JP4209949B2 - Fatty acid desaturase and its mutant sequence - Google Patents

Description

アミノ酸モチーフ及びそれらの対応する位置の保存（conservation）は、１つの種の中で突然変異し、非機能性デサチュラーゼを生成するδ−12又はδ−15脂肪酸デサチュラーゼの領域が、他の種由来の対応する領域中で突然変異し、その種内での非機能性δ−12デサチュラーゼ又はδ−15デサチュラーゼ遺伝子産物を生成し得ることを示す。
表１〜６の領域のいずれかにおける突然変異は、本発明の範囲内に特定的に含まれ、本明細書中に例示されるそれらの突然変異と実質的に同一であるが、但し、上記で考察されたように、その突然変異（又はそれらの突然変異）は、得られるデサチュラーゼ遺伝子産物を非機能性にする。
突然変異配列を含む核酸断片は、当業者に公知の技術によって生産され得る。そのような技術は、これらに限定されないが、野生型配列の部位特異的突然変異誘発及び自動DNA合成装置を用いる直接合成を含む。
突然変異配列を含む核酸断片は、例えば、エチルメタンスルホネート、Ｘ線又は他の突然変異原による、植物種子又は再生可能な植物組織の突然変異誘発によっても生産され得る。突然変異誘発によって、種子中に望ましい脂肪酸表現型を有する突然変異植物は、公知技術によって同定され、望ましい突然変異を含む核酸断片は、突然変異系列のゲノムDNA又はRNAから単離される。そして、特異的な突然変異の部位は、δ−12デサチュラーゼ又はδ−15デサチュラーゼ遺伝子のコード領域の配列決定によって決定される。あるいは、所望の突然変異イベントに特異的な、標識された核酸プローブを用いて、突然変異誘発された集団を迅速にスクリーニングすることができる。
開示された方法は、全ての脂肪種子のアブラナ属種に、並びに各種内の春成熟種及び冬成熟種の両者に適用され得る。これらに限定されないが、Ｘ線、紫外線及び染色体損傷を引き起こす他の物理的処理を含む、物理的突然変異原、及びこれらに限定されないが、臭化エチジウム、ニトロソグアニジン、ジエポキシブタン等を含む他の化学突然変異原を、突然変異の誘導に使用してもよい。突然変異誘発処理は、これらに限定されないが、細胞培養物、胚（embryos）、小胞子（microspores）及び茎頂（shoot apices）を含む、植物の他の成長段階に適用してもよい。
本明細書中で用いられる「安定突然変異（stable mutations）」は、ひとまとめにされた（bulked together）最少10個の無作為に選択された種子のガスクロマトグラフィー分析によって決定される、畑地条件（field conditions）下で、最少２世代を含み、最少２世代の確立された統計的閾値を超える、最少３世代の選択され、改変された脂肪酸プロフィールを維持する、Ｍ₅以上に進んだ系列として定義される。あるいは、安定性を、続く世代との比較によって同様に測定してもよい。続く世代において、安定性は、実質的に類似の条件下に生育した前の又は続く世代のものと類似の脂肪酸プロフィールを種子中に有するものとして定義される。
突然変異育種は、対象の特徴に加えて、複合的な、有害な特徴、例えば低下した植物生長力及び低下した繁殖力を有する植物を伝統的に製造してきた。そのような特徴は、脂肪酸組成に間接的に影響して不安定な突然変異を製造し；及び／又は収穫量を低下させ、それによって本発明の商業的有用性を低下させるかもしれない。有害な突然変異の発生を無くし、植物が有する突然変異の負荷を減らすためには、種子処理において、突然変異原の低用量を用い、LD30集団を生成させる。これは、許容可能な収穫量を製造する作物学のバックグラウンドにおける脂肪酸の特徴による単一遺伝子突然変異を迅速に選択することを可能とする。
幾つかの異なる植物系列の種子は、American Type Culture Collectionに寄託され、以下の受託番号を有している。
系列 受託番号 寄託日
A129.5 40811 1990年５月25日
A133.1 40812 1990年５月25日
M3032.1 75021 1991年６月７日
M3062.8 75025 1991年６月７日
M3028.10 75026 1991年６月７日
IMC130 75446 1993年４月16日
Q4275 97569 1996年５月10日
幾つかの植物種又は品種において、内因性のミクロソームδ−12デサチュラーゼの１つ以上の形態が見出され得る。複２倍体においては、各形態は、検討中の（under consideration）種を形成する親ゲノムの１つに由来していてもよい。両方の形態に突然変異を有する植物は、１つの形態のみに突然変異を有する植物とは異なる脂肪酸プロフィールを有する。そのような植物の例は、δ−12デサチュラーゼの突然変異Ｄ形態（配列番号11）及びδ−12デサチュラーゼの突然変異Ｆ形態（配列番号15）を含む、二重に突然変異誘発された系列であるブラッシカ・ナプス（Brassica napus）系列Q508である。もう１つの例は、系列Q4275であり、そしてそれは、δ−12デサチュラーゼの突然変異Ｄ形態（配列番号11）及びδ：−12デサチュラーゼの突然変異Ｆ形態（配列番号17）を含む。図２〜３参照。
本発明の核酸断片の導入のための好ましい宿主又は受容生物は、ダイズ（Glycine max）、菜種（例えば、ブラッシカ・ナプス（Brassica napus）、ブラッシカ・ラパ（B. rapa）及びブラッシカ・ジュンシー（B. juncea））、ヒマワリ（Helianthus annus）、トウゴマの種子（castor bean）（トウゴマ（Ricinuscommunis））、トウモロコシ（Zea mays）、及びベニバナ（ベニバナ（Carthamustinctorius））などの油製造（oil-producing）種である。
本発明の核酸断片は、さらに追加の核酸を含んでいてもよい。例えば、分泌又はリーダーアミノ酸配列をコードする核酸は、該分泌又はリーダー配列が突然変異δ−12又はδ−15デサチュラーゼポリペプチドのアミノ末端にフレーム内融合（fused in-frame）されるように突然変異デサチュラーゼ核酸断片に連結することができる。本明細書中に開示される突然変異デサチュラーゼポリペプチドにフレーム内融合するのに有用なアミノ酸配列をコードする、他の核酸断片は当業界で知られている。例えば、引用によって本明細書中に組み込まれる米国第5,629,193号参照。核酸断片は、また、それに機能可能なように連結された１つ以上の調節エレメントを有していてもよい。
本発明は、また、突然変異デサチュラーゼ配列に選択的にハイブリダイズする核酸断片を含む。そのような核酸断片は、典型的に少なくとも15個のヌクレオチドの長さである。ハイブリダイゼーションは、典型的に、サザン分析（サザンブロット）、すなわち標識オリゴヌクレオチド又はDNA断片プローブへのハイブリダイゼーションによって、標的核酸混合物中のDNA配列の存在を同定する方法を含む。サザン分析は、典型的に、アガロースゲル上でのDNA消化物の電気泳動分離、電気泳動分離後のDNAの変性、及びニトロセルロース、ナイロン又はSambrookら、（1989）Molecular Cloning、第２版、Cold Spring Harbor Laboratory,Plainview;NYの第9.37-9.52節に記載されている、放射標識された、ビオチン化された、又は酵素標識されたプローブによる分析のためのもう１つの好適な膜支持体へのDNAの移送を含む。
核酸断片は、中程度ストリンジェント条件下又は、好ましくは、高ストリンジェント条件下で、突然変異デサチュラーゼ配列にハイブリダイズできる。高ストリンジェント条件は、プローブと高い相同性を有する核酸を同定するのに使用される。高ストリンジェント条件は、洗浄のための低いイオン強度及び高い温度、例えば、0.015M塩化ナトリウム／0.0015Mクエン酸ナトリウム（0.1×SSC）；50-65℃で、0.1％ラウリル硫酸ナトリウム（SDS）を使用することを含み得る。あるいは、ハイブリダイゼーションの間、ホルムアミドなどの変性試薬、例えば、0.1％ウシ血清アルブミン／0.1％Ficoll／0.1％ポリビニルピロリドン／50mMリン酸ナトリウムバッファー、750mM NaClによるpH6.5、75mMクエン酸ナトリウムと共に、42℃で50％ホルムアミドを用いることができる。もう１つの例は、50％ホルムアミド、5×SSC（0.75M NaCl、0.075Mクエン酸ナトリウム）、50mMリン酸ナトリウム（pH6.8）、0.1％ピロリン酸ナトリウム、5×Denhardtの溶液、超音波分解されたサケの精子DNA（50μg/ml）、0.1％SDS、及び10％硫酸デキストランの42℃での使用（42℃で0.2×SSC及び0.1％SDSで洗浄）である。
中程度ストリンジェント条件は、高ストリンジェント条件下で同定するよりも、プローブへの低い同一性の程度を有する核酸を同定するために使用されるハイブリダイゼーション条件をいう。中程度ストリンジェント条件は、高ストリンジェントハイブリダイゼーションに用いられるイオン強度及び温度に比べて、ハイブリダイゼーション膜の洗浄のための、より高いイオン強度及び／又はより低い温度を使用することを含むことができる。例えば、0.060M塩化ナトリウム／0.0060Mクエン酸ナトリウム（4×SSC）及び0.1％ラウリル硫酸ナトリウム（SDS）を含む洗浄溶液−を50℃で、最後の洗浄では1×SSC、65℃で使用することができる。あるいは、1×SSC、37℃でのハイブリダイゼーション洗浄を使用できる。
ハイブリダイゼーションは、また、ノーザン分析（ノーザンブロット）、すなわちオリゴヌクレオチド、DNA断片、cDNA若しくはその断片、又はRNA断片などの既知のプローブにハイブリダイズするRNA類を同定するのに用いられる方法によって行うこともできる。プローブは、³²Pなどの放射性同位体を用いて、ビオチン化によって、又は酵素によって標識される。分析されるRNAは、通常、アガロース又はポリアクリルアミドゲル上で電気泳動によって分離され、ニトロセルロース、ナイロン、又は他の好適な膜に移され、そして上記Sambrookらの第7.39-7.52節に記載されているような、当業界で周知の標準技術を用いて、プローブとハイブリダイズされ得る。
本発明のポリペプチドは、突然変異アミノ酸配列を有する単離されたポリペプチドだけでなくその誘導体及び類似体を含む。例えば、図３の突然変異アミノ酸配列参照。「単離された」によって意味されるのは、ポリペプチドが天然に発現され製造される環境以外の環境で発現され製造される該ポリペプチドである。例えば、植物ポリペプチドは、細菌類又は真菌類中で発現され製造されたときに単離される。本発明のポリペプチドは、上記で考察したように、本明細書中に開示される突然変異デサチュラーゼポリペプチドの品種をも含む。
請求の範囲に記載した発明の１つの実施態様においては、植物は、δ−12デサチュラーゼ突然変異及びδ−15デサチュラーゼ突然変異の両者を含む。そのような植物は、非常に高いオレイン酸濃度及び非常に低いα−リノレン酸濃度を含む脂肪酸組成を有することができる。δ−12デサチュラーゼ及びδ−15デサチュラーゼ中の突然変異は、δ−12デサチュラーゼ及びδ−15デサチュラーゼの単一突然変異系列の間で遺伝的交雑を為すことによって、植物の中で組み合わされてもよい。δ−12脂肪酸デサチュラーゼ中に突然変異を有する植物は、δ−15脂肪酸デサチュラーゼ中に突然変異を有する第２の植物と交雑され、交配される。交雑によって製造された種子を植え、得られる植物は、子孫種子を得るために自家（受粉）される。そして、これらの子孫種子は、両方の突然変異遺伝子を担う種子を同定するためにスクリーニングされる。
あるいは、δ−12デサチュラーゼ突然変異又はδ−15デサチュラーゼ突然変異のいずれかを有する系列を突然変異誘発に付し、δ−12デサチュラーゼ及びδ−15デサチュラーゼの両者に突然変異を有する植物又は植物系列を生産することができる。例えば、IMC 129系列は、ミクロソームδ−12デサチュラーゼ構造遺伝子のＤ形態のコード領域中の突然変異（Glu₁₀₆からLys₁₀₆への）を有する。この系列の細胞（例えば、種子）は、突然変異誘発され、δ−15デサチュラーゼ遺伝子中の突然変異を誘導し、δ−12脂肪酸デサチュラーゼ遺伝子中の突然変異及びδ−15脂肪酸デサチュラーゼ遺伝子中の突然変異を有する植物又は植物系列をもたらすことができる。
子孫は、特定の植物又は植物系列の子孫を含み、例えば、その（instant）植物上に発育した種子は子孫である。その植物の子孫は、F₁植物、F₂植物、F₃植物、及び続く世代の植物上に形成された種子、又はBC₁植物、BC₂植物、BC₃植物及び続く世代の植物上に形成された種子を含む。
本発明による植物は、好ましくは、改変された脂肪酸組成を含む。例えば、そのような植物の種子から得られた油は、その種子の全脂肪酸組成に基づいて、約69〜約90％のオレイン酸を有する。そのような油は、好ましくは、約74％〜約90％のオレイン酸、より好ましくは、約80〜約90％のオレイン酸を有する。幾つかの実施態様においては、本発明の植物によって生産された種子から得られた油は、その種子の全脂肪酸組成に基づいて、約2.0％〜約5.0％の飽和脂肪酸を有していてもよい。幾つかの実施態様においては、本発明の種子から得られた油は、約1.0％〜約14.0％のリノール酸、又は約0.5％〜約10.0％のα−リノレン酸を有していてもよい。
油組成は、一般に、ひとかたまりの（バルク）種子サンプル（例えば、10個の種子）を圧搾し、それから脂肪酸を抽出することによって分析される。種子中の脂肪酸トリグリセリドは、加水分解され、脂肪酸メチルエステルに変換される。改変された脂肪酸組成を有するそれらの種子は、当業者に公知の技術、例えば、バルク種子サンプルの、又は１つの半分の種子（single half-seed）のガス液体クロマトグラフィー（GLC）分析によって同定されてもよい。胚の生存能が維持されており、それ故所望の脂肪酸プロフィールを有するそれらの種子が植えられて、次の世代を形成し得るので、半種子分析が有用であることは当業界において周知である。しかしながら、半種子分析は、また、分析される種子の遺伝子型の不正確な表現であることも知られている。バルク種子分析は、一般に、与えられた遺伝子型の脂肪酸プロフィールのより正確な表現をもたらす。脂肪酸組成は、また、より大量のサンプル、例えば、試験的施設（plant）又は種子中の内因性の油の商業的規模の精製、漂白及び脱臭によって得られる油に関しても決定され得る。
本発明の核酸断片は、植物遺伝子地図及び植物育種プログラムにおけるマーカーとして使用できる。そのようなマーカーは、例えば制限断片長多型（RFLP）、ランダム増幅多型検出（RAPD）、ポリメラーゼ連鎖反応（PCR）又は自己持続配列複製（3SR）マーカーを含んでいてもよい。マーカー補助による育種技術は、育種過程の間の所望の脂肪酸組成を同定し、追跡するために使用してもよい。マーカー補助による育種技術は、他の種類の同定技術に追加して、又はそれとは異なるものとして使用してもよい。マーカー補助育種の例は、δ−12デサチュラーゼ又はδ−15デサチュラーゼ中の所望の突然変異を含む配列を特異的に増幅するPCRプライマーの使用である。
本発明による方法は、得られる植物及び植物系列が望ましい種子脂肪酸組成だけでなく、改変された種子脂肪酸組成を有する公知の系列と比べてより優れた作物学的特性を有していることにおいて有用である。より優れた作物学的特徴は、例えば、増加した種子発芽パーセンテージ、増加した苗木生長力、苗菌病（seedling fungal diseases）（立枯病、根腐れなど）に対する増加した耐性、増加した収穫量、及び改善された安定性を含む。
本発明は、種々の修飾及び代替形態が可能であるが、そのある特異的な実施態様が、一般的な方法及び以下に述べる実施例において記載されている。例えば、発明は、ブラッシカ・ラパ（B.rapa）、ブラッシカ・ジュンシー（B.juncea）、及びブラッシカ・ヒルタ（B.hirta）を含む全てのアブラナ属（Brassica）種に適用され、実質的に類似の結果を与え得る。しかしながら、これらの実施例は、本発明を、開示された特定の形態に限定することを意図してはおらず、それどころか、本発明は全ての修飾、同等物及び本発明の範囲内に入る改変をカバーすると理解すべきである。これは、ソマクローナル変異；植物部分の物理的又は化学的突然変異誘発；葯、小胞子又は子房培養に続く染色体倍化；又は脂肪酸特性を伝達するための、単独又は他の特性との組み合わせで、新たなアブラナ属系列を発達させるための自家又は交雑受粉の使用を含む。
実施例１
突然変異誘発
カナディアン（ブラッシカ・ナプス）スプリング・キャノーラ（spring canola）品種である、ウェスター（Westar）の種子を、化学突然変異誘発に付した。ウェスターは、キャノーラ品質を有する、登録されたカナディアン・スプリング品種である。畑地生育ウェスターの脂肪酸組成、3.9％Ｃ_16:0、1.9％Ｃ_18:0、67.5％Ｃ_18:1、17.6％Ｃ_18:2、7.4％Ｃ_18:3、<2％Ｃ_20:1＋Ｃ_22:1は、<±10％偏差をもって、1982年から、商業的製造下で安定である。
突然変異誘発前に、ブラッシカ・ナプス シーブイ ウェスター（B.napus cv.Westar）種子30,000個を、300個の種子のロットで、湿った濾紙の上で２時間予め吸湿させ、種皮を柔らかくした。予め吸湿させた種子を、80mMエチルメタンスルホネート（EMS）中に４時間置いた。突然変異誘発に続いて、種子を蒸留水で３回すすいだ。その種子を、Pro-Mixを含む48ウエル平板（48-well flats）中に蒔いた。突然変異誘発された種子の68％が発芽した。その植物を、25℃／15℃、14／10時間・昼／夜の条件で、温室中で維持した。開花期に、各植物を個々に自家受粉させた。
個々の植物由来のＭ₂種子を、個別に分類して保存し、約15,000個のＭ₂系列を、マニトバ州、カーマン（Carman, Manitoba）で、夏の苗床に植えた。各自家植物由来の種子を、６インチの列間隔で、３メートルの列に植えた。ウェスターを、照合基準品種（check variety）として植えた。畑地で選択された系列を、各植物の主総状花序に袋がけすることによって自家させた。成熟期に、自家された植物を、個々に刈り取り、種子を分類して保存し、種子の起源を知る裏付けとした。
自家受粉されたＭ₃種子及びウェスター対照を、10個の種子バルクサンプルで、ガスクロマトグラフィーによる脂肪酸組成を分析した。各脂肪酸成分の統計的閾値を、10,000分の１のストリンジェントレベルでのＺ分布を用いて確定した。平均及び標準偏差値を、畑地の突然変異誘発されていないウェスター対照から決定した。各脂肪酸の上位及び下位の統計的閾値を、Ｚ分布によって複合された、集団の平均値±標準偏差から決定した。10,000個の集団規模に基づく信頼区間は、99.99％である。
選択されたＭ₃種子を、、温室中に、ウェスター対照に沿って植えた。種子を、Pro-Mix土を含む４インチのポットに蒔き、その植物を、温室中で25℃／15℃、14／10時間・昼／夜のサイクルに維持した。開花期に、末端の総状花序を、袋がけによって自家受粉させた。成熟期に、自家Ｍ₄種子を、各植物から個々に刈り取り、標識し、保存して、種子の起源を知る裏付けとした。
Ｍ₄種子を、10個の種子バルクサンプルで分析した。各脂肪酸成分の統計的閾値を、800分の１のＺ分布を用いて259個の対照サンプルから確定した。選択されたＭ₄系列を、６インチの間隔で３メートルの列で、マニトバ州カーマンでの畑地試験場に植えた。各列の10個のＭ₄植物を、自家受粉のために袋がけした。成熟期に、自家植物を、個々に刈り取り、その列の解放受粉された（open pollinated）植物をバルクで刈り取った。単一植物選択由来のＭ₅種子を、10個の種子バルクサンプルで分析し、バルク列刈り取りのものは、50個の種子バルクサンプルで分析した。
選択されたＭ₅系列を、温室中でウェスター対照に沿って植えた。種子を、前記のとおりに育成した。開花期に、末端の総状花序を袋がけによって自家受粉させた。成熟期に、自家Ｍ₆種子を各植物から個別に刈り取り、10個の種子のバルクサンプルで、脂肪酸組成を分析した。
選択されたＭ₆系列を、東アイダホ（Eastern Idaho）の畑地試験に参加させた（entered）。育成条件の変化性の広い４つの試験場所を選択した。場所は、バーレイ（Burley）、テトニア（Tetonia）、ラモント（Lamont）及びシェリー（Shelley）を含む（表７）。その系列を、８インチ間隔で、４本の３メートルの列に植え、各小区画（plot）を、４回複製した（replicated）。播種デザインを、無作為完全ブロックデザイン（Randomized Complete Block Designed）を用いて決定した。商業的栽培品種（cultivar）のウェスターを照合基準栽培品種として用いた。成熟期に、その小区画を刈り取り、収穫量を決定した。試験に参加したものの収穫量を、４回の複製の統計的平均を採ることによって決定した。最少有意差試験（The Least Significant Difference Test）を用いて、無作為完全ブロックデザインに参加したものをランク付けした。

