JP2017519519A - 植物体におけるニコチンからノルニコチンへの変換の低減 - Google Patents
植物体におけるニコチンからノルニコチンへの変換の低減 Download PDFInfo
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- JP2017519519A JP2017519519A JP2017510762A JP2017510762A JP2017519519A JP 2017519519 A JP2017519519 A JP 2017519519A JP 2017510762 A JP2017510762 A JP 2017510762A JP 2017510762 A JP2017510762 A JP 2017510762A JP 2017519519 A JP2017519519 A JP 2017519519A
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Abstract
Description
本発明の態様および実施形態は、付属されている請求の範囲に記載している。
さらなる態様で、本明細書で説明した植物体に由来するバイオマス、種子、幹または葉を含む植物材料が説明されている。NND3は、植物材料の花(例えば、未熟な花、成熟した花、未熟なさく果、乾燥したさく果)および根で発現されることが適切である。NND3は、植物材料の花(例えば、未熟な花、成熟した花、未熟なさく果、乾燥したさく果)および根で排他的または特異的に発現されることが適切である。
本明細書の範囲内で使用される技術的な用語および表現には一般に、植物体および分子生物学の関連分野でそれらに共通して適用される意味が与えられる。以下の用語の定義はどれも、本明細書の全内容に適用される。「含む、備える(comprising)」という語は、それ以外の要素または工程を除外するものではなく、また不定冠詞「a」もしくは「an」は、複数であることを除外するものではない。ある一つの工程が、請求の範囲に列挙された複数の特徴の機能を実現することもある。所定の数値または範囲の文脈での「約」、「本質的に」および「およそ」という用語は、所定の値または範囲の20%以内、10%以内、または5%、4%、3%、2%または1%以内である値または範囲を意味する。
一つの実施形態で、配列リストに示す任意のポリヌクレオチドを含めた、本明細書で説明した任意の配列との少なくとも95%の配列同一性を持つポリヌクレオチド配列を含むか、それから成るか、それから実質的に成る単離ポリヌクレオチドが提供されている。単離ポリヌクレオチドは、それに対する少なくとも95%、96%、97%、98%、99%または100%の配列同一性を持つ配列を含むか、それから成るか、それから実質的に成ることが適切である。
ニコチン変換におけるNND3の機能およびその役割を決定するために、NND3の発現が分析される。この目的で、温室で成長したN. tabacum品種TN90の異なる組織 が収穫され、定量的PCRで分析される。
予想されたタンパク質配列は、CYP82E4、CYP82E5およびCYP82E10などの周知のデメチラーゼに対して高いレベルの相同性を提示するが、その機能は相同性からは推察できない。これはシトクロムP450ファミリーでは、少しのアミノ酸の違いが基体の特異性を変化または軽減するのに十分であることが示されているためである。
サイレンシング構築物は、機能的CYP82E4、CYP82E5およびCYP82E10遺伝子を欠く超低コンバーターのバーレー種植物環境におけるMMVプロモーターの制御下で構成的に発現するように設計されている。サイレンシング構築物について、イントロン配列(配列番号4)によって分離された100 bp配列が順方向および逆方向に使用される。結果は、花におけるNND3のサイレンシングが、ニコチン変換の低減につながることを示し、NND3酵素が機能的であるだけでなく、植物におけるニコチン変換にも貢献することを実証するものである。
N. tabacum品種Stellaの葉における異なる乾燥処理の時点でのNND3および関連する機能的CYP82E遺伝子の相対的発現レベルが分析される。試料は、収穫時に「緑色の」葉(茎の上部の位置)および「成熟」葉(茎の下の位置)から採られる。葉が空気乾燥処理小屋に移され、試料が乾燥開始時(「0 h」)、さらに12時間後(「12 h」)、24時間後(「24 h」)および48時間後(「48 h」)に採られる。試料はいくつかの葉のプールから採られる。2つのプールが生物学的複製(複製1(図3a)および2(図3b))として分析される。結果を図3に示した。バーは、3つの技術的複製の平均±SDを示す。異なるCYP82E遺伝子の発現レベルは、独立的にのみ考慮でき、異なるPCR効率のため直接的な比較によるものではない。CYP82E5およびCYP82E10は、すべての葉試料において類似した範囲で発現された。乾燥処理条件の下で老齢の葉においてCYP82E4の発現が活性化され増大した。NND3は、分析されたすべての条件下の葉では発現されなかった。
