JP5466224B2 - 病原体を制御するためのアミノ酸及び核酸配列 - Google Patents
病原体を制御するためのアミノ酸及び核酸配列 Download PDFInfo
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
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Description
植物材料の調製、ニコチアナ・メガロシホンからの、抗病原体タンパク質NmDef−02をコードしているDNAの単離及びクローニング
N.メガロシホン(N.megalosiphon)種を、ブラッククラウド(black crowd)とコメの殻を4:1の比率で含有する6インチのポットで生長させ、23℃の温室条件下で維持した。ハバナのタバコ畑で採取したペロノスポラ・ヒオシアミ 品種タバシナの単離体を接種に用いた。接種は、6週齢のこの種の植物に、1ml当たり胞子5×103個の濃度の液滴10μlをいくつか置いて行った。植物を湿度が高い黒いプラスチックの袋の中に12時間置き、感染を促進した(湿度は、袋の内部に水を噴霧して実現した)。
抗病原体タンパク質NmDef−02の遺伝子を用いた植物の形質転換
タバコトランスジェニック植物の作出:バイナリーベクターの構築
本試験において、抗病原体タンパク質をコードしている遺伝子の完全なcDNAを、配列番号2及び配列番号3のオリゴヌクレオチドを用いて単離し、HindIII/PstI制限部位において形質転換ベクター「pCambia 2300」にクローニングした(図1A)。タバコ植物の遺伝子形質転換を、Zambryski et al.(1983)EMBO Journal,2:2143−2150から得た方法によって行った。この計画のために、アグロバクテリウム・ツメファシエンス(Agrobacterium tumefaciens)のAT2260株を、開発したバイナリーベクターと液体窒素法(Hofgen and Willmitzer(1988)Nucl.Acids Res.16:9877)に使用した。in vitroで培養した変種プチ・ハバナ(Petit Havana)SR1のタバコ(N.tabacum)植物の葉の円盤状切片を形質転換した。マーカー剤として100mg/Lのカナマイシンを用いた。葉の円盤状切片を、ムラシゲ・スクーグ(MS)液体培地中で48時間、組換えアグロバクテリウムと共培養した。タバコ植物の再分化(4〜6週間)を、ショ糖25g/L、6−ベンジルアミノプリン(BAP)1mg/L、ナフタリン酢酸(ANA)0.1mg/L、カナマイシン100mg/L及びクラフォラン(Claf)500mg/Lを含有するMS培地において行った。植物の発根(1〜3週間)を、ショ糖30g/L、カナマイシン100mg/L及びClaf500mg/Lを含有するMSにおいて行った。
本試験において、抗病原体タンパク質をコードしている遺伝子の完全なcDNAを、配列番号2及び配列番号3のオリゴヌクレオチドを用いて単離し、HindIII/PstI制限部位において形質転換ベクター「pCambia 3300」にクローニングした(図1A)。組織培養及び形質転換の試験に使用した植物材料は、ジャガイモ栽培品種「Desiree」由来の植物からin vitroで取得した。植物を試験管内でMS培地において生長させた。培地のpHは5.7に調整した。4週間培養した植物を用い、25℃、照明が2000ルクスの部屋で維持した。再分化試験及び形質転換のベースとして使用した培地はSC(MS塩、ビタミンB1 0.4mg/L、ミオイノシトール100mg/L、ショ糖20g/L、BAP3.5mg/L、ANA0.01mg/L、植物寒天(phytoagar)6g/L)、SB(MS塩、ミオイノシトール100mg/L、ショ糖20g/L、AG33.5mg/L、植物寒天6g/L)及びPP(MS塩、ビタミンB1 0.4mg/L、ミオイノシトール100mg/L、パントテン酸カルシウム2mg/L、ショ糖30g/L、植物寒天6g/L、活性炭5g/L及び硝酸銀−チオ硫酸ナトリウム1mg/L(STS))であった。