参加したものの脂肪酸プロフィールを決定するため、各小区画の植物を、自家受粉のために袋がけした。単一植物由来のＭ₇種子を、10個の種子バルクサンプルで脂肪酸分析した。
選択された脂肪酸の遺伝子的関係を決定するため、突然変異体交雑を行った。Ｍ₆又はそれより後の世代の突然変異の花を、交雑に用いた。Ｆ₁種子を、刈り取り、脂肪酸組成を分析して、遺伝子動作（action）の様式（mode）を決定した。Ｆ₁子孫を、温室に植えた。得られた植物を、自家受粉させ、Ｆ₂種子を刈り取り、対立性（allelism）研究のために脂肪酸組成を分析した。Ｆ₂種子及び親系列種子を温室内に植え、個々の植物を自家受粉させた。個々の植物のＦ₃種子を、前記の10個の種子バルクサンプルを用いて脂肪酸組成を試験した。
幾つかの遺伝子的関係の分析において、二ゲノム性半数体集団が、Ｆ₁雑種の小胞子から形成された。二ゲノム性半数体植物由来の自家受粉種子を、前記の方法を用いて脂肪酸分析した。
化学分析のために、10個の種子バルクサンプルを、15mlのポリプロピレンチューブ中でガラス棒ですりつぶし、1:1ジエチルエーテル／メタノール中0.25Nの水酸化カリウム1.2mlで抽出した。そのサンプルを30秒間攪拌し、60℃の温浴中で60秒間加熱した。飽和塩化ナトリウム4ml及びイソ−オクタン2.4mlを加え、混合物を再度攪拌した。層分離後、上部の有機層600μlを、ピペットで個々のバイアルに入れ、−５℃で窒素雰囲気下に保存した。サンプル1μlを、Supelco SP-2330融解シリカ毛細管カラム（0.25mm ID、長さ30M、0.20μm df）中に注入した。
ガスクロマトグラフィーを、180℃、5.5分に設定し、そして２℃／分で212℃まで増加するようプログラムし、この温度に1.5分保持した。全工程時間（total run time）は23分であった。クロマトグラフィーの設定は、カラム頭圧力15psi、カラムフロー（ヘリウム）0.7ml／分、補助（Auxiliary）及びカラムフロー33ml／分、水素フロー33ml／分、エアーフロー400ml／分、注入器温度250℃、検出器温度300℃、分割通風（Split vent）1／15であった。
興味ある各脂肪酸の上位及び下位の統計的閾値を表８に記載する。

実施例２
高オレイン酸キャノーラ系統
実施例１の研究において、M₃世代では、31系統がオレイン酸についての統計的上限値を超えていた（≧71.0％）。W7608.3系統は71.2％のオレイン酸を含んでいた。M₄世代では、その自殖子孫（W7608.3.5、以後A129.5と呼ぶ）は78.8％のオレイン酸を含むC_18:1についての統計的上限値を超え続けた。A129.5系統（ATCC40811）の５つの自家受粉植物のM₅種子は平均すると75.0％のオレイン酸を含んでいた。単独植物選択物（single plant selection）A129.5.3は75.6％のオレイン酸を含んでいた。この高オレイン酸突然変異体の脂肪酸組成は、M₇世代まで圃場（field）および温室条件下の両方で安定であり、それを表9にまとめる。この系統は、多数の場所における圃場試験においてもM₇世代までその突然変異体脂肪酸組成を安定的に維持した。全ての場所において、自家受粉植物（A129）は平均して78.3％のオレイン酸を含んでいた。各アイダホ試験場についてのA129の脂肪酸組成を表10にまとめる。多場所複製収穫量試験（multiple location replicated yield trials）では、A129は親栽培品種Westarと収穫量において有意な差はなかった。
商業的な加工処理後のA129のキャノーラ油は、脂肪安定性についての促進酸素法（Accelerated Oxygen Method, AOM）,American Oil Chemist’ Society Official Method Cd 12-57；活性酸素法（1989年改訂）によって測定した場合、Westarと比べて優れた酸化安定性を有することが分かった。WestarのAOMは18 AOM時間であり、A129については30 AOM時間であった。

高オレイン酸突然変異体A129とその他のオレイン酸デサチュラーゼの遺伝子的関係を、市販のキャノーラ栽培品種および低リノレン酸突然変異体になされた交雑において実証した。A129を市販の栽培品種Global（C_16:0−4.5％、C_18:0−1.5％、C_18:1−62.9％、C_18:2 20.0％、C_18:3−7.3％）と交雑した。約200のF₂個体を脂肪酸組成について分析した。その結果を表11にまとめる。単一相互優性（single co-dominant）遺伝子を示唆する分離適合度（segregation fit）比1:2:1は、高オレイン酸表現型の遺伝を制御した。

A129と低リノール酸品種であるIMC01（C_16:0−4.1％、C_18:0−1.9％、C_18:1−66.4％、C_18:2 18.1％、C_18:3−5.7％）との交雑を行ってオレイン酸デサチュラーゼとリノール酸デサチュラーゼの遺伝を調べた。F₁ハイブリッドにおいて、オレイン酸およびリノール酸デサチュラーゼ遺伝子は、相互優性遺伝子作用の指標となる両親の中間値（mid-parent value）に近づいた。F₂個体の脂肪酸分析により、２つの別個の相互優性遺伝子の1:2:1:2:4:2:1:2:1分離が確認された（表12）。A129とIMC01の交雑から系統を選択し、米国特許出願No.08/425,108（参照により本明細書に含まれる）に記載されたようにIMC130（ATCC寄託No.75446）と名付けた。

A128.3と呼ばれるその他の高オレイン酸系統も、開示された方法により産生させた。この系統の50-種子バルク分析により下記の脂肪酸組成：C_16:0−3.5％、C_18:0−1.8％、C_18:1−77.3％、C_18:2 9.0％、C_18:3−5.6％、FDA Sats−5.3％、全Sats−6.4％であることが分かった。この系統も、M₇世代までその突然変異体脂肪酸組成を安定的に維持した。多場所複製収穫量試験では、A128はその親栽培品種Westarと収穫量において有意な差はなかった。
A129を対立性研究のためにA128.3と交雑した。F₂種子の脂肪酸組成により、２つの系統が対立遺伝子的であることが分かった。A129およびA128.3における突然変異発生が、起源は異なるが、同一遺伝子内にあった。
M3028.-10（ATCC 75026）と呼ばれる別の高オレイン酸系統も、実施例１に開示した方法により産生させた。この系統の10-種子バルク分析により下記の脂肪酸組成：C_16:0−3.5％、C_18:0−1.8％、C_18:1−77.3％、C_18:2 9.0％、C_18:3−5.6％、FDA飽和物−5.3％、全飽和物−6.4％であることが分かった。単場所（single location）複製収穫量試験では、M3028.10はその親栽培品種Weatarと収穫量において有意な差はなかった。
実施例３
低リノール酸キャノーラ
実施例１の研究において、M₃世代では80系統がリノール酸についての統計的下限を超えていた（≦13.2％）。W12638.8系統は9.4％のリノール酸を含んでいた。M₄およびM₅世代では、その自殖子孫（W12638.8、以後A133.1（ATCC 40812）と呼ぶ）は、リノール酸レベルがそれぞれ10.2％および8.4％の低C_18:2についての統計的閾値を超え続けた。この低リノール酸突然変異体の脂肪酸組成は、圃場および温室条件下の両方においてM₇世代まで安定であり、それを表13にまとめる。多場所複製収穫量試験では、A133は親栽培品種Westarと収穫量において有意な差はなかった。M3062.8（ATCC 75025）と呼ばれる別の低リノール酸系統も、開示した方法により産生させた。この系統の10-種子バルク分析により下記の脂肪酸組成：C_16:0−3.8％、C_18:0−2.3％、C_18:1−77.1％、C_18:2 8.9％、C_18:3−4.3％、FDA飽和物−6.1％であることが分かった。この系統も、圃場および温室においてその突然変異体脂肪酸組成を安定的に維持していた。

実施例４
低リノレン酸およびリノール酸キャノーラ
実施例１の研究において、M₃世代では、57系統がリノレン酸についての統計的下限値を超えた（≦5.3％）。W14749.8系統は、5.3％のリノレン酸および15.0％のリノール酸を含んでいた。M₄およびM₅世代では、その自殖子孫（W14749.8、以後M3032（ATCC 75021）と呼ぶ）は、リノレン酸レベルがそれぞれ2.7％および2.3％の低C_18:3についての統計的閾値を超え続け、また総量がそれぞれ11.8％および12.5％である少量のリノレン酸およびリノール酸についての統計的閾値を超え続けた。この低リノレン酸＋リノール酸突然変異体の脂肪酸組成は、圃場および温室条件下の両方においてM₅世代まで安定であり、それを表14にまとめる。単場所複製収穫量試験では、M3032は親栽培品種（Westar）と収穫量において有意な差はなかった。