[配列表]
配列番号 1(N. tabacum TN90 NND3-T遺伝子のポリヌクレオチド配列。イントロンは小文字で示し、コード配列は下線付きである。開始コドンおよび終止コドンは灰色で強調表示されている。)
ATGGTTTTTCCCATAGAAGCCATTGTAGGACTAGTAACCTTCACATTTCTCTTCTACTTCCTATGGACAAAAAAATCTCAAAAACCTTCAAAACCCTTACCACCGAAAATCCCCGGAGGATGGCCGGTAATTGGCCATCTTTTCCACTTCAATGACGACGGCAACGACCGTCCATTAGCTCGAAAACTCGGAGACTTAGCTGACAAATACGGCCCCGTTTTCACTTTTCGGCTAGGCCTTCCCCTTGTGTTAGTTGTAAGCAGTTACGAAGCTATAAAAGACTGTTTCTCTACAAATGATGCCATTTTCTCTAATCGTCCAGCTTTTCTTTACGGCGAATACCTTGGCTACAATAATGCCATGCTATTTTTGGCAAATTACGGACCTTACTGGCGAAAAAATCGTAAATTAGTTATTCAGGAAGTTCTCTCAGCTAGTCGTCTCAAAAAATTCAAACACGTGAGATTCGCCAGAATTCAAACGAGCATTAAGAATTTATACACTCGAATTGATAGAAATTCGAGTACGATAAATTTAACTGATTGGTTAGAAGAATTGAATTTTGGTCTCATCGTGAAGATGATAGCTGGGAAAAATTATGAATCCGGTAAAGGAGATGAACAAGTGGAGAGATTTAAGAAAGCGTTTAAGGATTTTATGATTATATCAATGGAGTTTGTGTTATGGGATGCATTTCCAATTCCATTATTTAAATGGGTGGATTTTCAAGGGCATGTTAAGGCTATGAAAAGGACATTTAAGGATATAGATTCTGTTTTTCAGAATTGGTTAGAGGAACATATTAACAAAAGAGAAAAAATGGAGGTTAATGCAGAAGGGAATGAACAAGATTTCATTGATGTGGTGCTTTCAAAAATGAGTAATGAATATCTTGGTGAAGGTTACTCTCGTGATACTGTCATAAAAGCAACAGTTTTTgtaagttcatctgtcactcccgaattctgcttgaatgcagacaccgagttgcctttttattagactatctaaatattaaggatgattatatatagcaaaaatatagtaggatttctcgggctttgggatagaagaatattcatcattatataacttttgatggtaaaagatgagatctaacctcttataattgcccaaccacgttgatatatagaacaaaacctttttactcccattgagcataacgaaaatgaaagcaaagggacttcttctcttttttaggaaaaaaatctttgattgcttgttgaatatacattcatgtttttctttttctatttctaataataatggtgcttgaatcaggttgcgtgcctcgtgacttttgagaaaaaaaaacaaattcattagtataatgaggtgtgcatacttgacaactactatactaactagaacaaggttcggcagataatgacgctaacctacttttatattgaattatcatttgtatttaactgtattctattttgtccacagAGTTTGGTCTTGGATGCTGCGGACACAGTTGCTCTTCACATAAATTGGGGTATGGCATTACTGATCAACAATCAAAATGCCTTGAAGAAAGCACAAGAAGAGATAGACACAAAAGTTGGCAAGGATAGATGGGTAGAAGAGAGTGATATTAAGGATTTGGTGTACCTCCAAGCTATTGTTAAAGAAGTGTTACGATTATATCCACCGGGACCTTTGTTAGTACCACATGAAAATATAGAGGATTGTGTTGTTAGTGGATATTACATTTCTAAAGGGACTAGACTATTCGCAAATGTTATGAAACTGCAGCGCGATCCTAAACTCTGGCCAAATCCTGATAATTTCGATCCAGAGAGATTTGTCGCTGCAGGTATTGACTTTCGTGGTCAGCATTATGAGTATATCCCGTTTGGTTCTGGAAGACGATCTTGTCCGGGGATGACTTATGCATTGCAAGTGGAACACTTAACAATGGCACATTTGATCCAGGGTTTCAATTACAGCACTCCAAATGACGAGCCCTTGGATATGAAGGAAGGTGCAGGTATAACTATACGTAAGGTAAATCCCGTGGAAGTGATAATTATGCCTCGCCTGGCACCTGAGCTTTATTAA
