharts reagent, 0.5% SDS] [20 × SSPE: 3.6 M NaCl, 200 mM potassium dihydrogen phosphate, 20 mM EDTA (pH 7.4); 50 × Denharts reagent: 10 ml of 1.0% Ficoll, 1.0% polyvinylpyrrolidone, 1.0% BSA] was added, and 1/100 amount of heat-denatured salmon sperm DNA was added. The membrane was placed in a petri dish so that air bubbles would not enter, sealed with vinyl tape, and then incubated at 65 ° C. for 3 hours or more.
[0081]
(6) Hybridization
³²The P-labeled probe DNA was heat denatured at 95 ° C. for 3 minutes and then allowed to cool on ice for 5 minutes. For membranes after prehybridization³²P-labeled probe DNA was added and incubated at 55 ° C. for 6 hours or longer.
[0082]
(7) Cleaning
The hybridization buffer was discarded, and washing was performed twice at 42 ° C. for 20 minutes with 30 ml of washing buffer [6 × SSC, 0.1% SDS]. A survey meter was used to count several locations on the membrane, and it was confirmed that the target band was not counted except at a certain position. Before the membrane dried, it was placed in a Hybribag to seal the periphery and exposed to a Molecular imager (BioRAD) screen. After the exposure, it was confirmed that hybridization had occurred in the target band with a Molecular imager (BioRAD), and the film was developed by exposing to an X-ray film (Fujifilm: XR-1).
[0083]
(8) Results
The results are shown in FIG. A band of the target gene was detected only in the transformed lines α-2, α-3, and α-4, and no band was detected in the wild-type genomic DNA as the negative control. For the transformed lines, 1 to 3 bands were detected for genomic DNA digested with 4 types of restriction enzymes, and none of the transformed lines gave only a single band with all restriction enzymes. This suggests that multiple copies of the target gene have been introduced into these transformed lines.
[0084]
5.3 Expression analysis of recombinant protein in transformed rice
5.3.1 Analysis by ELISA
(1) Protein extraction
For callus, roots and leaves, 1.0 ml of cell lysis buffer was added to 300 mg of tissue and homogenized, and then centrifuged, and the supernatant was used as a sample. For seeds (ripening seeds and mature seeds), 500 μl of cell lysis buffer was added to 50 mg of tissue and treated in the same manner as above, and the supernatant was used as a sample. Further, the callus obtained above was further examined for the expression of human interferon α protein after liquid doubling.
[0085]
(2) ELIZA
100 μl / well of unlabeled antibody at a concentration of 1 μl / ml was placed in a 96-well plate for ELIZA and incubated at 37 ° C. for 1 hour. After removing the antibody, water was wiped off and PBS containing 1% bovine serum albumin was added at 350 μl / well and incubated at 37 ° C. for 1 hour. After discarding the block solution, the moisture was wiped off and washed once with TTBS. Samples diluted with interferon standard and BPS containing 1% bovine serum albumin were added to wells at 100 μl / well, and incubated at 37 ° C. for 1 hour. The sample was discarded and washed 3 times with TTBS. 1 μg / ml HRP-labeled anti-interferon α antibody was added at 100 μl / well and incubated at 37 ° C. for 1 hour. After discarding the secondary antibody, it was washed 3 times with TTBS. Add 50 μl / well of HRP substrate (TM blue) and allow to develop for about 5 minutes at room temperature. Thereafter, the reaction was stopped by adding 50 μl / well of 0.1 N sulfuric acid. Absorbance at 450 nm was measured with a plate reader, and the concentration in the sample was calculated from a calibration curve prepared in advance for a standard product.
[0086]
(2) Results
The results for each organization are summarized in FIG.
[0087]
Human interferon alpha protein level in transformed individuals (ELIZA)
[Table 2]

[0088]
The results are shown as the amount of human interferon α protein in 50 mg of rice tissue. As shown in the figure and table, high expression levels were obtained in callus and seeds at the ripening stage. Although only a small amount of expression was observed in the ripe seed, human interferon α was accumulated in the protein body of the ripe seed because the cell lysis buffer used for the operation did not contain a substance that dissolves the protein body. There is a possibility. Immunoelectron microscopic observations suggest that human interferon alpha protein is accumulated in PB-I in the seed (data not shown).
[0089]
The results for the liquid cultured callus are shown in Table 3 below. From the pair with the above table, it can be seen that callus cultured in an individual medium and almost identified human interferon α protein are also produced by liquid culture.
[0090]
[Table 3]

[0091]
5.3.2 Antiviral activity analysis
The antiviral activity of the expressed human interferon alpha protein was evaluated.
(1) Method
The protein sample used was prepared in the same procedure as ELIZA. However, callus was not used. Using vesicular stomatitis virus (VSV), human leukocyte interferon α standard, and FL5-1 cells (human amnion-derived cells), the procedure was performed according to a normal procedure by a 50% CPE reduction method (dye uptake method).
[0092]
(2) Results
The results are summarized in FIG.
[0093]
[Table 4]