実施例５
キャノーラ系統Q508およびQ4275
B.napus系統IMC-129の種子を、メチルN-ニトロソグアニジン（MNNG）で突然変異誘発した。MNNG処理は、３つの要素：予備浸漬、突然変異原添加および洗浄から構成された。0.05MのSorenson’sリン酸緩衝液を用いて予備浸漬および突然変異原処理をpH6.1に維持した。200個の種子をペトリ皿（100mm×15mm）内の濾紙（Whatman#3M）上で一度に処理した。種子を、15mlの0.05M Sorenson’s緩衝液中で、pH6.1にて2時間連続攪拌しながら予備浸漬した。予備浸漬期間の終わりに緩衝液を皿から除去した。
使用前に、0.05M Sorenson’s緩衝液にMNNGを10mM濃度で加えた溶液（pH6.1）を調製した。15mlの10m MNNGを各皿内の種子に加えた。種子を暗所にて22℃±3℃で4時間一定攪拌下でインキュベートした。インキュベーション期間の終わりに突然変異原溶液を除去した。
種子を、蒸留水を10分間隔で3回変更することにより洗浄した。4番目の洗浄は30分間行った。この処理養生法によりLD60集団が生じた。
処理種子を標準温室鉢植え用土壌（potting soil）にまいて、環境制御された温室に置いた。光の下で16時間植物を成長させた。花成期に、総状花序を袋に入れて自殖種子を産生させた。成熟期に、M2種子を採取した。各M2系統に識別番号を付した。全MNNG処理種子集団をQ系列と名付けた。
採取したM2種子を温室にまいた。成長条件は以前記載したとおりに維持した。総状花序を花成期に自殖のために袋に入れた。成熟期に、自殖M3種子を採取し、脂肪酸組成について分析した。各M3種子系統について、約10〜15個の種子を実施例１に記載したようなバルクで分析した。
高オレイン酸−低リノール酸M3系統を、＞82％オレイン酸および＜5.0％リノール酸のカットオフを用いてM3集団から選択した。脂肪酸組成についてスクリーニングした最初の1600のM3系統から、Q508を同定した。Q508 M3世代を温室内でM4世代に進ませた。表15にQ508およびIMC129の脂肪酸組成を示す。M4自殖種子は選択された高オレイン酸−低リノール酸表現型を維持していた（表16）。

M₄世代Q508植物は、圃場においてはWestarと比べて作物栽培学的品質が低かった。典型的な植物は、Westarと比べて成長が遅く、早期栄養活力に欠け、背丈が低く、退緑の傾向にあり、さやが短かった。Q508の収穫量はWestarと比べてかなり低かった。
温室内のM₄世代Q508植物は、Westarと比べて活力が低い傾向にあった。しかしながら、温室内におけるQ508収穫量は、圃場におけるQ508収穫量よりも高かった。

9個のその他のM4高オレイン酸−低リノール酸系統：Q3603、Q3733、Q4249、Q6284、Q6601、Q6761、Q7415、Q4275、およびQ6676も同定された。これらの系統のいくつかは約80％〜約84％の種子において良好な作物栽培学的特性を有しており、高レベルのオレイン酸を含んでいた。
Q4275を変種Cycloneと交雑した。7世代の自殖後、成熟した種子を、Q4275 Cyclone交雑の子孫系統93GS34-179から採取した。表17によれば、バルク種子試料の脂肪酸組成により、93GS34がQ4275の種子脂肪酸組成を維持していたことが分かる。93GS34-179は作物栽培学的に望ましい特性も維持していた。
7世代以上のQ4275の自殖の後、Q4275、IMC129および93GS34の植物を夏の間圃場成長させた。ランダム化ブロックデザイン（randomized block design）における４つの複製区画（replicated plots）（5フィート×20フィート）にて選択物を試験した。植物を放任受粉させた。自殖種子は産生されなかった。各区画は成熟期に収穫され、各系統から得たバルク採取種子の試料を上記のように脂肪酸組成について分析した。選択した系統の脂肪酸組成を表17に示す。

表17に示した結果から、Q4275は圃場条件下で選択された高オレイン酸−低リノール酸表現型を維持していたことが分かる。Q4275植物の作物栽培学的特性はQ508のものよりも優れていた。
M₄世代Q508植物を、雌親として働くWestarとともにWestarの二ゲノム性半数体選択物（dihaploid selection）と交雑させた。得られたF1種子を92EF集団と名付けた。Q508親よりも良好な作物栽培学的特性を有すると思われる約126のF1個体を自殖用に選択した。かかる個体から得られたF₂種子の一部を圃場に再びまいた。各F2植物を自殖させ、得られたF3種子の一部を脂肪酸組成について分析した。F₃種子のオレイン酸の含量は59〜79％の範囲であった。良好な作物栽培学的タイプの高オレイン酸（＞80％）個体は回収されなかった。
92EF集団のF₂種子の一部を温室にまいてQ508系統の遺伝学を分析した。380 F2個体から得たF₃種子を分析した。温室実験から得たF₃種子のC_18:1レベルを図１に示す。Q508が半優性であり相加的である２つの突然変異体遺伝子：元のIMC129突然変異ならびに一の追加の突然変異を含むという仮説に対してこのデータを試験した。また、この仮説はホモ接合性Q508がオレイン酸を85％以上含み、ホモ接合性Westarは62〜67％のオレイン酸を含むということも推測するものである。WestarによるQ508の交雑における各遺伝子の可能性のある遺伝子型は、
AA = Westar Fad2^a
BB = Westar Fad2^b
aa = Q508 Fad2^a-
bb = Q508 Fad2^b-
と示され得る。独立分離と仮定すると、1:4:6:4:1の割合の表現型が予測される。ヘテロ接合性植物の表現型は区別がつかないと仮定され、よってデータを、1:1:4:1の割合のホモ接合性Westar：ヘテロ接合性植物：ホモ接合性Q508に対する適合性について試験した。

カイ平方分析を用いると、オレイン酸データは1:14:1の割合に適合する。Q508は、半優性で相加的であり、独立に分離する２つの主要な遺伝子によりWestarとは相違するという結論が下された。比べると、IMC129の遺伝子型はaaBBである。
種子油中にオレイン酸を85％以上含む代表的なF3個体の脂肪酸組成を表18に示す。かかる植物においては、飽和脂肪酸のレベルがWestarと比べて低下していることが分かる。

実施例６
キャノーラ系統IMC-129、Q508、およびWestarの葉および根脂肪酸特性
Q508、IMC129およびWestarの植物を温室内で成長させた。成熟葉、一次発達中の葉、葉柄および根を６〜８葉段階で採取し、液体窒素中で凍結させ、−70℃で貯蔵した。実施例１に記載したようにして脂質抽出物をGLCにより分析した。脂肪酸特性データを表19に示す。
表19のデータは、Q508の全葉脂質は、C_18:2＋C_18:3含量よりもC_18:1含量が高いということを示すものである。WestarおよびIMC129はその逆である。Q508とIMC129間の全葉脂質の相違は、第2のFad2遺伝子がQ508において突然変異しているという仮説と一致するものである。
全脂質画分におけるC_16:3含量は3系統全てについてほぼ同一であり、これは色素体FadC遺伝子産物はQ508突然変異により影響されないということを示唆するものである。FadC遺伝子が突然変異されなかったことを確認するために、葉緑体脂質を分離し、分析した。３つの系統において、葉緑体C_16:1、C_16:2またはC_16:3脂肪酸における変化は検出されなかった。Q508、WestarおよびIMC129の中の色素体葉脂質の類似性は、Q508における第2の突然変異はミクロソームFad2遺伝子に影響を及ぼすが色素体FadC遺伝子には影響を及ぼさないという仮設と一致するものである。

実施例７
B.napus由来の突然変異体および野生型Δ-12脂肪酸デサチュラーゼの配列
FAD2構造遺伝子に特異的なプライマーを用いて逆転写酵素ポリメラーゼ連鎖反応（RT-PCR）によってDおよびF Δ-12デサチュラーゼ遺伝子の全オープンリーディングフレーム（ORF）をクローニングした。IMC129、Q508およびWestar植物の種子からRNAを標準的な方法により単離し、鋳型として用いた。RT増幅断片をヌクレオチド配列決定に用いた。各系統由来の各遺伝子のDNA配列を標準ジデオキシ配列決定法によって両方の鎖から決定した。
配列分析により、Westarの配列と比べた場合、IMC129とQ508の両方においてD遺伝子のヌクレオチド316（翻訳開始コドン由来）がGからAにトランスバージョンしていることが判明した。このトランスバージョンによりかかる位置におけるコドンがGAGからAAGに変化し、塩基性残基であるリシンが酸性残基であるグルタミン酸に非保存的置換されることになる。Q508系統はIMC129から誘導されるので、両系統において同一の突然変異の存在が予測された。同一の塩基の変化は、葉組織由来のRNAを鋳型として用いる場合にもQ508およびIMC129において検出された。
ヌクレオチド316におけるGからAへの突然変異は、IMC129およびWestarのゲノムDNAから直接増幅した断片を含むいくつかの独立のクローンを配列決定することにより確認された。これらの結果により、RT-PCRプロトコルにおける逆転写およびPCR中に導入される珍しい突然変異の可能性は排除された。IMC129突然変異体は、菜種ミクロソームΔ-12デサチュラーゼのD遺伝子のコード領域内のヌクレオチド316における1塩基トランスバージョンによるものであるという結論が下された。
F遺伝子のヌクレオチド515におけるTからAへの1塩基変化が、Q508において、Westar配列と比べた場合に検出された。この突然変異によりこの位置におけるコドンがCTCからCACに変化し、得られる遺伝子産物において極性残基ヒスチジンが非極性残基ロイシンに非保存的に置換されることになる。IMC129のF遺伝子配列においては、WestarのF遺伝子配列と比べた場合、突然変異は見出されなかった。
これらのデータは、Δ-12デサチュラーゼ遺伝子配列における突然変異がそのような突然変異遺伝子を含む植物の脂肪酸特性を変更させるという結論を支持するものである。さらに、このデータは、植物系統または種が2個のΔ-12デサチュラーゼ遺伝子座を含む場合、2個の突然変異遺伝子座を有する個体の脂肪酸特性は、1個の突然変異遺伝子座を有する個体の脂肪酸特性とは異なるということを示すものである。
保存アミノ酸モチーフ（His-Xaa-Xaa-Xaa-His）を有する領域にマッピングされたIMC129およびQ508のD遺伝子における突然変異が、クローン化Δ-12およびΔ-15膜結合デサチュラーゼにおいて見出された（表20）。

実施例８
ミクロソームΔ-12脂肪酸デサチュラーゼの転写および翻訳
in vivo転写をII段階およびIII段階発達中の種子および葉組織のRT-PCR分析により分析した。示した組織由来のΔ-12デサチュラーゼF遺伝子RNAを特異的に増幅させるのに用いたプライマーは、センスプライマー5’-GGATATGATGATGGTGAAAGA-3’およびアンチセンスプライマー5’TCTTTCACCATCATCATATCC−3’であった。示した組織由来のΔ-12デサチュラーゼD遺伝子RNAを特異的に増幅させるのに用いたプライマーは、センスプライマー5’-GTTATGAAGCAAAGAAGAAAC−3’およびアンチセンスプライマー5’-GTTTCTTCTTTGCTTCATAAC−3’であった。この結果から、DおよびF遺伝子のmRNAが、IMC129、Q508および野生型Westar植物の種子および葉組織において発現していることが分かった。
in vitro転写および翻訳分析により、約46kDのペプチドが製造されていることが分かった。これは、各遺伝子の推定アミノ酸配列およびミクロソーム膜ペプチドの同時翻訳付加の合計に基づき、D遺伝子産物およびF遺伝子産物両方について予測された大きさである。
これらの結果は、末端切断ポリペプチド遺伝子産物が生じる非センスまたはフレームシフト変異が突然変異体D遺伝子または突然変異体F遺伝子に存在しているという可能性を排除するものである。このデータは、実施例７のデータとともに、Q508およびIMC129における突然変異が、調節遺伝子内ではなく、デサチュラーゼ酵素をコードするΔ-12脂肪酸デサチュラーゼ構造遺伝子内にあるということを支持するものである。
実施例９
遺伝子特異的PCRマーカーの開発
上記のIMC129の突然変異体D遺伝子における1塩基変化に基づき、2個の5’PCRプライマーをデザインした。このプライマーのヌクレオチド配列は、3’末端の塩基（WestarではG、IMC129ではA）のみが異なっていた。このプライマーにより、DNA主体のPCRアッセイにおいて突然変異体fad2-Dと野生型Fad2-D対立遺伝子とを区別することが可能になる。5’PCRプライマーにおいては1個の塩基が異なるのみであるので、PCRアッセイはアニーリング温度、サイクル数、量および用いられるDNA鋳型の純度のようなPCR条件に非常に敏感である。IMC129および野生型植物由来のゲノムDNAを鋳型として用いて、突然変異体遺伝子と野生型遺伝子とを区別するアッセイ条件が確立されている。可変天然DNAサンプルを用いるなどしてPCRパラメーターを変化させると、条件をさらに最適化し得る。IMC129における1塩基突然変異を野生型遺伝子と区別するPCRアッセイは、脂肪酸組成分析とともに、Δ-12脂肪酸デサチュラーゼ突然変異を有する植物を含む遺伝子交雑の分離および選択分析を単純化させるための手段を提供する。
実施例１０
突然変異体および野生型Fad3遺伝子による形質転換
B.napus栽培品種Westarを、突然変異体および野生型Fad3遺伝子で形質転換してキャノーラ細胞質リノール酸デサチュラーゼΔ-15デサチュラーゼについての突然変異体Fad3遺伝子が非機能性であることを証明した。形質転換および再生は、本質的にWO94/11516に記載された手順にしたがって安全化（disarmed）Agrobacterium tumefaciensを用いて行った。
２つの安全化Agrobacterium株を、それぞれ、種子特異的プロモーターおよび対応する終結配列に適切に連結された遺伝子を有するTiプラスミドを含むように操作した。第1のプラスミドpIMC110を、安全化Tiベクターに全長野生型Fad3遺伝子をセンス方向（WO93/11245の配列番号６のヌクレオチド208〜1336）に挿入することにより調製した。全長野生型Fad3遺伝子には、Fad3遺伝子の5’に配置したnapinプロモーター配列およびFad3遺伝子の3’に配置したnapin終結配列が隣接していた。菜種napinプロモーターはEP0255378に記載されている。
第2のプラスミド、pIMC205は、突然変異したFad3遺伝子を安全化Tiベクターにセンス方向に挿入することによって調製した。突然変異体配列は、WO93/11245に記載されたミクロソームFad3遺伝子のヌクレオチド411および413に突然変異を含んでおり、したがってコドン96の配列がGACからAAGに変更されていた。よって遺伝子産物のコドン96におけるアミノ酸は、アスパラギン酸からリシンに変更された。表20を参照されたい。5’-TGGTCTTTTGGT-3’で始まる495塩基対の豆（Phaseolus vulgaris）ファゼオリン（7S種子貯蔵タンパク質）プロモーター断片を、突然変異体Fad3遺伝子の5’に配置し、ファゼオリン末端配列を突然変異体Fad3遺伝子の3’に配置した。ファゼオリン配列は、Doyleら，（1986）J. Biol. Chem. 261:9228-9238およびSlightomら，（1983）Proc. Natl. Acad. Sci. USA 80:1897-1901に記載されている。
適当なプラスミドをAgrobacterium株LBA4404に別個に操作し、移入した。各操作株を同時培養によるWestar種子由来の胚軸外植体の5mm切片の感染に用いた。感染胚軸をカルス培地に移し、その後再生培地に移した。カルスから識別可能な茎が生じたら、シュートを切除して伸長培地に移した。伸長したシュートを切断し、Rootone^TMに浸し、寒天培地に根付かせ、鉢植え用土壌に移植して結実能力のあるT1植物を得た。得られたT1植物を自家受粉させることによりT2種子を得た。
T2種子の脂肪酸分析を上記のようにして行った。結果を表21にまとめる。pIMC110プラスミドを用いて得られた40個の形質転換体の中で、17個の植物が野生型脂肪酸特性を示し、16個は過剰発現を示した。形質転換体の一部は、機能遺伝子が植物にセンス方向に形質転換されている場合、過剰発現表現型を示すものと予測される。
pIMC205遺伝子を有する307個の形質転換植物の中で、過剰発現の指標となる脂肪酸組成を示したものはなかった。形質転換体のいくつかは遺伝子産物が機能的である場合過剰発現表現型を示したので、この結果は突然変異体fad3遺伝子産物は非機能的であるということを示すものである。