配列番号 2 (MMVプロモーターの制御下で過剰発現構築物で使用されたNND3のポリヌクレオチドのコード配列)。
ATGGTTTTTCCCATAGAAGCCATTGTAGGACTAGTAACCTTCACATTTCTCTTCTACTTCCTATGGACAAAAAAATCTCAAAAACCTTCAAAACCCTTACCACCGAAAATCCCCGGAGGATGGCCGGTAATTGGCCATCTTTTCCACTTCAATGACGACGGCAACGACCGTCCATTAGCTCGAAAACTCGGAGACTTAGCTGACAAATACGGCCCCGTTTTCACTTTTCGGCTAGGCCTTCCCCTTGTGTTAGTTGTAAGCAGTTACGAAGCTATAAAAGACTGTTTCTCTACAAATGATGCCATTTTCTCTAATCGTCCAGCTTTTCTTTACGGCGAATACCTTGGCTACAATAATGCCATGCTATTTTTGGCAAATTACGGACCTTACTGGCGAAAAAATCGTAAATTAGTTATTCAGGAAGTTCTCTCAGCTAGTCGTCTCAAAAAATTCAAACACGTGAGATTCGCCAGAATTCAAACGAGCATTAAGAATTTATACACTCGAATTGATAGAAATTCGAGTACGATAAATTTAACTGATTGGTTAGAAGAATTGAATTTTGGTCTCATCGTGAAGATGATAGCTGGGAAAAATTATGAATCCGGTAAAGGAGATGAACAAGTGGAGAGATTTAAGAAAGCGTTTAAGGATTTTATGATTATATCAATGGAGTTTGTGTTATGGGATGCATTTCCAATTCCATTATTTAAATGGGTGGATTTTCAAGGGCATGTTAAGGCTATGAAAAGGACATTTAAGGATATAGATTCTGTTTTTCAGAATTGGTTAGAGGAACATATTAACAAAAGAGAAAAAATGGAGGTTAATGCAGAAGGGAATGAACAAGATTTCATTGATGTGGTGCTTTCAAAAATGAGTAATGAATATCTTGGTGAAGGTTACTCTCGTGATACTGTCATAAAAGCAACAGTTTTTAGTTTGGTCTTGGATGCTGCGGACACAGTTGCTCTTCACATAAATTGGGGTATGGCATTACTGATCAACAATCAAAATGCCTTGAAGAAAGCACAAGAAGAGATAGACACAAAAGTTGGCAAGGATAGATGGGTAGAAGAGAGTGATATTAAGGATTTGGTGTACCTCCAAGCTATTGTTAAAGAAGTGTTACGATTATATCCACCGGGACCTTTGTTAGTACCACATGAAAATATAGAGGATTGTGTTGTTAGTGGATATTACATTTCTAAAGGGACTAGACTATTCGCAAATGTTATGAAACTGCAGCGCGATCCTAAACTCTGGCCAAATCCTGATAATTTCGATCCAGAGAGATTTGTCGCTGCAGGTATTGACTTTCGTGGTCAGCATTATGAGTATATCCCGTTTGGTTCTGGAAGACGATCTTGTCCGGGGATGACTTATGCATTGCAAGTGGAACACTTAACAATGGCACATTTGATCCAGGGTTTCAATTACAGCACTCCAAATGACGAGCCCTTGGATATGAAGGAAGGTGCAGGTATAACTATACGTAAGGTAAATCCCGTGGAAGTGATAATTATGCCTCGCCTGGCACCTGAGCTTTATTAA
配列番号 3 (N. tabacum TN90 NND3タンパク質配列のポリペプチド配列)
