JP5336428B2 - プレニルキノン生合成能の高い形質転換植物 - Google Patents
プレニルキノン生合成能の高い形質転換植物 Download PDFInfo
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- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- 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
- C12N15/8274—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 herbicide resistance
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
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Description
(1)植物中で機能的でありPDH酵素を過発現させる遺伝子、
(2)植物中で機能的でありHPPD酵素を過発現させる遺伝子
を含むことを特徴とする形質転換植物に関する。
(1)植物中で機能的でありPDH酵素を過発現させる遺伝子、
(2)植物中で機能的でありHPPD酵素を過発現させる遺伝子
を含み、植物中で機能的でありフィチル/プレニルトランスフェラーゼ酵素を過発現させる遺伝子は除くことを特徴とする形質転換植物に関する。
(1)植物中で機能的でありPDH酵素を過発現させる遺伝子、および
(2)植物中で機能的でありHPPD酵素を過発現させる遺伝子
で形質転換された植物からなることを特徴とする形質転換植物に関する。
(1)植物中で機能的でありPDH酵素を過発現させる遺伝子、
(2)植物中で機能的でありHPPD酵素を過発現させる遺伝子
で形質転換することを特徴とする方法にも関する。
(1)植物中で機能的でありPDH酵素を過発現させる遺伝子、
(2)植物中で機能的でありHPPD酵素を過発現させる遺伝子
で形質転換することを特徴とする方法にも関する。
Garciaらの文献(1997年、Biochem.J.第325巻、761〜769頁)またはGarciaらの文献(1999年、Plant Physiol.第119巻、1507〜1516頁)に記載の方法によりHPPD活性を測定することができる。
プレフェナート脱水素酵素活性は、50mMのTris−HCl、pH8.6、300μMのプレフェナートおよび1mMのNADまたはNADPを含み合計体積が200μlである溶液中でNADHまたはNADPHの形成を25℃で、340nmでの分光光度モニターにより測定する。
HPPD阻害性除草剤への抵抗性を植物に付与するためにHPPDを過発現させるキメラ遺伝子を構築した。
キメラ遺伝子過発現PDHの構築は、翻訳の方向において、欧州特許出願第0 507 698号に記載のような「二重ヒストン」プロモーター(PdH4)、CarringtonおよびFreedの文献(1990年;J.Virol.第64巻:1590〜1597頁)に記載のタバコエッチウイルス翻訳エンハンサー(TEV)配列、欧州特許出願第0 508 909号に記載のような最適化トランジットペプチド(OTP)をコードする配列、Mannhauptらの文献(1989年、Gene第85巻、303〜311頁)に記載の酵母PDH遺伝子のコード部分、およびBevanらの文献(1983年、Nucleic Acids Res.第11(2)巻、369〜385頁)に記載のノパリンシンターゼ遺伝子のnosターミネーターを組み立てることからなる。次に、アセンブリを、カナマイシン抵抗性遺伝子(NPTII)を含むバイナリーベクターpRD224にクローニングしてベクターpRD224−PDHを得る。このベクターは以下の構造を有する。
PBD6−ARA9タバコ植物は、実施例3に記載のようなキメラ遺伝子で形質転換したタバコ植物であり、特許出願WO96/38567に記載のA.thaliana HPPDを過発現している。PBD6−ARA9タバコ植物を得るための方法が、Garciaらの文献(1999年、Plant Physiol.第119巻、1507〜1516頁)に記載されている。実施例4に記載のようなPDHを過発現しているキメラ遺伝子で形質転換されたPBD6−ARA9系を、ARA9−PDH系と呼ぶ。
foliar disk技術(Horschら著、1985年、Science第227巻:1229〜1231頁)に従って、非発癌遺伝子であるAgrobacterium tumefaciens菌株EHA105−pRD224−PDHを用いて形質転換を行う。
30g/lのスクロースおよび350mg/lのセフォタキシムおよび200mg/mlのカナマイシンを含むMurashigeおよびSkoog(MS)基礎培地上で、外植葉からARA9−PDHタバコ植物を再生する。外植葉は、温室中の植物から得、foliar disk技術(Horschら著、1985年、Science第227巻:1229〜1231頁)に従って3つの連続工程で再生する:
・第1の工程は、30g/lのスクロースと0.05mg/lのナフチル酢酸(ANA)および2mg/lのベンジルアミノプリン(BAP)も含むMS基礎培地上で15日間、そして200mg/mlのカナマイシンで若枝を誘導することを含む。
6.1.ジケトニトリル(DKN)への耐性
13個のARA9−PDH系および、形質転換用の出発材料として用いたPBD6−ARA9系を、DKNの濃度が5、10および32ppmと増加するように蒔いた。
HPPD阻害剤スルコトリオンおよびメソトリオンを用いて同じ実験を行った。ARA9−PDH18系は、3μMのメソトリオンおよび6μMのスルコトリオンに耐性であるが、Petit Havana型の野生型タバコ系は、0.375μMのこれら二つの化合物に感受性であることが分かった。
分析した植物の各々の中間葉および幼若葉のサンプルから、Folchの方法(Folchら著、1957頁、J.Biol.Chem.、226〜497頁)により脂質抽出物を得る。次に、それらのトコフェロールおよびトコトリエノール含量の分析を、Frazerらの方法(2000年、Plant J.第24巻:551〜558頁)に従ったHPLCにより行う。次に、これらの含量を、対照産物に関して定量し、次に、固形分1g当たりのμgで表す。結果を表1に示す。
Claims (5)