代表的な形質転換植物の脂肪酸組成を表22に示す。652−09系統および663-40系統は、pIMC110を含み、それぞれ過剰発現および同時抑制表現型を示す植物の代表例である。205-284系統は、pIMC205を含み、突然変異体fad3遺伝子を有する植物の代表例である。

実施例11
野生型および突然変異体Fad2-DおよびFad2-Fの配列
高分子量ゲノムDNAをQ4275植物の葉（実施例５）ならびにWestarおよびBridgerキャノーラ植物から単離した。このDNAをポリメラーゼ連鎖反応（PCR）によるFad2-DおよびFad2-F遺伝子の増幅用の鋳型として用いた。PCR増幅を、0.3μgのゲノムDNA、200μMのデオキシリボヌクレオシド3リン酸、３mMのMgSO₄、１〜２単位のDNAポリメラーゼおよび１×緩衝液（DNAポリメラーゼ製造者により供給されたもの）を含む、合計容量100μl中で行った。サイクル条件は、95℃で1分間を1サイクル、次いで94℃で1分間、55℃で2分間および73℃で3分間を30サイクルであった。
Fad2-D遺伝子をElongase（登録商標）（Gibco-BRL）を用いて1回増幅した。PCRプライマーは、遺伝子の5’末端および3’末端に対して、それぞれ、CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG（配列番号23）およびCUACUACUACUATCATAGAAGAGAAAGGTTCAG（配列番号24）であった。
Fad2-F遺伝子を独立に4回、すなわちElongase（登録商標）で2回およびTaqポリメラーゼ（Boehringer Mannheim）で2回増幅させた。用いたPCRプライマーは、遺伝子の5’末端および3’末端に対して、それぞれ、5’CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3’（配列番号25）および5’CUACUACUACUATCTTTCACCATCATCATATCC3’（配列番号26）であった。
増幅DNA産物をアガロースゲル上で分離させ、JetSorb（登録商標）で精製し、その後プライマーの末端にある（CAU）₄および（CUA）₄配列を介してpAMP1（Gibco-BRL）にアニールし、E.coli DH5αに形質転換した。
Fad2-DおよびFad2-F挿入物を、合成プライマーAmpliTaq（登録商標）DNAポリメラーゼおよび色素ターミネーターを用いて、ABI PRISM310自動シーケンサー（Perkin-Elmer）にて製造業者の指示にしたがって両方の鎖について配列決定した。
Fad2-D遺伝子は、ATG開始コドンの上流にあるイントロン様配列（配列番号30および配列番号31）を有することが分かった。予想したように、IMC129由来の遺伝子のコード配列はヌクレオチド316においてGからAの突然変異を含んでいた（図２）。
ヌクレオチド908におけるGからAへの1塩基トランスバージョンは、野生型F遺伝子配列と比較した場合、Q4275増幅産物のF遺伝子配列において検出された（図２）。この突然変異は、アミノ酸303におけるコドンをGGAからGAAに変化させるものであり、グリシン残基がグルタミン酸残基に非保存的に置換されることになる（表３および図３）。植物における突然変異体Q4275 Fad2-F Δ-12デサチュラーゼ遺伝子の発現は、上記のように、脂肪酸組成を変化させる。
実施例12
野生型Fad2-Uの配列
高分子量ゲノムDNAを標準法によりBridgerおよびWestar Brassica植物の葉から単離した。Fad2-U遺伝子を、各1μgのプライマー、0.3μgのゲノムDNA、200μMのdNTP、３mMのMgSO₄、１×緩衝液（DNAポリメラーゼ製造者により供給されたもの）、および１〜２単位のElongaseDNAポリメラーゼ（BRL）を含む、100μlの合計反応液中で増幅させた。増幅条件は、95℃で1分間を1サイクル、94℃で1分間の変性、55℃で2分間のアニーリングおよび72℃で3分間の伸長を30サイクル含んでいた。その後、反応液をさらに10分間72℃でインキュベートした。Fad2U遺伝子を、下記のプライマー：

を用いてWestarから2回、BridgerゲノムDNAから2回増幅させた。
増幅したDNA産物を実施例11に記載したようにして精製し、配列決定した。Fad2-U配列は、ATG開始コドンの上流にイントロン様配列（配列番号28）を含む。
本明細書に記載され、説明された種々の特定の実施態様の任意の一つをまだ示していない範囲まで、さらに変形することにより、その他の特定の実施態様に示された特徴を組み込み得るということは当業者には理解されるであろう。
添付請求の範囲から逸脱することなくいくつかの変形が当業者には明白であるので、上記の詳細な説明は、本発明をより理解するためだけに提供されたものであり、そこから不必要な限定を意図するものではない。
配列表
配列番号：1
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：DNA
ハイポセティカル：YES
アンチセンス：NO
起源
生物名：Brassica napus
配列の特徴
他の情報：野生型Fad2
配列

配列番号：2
配列の長さ：384アミノ酸
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
配列

配列番号：3
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：DNA
ハイポセティカル：YES
アンチセンス：NO
起源
生物名：Brassica napus
配列の特徴
他の情報：ヌクレオチド316におけるGからAへのトランスバージョン突然変異
配列

配列番号：4
配列の長さ：384アミノ酸
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
配列

配列番号：5
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：DNA
ハイポセティカル：YES
アンチセンス：NO
起源
生物名：Brassica napus
配列の特徴
他の情報：野生型Fad2
配列

配列番号：6
配列の長さ：384アミノ酸
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
配列

配列番号：7
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：DNA
ハイポセティカル：YES
アンチセンス：NO
起源
生物名：Brassica napus
配列の特徴
他の情報：ヌクレオチド515におけるTからAへのトランスバージョン突然変異
配列

配列番号：8
配列の長さ：384アミノ酸
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
配列

配列番号：9
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列の特徴
特徴を表す記号：コード配列（Coding Sequence）
存在位置：1...1152
他の情報：
配列

配列番号：10
配列の長さ：384アミノ酸
配列の型：アミノ酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列

配列番号：11
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列の特徴
特徴を表す記号：コード配列（Coding Sequence）
存在位置：1...1152
他の情報：
配列

配列番号：12
配列の長さ：384アミノ酸
配列の型：アミノ酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列

配列番号：13
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列の特徴
特徴を表す記号：コード配列（Coding Sequence）
存在位置：1...1152
他の情報：
配列

配列番号：14
配列の長さ：384アミノ酸
配列の型：アミノ酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列

配列番号：15
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列の特徴
特徴を表す記号：コード配列（Coding Sequence）
存在位置：1...1152
他の情報：
配列

配列番号：16
配列の長さ：384アミノ酸
配列の型：アミノ酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列

配列番号：17
配列の長さ：1155塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列の特徴
特徴を表す記号：コード配列（Coding Sequence）
存在位置：1...1152
他の情報：
配列

配列番号：18
配列の長さ：384アミノ酸
配列の型：アミノ酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント、
配列

配列番号：19
配列の長さ：21塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：20
配列の長さ：21塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：21
配列の長さ：21塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：22
配列の長さ：26塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：23
配列の長さ：32塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：24
配列の長さ：33塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：25
配列の長さ：32塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：26
配列の長さ：33塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：27
配列の長さ：33塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸
配列

配列番号：28
配列の長さ：2168塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列の特徴
特徴を表す記号：コード配列（Coding Sequence）
存在位置：1014...2165
他の情報：
配列

配列番号：29
配列の長さ：384アミノ酸
配列の型：アミノ酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列

配列番号：30
配列の長さ：1132塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列

配列番号：31
配列の長さ：1135塩基対
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：Genomic DNA
配列