MVFPIEAIVGLVTFTFLFYFLWTKKSQKPSKPLPPKIPGGWPVIGHLFHFNDDGNDRPLARKLGDLADKYGPVFTFRLGLPLVLVVSSYEAIKDCFSTNDAIFSNRPAFLYGEYLGYNNAMLFLANYGPYWRKNRKLVIQEVLSASRLKKFKHVRFARIQTSIKNLYTRIDRNSSTINLTDWLEELNFGLIVKMIAGKNYESGKGDEQVERFKKAFKDFMIISMEFVLWDAFPIPLFKWVDFQGHVKAMKRTFKDIDSVFQNWLEEHINKREKMEVNAEGNEQDFIDVVLSKMSNEYLGEGYSRDTVIKATVFSLVLDAADTVALHINWGMALLINNQNALKKAQEEIDTKVGKDRWVEESDIKDLVYLQAIVKEVLRLYPPGPLLVPHENIEDCVVSGYYISKGTRLFANVMKLQRDPKLWPNPDNFDPERFVAAGIDFRGQHYEYIPFGSGRRSCPGMTYALQVEHLTMAHLIQGFNYSTPNDEPLDMKEGAGITIRKVNPVEVIIMPRLAPELY
配列番号 4 (100bpの唯一の配列NND3を、IPMS2遺伝子にあるイントロン配列で分離された、順方向および逆方向で使用するRNAi構築物のポリヌクレオチド配列。イントロン配列は小文字で示されている。)
GCGATCCTAAACTCTGGCCAAATCCTGATAATTTCGATCCAGAGAGATTTGTCGCTGCAGGTATTGACTTTCGTGGTCAGCATTATGAGTATATCCCGTTtggtaacctttaatgtttaaccgttcacatttctaatatttacttatttgtaacatgtcgtcacgtgttagtttcattctttttatgaaccaaacatgcatgcaaagatatttttagatatttggacggcgagtgagatttgaaactaggaccgtttgcctgatacaatattaaaatatgtaaccattttatgtacaagtttaaactgttgatagtagcatattttttacttttatttaagtatactatattccaacaggtaagttaacAACGGGATATACTCATAATGCTGACCACGAAAGTCAATACCTGCAGCGACAAATCTCTCTGGATCGAAATTATCAGGATTTGGCCAGAGTTTAGGATCGC
Claims (21)
- 突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞であって、
(i)機能的ニコチンN−デメチラーゼをコードし、かつ配列番号2との少なくとも95%の配列同一性を有する配列を含む、それから構成される、またはそれから実質的に構成されるポリヌクレオチド、
(ii)(i)に記載した前記ポリヌクレオチドによりコードされるポリペプチド、
(iii)ニコチンN−デメチラーゼをコードし、かつ配列番号3との少なくとも95%の配列同一性を有する配列を含む、それから構成される、またはそれから実質的に構成されるポリペプチド、または
(iv)(i)に記載した前記単離ポリヌクレオチドを含む構築物、ベクターまたは発現ベクターを含み、
かつ前記ニコチンデメチラーゼの前記発現または活性が、前記ニコチンデメチラーゼの前記発現または活性が低減されなかった対照のたばこ植物体細胞と比較して低減される、たばこ植物体細胞。 - 前記たばこ植物体細胞が前記ニコチンN−デメチラーゼの前記発現または活性を減少させる1つ以上の突然変異を含む、請求項1に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞。
- 請求項1または請求項2に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞であって、CYP82E4ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異を含み、ここで前記突然変異は結果的に前記CYP82E4ニコチンデメチラーゼの発現または機能の低減をもたらすが、好ましくは、前記CYP82E4ニコチンデメチラーゼが、配列番号12〜16またはそれらのうちの二つ以上の組み合わせから成る群から選択され、好ましくは、前記突然変異が結果的に前記CYP82E4ニコチンデメチラーゼの変性をもたらし、かつ配列番号5のアミノ酸残基329、364、376、382、および458またはそれらのうちの二つ以上の組み合わせから成る群から選択される位置で発生し、好ましくは、前記突然変異が、a)配列番号5の位置329にあるトリプトファン残基のための終止コドン置換、b)配列番号5の位置364にあるリジン残基のためのアスパラギン置換、c)配列番号5の位置376にあるバリン残基のためのメチオニン置換、d)配列番号5の位置382にあるプロリン残基のためのセリン置換、d)配列番号5の位置458にあるプロリン残基のためのセリン置換、およびe)それらのうちの二つ以上の任意の組み合わせから成る群から選択される、たばこ植物体細胞。
