JP2012500003A - 形質転換植物 - Google Patents
形質転換植物 Download PDFInfo
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- JP2012500003A JP2012500003A JP2011522524A JP2011522524A JP2012500003A JP 2012500003 A JP2012500003 A JP 2012500003A JP 2011522524 A JP2011522524 A JP 2011522524A JP 2011522524 A JP2011522524 A JP 2011522524A JP 2012500003 A JP2012500003 A JP 2012500003A
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Abstract
Description
配列番号1
TCGAGACCCGATTGTTATTTTTAGACTGAGACAAAAAAGTAGAATCGTTGATTGTTAAAATTTAAAATTAGTTTCATTACGTTTCGATAAAAAAATGATTAGTTTATCATAGCTTAATTATAGCATTGATTTCTAAATTTGTTTTTTGACCACCCTTTTTTCTCTCTTTGGTGTTTTCTTAACATTAGAAGAACCCATAACAATGTACGTTCAAATTAATTAAAAACAATATTTCCAAGTTTTATATACGAAACTTGTTTTTTTTAATGAAAACAGTTGAATAGTTGATTATGAATTAGTTAGATCAATACTCAATATATGATCAATGATGTATATATATGAACTCAGTTGTTATACAAGAAATGAAAATGCTATTTAAATACAGATCATGAAGTGTTAAAAAGTGTCAGAATATGACATGAAGCGTTTTGTCCTACCGGGTATTCGAGTTATAGGTTTGGATCTCTCAAGAATATTTTGGGCCATACTAGTTATATTTGGGCTTAAGCGTTTTGCAAAGAGACGAGGAAGAAAGATTGGGTCAAGTTAACAAAACAGAGACACTCGTATTAGTTGGTACTTTGGTAGCAAGTCGATTTATTTGCCAGTAAAAACTTGGTACACAACTGACAACTCGTATCGTTATTAGTTTGTACTTGGTACCTTTGGTTCAAGAAAAAGTTGATATAGTTAAATCAGTTGTGTTCATGAGGTGATTGTGATTTAATTTGTTGACTAGGGCGATTCCTTCACATCACAATAACAAAGTTTTATAGATTTTTTTTTTATAACATTTTTGCCACGCTTCGTAAAGTTTGGTATTTACACCGCATTTTTCCCTGTACAAGAATTCATATATTATTTATTTATATACTCCAGTTGACAATTATAAGTTTATAACGTTTTTACAATTATTTAAATACCATGTGAAGATCCAAGAATATGTCTTACTTCTTCTTTGTGTAAGAAAACTAACTATATCACTATAATAAAATAATTCTAATCATTATATTTGTAAATATGCAGTTATTTGTCAATTTTGAATTTAGTATTTTAGACGTTATCACTTCAGCCAAATATGATTTGGATTTAAGTCCAAAATGCAATTTCGTACGTATCCCTCTTGTCGTCTAATGATTATTTCAATATTTCTTATATTATCCCTAACTACAGAGCTACATTTATATTGTATTCTAATGACAGGGAAACCTTCATAGAGATTCAGATAGATGAAATTGGTGGGAAACATCATTGAACAGGAAACTTTTAGCAAATCATATCGATTTATCTACAAAAGAATACGTAGCGTAATGAAGTCCACTTGTTGTGAATGACTATGATTTGATCAAATTAGTTAATTTTGTCGAATCATTTTTCTTTTTGATTTGATTAAGCTTTTAACTTGCACGAATGGTTCTCTTGTGAATAAACAGAATCTTTGAATTCAAACTATTTGATTAGTGAAAAGACAAAAGAAGATTCCTTGTTTTTATGTGATTAGTGATTTTGATGCATGAAAGGTACCTACGTACTACAAGAAAAATAAACATGTACGTAACTACGTATCAGCATGTAAAAGTATTTTTTTCCAAATAATTTATACTCATGATAGATTTTTTTTTTTTGAAATGTCAATTAAAAATGCTTTCTTAAATATTAATTTTAATTAATTAAATAAGGAAATATATTTATGCAAAACATCATCAACACATATCCAACTTCGAAAATCTCTATAGTACACAAGTAGAGAAATTAAATTTTACTAGATACAAACTTCCTAATCATCAAATATAAATGTTTACAAAACTAATTAAACCCACCACTAAAATTAACTAAAAATCCGAGCAAAGTGAGTGAACAAGACTTGATTTCAGGTTGATGTAGGACTAAAATGACTACGTATCAAACATCAACGATCATTTAGTTATGTATGAATGAATGTAGTCATTACTTGTAAAACAAAAATGCTTTGATTTGGATCAATCACTTCATGTGAACATTAGCAATTACATCAACCTTATTTTCACTATAAAACCCCATCTCAGTACCCTTCTGAAGTAATCAAATTAAGAGCAAAAGTCATTTAACTTAGG
従って、本発明の構築物におけるプロモーターは、実質的に配列番号1に示されているようなヌクレオチド配列またはその機能的な変異体もしくは機能的な断片を含んでいてもよい。SAG12プロモーター配列は、米国特許第5,689,042号に記載されているようなシロイヌナズナ(Arabidopsis thaliana)から得られたものであってもよい。プロモーターがSAG12である実施形態では、当然のことながら、プロモーターは、配列番号1の1〜2093の各塩基を含んでいてもよい。ただし、プロモーターの機能的な変異体もしくは機能的な断片が本発明の遺伝子構築物に使用されてもよい。
配列番号2
ATGGCGACTCTGAAGCCGTCATTTACTGTTTCTCCGCCGAATAGTAATCCGATTAGATTTGGAAGTTTTCCGCCGCAATGCTTTCTCCGTGTTCCGAAACCGCGGCGACTTATATTGCCTAGGTTTCGGAAGACGACTGGTGGTGGCGGCGGCTTGATTCGATGTGAGCTTCCAGATTTTCATCTATCAGCAACAGCAACTACTGTATCAGGTGTATCGACGGTGAATTTAGTGGATCAAGTTCAGATTCCTAAAGGTGAAATGTGGAGTGTTCACAAGTTTGGTGGGACTTGTGTGGGAAACTCTCAGAGGATCAGAAATGTAGCAGAGGTTATAATCAATGATAATTCCGAAAGAAAACTTGTGGTTGTCTCGGCGATGTCGAAGGTTACGGACATGATGTATGACTTAATCCGCAAGGCACAATCACGAGATGATTCTTATTTATCCGCGTTGGAAGCTGTCTTGGAAAAGCATCGTTTAACAGCTCGTGACCTTCTCGATGGAGATGATCTCGCTAGTTTCTTGTCACATTTGCATAATGATATTAGTAATCTTAAAGCAATGCTTCGTGCTATATACATAGCTGGCCATGCATCAGAGTCGTTTTCAGATTTTGTTGCAGGACATGGGGAGCTTTGGTCTGCTCAGATGCTATCATATGTTGTCAGAAAGACTGGGCTTGAGTGCAAGTGGATGGATACTAGAGACGTGCTCATTGTTAATCCCACCAGCTCTAATCAGGTTGATCCTGATTTTGGTGAATCTGAGAAGAGACTCGATAAATGGTTCTCCTTAAATCCGTCGAAAATTATTATTGCGACTGGGTTTATTGCTAGCACTCCGCAAAATATTCCAACAACTTTGAAAAGAGATGGGAGTGATTTCTCAGCAGCTATTATGGGTGCTTTATTGAGAGCTCGTCAAGTAACCATTTGGACAGATGTTGATGGTGTATACAGTGCGGATCCTCGTAAAGTTAATGAGGCAGTGATACTCCAGACACTTTCTTATCAAGAGGCCTGGGAAATGTCTTATTTTGGAGCAAATGTGTTACATCCTCGCACCATCATTCCTGTGATGCGATATAATATTCCGATTGTGATTAGAAATATTTTCAATCTCTCTGCACCGGGAACAATAATCTGTCAACCTCCTGAAGATGATTATGACCTTAAACTGACAACTCCTGTCAAAGGGTTTGCAACTATTGACAATTTGGCCCTCATAAATGTTGAAGGTACTGGAATGGCTGGTGTACCCGGTACTGCAAGTGACATTTTTGGCTGTGTAAAAGATGTTGGAGCTAATGTGATTATGATATCAGCTGCTAGCAGTGAGCATTCTGTGTGCTTTGCTGTGCCTGAGAAGGAAGTAAACGCAGTCTCTGAGGCATTGCGGTCGAGATTTAGTGAAGCTTTACAAGCGGGACGTCTTTCTCAGATTGAGGTGATACCAAACTGTAGCATCTTAGCTGCAGTCGGCCAGAAAATGGCTAGTACACCTGGAGTTAGTTGTACACTTTTCAGTGCTTTGGCGAAGGCTAATATTAATGTCCGAGCTATATCTCARGGTTGTTCTGAGTACAATGTTACTGTCGTTATTAAACGTGAAGATAGCGTTAAGGCGTTAAGAGCTGTACACTCGAGGTTTTTCTTGTCAAGAACAACATTAGCAATGGGAATCGTAGGACCGGGCTTGATTGGTGCAACATTACTTGACCAGCTGCGGGATCAGGCTGCTGTTCTCAAACAAGAATTTAACATTGATCTGCGTGTTTTGGGAATCACTGGTTCAAAGAAGATGTTATTGAGTGACATTGGTATTGATTTGTCGAGATGGAGAGAACTTCTAAACGAGAAGGGAACAGAGGCGGATTTGGATAAATTCACTCAACAAGTGCATGGAAATCATTTTATCCCCAACTCTGTAGTGGTTGATTGTACAGCAGACTCTGCTATTGCAAGCCGTTACTATGATTGGTTACGAAAGGGAATTCATGTCATTACCCCAAATAAAAAGGCTAACTCAGGTCCCCTCGATCAGTACTTGAAACTGAGAGATCTTCAAAGGAAATCCTACACTCATTACTTCTACGAAGCCACTGTTGGAGCTGGTCTTCCAATTATCAGCACTTTACGTGGTCTCCTTGAGACAGGAGATAAGATACTACGCATAGAGGGCATTTGCAGTGGAACTTTGAGTTATCTATTCAACAATTTTGTTGGAGATCGAAGTTTCAGCGAGGTTGTCACTGAAGCAAAGAACGCAGGTTTCACTGAGCCTGATCCAAGAGATGATTTATCTGGAACTGATGTTGCAAGGAAGGTGATTATCCTCGCTCGAGAATCTGGACTGAAATTGGACCTCGCTGATCTCCCCATTAGAAGTCTCGTACCAGAACCTCTAAAAGGATGCACTTCTGTTGAAGAATTCATGGAGAAACTCCCACAGTACGATGGAGACCTAGCAAAAGAAAGGCTAGATGCTGAAAACTCTGGGGAAGTTCTGAGATATGTTGGAGTGGTGGACGCTGTTAACCAAAAGGGAACAGTTGAACTTCGAAGATACAAGAAAGAACATCCATTTGCGCAGCTCGCAGGTTCAGACAACATAATAGCCTTCACAACGACAAGGTACAAGGATCATCCACTTATAGTCCGAGGACCTGGAGCTGGTGCTCAAGTCACGGCCGGTGGTATATTCAGCGACATACTAAGGCTTGCATCTTATCTCGGTGCACCGTCTTAA
配列番号2(すなわち、Q443A)では、強調されているGCTはアラニンをコードする突然変異型Gln443に対応し、強調されているCARは野生型Gln524に対応する。ここで、RはGまたはAのいずれであってもよい。
配列番号3