疾患抵抗性に対する抗病原体タンパク質NmDef−02の効果の評価
ペロノスポラ・ヒオシアミ 品種タバシナに対するタバコの疾患抵抗性試験
抗生物質カナマイシンに対する抵抗性を有し、抗病原体タンパク質の遺伝子を有する根付きの植物を、45日間、温室条件下で適応させるためにポットに植えた。45日後、6週齢のトランスジェニッククローン100体からなる群に、ペロノスポラ・ヒオシアミ 品種タバシナの懸濁液を、胞子5×103個/mlの濃度の液滴10μlをいくつか置いて接種した。その植物を12時間、黒いプラスチックの袋の中に置いて水を噴霧し、感染を促進した。1週間後に疾患の症状がある葉の割合を測定して感受性の評価を行った(図3A)。図3Aから分かるように、接種した対照と種々のクローンの間で症状がある葉の割合を比較した場合、この病原体に対する高い抵抗性レベルが実現し、これは、この重要な病原体を制御するために用いた抗病原体タンパク質の有用性を示している。
抗生物質カナマイシンに対する抵抗性を有し、抗病原体タンパク質の遺伝子を有する根付きの植物を、45日間、温室条件下で適応させるためにポットに植えた。45日後、6週齢のトランスジェニッククローン100体からなる群に、P.パラシティカ(P.parasitica)を接種し、1カ月後に疾患の症状がある茎の割合を評価した。
この試験は、5週齢のジャガイモトランスジェニック植物に、明るさ、温度及び相対湿度の制御条件下でジャガイモ疫病菌を接種することにある。抗病原体タンパク質を発現している5週齢のクローン約100体を、遊走子106個/mlの懸濁液で拡散させた。このクローンを、相対湿度85〜95%及び温度23℃の制御条件下で維持した。接種した1週間後の、症状がある葉の割合を、この病原体に対する感受性の尺度として用いた(図3C)。この病原体は制御するのが極めて困難であるので、これは予想外の結果であった。図3Cにおいて、抗病原体タンパク質を発現しているクローンは、対照と比較して高い抵抗性レベルを示した。この結果は、この病原体の制御においてこのタンパク質を用いることの可能性、とりわけ世界的なレベルでの重要性について指摘している。
真菌アルテルナリア・ソラニを、明るさ、温度及び相対湿度の制御条件下で、5週齢のジャガイモトランスジェニック植物100体に接種した。クローンに、胞子106個/mlの懸濁液を拡散させた。このクローンを、相対湿度85〜95%及び温度20℃の制御条件下で維持した。接種した1週間後の、症状がある葉の割合を、この病原体に対する感受性の尺度として用いた(図3D)。3種の分析クローンがこの病原体に対する高い抵抗性レベルを示し、制御するためにこのタンパク質を用いることの可能性が初めて提供される。
ピキア・パストリス培養物の上清における、細胞外での抗病原体タンパク質NmDef−02の発現ベクターの構築
N.メガロシホン(N.megalosiphon)由来の抗病原体タンパク質をコードしている遺伝子を、配列番号4及び配列番号5に対応する特異的オリゴヌクレオチドを用いて単離し、発現ベクターpPIC9kのクローニングに必要な制限酵素部位XhoI/EcoRIを有する、抗病原体タンパク質NmDef−02をコードしている遺伝子の完全な配列を得た。このクローニング戦略により、得られるタンパク質が配列番号7に属するよう、対象のタンパク質のアミノ末端にサッカロマイセス・セレビシエのアルファ因子のシグナルペプチドが付加される(図1B)。P.パストリスのGS115株を形質転換する前にプラスミドをBgIIIで直鎖化した。電気穿孔法によって形質転換を行った。GS115株は、形質転換後にHis+表現型を獲得するhis3栄養要求性変異株である。His+クローンをグルコース最小培地で選択し、そのクローンをグリセロール最小培地で培養し、28℃で126時間、メタノールを用いて誘導した。
抗病原体タンパク質の精製及びその生物活性のアッセイ