- 請求項1〜3に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞であって、CYP82E5ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異を含むが、ここで前記突然変異は結果的に前記CYP82E5ニコチンデメチラーゼの発現または機能の低減をもたらし、好ましくは、前記CYP82E5ニコチンデメチラーゼが配列番号24または25またはその組み合わせから選択され、好ましくは、前記突然変異が結果的に前記CYP82E5ニコチンデメチラーゼの変性をもたらし、かつ配列番号17のアミノ酸残基422または449またはその組み合わせで発生し、好ましくは前記突然変異は、a)配列番号17の位置422にあるトリプトファン残基のための終止コドン置換、b)配列番号17の位置449にあるプロリン残基のためのロイシン置換、およびc)その組み合わせから成る群から選択される、たばこ植物体細胞。
- 請求項1〜4のいずれか1項に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞であって、CYP82E10ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異を含むが、ここで前記突然変異は結果的に前記CYP82E10ニコチンデメチラーゼの発現または機能の低減をもたらし、好ましくは前記CYP82E10ニコチンデメチラーゼが、配列番号32〜35またはそれらのうちの二つ以上の組み合わせから成る群から選択され、好ましくは、前記突然変異が結果的に前記CYP82E10ニコチンデメチラーゼの変性をもたらし、かつ配列番号26のアミノ酸残基79、107、382、419またはそれらのうちの二つ以上の組み合わせから成る群から選択される位置で発生し、好ましくは、前記突然変異は、a)配列番号26の位置79にあるグリシン残基のためのセリン置換、b)配列番号26の位置107にあるプロリン残基のためのセリン置換、c)配列番号26の位置382にあるプロリン残基のためのセリン置換、d)配列番号26の位置419にあるプロリン残基のためのセリン置換、およびe)その任意の組み合わせから成る群から選択される、たばこ植物体細胞。
- 請求項1または請求項2に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞であって、
(i)CYP82E4ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異を含み、ここで前記突然変異は結果的に前記CYP82E4ニコチンデメチラーゼの発現または機能の低減をもたらすが、好ましくは、前記CYP82E4ニコチンデメチラーゼが、配列番号12〜16またはそれらのうちの二つ以上の組み合わせから成る群から選択され、好ましくは、前記突然変異が結果的に前記CYP82E4ニコチンデメチラーゼの変性をもたらし、かつ配列番号5のアミノ酸残基329、364、376、382、および458またはそれらのうちの二つ以上の組み合わせから成る群から選択される位置で発生し、好ましくは、前記突然変異が、a)配列番号5の位置329にあるトリプトファン残基のための終止コドン置換、b)配列番号5の位置364にあるリジン残基のためのアスパラギン置換、c)配列番号5の位置376にあるバリン残基のためのメチオニン置換、d)配列番号5の位置382にあるプロリン残基のためのセリン置換、d)配列番号5の位置458にあるプロリン残基のためのセリン置換、およびe)それらのうちの二つ以上の任意の組み合わせから成る群から選択されるものと、
(ii)CYP82E5ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異を含むが、ここで前記突然変異は結果的に前記CYP82E5ニコチンデメチラーゼの発現または機能の低減をもたらし、好ましくは、前記CYP82E5ニコチンデメチラーゼが配列番号24または25またはその組み合わせから選択され、好ましくは、前記突然変異が結果的に前記CYP82E5ニコチンデメチラーゼの変性をもたらし、かつ配列番号17のアミノ酸残基422または449またはその組み合わせで発生し、好ましくは前記突然変異は、a)配列番号17の位置422にあるトリプトファン残基のための終止コドン置換、b)配列番号17の位置449にあるプロリン残基のためのロイシン置換、およびc)その組み合わせから成る群から選択されるものと、