Technical field
The present invention relates to nucleic acids encoding fatty acid desaturases and desaturase proteins. More particularly, the present invention relates to nucleic acids encoding delta-12 and delta-15 fatty acid desaturase proteins that affect fatty acid composition in plants, polypeptides produced from such nucleic acids, and plants that express such nucleic acids.
Background of the Invention
Many breeding studies have been conducted to improve the fatty acid profile of Brassica varieties. Pleines and Freidt, Fat Sci. Technol., 90 (5), 167-171 (1988), combined with high oleic acid content (73-79%)_{18: 3}Plant lines with reduced levels (2.5-5.8%) are described. Rakow and McGregor, J. Amer. Oil. Chem Soc., 50, 400-403 (October 1973) discusses problems associated with the selection of linoleic and linolenic acid variants. Can. J. Plant Sci., 68, 509-511 (April 1988) discloses Stellar summer rape that produces seed oils containing 3% linolenic acid and 28% linoleic acid. ing. In Roy and Tarr, Z. Pflanzenzuchtg, 95 (3), 201-209 (1985) napus) teaches gene transfer by interspecific crossing. Roy and Tarr, Plant Breeding, 98, 89-96 (1987) discuss the potential for the development of B. napus L. with increased linolenic acid and linolenic acid content. European Patent Application No. 323,753 published July 12, 1989 discloses seeds and oils having more than 79% oleic acid in combination with less than 3.5% linolenic acid. Canvin, Can. J. Botany, 43, 63-69 (1965) discusses the effect of temperature on the fatty acid composition of oils from several seed crops including rapeseed.
Typically, mutations are induced with very high radiation doses and / or chemical mutagens (Gaul, H. Radiation Botany (1964) 4: 155-232). High dose levels above LD50 and typically reaching LD90 resulted in the maximum mutation rate that could be achieved. In mutation breeding of Brassica varieties, only a limited number of fatty acid mutations were induced by high levels of chemical mutagens alone or in combination with radiation (Rakow, G. Z. Pflanzenzuchtg (1973) 69: 62-82). Low α-linolenic acid mutations derived from the Rakow mutation breeding program were not suitable for commercial application due to low seed yield. The first commercial cultivar using the low α-linolenic acid mutation induced in 1973 was published in 1988 as a stellar variety (Scarth, R. et al., Can. J. Plant Sci., (1988) 68. , 509-511). Stellar yielded 20% lower than the commercial cultivars at the time of the announcement.
Changes in the fatty acid composition of vegetable oils are desirable to suit special food and industrial uses. For example, brassica varieties with increased monounsaturated oil levels (oleic acid) in seed oils and products derived from such oils will improve lipid nutrition. Canola systems with low polyunsaturated fatty acids and high oleic acid tend to have high oxidative stability, a useful property for the retail food industry.
Delta-12 fatty acid desaturase (also known as oleate desaturase) is involved in the enzymatic conversion of oleic acid to linoleic acid. Delta-15 fatty acid desaturase (also known as linoleic acid desaturase) is involved in the enzymatic conversion of linoleic acid to α-linolenic acid. Microsomal delta-12 desaturase was cloned and identified using a T-DNA tag. Okuley, et al., Plant Cell 6: 147-158 (1994). The nucleotide sequence of a higher plant gene encoding microsomal delta-12 fatty acid desaturase is described in Lightner et al., WO94 / 11516. The sequences of higher plant genes encoding microsomes and plastid delta-15 fatty acid desaturases are described in Yadav, N., et al., Plant Physiol., 103: 467-476 (1993), WO93 / 11245 and Arondel, V. et al., Science, 258: 1353-1355 (1992). However, there is no teaching disclosing mutations in plant-derived delta-12 or delta-15 fatty acid desaturase coding sequences. More efficient methods are needed in the art to develop plant lines containing delta-12 or delta-15 fatty acid desaturase gene sequence mutations that are effective in altering the fatty acid composition of seeds.
Summary of the Invention
The present invention comprises Brassicaceae or Helianthus seeds, plants and plant lines having at least one mutation that controls the level of unsaturated fatty acids in the plant. One embodiment of the invention is an isolation comprising a nucleotide sequence encoding a mutation from a mutant delta-12 fatty acid desaturase that alters the fatty acid composition in the seed when the fragment is present in the plant. Nucleic acid fragment. Preferred sequences comprise mutated sequences as shown in FIG. Another embodiment of the invention is an isolated nucleic acid fragment comprising a nucleotide sequence encoding a mutation from a mutant delta-15 fatty acid desaturase. The plant of this embodiment may be soybean, oilseed Brassica species, sunflower, castor bean or corn. The mutated sequence may be derived from, for example, the Brassica napus, Brassica rapa, Brassica juncea or sunflower delta-12 or delta-15 desaturase genes.
Another embodiment of the present invention is:
(a) Inducing mutations in cells of the starting varieties of Brassicaceae or Sunflower species; (b) obtaining progeny plants from the mutagenized cells; (c) mutations in the delta-12 or delta-15 fatty acid desaturase genes A method for producing a Brassicaceae or sunflower plant line comprising the step of: (d) producing a plant line by self-breeding or crossing. The resulting plant line may be mutagenized to obtain lines having both delta-12 desaturase mutations and delta-15 desaturase mutations.
Yet another embodiment of the present invention is:
(a) crossing a first plant with a second plant having a mutant delta-12 or delta-15 fatty acid desaturase; (b) obtaining seeds from the crossing of step (a); (c) such seeds (D) obtaining progeny seeds from the plant of step (c); then (e) identifying those seeds from progeny with altered fatty acid composition, A method of producing a plant line comprising an altered fatty acid composition is included. Suitable plants include soybean, rapeseed, sunflower, safflower, castor bean or corn. Preferred plants are rapeseed and sunflower.
The present invention is also embodied in a vegetable oil having an altered fatty acid composition obtained from the plants disclosed herein.
Brief description of sequence listing
SEQ ID NO: 1 shows the hypothetical DNA sequence of Brassica Fad2 gene. SEQ ID NO: 2 is the deduced amino acid sequence of SEQ ID NO: 1.
SEQ ID NO: 3 shows the hypothetical DNA sequence of Brassica Fad2 gene having a mutation at nucleotide position 316. SEQ ID NO: 4 is the deduced amino acid sequence of SEQ ID NO: 3.
SEQ ID NO: 5 shows the hypothetical DNA sequence of Brassica Fad2 gene. SEQ ID NO: 6 is the deduced amino acid sequence of SEQ ID NO: 5.
SEQ ID NO: 7 shows the hypothetical DNA sequence of Brassica Fad2 gene having a mutation at nucleotide position 515. SEQ ID NO: 8 is the deduced amino acid sequence of SEQ ID NO: 7.
SEQ ID NO: 9 shows the DNA sequence of the coding region of the wild type Brassica Fad2-D gene. SEQ ID NO: 10 is the deduced amino acid sequence of SEQ ID NO: 9.
SEQ ID NO: 11 shows the DNA sequence of the coding region of the IMC129 mutant brassica Fad2-D gene. SEQ ID NO: 12 is the deduced amino acid sequence of SEQ ID NO: 11.
SEQ ID NO: 13 shows the DNA sequence of the coding region of the wild type Brassica Fad2-F gene. SEQ ID NO: 14 shows the deduced amino acid sequence of SEQ ID NO: 13.
SEQ ID NO: 15 shows the DNA sequence of the coding region of the Q508 mutant brassica Fad2-F gene. SEQ ID NO: 16 shows the deduced amino acid sequence of SEQ ID NO: 15.
SEQ ID NO: 17 shows the DNA sequence of the coding region of the Q4275 mutant brassica Fad2-F gene. SEQ ID NO: 18 shows the deduced amino acid sequence of SEQ ID NO: 17.
SEQ ID NOs: 19-27 show the oligonucleotide sequences.
SEQ ID NO: 28 shows the genomic DNA sequence of the Brassica-derived Fad2-U gene.
SEQ ID NOs: 30 to 31 show genomic sequences located upstream from the start codon of Brassica Fad2-D gene.
[Brief description of the drawings]
Figure 1 shows seed oil oleic acid (C) in a segregated population of Q508X Wester mating_{18: 1}) A histogram showing the frequency distribution of content. The bar labeled WSGA 1A is Wester's parent C_{18: 1}It represents the content. The bar labeled Q508 is the Q508 parent C_{18: 1}It represents the content.
Figure 2 shows Brassica Fad2-D wild type gene (Fad2-D wt), IMC129 mutant type gene (Fad2-D GA316 IMC129), Fad2-F wild type gene (Fad2-F wt), Q508 mutant type gene (Fad2- F TA515 Q508) and Q4275 mutant gene (Fad2-F GA908 Q4275).
FIG. 3 shows the deduced amino acid sequence of the polynucleotide of FIG.
DESCRIPTION OF PREFERRED EMBODIMENTS
All% fatty acids herein are weight percentages in the oil in which the fatty acid is a component.
As used herein, a “line” is a group of plants that exhibits little or no genetic variation between individuals in at least one characteristic. Such lines are produced by asexual reproduction from a single parent using several generations of self-pollination and selection or tissue culture techniques or cell culture techniques. As used herein, the phrase “variety” refers to a series used for commercial manufacture.
The phrase “mutagenesis” refers to inducing random gene mutations within a population using a mutagenic agent. The treated population, or the generation that follows the population, is then screened for useful characteristics brought about by the mutation. A “population” is any group of individuals that share a common gene pool. As used herein, “M₀"Is untreated seed. As used herein, “M₁"Is the seed (and resulting plant) that has been exposed to the mutagenic agent, while" M₂”Is self-pollinated M₁Plant descendants (seed and plant)_Three”Is self-pollinated M₂A descendant of the plant, and "M_Four”Is self-pollinated M_ThreeA descendant of the plant. "M_Five”Is self-pollinated M_FourA descendant of the plant. "M₆"," M₇"Etc. are the respective descendants of the self-pollinated plant of the previous generation. As used herein, the phrase “selfed” means self-pollination.
As used herein, “stability” or “stable” refers to a fatty acid component that is at substantially the same level for at least two generations, preferably at least three generations, For example, it is preferably maintained at ± 5% from generation to generation. The method of the present invention can produce a series with stable and improved fatty acid composition from generation to generation up to ± 5%. The stability can be affected by temperature, location, stress and planting time. Thus, comparison of fatty acid profiles should be made from seeds produced under similar growth conditions. Stability can be measured based on previous generation information.
Intensive breeding produced Brassica plants whose seed oil contained less than 2% erucic acid. The breed is also bred so that its defatted meal contains less than 30 μmol / g glucosinolates. As used herein, “Canola” refers to less than 2% erucic acid (C_{22: 1}) And plant varieties seeds or oils containing meals with less than 30 μmol / g glucosinolate.
Applicants have discovered plants with mutations in the δ-12 fatty acid desaturase gene. These plants have useful modifications to the fatty acid composition of the seed oil. Such mutations result in, for example, an increase in oleic acid content, a decrease in linoleic acid content, stabilization, or both an increase in oleic acid and a decrease in linoleic acid content, stabilization.
Applicants have also discovered plants with mutations in the δ-15 fatty acid desaturase gene. These plants have useful modifications in the fatty acid composition of the seed oil, such as a reduction in α-linolenic acid content, stabilization.
Applicants have further discovered an isolated nucleic acid fragment (polynucleotide) comprising a sequence having a mutation in the coding sequence of a δ-12 or δ-15 fatty acid desaturase. The mutation provides a desirable modification to the fatty acid concentration in the seed oil of the plant carrying the mutation. δ-12 fatty acid desaturase is also known as ω-6 fatty acid desaturase and may be referred to herein as Fad2 or 12-DES. δ-15 fatty acid desaturase is also known as ω-3 fatty acid desaturase, and may be referred to herein as Fad3 or 15-DES.
The nucleic acid fragments of the present invention can be in the form of RNA or DNA, such as cDNA, synthetic DNA or genomic DNA. DNA can be double-stranded or single-stranded, and if single-stranded can be either the coding strand or the non-coding strand. The RNA analog can be, for example, mRNA or a combination of ribonucleotides and deoxyribonucleotides. A specific example of the nucleic acid fragment of the present invention is the mutated sequence shown in FIG.
The nucleic acid fragments of the invention comprise a mutation in the sequence encoding microsomal δ-12 fatty acid desaturase or a mutation in the sequence encoding microsomal δ-15 fatty acid desaturase. Such mutations render the resulting desaturase gene product non-functional in the plant compared to the function of the gene product encoded by the wild type sequence. The non-functionality of the δ-12 desaturase gene product is a reaction product in plant tissue expressing the mutant sequence compared to the corresponding concentration in plant tissue expressing the wild type sequence. It can be inferred from the reduced concentration of (linoleic acid) and the increased concentration of substrate (oleic acid). Defunctionalization of the δ-15 desaturase gene product results in a reaction product (α-linolenic acid) in the plant tissue expressing the mutant sequence compared to the corresponding concentration in the plant tissue expressing the wild type sequence. ) And decreased concentration of the substrate (linoleic acid).
A nucleic acid fragment of the invention can comprise a portion of a coding sequence, eg, at least about 10 nucleotides, provided that the fragment comprises at least one mutation in the coding sequence. The desired fragment length depends on the intended use of the fragment, eg, PCR primers, site-directed mutagenesis, etc. In one embodiment, a nucleic acid fragment of the invention comprises the full length coding sequence of a mutant δ-12 or mutant δ-15 fatty acid desaturase, eg, the mutant sequence of FIG. In other embodiments, the nucleic acid fragment is about 20 to about 50 nucleotides (or base pairs, bp) in length, or about 50 to about 500 nucleotides, or about 500 to about 1200 nucleotides. It is a nucleotide.
In another embodiment, the present invention provides an isolation consisting of at least 50 nucleotides in length having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 30 or SEQ ID NO: 31. It is related with the made nucleic acid fragment. In some embodiments, such a nucleic acid fragment has at least 80% or 90% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 31. The sequence identity of these and other nucleic acids disclosed herein can be determined, for example, using Blast 2.0.4 (February 24, 1998) using nr databases (non-redundant GenBank, EMBL, DDBT and PDB). It can be determined by searching. BLAST 2.0.4 is the National Center for Biotechnology (http://www.ncbi.nlm.nih.gov). Altschul, S.F., et al.Nucleic Acids Res.25: 3389-3402 (1997). Alternatively, MEGALIGN® (DNASTAR, Madison, WI) sequence alignment software can be used to determine sequence identity by the Clustal algorithm. In this method, sequences are grouped into clusters by testing the distance between all pairs. Arrange clusters in pairs to form a group. The Jotun Hein algorithm can also be used with MEGALIGN®. The nucleotide sequences of SEQ ID NOs: 30 and 31 are approximately 85% identical using the Clustal algorithm with default parameters.
The nucleotide sequences of SEQ ID NO: 30 and SEQ ID NO: 31 are located upstream of the ATG start codon of the Fad2-D gene and can be isolated from Bridger and Westar canola plants, respectively. These upstream elements contain intron-like features.
The invention also relates to an isolated nucleic acid fragment comprising a sequence consisting of at least 200 nucleotides. The fragment has at least 70% identity to the first to about 1012 nucleotides of SEQ ID NO: 28. In some embodiments, the fragment has 80% or at least 90% sequence identity to the first to about 1012 nucleotides of SEQ ID NO: 28. This part of SEQ ID NO: 28 is located upstream of the ATG start codon and has intron-like characteristics.
Mutations in the nucleic acid fragments of the invention can be any part of the coding sequence that renders the resulting gene product non-functional. Suitable types of mutations include, but are not limited to, nucleotide insertions, nucleotide deletions, or transition and transversion mutations in the wild-type coding sequence. Such mutations result in insertion of one or more amino acids, deletion of one or more amino acids, and non-conservative amino acid substitutions in the corresponding gene product. In some embodiments, the sequence of the nucleic acid fragment can include one or more mutations or one or more mutations.
Insertion or deletion of amino acids in the coding sequence can, for example, disrupt the essential α-helical or β-pleated sheet region structure of the resulting gene product. Amino acid insertions or deletions can also disrupt binding or catalytic sites important for gene product activity. It is known in the art that insertions or deletions of a large number of adjacent amino acids tend to render a gene product more non-functional than fewer inserted or deleted amino acids.
Non-conservative amino acid substitutions can replace one class of amino acids with a different class of amino acids. Non-conservative substitutions can substantially change the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions can also substantially change the bulk of the residue side chain, for example by replacing an alanine residue with an isoleucine residue.
Examples of non-conservative substitutions include substitution of basic amino acids with nonpolar amino acids, or substitution of polar amino acids with acidic amino acids. Since only 20 amino acids are encoded in a gene, substitutions that result in non-functional gene products are determined by routine experimentation that introduces different classes of amino acids in the region of the gene product targeted for mutation. it can.
Preferred mutations include amino acid sequence motifs such as His-Xaa-Xaa-Xaa-His motifs (Tables 1-3) that are conserved among multiple δ-12 fatty acid desaturases or multiple δ-15 fatty acid desaturases. It is in the region of the nucleic acid that it encodes. Examples of suitable regions include, for example, nucleotides corresponding to amino acids 105 to 109 of the Arabidopsis and Brassica δ-12 desaturase sequences, from 101 to 105 of the soybean δ-12 desaturase sequence. It has a conserved HECGH motif found in the nucleotide corresponding to the first amino acid and in the nucleotide corresponding to the 111th to 115th amino acids of the maize δ-12 desaturase sequence. See, for example, International Publication No. WO94 / 115116; Okuley et al., Plant Cell 6: 147-158 (1994). The single letter amino acid notation used herein is described in Alberts, B. et al., Molecular Biology of the Cell, 3rd edition, Garland Publishing, New York, 1994. The amino acids adjacent to this motif are also very conserved in δ-12 desaturases and δ-15 desaturases and are also good candidates for mutations in the fragments of the invention.
Illustrative embodiments of mutations in the nucleic acid fragments of the invention include substitution of Glu to Lys in the HECGH motif of the Brassica microsomal δ-12 desaturase sequence, either in the D form or the F form. It is. This mutation, as can be seen by comparing SEQ ID NO: 10 (wild type D form) with SEQ ID NO: 12 (mutant D form)ECGH HKBring change to CGH. Similar mutations in other Fad-2 are contemplated, resulting in non-functional gene products. (Compare SEQ ID NO: 2 and SEQ ID NO: 4)
Similar motifs can be found at amino acids 101 to 105 of the Arabidopsis microsomal δ-15 fatty acid desaturase and in the corresponding rape blossom desaturase and soybean desaturase (Table 5). See, eg, International Publication No. WO 93/11245; Arondel, V. et al., Science, 258: 1153-1155 (1992); Yadav, N. et al., Plant Physiol., 103: 467-476 (1993). . Plastid δ-15 fatty acids have a similar motif (Table 5).
Among the types of mutations in the HECGH motif that render the resulting gene product non-functional are non-conservative substitutions. An example of a non-conservative substitution is the substitution of a glycine residue with either the first or second histidine. Such substitution replaces the charged residue (histidine) with a nonpolar residue (glycine). Another type of mutation that renders the resulting gene product non-functional is an insertion mutation, for example, the insertion of glycine between a cysteine residue and a glutamic acid residue in the HECGH motif.
Other regions with suitable conserved amino acid motifs include the HRRHH motif shown in Table 2, the HRTHH motif shown in Table 6, and the HVAHH motif shown in Table 3. See, e.g., International Publication No. WO 94/115116; Hitz, W. et al., Plant Physiol., 105: 635-641 (1994); Okuley, J. et al., Above; Yadav, N. et al. An example of a mutation in the region shown in Table 3 is a mutation at the nucleotide corresponding to the codon corresponding to glycine (the 303rd amino acid of Brassica napus). The non-conservative Gly to Glu substitution is the sequence DRDY of the amino acid sequence DRDYGILNKV.EThis results in a change to ILNKV (compare FIG. 3, wild-type F form of SEQ ID NO: 14 with mutant Q4275 of SEQ ID NO: 18).
Another region suitable for mutation in the δ-12 desaturase sequence contains the motif KYLNNP at the nucleotide corresponding to amino acids 171 to 175 of the Brassica desaturase sequence. An example of a mutation in this region is the substitution of Leu for His and the amino acid sequence (Table 4) KYHNN (compare the wild type Fad2-F of SEQ ID NO: 14 with the mutant of SEQ ID NO: 16). Similar mutations in other Fad-2 amino acid sequences are contemplated, resulting in non-functional gene products. (Compare SEQ ID NO: 6 and SEQ ID NO: 8)