- 形質転換植物を栽培する方法であって、前記植物の栽培に適した耕地の領域に前記形質転換植物の種子を播くことと、前記形質転換種子または前記形質転換植物に実質的に影響を与えることなく前記耕地の前記領域にHPPD阻害剤を含む少なくとも一種の除草剤組成物を適用することと、次に、所望の成熟度に達したときに栽培した植物を採取することと、任意に、採取した植物から種子を分離することとを具備することを特徴とする方法であって、
前記形質転換植物は、
(1)植物中で機能的でありPDH酵素を過発現させる遺伝子、
(2)植物中で機能的でありHPPD酵素を過発現させる遺伝子
を含むことを特徴とする形質転換植物である、前記方法。 - 植物中で機能的でありPDHを過発現させる遺伝子が、酵母PDHをコードする遺伝子のコード配列を含むことを特徴とする、請求項1に記載の方法。
- 酵母PDHをコードする遺伝子のコード配列が、サッカロミセル・セレビシエ(Saccharomyces cereviseae)遺伝子のコード配列であることを特徴とする、請求項2に記載の方法。
- 植物中で機能的でありHPPDを過発現させる遺伝子が、植物HPPDをコードする遺伝子のコード配列を含むことを特徴とする、請求項1から3のいずれか一項に記載の方法。
- 植物HPPDをコードする遺伝子のコード配列が、アラビドプシス・タリアナ(Arabidopsis thaliana)遺伝子のコード配列であることを特徴とする、請求項4に記載の方法。
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FR0211209 | 2002-09-11 | ||
FR0211209A FR2844142B1 (fr) | 2002-09-11 | 2002-09-11 | Plantes transformees a biosynthese de prenylquinones amelioree |
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JP5336428B2 true JP5336428B2 (ja) | 2013-11-06 |
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JP2010126784A Expired - Fee Related JP5336428B2 (ja) | 2002-09-11 | 2010-06-02 | プレニルキノン生合成能の高い形質転換植物 |
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US (1) | US10138490B2 (ja) |
EP (1) | EP1537216B1 (ja) |
JP (2) | JP4683923B2 (ja) |
KR (1) | KR20050046764A (ja) |
CN (1) | CN100335641C (ja) |
AT (1) | ATE532871T1 (ja) |
AU (1) | AU2003278294B2 (ja) |
BR (2) | BRPI0306432B1 (ja) |
ES (1) | ES2375718T3 (ja) |
FR (1) | FR2844142B1 (ja) |
NZ (1) | NZ538753A (ja) |
WO (1) | WO2004024928A2 (ja) |
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- 2003-09-10 EP EP03769603A patent/EP1537216B1/fr not_active Expired - Lifetime
- 2003-09-10 WO PCT/FR2003/002684 patent/WO2004024928A2/fr active Application Filing
- 2003-09-10 BR BRPI0306432-8A patent/BRPI0306432B1/pt unknown
- 2003-09-10 CN CNB038243261A patent/CN100335641C/zh not_active Expired - Fee Related
- 2003-09-10 KR KR1020057004192A patent/KR20050046764A/ko active IP Right Grant
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Also Published As
Publication number | Publication date |
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CN100335641C (zh) | 2007-09-05 |
NZ538753A (en) | 2007-02-23 |
FR2844142B1 (fr) | 2007-08-17 |
CN1688700A (zh) | 2005-10-26 |
JP2010246552A (ja) | 2010-11-04 |
FR2844142A1 (fr) | 2004-03-12 |
BR0306432A (pt) | 2004-10-26 |
ES2375718T3 (es) | 2012-03-05 |
KR20050046764A (ko) | 2005-05-18 |
US10138490B2 (en) | 2018-11-27 |
EP1537216A2 (fr) | 2005-06-08 |
ATE532871T1 (de) | 2011-11-15 |
AU2003278294A1 (en) | 2004-04-30 |
AU2003278294B2 (en) | 2010-05-27 |
WO2004024928A2 (fr) | 2004-03-25 |
BRPI0306432B1 (pt) | 2019-04-02 |
US20050257283A1 (en) | 2005-11-17 |
WO2004024928A3 (fr) | 2004-04-22 |
JP4683923B2 (ja) | 2011-05-18 |
JP2005537808A (ja) | 2005-12-15 |
EP1537216B1 (fr) | 2011-11-09 |
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