(iii)CYP82E10ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異を含むが、ここで前記突然変異は結果的に前記CYP82E10ニコチンデメチラーゼの発現または機能の低減をもたらし、好ましくは前記CYP82E10ニコチンデメチラーゼが、配列番号32〜35またはそれらのうちの二つ以上の組み合わせから成る群から選択され、好ましくは、前記突然変異が結果的に前記CYP82E10ニコチンデメチラーゼの変性をもたらし、かつ配列番号26のアミノ酸残基79、107、382、419またはそれらのうちの二つ以上の組み合わせから成る群から選択される位置で発生し、好ましくは、前記突然変異は、a)配列番号26の位置79にあるグリシン残基のためのセリン置換、b)配列番号26の位置107にあるプロリン残基のためのセリン置換、c)配列番号26の位置382にあるプロリン残基のためのセリン置換、d)配列番号26の位置419にあるプロリン残基のためのセリン置換、およびe)その任意の組み合わせから成る群から選択されるものとをさらに含む、たばこ植物体細胞。 - 請求項1または請求項2に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞であって、
(i)CYP82E4ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異、CYP82E5ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異およびCYP82E10ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異であって、前記CYP82E4ニコチンデメチラーゼが配列番号13(W329Stop)に規定された配列を含み、前記CYP82E5ニコチンデメチラーゼが配列番号24(W422Stop)に規定された配列を含み、前記CYP82E10ニコチンデメチラーゼが配列番号33(G79S)に規定された配列を含むものであるか、または
(ii)CYP82E4ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異、CYP82E5ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異およびCYP82E10ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異であって、前記CYP82E4ニコチンデメチラーゼが配列番号13(W329Stop)に規定された配列を含み、前記CYP82E5ニコチンデメチラーゼが配列番号24(W422Stop)に規定された配列を含み、前記CYP82E10ニコチンデメチラーゼが配列番号34(P107S)に規定された配列を含むものであるか、または
(iii)CYP82E4ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異、CYP82E5ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異およびCYP82E10ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異であって、前記CYP82E4ニコチンデメチラーゼが配列番号13(W329Stop)に規定された配列を含み、前記CYP82E5ニコチンデメチラーゼが配列番号24(W422Stop)に規定された配列を含み、前記CYP82E10ニコチンデメチラーゼが配列番号35(P382S)に規定された配列を含むもの。
(iv)CYP82E4ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異、CYP82E5ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異およびCYP82E10ニコチンデメチラーゼをコードする遺伝子における1つ以上の突然変異であって、前記CYP82E4ニコチンデメチラーゼが配列番号13(W329Stop)に規定された配列を含み、前記CYP82E5ニコチンデメチラーゼが配列番号24(W422Stop)に規定された配列を含み、前記CYP82E10ニコチンデメチラーゼが配列番号32(P419S)に規定された配列を含むものをさらに備えた、たばこ植物体細胞。 - 前記突然変異がヘテロ接合性またはホモ接合性の突然変異である、請求項1〜7のいずれか1項に記載の、突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞。
- 前記植物体細胞が1%未満のニコチンからノルニコチンへの変換を持つ、請求項1〜8のいずれか1項に記載の突然変異体、非天然、または遺伝子組み換えのたばこ植物体細胞。