Conservation of amino acid motifs and their corresponding positions is mutated in one species and a region of δ-12 or δ-15 fatty acid desaturase that produces a non-functional desaturase is derived from another species. It shows that it can be mutated in the corresponding region to produce a non-functional δ-12 desaturase or δ-15 desaturase gene product within that species.
Mutations in any of the regions of Tables 1-6 are specifically included within the scope of the present invention and are substantially the same as those mutations exemplified herein, provided that As discussed in, the mutation (or mutation thereof) renders the resulting desaturase gene product non-functional.
Nucleic acid fragments containing mutated sequences can be produced by techniques known to those skilled in the art. Such techniques include, but are not limited to, site-directed mutagenesis of wild type sequences and direct synthesis using automated DNA synthesizers.
Nucleic acid fragments containing mutated sequences can also be produced by mutagenesis of plant seeds or renewable plant tissues, for example with ethyl methanesulfonate, X-rays or other mutagens. By mutagenesis, mutant plants having the desired fatty acid phenotype in the seed are identified by known techniques, and nucleic acid fragments containing the desired mutation are isolated from the genomic DNA or RNA of the mutant line. The site of the specific mutation is then determined by sequencing the coding region of the δ-12 desaturase or δ-15 desaturase gene. Alternatively, a mutagenized population can be rapidly screened using a labeled nucleic acid probe specific for the desired mutation event.
The disclosed method can be applied to all oilseed Brassica species, as well as both spring mature and winter mature species. Physical mutagens, including but not limited to x-rays, ultraviolet light and other physical processes that cause chromosomal damage, and others including but not limited to ethidium bromide, nitrosoguanidine, diepoxybutane, etc. These chemical mutagens may be used to induce mutations. Mutagenesis treatments may be applied to other growth stages of the plant, including but not limited to cell cultures, embryos, microspores and shoot apices.
As used herein, “stable mutations” are field conditions (determined by gas chromatographic analysis of a minimum of 10 randomly selected seeds bulked together ( maintaining a selected and modified fatty acid profile of at least three generations under field conditions), including at least two generations and exceeding the established statistical threshold of at least two generations, M_FiveIt is defined as a series that has advanced above. Alternatively, stability may be similarly measured by comparison with subsequent generations. In subsequent generations, stability is defined as having a fatty acid profile in the seed similar to that of the previous or subsequent generation grown under substantially similar conditions.
Mutation breeding has traditionally produced plants with complex and deleterious characteristics, such as reduced plant growth and reduced fertility, in addition to subject characteristics. Such features may indirectly affect fatty acid composition to produce unstable mutations; and / or reduce yield and thereby reduce the commercial utility of the present invention. In order to eliminate the occurrence of deleterious mutations and reduce the mutation burden that plants have, a low dose of mutagen is used in seed treatment to generate an LD30 population. This allows for the rapid selection of single gene mutations due to the characteristics of fatty acids in the agronomical background producing acceptable yields.
Several different plant line seeds have been deposited with the American Type Culture Collection and have the following accession numbers.
series Accession number Deposit date
A129.5 40811 May 25, 1990
A133.1 40812 May 25, 1990
M3032.1 75021 June 7, 1991
M3062.8 75025 June 7, 1991
M3028.10 75026 June 7, 1991
IMC130 75446 April 16, 1993
Q4275 97569 May 10, 1996
In some plant species or varieties, one or more forms of endogenous microsomal δ-12 desaturase can be found. In diploids, each form may be derived from one of the parental genomes that forms the species under consideration. Plants with mutations in both forms have a different fatty acid profile than plants with mutations in only one form. Examples of such plants are doubly mutagenized lines, including the mutant D form of δ-12 desaturase (SEQ ID NO: 11) and the mutant F form of δ-12 desaturase (SEQ ID NO: 15). A Brassica napus series Q508. Another example is the series Q4275, which includes the mutant D form of δ-12 desaturase (SEQ ID NO: 11) and the mutant F form of δ: -12 desaturase (SEQ ID NO: 17). See FIGS.
Preferred hosts or recipient organisms for introduction of the nucleic acid fragments of the present invention are soybean (Glycine max), rapeseed (eg, Brassica napus, B. rapa) and Brassica junsi (B. juncea), sunflower (Helianthus annus), castor bean (Ricinuscommunis), corn (Zea mays), and safflower (Carthamustinctorius) oil-producing species .
The nucleic acid fragment of the present invention may further contain an additional nucleic acid. For example, a nucleic acid encoding a secreted or leader amino acid sequence is mutated such that the secreted or leader sequence is fused in-frame to the amino terminus of the mutant δ-12 or δ-15 desaturase polypeptide. It can be linked to a desaturase nucleic acid fragment. Other nucleic acid fragments encoding amino acid sequences useful for in-frame fusion to the mutant desaturase polypeptides disclosed herein are known in the art. See, for example, US Pat. No. 5,629,193, which is incorporated herein by reference. A nucleic acid fragment may also have one or more regulatory elements operably linked thereto.
The invention also includes nucleic acid fragments that selectively hybridize to mutant desaturase sequences. Such nucleic acid fragments are typically at least 15 nucleotides in length. Hybridization typically includes a method of identifying the presence of a DNA sequence in a target nucleic acid mixture by Southern analysis (Southern blot), ie, hybridization to a labeled oligonucleotide or DNA fragment probe. Southern analysis is typically performed by electrophoretic separation of DNA digests on agarose gels, denaturation of DNA after electrophoretic separation, and nitrocellulose, nylon or Sambrook et al. (1989) Molecular Cloning, 2nd edition, Cold. Spring Harbor Laboratory, Plainview; to another suitable membrane support for analysis with radiolabeled, biotinylated, or enzyme labeled probes as described in NY Sections 9.37-9.52. Includes DNA transfer.
The nucleic acid fragment can hybridize to the mutant desaturase sequence under moderately stringent conditions, or preferably under high stringency conditions. High stringency conditions are used to identify nucleic acids with high homology with the probe. High stringency conditions include low ionic strength and high temperature for washing, eg, 0.015 M sodium chloride / 0.0015 M sodium citrate (0.1 × SSC); 50% to 65 ° C., 0.1% sodium lauryl sulfate (SDS) Using. Alternatively, during hybridization, a denaturing reagent such as formamide, eg, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5 with 750 mM NaCl, 75 mM sodium citrate, 50% formamide can be used at 0 ° C. Another example is 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5x Denhardt solution, sonication Salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C. (washed at 42 ° C. with 0.2 × SSC and 0.1% SDS).
Moderate stringent conditions refer to hybridization conditions used to identify nucleic acids that have a lower degree of identity to the probe than to identify under high stringency conditions. Medium stringency conditions may include using higher ionic strength and / or lower temperature for washing of the hybridization membrane compared to ionic strength and temperature used for high stringency hybridization. it can. For example, use a wash solution containing 0.060M sodium chloride / 0.0060M sodium citrate (4 x SSC) and 0.1% sodium lauryl sulfate (SDS) at 50 ° C and 1x SSC at 65 ° C for the final wash. Can do. Alternatively, a 1 × SSC, 37 ° C. hybridization wash can be used.
Hybridization is also performed by Northern analysis (Northern blot), a method used to identify RNAs that hybridize to known probes such as oligonucleotides, DNA fragments, cDNA or fragments thereof, or RNA fragments. You can also. The probe³²It is labeled by biotinylation with a radioisotope, such as P, or by an enzyme. The RNA to be analyzed is usually separated by electrophoresis on an agarose or polyacrylamide gel, transferred to nitrocellulose, nylon, or other suitable membrane, and described in Sambrook et al., Sections 7.39-7.52 above. Can be hybridized with the probe using standard techniques well known in the art.
The polypeptides of the present invention include not only isolated polypeptides having mutated amino acid sequences, but also derivatives and analogs thereof. See, for example, the mutated amino acid sequence in FIG. By “isolated” is meant a polypeptide that is expressed and produced in an environment other than the environment in which the polypeptide is naturally expressed and produced. For example, a plant polypeptide is isolated when expressed and produced in bacteria or fungi. The polypeptides of the present invention also include varieties of mutant desaturase polypeptides disclosed herein, as discussed above.
In one embodiment of the claimed invention, the plant contains both a δ-12 desaturase mutation and a δ-15 desaturase mutation. Such plants can have a fatty acid composition that includes very high oleic acid concentrations and very low α-linolenic acid concentrations. Mutations in the δ-12 desaturase and δ-15 desaturase may be combined in plants by making a genetic cross between a single mutant line of δ-12 desaturase and δ-15 desaturase . A plant having a mutation in the δ-12 fatty acid desaturase is crossed and crossed with a second plant having a mutation in the δ-15 fatty acid desaturase. The seeds produced by crossing are planted and the resulting plants are self-pollinated to obtain offspring seeds. These progeny seeds are then screened to identify seeds bearing both mutant genes.
Alternatively, a line having either a δ-12 desaturase mutation or a δ-15 desaturase mutation is subjected to mutagenesis, and a plant or plant line having a mutation in both δ-12 desaturase and δ-15 desaturase is obtained. Can be produced. For example, the IMC 129 series includes mutations in the D-form coding region of the microsomal δ-12 desaturase structural gene (Glu₁₀₆To Lys₁₀₆To have). Cells of this lineage (eg, seeds) are mutagenized to induce mutations in the δ-15 desaturase gene, mutations in the δ-12 fatty acid desaturase gene, and mutations in the δ-15 fatty acid desaturase gene. Can result in plants or plant lines having
A progeny includes a specific plant or plant line progeny, for example, a seed that has grown on an instant plant is a progeny. The offspring of the plant is F₁Plant, F₂Plant, F_ThreeSeeds formed on plants and subsequent generations of plants, or BC₁Plant, BC₂Plant, BC_ThreeIncludes seeds formed on plants and subsequent generations of plants.
The plant according to the invention preferably comprises a modified fatty acid composition. For example, oils obtained from the seeds of such plants have about 69 to about 90% oleic acid, based on the total fatty acid composition of the seeds. Such oils preferably have about 74% to about 90% oleic acid, more preferably about 80 to about 90% oleic acid. In some embodiments, the oil obtained from seeds produced by the plants of the present invention may have from about 2.0% to about 5.0% saturated fatty acids, based on the total fatty acid composition of the seeds. Good. In some embodiments, the oil obtained from seeds of the present invention may have from about 1.0% to about 14.0% linoleic acid, or from about 0.5% to about 10.0% α-linolenic acid. .
The oil composition is generally analyzed by pressing a batch (bulk) seed sample (eg, 10 seeds) and extracting the fatty acids therefrom. Fatty acid triglycerides in seeds are hydrolyzed and converted to fatty acid methyl esters. Those seeds having modified fatty acid composition are identified by techniques known to those skilled in the art, for example, gas liquid chromatography (GLC) analysis of bulk seed samples or of a single half-seed. May be. It is well known in the art that half-seed analysis is useful because embryo viability is maintained and therefore those seeds with the desired fatty acid profile can be planted to form the next generation . However, half-seed analysis is also known to be an inaccurate representation of the genotype of the seed being analyzed. Bulk seed analysis generally provides a more accurate representation of the fatty acid profile of a given genotype. Fatty acid composition can also be determined for oils obtained by commercial scale purification, bleaching and deodorization of endogenous oils in larger samples, eg, test plants or seeds.
The nucleic acid fragments of the present invention can be used as markers in plant genetic maps and plant breeding programs. Such markers may include, for example, restriction fragment length polymorphism (RFLP), random amplification polymorphism detection (RAPD), polymerase chain reaction (PCR) or self-sustained sequence replication (3SR) markers. Marker-assisted breeding techniques may be used to identify and track the desired fatty acid composition during the breeding process. Marker-assisted breeding techniques may be used in addition to or different from other types of identification techniques. An example of marker assisted breeding is the use of PCR primers that specifically amplify sequences containing the desired mutation in δ-12 desaturase or δ-15 desaturase.
The method according to the invention is useful in that the resulting plant and plant line have not only the desired seed fatty acid composition but also better agronomic characteristics compared to known lines with modified seed fatty acid composition It is. Better agronomic characteristics include, for example, increased seed germination percentage, increased seedling vigor, increased resistance to seedling fungal diseases (withering, root rot, etc.), increased yield, And improved stability.
While the invention is susceptible to various modifications and alternative forms, certain specific embodiments thereof are described in the general methods and examples set forth below. For example, the invention applies to and substantially similar to all Brassica species including B.rapa, B.juncea, and B.hirta. Can give the result. However, these examples are not intended to limit the invention to the particular forms disclosed, but rather, the invention is intended to cover all modifications, equivalents, and variations that fall within the scope of the invention. It should be understood to cover. This can include somaclonal mutations; physical or chemical mutagenesis of plant parts; chromosome doubling following sputum, microspore or ovary culture; or in combination with other properties alone to convey fatty acid properties , Including the use of self- or cross-pollination to develop new Brassica lineages.
Example 1
Mutagenesis
The seeds of the Canadian (Brassica napus) spring canola variety, Westar, were subjected to chemical mutagenesis. Wester is a registered Canadian Spring variety with canola quality. Fatty acid composition of field growing Wester, 3.9% C_{16: 0}1.9% C_{18: 0}67.5% C_{18: 1}17.6% C_{18: 2}7.4% C_{18: 3}, <2% C_{20: 1}+ C_{22: 1}Is stable under commercial manufacture since 1982 with <± 10% deviation.
Prior to mutagenesis, 30,000 B. napus cv. Westar seeds were pre-absorbed on a wet filter paper for 2 hours in a 300 seed lot to soften the seed coat. Pre-moistured seeds were placed in 80 mM ethyl methanesulfonate (EMS) for 4 hours. Following mutagenesis, the seeds were rinsed 3 times with distilled water. The seeds were sown in 48-well flats containing Pro-Mix. 68% of the mutagenized seeds germinated. The plants were maintained in a greenhouse at 25 ° C / 15 ° C, 14/10 hours day / night. During the flowering period, each plant was individually self-pollinated.
M from individual plants₂Seeds are classified and stored individually, about 15,000 M₂Lines were planted in summer nurseries in Carman, Manitoba, Manitoba. Seeds from each indigenous plant were planted in 3 meter rows with 6 inch row spacing. Wester was planted as a check variety. Lines selected in the field were made private by bagging the main gross inflorescence of each plant. In the maturity period, the self-grown plants were cut individually, and the seeds were classified and stored to support the origin of the seeds.
Self-pollinated M_ThreeSeed and Wester controls were analyzed for fatty acid composition by gas chromatography on 10 seed bulk samples. Statistical thresholds for each fatty acid component were established using the Z distribution at a stringency level of 1 / 10,000. Mean and standard deviation values were determined from unmutated Wester controls in the field. The upper and lower statistical thresholds for each fatty acid were determined from the population mean ± standard deviation combined by the Z distribution. The confidence interval based on the population size of 10,000 is 99.99%.
Selected M_ThreeSeeds were planted in the greenhouse along the Wester control. Seeds were planted in 4 inch pots containing Pro-Mix soil and the plants were maintained in a greenhouse at 25 ° C / 15 ° C, 14/10 hours day / night cycle. At the flowering stage, the end inflorescences were self-pollinated by bagging. In maturity, self M_FourSeeds were individually cut from each plant, labeled, and stored to support the origin of the seeds.
M_FourSeeds were analyzed on 10 seed bulk samples. Statistical thresholds for each fatty acid component were established from 259 control samples using a 1/800 Z distribution. Selected M_FourLines were planted in a field trial site in Carman, Manitoba in 3 meter rows at 6 inch intervals. 10 M in each row_FourPlants were bagged for self-pollination. At maturity, the indigenous plants were cut individually and the rows of open pollinated plants were cut in bulk. M from single plant selection_FiveSeeds were analyzed with 10 seed bulk samples and those with bulk row harvest were analyzed with 50 seed bulk samples.
Selected M_FiveLines were planted along the Wester control in the greenhouse. Seeds were grown as described above. During the flowering period, the end inflorescences were self-pollinated by bagging. In maturity, self M₆Seeds were cut individually from each plant, and fatty acid composition was analyzed on a bulk sample of 10 seeds.
Selected M₆The lineage was entered into the Eastern Idaho field trial. Four test locations with a wide variety of growth conditions were selected. Locations include Burley, Tetonia, Lamont, and Shelley (Table 7). The line was planted in 4 3 meter rows at 8 inch intervals and each plot replicated 4 times. The sowing design was determined using Randomized Complete Block Designed. A commercial cultivar (cultivar) Wester was used as the reference cultivar. At maturity, the parcel was cut and the yield was determined. The yield of those participating in the study was determined by taking a statistical average of 4 replicates. The Least Significant Difference Test was used to rank those who participated in the random complete block design.

To determine the fatty acid profile of those who participated, plants in each subcompartment were bagged for self-pollination. M from a single plant₇Seeds were fatty acid analyzed on 10 seed bulk samples.
Mutant crosses were performed to determine the genetic relationship of selected fatty acids. M₆Alternatively, later generations of mutant flowers were used for crossing. F₁Seeds were cut and analyzed for fatty acid composition to determine the mode of gene action. F₁The offspring were planted in the greenhouse. The obtained plant is self-pollinated and F₂Seeds were cut and analyzed for fatty acid composition for allelism studies. F₂Seeds and parent line seeds were planted in a greenhouse and individual plants were self-pollinated. F of individual plants_ThreeSeeds were tested for fatty acid composition using the 10 seed bulk samples described above.
In the analysis of several genetic relationships, a bigenomic haploid population is₁Formed from hybrid microspores. Self-pollinated seeds derived from digenomic haploid plants were analyzed for fatty acids using the method described above.
For chemical analysis, 10 seed bulk samples were ground with a glass rod in a 15 ml polypropylene tube and extracted with 1.2 ml of 0.25 N potassium hydroxide in 1: 1 diethyl ether / methanol. The sample was stirred for 30 seconds and heated in a 60 ° C. bath for 60 seconds. 4 ml of saturated sodium chloride and 2.4 ml of iso-octane were added and the mixture was stirred again. After layer separation, 600 μl of the upper organic layer was pipetted into individual vials and stored at −5 ° C. under a nitrogen atmosphere. 1 μl of sample was injected into a Supelco SP-2330 fused silica capillary column (0.25 mm ID, 30 M length, 0.20 μm df).
The gas chromatography was set to 180 ° C., 5.5 minutes and programmed to increase to 212 ° C. at 2 ° C./minute and held at this temperature for 1.5 minutes. The total run time was 23 minutes. Chromatographic settings are: column head pressure 15 psi, column flow (helium) 0.7 ml / min, auxiliary and column flow 33 ml / min, hydrogen flow 33 ml / min, air flow 400 ml / min, injector temperature 250 ° C., The detector temperature was 300 ° C. and the split vent was 1/15.
The upper and lower statistical thresholds for each fatty acid of interest are listed in Table 8.