アルファ因子のシグナルペプチドに融合させた抗病原体タンパク質(NmDef−Plus)を、25mMの酢酸ナトリウム、pH4.5で透析し;透析の産物を、25mMの酢酸ナトリウム、pH4.5で平衡化した陽イオン交換樹脂CM−Sepharosaの高速流を通して使い;タンパク質を1Mの塩化ナトリウム、50mMのトリス、pH7.6を用いて溶出することによって、培地の上清から精製した。タンパク質含有画分を回収し、孔サイズ(カットオフ)が3kDaの膜を用いた超遠心システムを使用して濃縮した。254nmの波長を検出に用いた。SDS−PAGE、ドデシル硫酸ナトリウム−ポリアクリルアミドゲル電気泳動(15%ris−Glicne)を用いたポリアクリルアミドにおける電気泳動によって精製の確認を行い、銀染色法によってタンパク質を可視化した(図2)。
土壌中及び空気中の真菌病原体に対する、抗病原体タンパク質NmDef−02製剤による制御の実証
タバコ種の種子を、抗病原体タンパク質製剤(9μg/ml)及び5%アルギン酸ナトリウムで処理し、その後、その種子を、ブラッククラウド及びコメを4:1の比率で含有する6インチのポット内で発芽させ、23℃の温室で維持した。対照として、この処理に、10%次亜塩素酸ナトリウムで処理した種子及び何の処理もしていないタイプの種子を用いた。30日目に評価を行い、自然感染によって枯れた植物の割合を測定した(図5A)。
抗病原体タンパク質NmDef−02断片の生物活性
抗病原体タンパク質NmDef−02の断片を化学合成によって得た(配列番号8及び配列番号9)。病原体ジャガイモ疫病菌に対する効果を、報告されている情報(Terras et al.(1992)J.Biol.Chem.267:14301−15309)に従って、種々の濃度に関して評価した。それを図6に示す。得られた結果から、配列番号9のタンパク質画分を用いると、病原体の制御又は阻害を実現するためにタンパク質画分を使用することが可能になる。
Claims (15)
- 配列番号1として特定される核酸配列を含む核酸。
- a)配列番号6として特定されるアミノ酸配列、又はb)配列番号6として特定されるアミノ酸に対して1個又は数個のアミノ酸残基が除去、置換及び付加されており、病原物質による感染を制御する特性を維持しているアミノ酸配列を含むポリペプチドをコードしている核酸。
- 配列番号6を含む抗病原体タンパク質の構成的発現又は誘導された発現を導く、配列番号1として特定される核酸配列を含む核酸配列を用いた植物の遺伝子形質転換により、病原物質によって生じる植物疾患に対する抵抗性を増大させる方法。
- 配列番号6又は配列番号7として特定されるアミノ酸配列を含むポリペプチド。
- 組換えDNA技術又は化学合成によって得られる、請求項4に記載のポリペプチド。
- 組換えDNA技術による発現を、ピキア・パストリス(Pichia pastoris)において行う、請求項5に記載のポリペプチド。
- ピキア・パストリス(Pichia pastoris)の培養上清から得られる、請求項6に記載のポリペプチド。
- 配列番号6、配列番号7又は配列番号9として特定されるポリペプチドを含む、病原物質を制御するためのバイオプロダクト。
- ポリペプチドが遺伝子形質転換された宿主から精製され、又はそのような宿主の培養物の上清中に含有されたポリペプチドが直接使用される、請求項8に記載のバイオプロダクト。
- 宿主がP.パストリス(P.pastoris)である、請求項9に記載のバイオプロダクト。
- 病原物質が真菌病原体である、請求項8に記載のバイオプロダクト。
- ポリペプチドが1〜9μg/mlの濃度範囲にある、請求項8に記載のバイオプロダクト。
- 請求項8に記載のバイオプロダクトを植物に適用することを特徴とする、植物病原物質を制御する方法。
- 請求項8に記載のバイオプロダクトをバイオ農薬と混合して適用することを特徴とする、植物病原物質を制御する方法。
- 病原物質によって生じる植物疾患に対する抵抗性を増大させるために、配列番号1として特定される核酸配列を用いて遺伝子改変された植物。
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