[0094]
The figure and table reveal that the protein extracted from transformed rice has antiviral activity. In addition, the fact that the antiviral activity is correlated with the expression level of human interferon α protein confirmed in ELIZA indicates that this antiviral activity is due to human interferon α expressed in transformed rice. ing.
[0095]
6). Introduction of human interferon alpha gene into Camellia sinensis
6.1 Callus culture and somatic embryogenesis of Camellia sinensis
Immature seeds of Camellia sinensis were sterilized with a sodium hypochlorite (effective chlorine concentration 1%) solution for 10 minutes and then washed three times with sterilized water. Cotyledons were excised from sterilized immature seeds, divided into two, placed on 1/2 MS medium supplemented with 1.0 mg / l BA, and cultured under a 4,000 lux fluorescent light for 16 hours at 25 ° C. for 16 hours. Callus formation was observed after 6 weeks of culture. The obtained callus could be maintained by subculturing in a medium having the same composition. By transplanting to a hormone-free 1 / 2MS medium, seedlings were obtained from callus via somatic embryos.
[0096]
6.2 Production of transformed callus
PNPI132 was introduced into E. coli in the same manner as in “2.1 Introduction of plasmid into E. coli”, and a large amount of plasmid was prepared in the same manner as in “2.2 Large-scale preparation of plasmid”. The plasmid was introduced into Agrobacterium (Agrobacterium tumefaciens LBA4404) in the same manner as in “2.3 Introduction of Plasmid into Agrobacterium”. Next, the callus derived from the cotyledon of Camellia sinensis prepared above was sonicated (38 kHz, 5 seconds), then Agrobacterium tumefaciens having pNPI132 (Nippon Paper Industries Co., Ltd.) and the above-mentioned “3.3 Agrobacterium-infected gene The co-culture was carried out for 3 days according to “Introduction”. GUS activity was examined one month after the end of co-culture. As a result, GUS activity was observed in about 30% of callus. See FIG. 10 for the structure of pNPI132. In the figure, Rs is a DNA sequence recognized and acted on by a recombinant enzyme, R is a gene encoding the recombinant enzyme, ipt is a gene encoding isopentenyl transferase involved in cytokinin synthesis, and T is a terminator. .
[0097]
In the same way as in the experimental procedure for rice, pNPI132 is digested with Sna BI and Sac I, and a part of the GUS gene in pNPI132 is removed, and DNA encoding interferon is ligated instead. As a result, plasmid DNA incorporating the interferon gene is obtained. As in the case of rice, this was amplified in E. coli, transformed into Agrobacterium, and the transformed Agrobacterium was infected with Camellia sinensis callus, Camellia sinensis with interferon gene introduced can be obtained.
[0098]
7). Introduction of human interferon alpha gene into tea
Since Cha (Thea) is taxonically adjacent to Camellia, the GUS gene or human interferon α gene can be introduced for Cha by the same procedure as for Camellia sinensis. For example, an immature seed of tea is sterilized with a sodium hypochlorite (effective chlorine concentration 1%) solution, washed with sterilized water, a cotyledon is excised, divided into two, and a 1/2 MS medium supplemented with 1.0 mg / l BA And cultured under conditions of 25 ° C. for 16 hours under a 4,000 lux fluorescent lamp. The obtained callus is sonicated in the same manner as described above, and co-culturing with Agrobacterium tumefaciens having pNPI132 (Nippon Paper Industries Co., Ltd.) will be possible to obtain a tea incorporating the GUS gene. Therefore, if part of the GUS gene in pNPI132 is removed and removed, and the DNA encoding interferon is ligated instead, plasmid DNA incorporating the interferon gene can be obtained. In the same way as in the case of rice, the gene is amplified in E. coli, transformed into Agrobacterium, and the transformed Agrobacterium is infected with tea callus to interferon gene. It is reasonably presumed that a cha with the introduction of can be obtained.
[0099]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a process for preparing pINT-RP10sGUS.
FIG. 2 shows a process for preparing pIG121Hm.
FIG. 3 is a diagram showing incorporation of human interferon α gene sequence into pUC19.
FIG. 4 is a diagram showing the integration of pUC19 fragment incorporating human interferon α gene sequence into pIG121Hm.
FIG. 5 is a view showing a region containing human interferon α gene sequence in the constructed pIG-A11.
FIG. 6 is an amplification image by PCR showing introduction of a target gene in a transformed plant.
FIG. 7 shows a genomic Southern analysis showing introduction of a target gene in a transformed plant.
FIG. 8 is a graph showing the amount of human interferon α protein in transformed plant tissue.
FIG. 9 is a graph showing the antiviral activity of proteins in transformed plant tissues.
FIG. 10 shows the structure of pNPI132.

Claims (10)

Expressed in genomic DNA a base sequence encoding human interferon protein, in which the base sequence encoding the signal amino acid sequence that causes accumulation of prolamin in the protein body is linked upstream and the base sequence of the CaMV35S promoter is further upstream. A gramineous plant characterized by being incorporated.   The plant of claim 1 which is rice, wheat, barley, oats or corn. The plant of claim 1 which is rice.   The plant according to claim 1, wherein the human interferon is human interferon α or ω.   Callus of the plant according to any one of claims 1 to 4.   A plant somatic cell according to any one of claims 1 to 4. Recombinant plasmid DNA having a base sequence encoding human interferon protein, in which a base sequence encoding a signal amino acid sequence that causes accumulation of prolamin in a protein body is linked upstream and a base sequence of CaMV35S promoter is further linked upstream. And transforming Agrobacterium with the plasmid DNA, infecting the transformed Agrobacterium with a callus of the Gramineae plant, and selecting a callus expressing human interferon protein. A method for producing a Gramineae plant, characterized in that a nucleotide sequence encoding a human interferon protein is incorporated into a genomic DNA such that it can be expressed. The production method according to claim 7 , wherein the gramineous plant is rice, wheat, barley, oats or corn. The production method according to claim 7 , wherein the gramineous plant is rice . The production method according to any one of claims 7 to 9 , wherein the human interferon is human interferon α or ω.

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