Example 2
High oleic acid canola line
In the study of Example 1, M_ThreeIn the generation, 31 lines exceeded the statistical upper limit for oleic acid (≧ 71.0%). The W7608.3 line contained 71.2% oleic acid. M_FourIn the generation, its selfing offspring (W7608.3.5, hereafter referred to as A129.5) is C containing 78.8% oleic acid_{18: 1}Continued to exceed the statistical upper limit for. M of five self-pollinated plants of A129.5 line (ATCC40811)_FiveSeeds contained 75.0% oleic acid on average. Single plant selection A129.5.3 contained 75.6% oleic acid. The fatty acid composition of this high oleic acid mutant is M₇Stable to both generations under both field and greenhouse conditions, which are summarized in Table 9. This line is also suitable for field testing in many locations.₇The mutant fatty acid composition was stably maintained until generation. In all places, self-pollinated plants (A129) contained an average of 78.3% oleic acid. Table 10 summarizes the fatty acid composition of A129 for each Idaho test site. In multiple location replicated yield trials, A129 was not significantly different in yield from the parent cultivar Westar.
A129 canola oil after commercial processing is processed by the Accelerated Oxygen Method (AOM), American Oil Chemist 'Society Official Method Cd 12-57; Reactive Oxygen Method (revised in 1989) When measured, it was found to have superior oxidation stability compared to Westar. Westar's AOM was 18 AOM hours and A129 was 30 AOM hours.

The genetic relationship between the high oleic acid mutant A129 and other oleic acid desaturases was demonstrated in crosses made to commercial canola cultivars and low linolenic acid mutants. A129 is a commercially available cultivar Global (C_{16: 0}−4.5%, C_{18: 0}−1.5%, C_{18: 1}−62.9%, C_{18: 2} 20.0%, C_{18: 3}-7.3%). About 200 F₂Individuals were analyzed for fatty acid composition. The results are summarized in Table 11. A segregation fit ratio of 1: 2: 1 suggesting a single co-dominant gene controlled the inheritance of the high oleic acid phenotype.

A129 and IMC01 (C_{16: 0}-4.1%, C_{18: 0}-1.9%, C_{18: 1}−66.4%, C_{18: 2} 18.1%, C_{18: 3}-5.7%) and the genetics of oleate desaturase and linoleate desaturase were examined. F₁In the hybrid, the oleic acid and linoleic acid desaturase genes approached the mid-parent value of the parent, which is indicative of reciprocal dominant gene action. F₂Individual fatty acid analysis confirmed a 1: 2: 1: 2: 4: 2: 1: 2: 1 segregation of two distinct reciprocal genes (Table 12). A line was selected from the cross of A129 and IMC01 and named IMC130 (ATCC Deposit No. 75446) as described in US Patent Application No. 08 / 425,108 (included herein by reference).

Another high oleic acid line called A128.3 was also produced by the disclosed method. The 50-seed bulk analysis of this line shows the following fatty acid composition: C_{16: 0}−3.5%, C_{18: 0}−1.8%, C_{18: 1}−77.3%, C_{18: 2} 9.0%, C_{18: 3}-5.6%, FDA Sats -5.3%, total Sats -6.4%. This line is also M₇The mutant fatty acid composition was stably maintained until generation. In a multi-site replicate yield test, A128 was not significantly different in yield from its parent cultivar Westar.
A129 was crossed with A128.3 for antagonism studies. F₂The fatty acid composition of the seeds revealed that the two lines were allelic. Mutations in A129 and A128.3 were within the same gene, although of different origins.
Another high oleic acid line called M3028.-10 (ATCC 75026) was also produced by the method disclosed in Example 1. The 10-seed bulk analysis of this line shows the following fatty acid composition: C_{16: 0}−3.5%, C_{18: 0}−1.8%, C_{18: 1}−77.3%, C_{18: 2} 9.0%, C_{18: 3}-5.6%, FDA saturate -5.3%, total saturate -6.4%. In a single location replicate yield test, M3028.10 was not significantly different in yield than its parent cultivar WEATAR.
Example 3
Low linoleic acid canola
In the study of Example 1, M_ThreeIn the generation, 80 lines exceeded the statistical lower limit for linoleic acid (≦ 13.2%). Line W12638.8 contained 9.4% linoleic acid. M_FourAnd M_FiveIn the generation, its self-propelled progeny (W12638.8, hereafter referred to as A133.1 (ATCC 40812)) have low C with linoleic acid levels of 10.2% and 8.4%, respectively._{18: 2}The statistical threshold for continued to be exceeded. The fatty acid composition of this low linoleic acid mutant is determined by M in both field and greenhouse conditions.₇It is stable up to generations and is summarized in Table 13. In a multi-site replicate yield test, A133 was not significantly different from the parent cultivar Westar in yield. Another low linoleic acid line called M3062.8 (ATCC 75025) was also produced by the disclosed method. The 10-seed bulk analysis of this line shows the following fatty acid composition: C_{16: 0}-3.8%, C_{18: 0}-2.3%, C_{18: 1}−77.1%, C_{18: 2} 8.9%, C_{18: 3}It was found to be -4.3%, FDA saturate -6.1%. This line also stably maintained its mutant fatty acid composition in the field and greenhouse.

Example 4
Low linolenic and linoleic acid canola
In the study of Example 1, M_ThreeIn the generation, 57 strains exceeded the statistical lower limit for linolenic acid (≦ 5.3%). W14749.8 line contained 5.3% linolenic acid and 15.0% linoleic acid. M_FourAnd M_FiveIn the generation, its selfing offspring (W14749.8, hereafter referred to as M3032 (ATCC 75021)) have low C levels with linolenic acid levels of 2.7% and 2.3%, respectively._{18: 3}Continued to exceed the statistical threshold for small amounts of linolenic acid and linoleic acid with total amounts of 11.8% and 12.5%, respectively. The fatty acid composition of this low linolenic acid + linoleic acid mutant is determined by M in both field and greenhouse conditions._FiveIt is stable up to generations and is summarized in Table 14. In a single-site replicate yield test, M3032 was not significantly different from the parent cultivar (Westar) in yield.

Example 5
Canola lines Q508 and Q4275
B. napus line IMC-129 seeds were mutagenized with methyl N-nitrosoguanidine (MNNG). The MNNG treatment consisted of three elements: pre-soaking, mutagen addition and washing. Pre-soak and mutagen treatment was maintained at pH 6.1 using 0.05 M Sorenson's phosphate buffer. 200 seeds were treated at once on filter paper (Whatman # 3M) in a Petri dish (100 mm × 15 mm). The seeds were pre-soaked in 15 ml 0.05 M Sorenson's buffer at pH 6.1 for 2 hours with continuous stirring. Buffer was removed from the dish at the end of the pre-soak period.
Before use, a solution (pH 6.1) in which MNNG was added at a concentration of 10 mM to 0.05 M Sorenson's buffer was prepared. 15 ml of 10m MNNG was added to the seeds in each dish. Seeds were incubated in the dark at 22 ° C. ± 3 ° C. for 4 hours under constant agitation. The mutagen solution was removed at the end of the incubation period.
Seeds were washed by changing distilled water three times at 10 minute intervals. The fourth wash was for 30 minutes. This treatment regime resulted in an LD60 population.
Treated seeds were placed in a standard greenhouse potting soil and placed in an environmentally controlled greenhouse. Plants were grown for 16 hours under light. During the flowering period, the inflorescences were put in a bag to produce self-propagating seeds. M2 seeds were collected at maturity. Each M2 line was assigned an identification number. The whole MNNG treated seed population was named Q series.
The collected M2 seeds were spread in a greenhouse. Growth conditions were maintained as previously described. The inflorescences were put in a bag for selfing during the flowering period. At maturity, self-fertilized M3 seeds were collected and analyzed for fatty acid composition. For each M3 seed line, approximately 10-15 seeds were analyzed in bulk as described in Example 1.
High oleic acid-low linoleic acid M3 lines were selected from the M3 population using> 82% oleic acid and <5.0% linoleic acid cutoffs. Q508 was identified from the first 1600 M3 lines screened for fatty acid composition. Q508 The M3 generation was advanced to the M4 generation in the greenhouse. Table 15 shows the fatty acid composition of Q508 and IMC129. M4 self-grown seeds maintained the selected high oleic-low linoleic acid phenotype (Table 16).

M_FourGeneration Q508 plants had lower crop agronomic quality in the field compared to Westar. The typical plant was slower growing than Westar, lacked early vegetative vitality, had a low height, tended to fade, and had a short pod. The yield of Q508 was much lower than Westar.
M in the greenhouse_FourGeneration Q508 plants tended to be less active than Westar. However, the Q508 yield in the greenhouse was higher than the Q508 yield in the field.

Nine other M4 high oleic-low linoleic acid lines: Q3603, Q3733, Q4249, Q6284, Q6601, Q6761, Q7415, Q4275, and Q6676 were also identified. Some of these lines had good crop agronomic characteristics in about 80% to about 84% seed and contained high levels of oleic acid.
Q4275 was crossed with the variant Cyclone. After 7 generations of selfing, mature seeds were collected from the Q4275 Cyclone cross progeny line 93GS34-179. According to Table 17, it can be seen that 93GS34 maintained the seed fatty acid composition of Q4275 by the fatty acid composition of the bulk seed sample. 93GS34-179 also maintained desirable characteristics in crop agronomy.
After self-breeding of Q4275 for more than 7 generations, Q4275, IMC129 and 93GS34 plants were grown in the field during the summer. Selections were tested in 4 replicated plots (5 feet x 20 feet) in a randomized block design. The plant was pollinated. Self-propagating seeds were not produced. Each compartment was harvested at maturity and samples of bulk harvested seeds from each line were analyzed for fatty acid composition as described above. Table 17 shows the fatty acid composition of the selected lines.

From the results shown in Table 17, it can be seen that Q4275 maintained the selected high oleic acid-low linoleic acid phenotype under field conditions. The crop agronomic characteristics of Q4275 plants were superior to those of Q508.
M_FourGeneration Q508 plants were crossed with Westar's dihaploid selection with Westar acting as a female parent. The obtained F1 seeds were named 92EF population. Approximately 126 F1 individuals that appeared to have better crop agronomic characteristics than Q508 parents were selected for selfing. F obtained from such individuals₂Part of the seeds were sown again in the field. Each F2 plant was selfed, and a part of the obtained F3 seed was analyzed for fatty acid composition. F_ThreeSeed oleic acid content ranged from 59-79%. Good crop agronomic type high oleic acid (> 80%) individuals were not recovered.
92EF Group F₂Part of the seeds were placed in a greenhouse to analyze the genetics of the Q508 line. F from 380 F2 individuals_ThreeSeeds were analyzed. F from a greenhouse experiment_ThreeSeed C_{18: 1}The levels are shown in FIG. This data was tested against the hypothesis that Q508 contains two mutant genes that are semi-dominant and additive: the original IMC129 mutation as well as one additional mutation. This hypothesis also speculates that homozygous Q508 contains 85% or more oleic acid and homozygous Westar contains 62-67% oleic acid. The possible genotypes of each gene in the Q508 cross by Westar are
AA = Westar Fad2^a
BB = Westar Fad2^b
aa = Q508 Fad2^a-
bb = Q508 Fad2^b-
Can be shown. Assuming independent separation, a 1: 4: 6: 4: 1 ratio phenotype is predicted. The heterozygous plant phenotype was assumed to be indistinguishable, so the data was tested for suitability for a 1: 1: 4: 1 ratio of homozygous Westar: heterozygous plant: homozygous Q508 .

Using chi-square analysis, the oleic acid data fits a ratio of 1: 14: 1. It was concluded that Q508 is semi-dominant and additive and differs from Westar by two major genes that segregate independently. In comparison, the genotype of IMC129 is aaBB.
Table 18 shows the fatty acid composition of representative F3 individuals containing 85% or more oleic acid in seed oil. It can be seen that in such plants, the level of saturated fatty acids is reduced compared to Westar.

Example 6
Leaf and root fatty acid properties of canola lines IMC-129, Q508, and Westar
Q508, IMC129 and Westar plants were grown in a greenhouse. Mature leaves, primary developing leaves, petiole and roots were collected at the 6-8 leaf stage, frozen in liquid nitrogen and stored at -70 ° C. Lipid extracts were analyzed by GLC as described in Example 1. Fatty acid characteristic data is shown in Table 19.
The data in Table 19 show that Q508 whole leaf lipids are C_{18: 2}+ C_{18: 3}C than content_{18: 1}It indicates that the content is high. Westar and IMC129 are the opposite. The whole-leaf lipid difference between Q508 and IMC129 is consistent with the hypothesis that the second Fad2 gene is mutated in Q508.
C in the total lipid fraction_{16: 3}The content is nearly identical for all three lines, suggesting that the plastid FadC gene product is not affected by the Q508 mutation. To confirm that the FadC gene was not mutated, chloroplast lipids were isolated and analyzed. In three lines, chloroplast C_{16: 1}, C_{16: 2}Or C_{16: 3}No changes in fatty acids were detected. The similarity of plastid leaf lipids in Q508, Westar and IMC129 is consistent with the hypothesis that the second mutation in Q508 affects the microsomal Fad2 gene but not the plastid FadC gene. is there.

Example 7
Mutant and wild-type Δ-12 fatty acid desaturase sequences from B. napus
The entire open reading frame (ORF) of the D and F Δ-12 desaturase genes was cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using primers specific for the FAD2 structural gene. RNA was isolated from IMC129, Q508 and Westar plant seeds by standard methods and used as a template. RT amplified fragments were used for nucleotide sequencing. The DNA sequence of each gene from each line was determined from both strands by standard dideoxy sequencing.
Sequence analysis revealed that nucleotide 316 (derived from the translation initiation codon) of the D gene was transversioned from G to A in both IMC129 and Q508 when compared to the Westar sequence. This transversion changes the codon at such a position from GAG to AAG, resulting in a non-conservative substitution of ricin, a basic residue, with glutamic acid, an acidic residue. Since the Q508 line was derived from IMC129, the presence of the same mutation was predicted in both lines. The same base change was detected in Q508 and IMC129 when RNA from leaf tissue was used as a template.
The G to A mutation at nucleotide 316 was confirmed by sequencing several independent clones containing fragments directly amplified from IMC129 and Westar genomic DNA. These results eliminated the possibility of reverse transcription in the RT-PCR protocol and unusual mutations introduced during PCR. It was concluded that the IMC129 mutant was due to a single base transversion at nucleotide 316 within the coding region of the D gene of rapeseed microsomal Δ-12 desaturase.
A single base change from T to A at nucleotide 515 of the F gene was detected in Q508 when compared to the Westar sequence. This mutation changes the codon at this position from CTC to CAC, resulting in a non-conservative substitution of the polar residue histidine for the nonpolar residue leucine in the resulting gene product. No mutations were found in the IMC129 F gene sequence when compared to the Westar F gene sequence.
These data support the conclusion that mutations in the Δ-12 desaturase gene sequence alter the fatty acid properties of plants containing such mutant genes. Furthermore, this data shows that if a plant line or species contains two Δ-12 desaturase loci, the fatty acid profile of an individual with two mutant loci is that of an individual with one mutant locus. It shows that it is different from the fatty acid characteristics.
Mutations in the D gene of IMC129 and Q508 mapped to a region with a conserved amino acid motif (His-Xaa-Xaa-Xaa-His) were found in cloned Δ-12 and Δ-15 membrane-bound desaturases ( Table 20).