- 請求項1〜9のいずれか1項に記載の植物体細胞を含む、突然変異体、非天然、または遺伝子組み換えの植物体またはその構成要素またはその部分。
- 請求項10に記載の植物体に由来するバイオマス、種子、幹、花または葉を含む、植物材料。
- 請求項1〜9のいずれか1項に記載の植物体細胞、請求項10の植物体の少なくとも一部、または請求項11に記載の植物材料を含む、たばこ製品。
- ノルニコチンおよび/またはNNNのレベルが低減されたたばこ植物体を調製するための方法であって、前記方法が、
(a)(i)機能的ニコチンN−デメチラーゼをコードし、かつ配列番号2との少なくとも95%の配列同一性を有する配列を含む、それから構成される、またはそれから実質的に構成されるポリヌクレオチドを提供する工程と、
(b)1つ以上の突然変異を前記たばこ植物体の前記ポリヌクレオチドに挿入して突然変異体たばこ植物体を生成する工程と、
(c)随意に前記たばこ植物材料を乾燥処理する工程と、
(d)前記突然変異体たばこ植物体中のノルニコチンおよび/またはNNNの前記レベルを測定する工程とを含み、対照たばこ植物体と比較した前記突然変異体たばこ植物体中のノルニコチンおよび/またはNNNの前記レベルの低下が、前記突然変異体たばこ植物体における前記ノルニコチンおよび/またはNNNのレベルが低減されたことを示すものである、方法。 - 工程(b)における前記たばこ植物体が、突然変異体たばこ植物体であり、好ましくは、前記突然変異体たばこ植物体が1つ以上のさらなるニコチンN−デメチラーゼ遺伝子における1つ以上の突然変異を含む、請求項13に記載の方法。
- 前記突然変異体たばこ植物体が、CYP82E4、CYP82E5またはCYP82E10またはそれらのうちの二つ以上の組み合わせの群から構成される前記遺伝子における1つ以上のさらなる突然変異を持つ、請求項14に記載の方法。
- 前記植物または植物材料に由来する前記たばこ植物体、たばこ植物材料またはたばこの煙において、低いレベルのノルニコチンおよび/またはNNNと関連するたばこ植物体における1つ以上の突然変異を同定するための方法であって、前記方法が、
(a)対照たばこ植物体と比較して低いレベルのノルニコチンおよび/またはNNNを有するたばこ植物体を同定する工程と、
(b)工程(a)で同定された前記たばこ植物体に由来する核酸試料を提供する工程と、
(c)工程(b)での前記核酸試料を前記対照植物中には存在しない配列番号1の配列における1つ以上の突然変異の存在についてスクリーニングする工程と、
(d)工程(c)で同定された前記1つ以上の突然変異を、たばこ植物体中のノルニコチンおよび/またはNNNの前記レベルを低減させる周知の突然変異と随意に比較する工程と、
(e)低いレベルのノルニコチンおよび/またはNNNと関連した1つ以上の突然変異を同定する工程とを含む、方法。 - 対照植物材料と比較して低いレベルのノルニコチンを持つか、または対照植物材料と比較してその中に低いレベルの少なくともNNNを持つか、またはその組み合わせを持つ乾燥処理済み植物材料、好ましくは、乾燥処理済みの葉、または花を製作する方法であって、
(a)請求項10に記載の植物または請求項11に記載の前記植物材料を提供する工程と、
(b)随意に、前記植物材料をそこから収穫する工程と、
(c)少なくともノルニコチンまたはNNNの前記レベルが対照の乾燥処理済み植物材料よりも低くなるような期間だけ前記植物材料を乾燥処理する工程とを含む、方法。 - 単離ポリヌクレオチド配列であって、
(i)配列番号2の前記ヌクレオチド配列と、
(ii)配列番号2との少なくとも95%の配列同一性を有するヌクレオチド配列と、
(iii)(ii)によるヌクレオチド配列の断片であって、前記断片が配列番号2の少なくとも15個の隣接するヌクレオチドを含むものの中から選択される配列を含む、単離ポリヌクレオチド配列。 - 請求項18に記載のポリヌクレオチドによってコードされる単離ポリペプチド。
- 単離ポリペプチドであって、
(i)配列番号3の前記アミノ酸配列と、
(ii)配列番号3との少なくとも95%の配列同一性を持つアミノ酸配列と、
(iii)(ii)によるアミノ酸配列の断片であって、前記断片が配列番号3の少なくとも15個の隣接するアミノ酸を含み、前記15個のアミノ酸が、配列番号3の残基1〜15、330〜345、420〜450および480〜510に規定された少なくとも一つの配列と重なるものの中から選択される配列を含む、単離ポリペプチド。 - 請求項18に記載の単離ポリヌクレオチドを含む、構築物、ベクターまたは発現ベクター。
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