Example 8
Transcription and translation of microsomal Δ-12 fatty acid desaturase
In vivo transcription was analyzed by RT-PCR analysis of seed and leaf tissues during stage II and stage III development. The primers used to specifically amplify Δ-12 desaturase F gene RNA from the indicated tissues were sense primer 5'-GGATATGATGATGGTGAAAGA-3 'and antisense primer 5'TCTTTCACCATCATCATATCC-3'. The primers used to specifically amplify Δ-12 desaturase D gene RNA from the indicated tissues were sense primer 5'-GTTATGAAGCAAAGAAGAAAC-3 'and antisense primer 5'-GTTTCTTCTTTGCTTCATAAC-3'. From this result, it was found that mRNAs of D and F genes were expressed in seed and leaf tissues of IMC129, Q508 and wild type Westar plants.
In vitro transcription and translation analysis revealed that an approximately 46 kD peptide was produced. This is the predicted size for both the D and F gene products based on the deduced amino acid sequence of each gene and the sum of the cotranslational additions of the microsomal membrane peptide.
These results eliminate the possibility that a non-sense or frameshift mutation that results in a truncated polypeptide gene product is present in the mutant D gene or mutant F gene. This data, along with the data from Example 7, supports that mutations in Q508 and IMC129 are not in the regulatory gene but in the Δ-12 fatty acid desaturase structural gene encoding the desaturase enzyme.
Example 9
Development of gene-specific PCR markers
Two 5 'PCR primers were designed based on a single base change in the mutant D gene of IMC129. The nucleotide sequence of this primer differed only in the base at the 3 'end (G for Westar, A for IMC129). This primer makes it possible to distinguish between mutant fad2-D and wild-type Fad2-D alleles in DNA-based PCR assays. Since only one base is different in the 5 'PCR primer, the PCR assay is very sensitive to PCR conditions such as annealing temperature, number of cycles, amount and purity of the DNA template used. Assay conditions have been established to distinguish mutant genes from wild type genes using IMC129 and genomic DNA from wild type plants as templates. The conditions can be further optimized by changing the PCR parameters, such as by using a variable natural DNA sample. A PCR assay that distinguishes single-base mutations in IMC129 from wild-type genes, along with fatty acid composition analysis, provides a means to simplify the isolation and selection analysis of genetic crosses that include plants with Δ-12 fatty acid desaturase mutations To do.
Example 10
Transformation with mutant and wild-type Fad3 gene
B. napus cultivar Westar was transformed with mutant and wild-type Fad3 gene to prove that the mutant Fad3 gene for canola cytosolic linoleate desaturase Δ-15 desaturase is non-functional. Transformation and regeneration were performed using disarmed Agrobacterium tumefaciens essentially according to the procedure described in WO94 / 11516.
Two safety Agrobacterium strains were each engineered to contain Ti plasmids with genes appropriately linked to seed-specific promoters and corresponding termination sequences. The first plasmid pIMC110 was prepared by inserting the full-length wild-type Fad3 gene in the sense direction (nucleotides 208 to 1336 of SEQ ID NO: 6 of WO93 / 11245) into a safety Ti vector. The full-length wild-type Fad3 gene was flanked by a napin promoter sequence located 5 'of the Fad3 gene and a napin termination sequence located 3' of the Fad3 gene. The rapeseed napin promoter is described in EP0255378.
A second plasmid, pIMC205, was prepared by inserting the mutated Fad3 gene into a safety Ti vector in the sense orientation. The mutant sequence contained mutations at nucleotides 411 and 413 of the microsomal Fad3 gene described in WO93 / 11245, so the sequence of codon 96 was changed from GAC to AAG. Thus, the amino acid at codon 96 of the gene product was changed from aspartic acid to lysine. See Table 20. A 495 base pair bean (Phaseolus vulgaris) phaseolin (7S seed storage protein) promoter fragment beginning with 5'-TGGTCTTTTGGT-3 'is located 5' of the mutant Fad3 gene and the phaseolin terminal sequence is the mutant Fad3 gene Placed 3 '. Phaseolin sequences are described in Doyle et al. (1986) J. Biol. Chem. 261: 9228-9238 and Slightom et al. (1983) Proc. Natl. Acad. Sci. USA 80: 1897-1901.
Appropriate plasmids were separately manipulated and transferred into Agrobacterium strain LBA4404. Each engineered strain was used for infection of 5 mm sections of hypocotyl explants derived from Westar seeds by co-culture. Infected hypocotyls were transferred to callus medium and then transferred to regeneration medium. When discernable stems were generated from the callus, the shoots were excised and transferred to extension medium. Cut the stretched chute, rootone^TMSoaked in agar, rooted in agar, and transplanted to potting soil to obtain T1 plants with fruiting ability. T2 seeds were obtained by self-pollination of the obtained T1 plants.
Fatty acid analysis of T2 seed was performed as described above. The results are summarized in Table 21. Of the 40 transformants obtained with the pIMC110 plasmid, 17 plants showed wild-type fatty acid properties and 16 showed overexpression. Some of the transformants are expected to exhibit an overexpression phenotype when the functional gene is transformed into the plant in the sense direction.
None of the 307 transformed plants carrying the pIMC205 gene showed a fatty acid composition that was an indicator of overexpression. This result indicates that the mutant fad3 gene product is non-functional because some of the transformants showed an overexpression phenotype when the gene product was functional.

Table 22 shows the fatty acid composition of representative transformed plants. Lines 652-09 and 663-40 are representative examples of plants that contain pIMC110 and show overexpression and co-suppression phenotypes, respectively. Line 205-284 is a representative example of a plant containing pIMC205 and having a mutant fad3 gene.

Example 11
Sequences of wild type and mutants Fad2-D and Fad2-F
High molecular weight genomic DNA was isolated from leaves of the Q4275 plant (Example 5) and Westar and Bridger canola plants. This DNA was used as a template for amplification of Fad2-D and Fad2-F genes by polymerase chain reaction (PCR). PCR amplification was performed using 0.3 μg genomic DNA, 200 μM deoxyribonucleoside triphosphate, 3 mM MgSO._Four, In a total volume of 100 μl, containing 1-2 units of DNA polymerase and 1 × buffer (supplied by the DNA polymerase manufacturer). The cycling conditions were 95 ° C for 1 minute for 1 cycle, then 94 ° C for 1 minute, 55 ° C for 2 minutes and 73 ° C for 3 minutes for 30 cycles.
The Fad2-D gene was amplified once using Elongase® (Gibco-BRL). The PCR primers were CAUCAUCAUCAUCTTCTTCGTAGGGTTCATCG (SEQ ID NO: 23) and CUACUACUACUATCATAGAAGAGAAAGGTTCAG (SEQ ID NO: 24) for the 5 'end and 3' end of the gene, respectively.
The Fad2-F gene was amplified independently four times, ie twice with Elongase® and twice with Taq polymerase (Boehringer Mannheim). The PCR primers used were 5 'CAUCAUCAUCAUCATGGGTGCACGTGGAAGAA3' (SEQ ID NO: 25) and 5 'CUACUACUACUATCTTTCACCATCATCATATCC3' (SEQ ID NO: 26), respectively, with respect to the 5 'end and 3' end of the gene.
Amplified DNA products are separated on an agarose gel, purified with JetSorb®, and then at the end of the primer (CAU)_FourAnd (CUA)_FourIt was annealed to pAMP1 (Gibco-BRL) via the sequence and transformed into E. coli DH5α.
Fad2-D and Fad2-F inserts were sequenced on both strands on the ABI PRISM310 automated sequencer (Perkin-Elmer) using the synthetic primers AmpliTaq® DNA polymerase and dye terminator according to the manufacturer's instructions did.
The Fad2-D gene was found to have intron-like sequences (SEQ ID NO: 30 and SEQ ID NO: 31) upstream of the ATG start codon. As expected, the coding sequence of the gene from IMC129 contained a G to A mutation at nucleotide 316 (FIG. 2).
A 1 base transversion from G to A at nucleotide 908 was detected in the F gene sequence of the Q4275 amplification product when compared to the wild type F gene sequence (FIG. 2). This mutation changes the codon at amino acid 303 from GGA to GAA, resulting in a non-conservative substitution of a glycine residue for a glutamic acid residue (Table 3 and FIG. 3). Expression of the mutant Q4275 Fad2-F Δ-12 desaturase gene in plants changes the fatty acid composition as described above.
Example 12
Sequence of wild type Fad2-U
High molecular weight genomic DNA was isolated from leaves of Bridger and Westar Brassica plants by standard methods. Fad2-U gene, each 1 μg primer, 0.3 μg genomic DNA, 200 μM dNTP, 3 mM MgSO_FourAmplification was performed in a 100 μl total reaction containing 1 × buffer (supplied by the DNA polymerase manufacturer) and 1-2 units of Elongase DNA polymerase (BRL). Amplification conditions included 1 cycle at 95 ° C for 1 cycle, denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 2 minutes, and extension at 72 ° C for 3 minutes for 30 cycles. The reaction was then further incubated at 72 ° C. for 10 minutes. Fad2U gene with the following primers:

Was amplified twice from Westar and twice from Bridger genomic DNA.
The amplified DNA product was purified and sequenced as described in Example 11. The Fad2-U sequence contains an intron-like sequence (SEQ ID NO: 28) upstream of the ATG start codon.
The features shown in other specific embodiments may be incorporated by further modifications to the extent that any one of the various specific embodiments described and described herein has not yet been shown. This will be understood by those skilled in the art.
Since several variations will be apparent to those skilled in the art without departing from the scope of the appended claims, the foregoing detailed description has been provided only for a better understanding of the present invention and is not required therefrom. It is not intended to be limiting.
Sequence listing
SEQ ID NO: 1
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: DNA
Hypothetical: YES
Antisense: NO
origin
Name of organism: Brassica napus
Sequence features
Other information: Wild-type Fad2
Array

SEQ ID NO: 2
Sequence length: 384 amino acids
Sequence type: amino acid
Topology: Linear
Sequence type: Protein
Array

SEQ ID NO: 3
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: DNA
Hypothetical: YES
Antisense: NO
origin
Name of organism: Brassica napus
Sequence features
Other information: G to A transversion mutation at nucleotide 316
Array

SEQ ID NO: 4
Sequence length: 384 amino acids
Sequence type: amino acid
Topology: Linear
Sequence type: Protein
Array

SEQ ID NO: 5
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: DNA
Hypothetical: YES
Antisense: NO
origin
Name of organism: Brassica napus
Sequence features
Other information: Wild-type Fad2
Array

SEQ ID NO: 6
Sequence length: 384 amino acids
Sequence type: amino acid
Topology: Linear
Sequence type: Protein
Array

SEQ ID NO: 7
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: DNA
Hypothetical: YES
Antisense: NO
origin
Name of organism: Brassica napus
Sequence features
Other information: T to A transversion mutation at nucleotide 515
Array

SEQ ID NO: 8
Sequence length: 384 amino acids
Sequence type: amino acid
Topology: Linear
Sequence type: Protein
Array

SEQ ID NO: 9
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Sequence features
Symbols representing characteristics: Coding Sequence
Location: 1 ... 1152
Other information:
Array

SEQ ID NO: 10
Sequence length: 384 amino acids
Sequence type: amino acid
Number of chains: single chain
Topology: Linear
Sequence type: Protein
Fragment type: middle fragment
Array

SEQ ID NO: 11
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Sequence features
Symbols representing characteristics: Coding Sequence
Location: 1 ... 1152
Other information:
Array

SEQ ID NO: 12
Sequence length: 384 amino acids
Sequence type: amino acid
Number of chains: single chain
Topology: Linear
Sequence type: Protein
Fragment type: middle fragment
Array

SEQ ID NO: 13
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Sequence features
Symbols representing characteristics: Coding Sequence
Location: 1 ... 1152
Other information:
Array

SEQ ID NO: 14
Sequence length: 384 amino acids
Sequence type: amino acid
Number of chains: single chain
Topology: Linear
Sequence type: Protein
Fragment type: middle fragment
Array

SEQ ID NO: 15
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Sequence features
Symbols representing characteristics: Coding Sequence
Location: 1 ... 1152
Other information:
Array

SEQ ID NO: 16
Sequence length: 384 amino acids
Sequence type: amino acid
Number of chains: single chain
Topology: Linear
Sequence type: Protein
Fragment type: middle fragment
Array

SEQ ID NO: 17
Sequence length: 1155 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Sequence features
Symbols representing characteristics: Coding Sequence
Location: 1 ... 1152
Other information:
Array

SEQ ID NO: 18
Sequence length: 384 amino acids
Sequence type: amino acid
Number of chains: single chain
Topology: Linear
Sequence type: Protein
Fragment type: middle fragment,
Array

SEQ ID NO: 19
Sequence length: 21 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 20
Sequence length: 21 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 21
Sequence length: 21 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 22
Sequence length: 26 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 23
Sequence length: 32 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 24
Sequence length: 33 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 25
Sequence length: 32 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 26
Sequence length: 33 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 27
Sequence length: 33 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Other nucleic acids
Array

SEQ ID NO: 28
Sequence length: 2168 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Sequence features
Symbols representing characteristics: Coding Sequence
Location: 1014 ... 2165
Other information:
Array

SEQ ID NO: 29
Sequence length: 384 amino acids
Sequence type: amino acid
Number of chains: single chain
Topology: Linear
Sequence type: Protein
Fragment type: middle fragment
Array

SEQ ID NO: 30
Sequence length: 1132 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Array

SEQ ID NO: 31
Sequence length: 1135 base pairs
Sequence type: Nucleic acid
Number of chains: single chain
Topology: Linear
Sequence type: Genomic DNA
Array

Claims (5)

Including first and second delta-12 fatty acid desaturase genes, each of said genes having at least one mutation, at least one of the mutations encoding a His-Glu-Cys-Gly-His amino acid motif Each of the mutations changes the fatty acid composition of the plant seed , where the altered fatty acid composition is about 80% to about 90% oleic acid content and less than about 4% α-linolene Ru der those containing acid content, Brassicaceae or Helianthus plant. a) inducing mutations in cells of the starting varieties of Brassicaceae species;
b) obtaining one or more progeny plants from said cells;
c) identifying at least one progeny plant comprising a delta-12 fatty acid desaturase gene having at least one mutation, wherein the at least one mutation is in a region encoding a His-Glu-Cys-Gly-His amino acid motif;
d) producing a first plant line having at least one delta-12 gene mutation from at least one said progeny plant by self-pollination or cross-pollination;
e) inducing a mutation in cells of said first plant line;
f) obtaining one or more progeny plants from said first plant lineage cells;
g) comprising a second delta-12 fatty acid desaturase gene having at least one mutation, wherein the second gene mutation is in a region other than the region encoding the His-Xaa-Xaa-Xaa-His amino acid motif, Identifying one progeny plant of said first plant line;
h) producing a second plant line having the first delta-12 gene mutation and the second delta-12 gene mutation from the at least one plant line progeny plant by self-pollination or cross pollination A cruciferous plant line , wherein the second plant line produces seeds that yield oils having an oleic acid content of about 80% to about 90% and an α-linolenic acid content of less than about 4%. How to produce. Canola seed named Q4275 and represented by ATCC deposit number 97569. The progeny of seed according to claim 3 , which has a mutant delta-12 fatty acid desaturase present in the seed represented by ATCC accession number 97569. 5. The progeny of claim 4 , wherein the progeny is a Brassica napus plant.

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