ATGGCGACTCTGAAGCCGTCATTTACTGTTTCTCCGCCGAATAGTAATCCGATTAGATTTGGAAGTTTTCCGCCGCAATGCTTTCTCCGTGTTCCGAAACCGCGGCGACTTATATTGCCTAGGTTTCGGAAGACGACTGGTGGTGGCGGCGGCTTGATTCGATGTGAGCTTCCAGATTTTCATCTATCAGCAACAGCAACTACTGTATCAGGTGTATCGACGGTGAATTTAGTGGATCAAGTTCAGATTCCTAAAGGTGAAATGTGGAGTGTTCACAAGTTTGGTGGGACTTGTGTGGGAAACTCTCAGAGGATCAGAAATGTAGCAGAGGTTATAATCAATGATAATTCCGAAAGAAAACTTGTGGTTGTCTCGGCGATGTCGAAGGTTACGGACATGATGTATGACTTAATCCGCAAGGCACAATCACGAGATGATTCTTATTTATCCGCGTTGGAAGCTGTCTTGGAAAAGCATCGTTTAACAGCTCGTGACCTTCTCGATGGAGATGATCTCGCTAGTTTCTTGTCACATTTGCATAATGATATTAGTAATCTTAAAGCAATGCTTCGTGCTATATACATAGCTGGCCATGCATCAGAGTCGTTTTCAGATTTTGTTGCAGGACATGGGGAGCTTTGGTCTGCTCAGATGCTATCATATGTTGTCAGAAAGACTGGGCTTGAGTGCAAGTGGATGGATACTAGAGACGTGCTCATTGTTAATCCCACCAGCTCTAATCAGGTTGATCCTGATTTTGGTGAATCTGAGAAGAGACTCGATAAATGGTTCTCCTTAAATCCGTCGAAAATTATTATTGCGACTGGGTTTATTGCTAGCACTCCGCAAAATATTCCAACAACTTTGAAAAGAGATGGGAGTGATTTCTCAGCAGCTATTATGGGTGCTTTATTGAGAGCTCGTCAAGTAACCATTTGGACAGATGTTGATGGTGTATACAGTGCGGATCCTCGTAAAGTTAATGAGGCAGTGATACTCCAGACACTTTCTTATCAAGAGGCCTGGGAAATGTCTTATTTTGGAGCAAATGTGTTACATCCTCGCACCATCATTCCTGTGATGCGATATAATATTCCGATTGTGATTAGAAATATTTTCAATCTCTCTGCACCGGGAACAATAATCTGTCAACCTCCTGAAGATGATTATGACCTTAAACTGACAACTCCTGTCAAAGGGTTTGCAACTATTGACAATTTGGCCCTCATAAATGTTGAAGGTACTGGAATGGCTGGTGTACCCGGTACTGCAAGTGACATTTTTGGCTGTGTAAAAGATGTTGGAGCTAATGTGATTATGATATCACARGCTAGCAGTGAGCATTCTGTGTGCTTTGCTGTGCCTGAGAAGGAAGTAAACGCAGTCTCTGAGGCATTGCGGTCGAGATTTAGTGAAGCTTTACAAGCGGGACGTCTTTCTCAGATTGAGGTGATACCAAACTGTAGCATCTTAGCTGCAGTCGGCCAGAAAATGGCTAGTACACCTGGAGTTAGTTGTACACTTTTCAGTGCTTTGGCGAAGGCTAATATTAATGTCCGAGCTATATCTGCTGGTTGTTCTGAGTACAATGTTACTGTCGTTATTAAACGTGAAGATAGCGTTAAGGCGTTAAGAGCTGTACACTCGAGGTTTTTCTTGTCAAGAACAACATTAGCAATGGGAATCGTAGGACCGGGCTTGATTGGTGCAACATTACTTGACCAGCTGCGGGATCAGGCTGCTGTTCTCAAACAAGAATTTAACATTGATCTGCGTGTTTTGGGAATCACTGGTTCAAAGAAGATGTTATTGAGTGACATTGGTATTGATTTGTCGAGATGGAGAGAACTTCTAAACGAGAAGGGAACAGAGGCGGATTTGGATAAATTCACTCAACAAGTGCATGGAAATCATTTTATCCCCAACTCTGTAGTGGTTGATTGTACAGCAGACTCTGCTATTGCAAGCCGTTACTATGATTGGTTACGAAAGGGAATTCATGTCATTACCCCAAATAAAAAGGCTAACTCAGGTCCCCTCGATCAGTACTTGAAACTGAGAGATCTTCAAAGGAAATCCTACACTCATTACTTCTACGAAGCCACTGTTGGAGCTGGTCTTCCAATTATCAGCACTTTACGTGGTCTCCTTGAGACAGGAGATAAGATACTACGCATAGAGGGCATTTGCAGTGGAACTTTGAGTTATCTATTCAACAATTTTGTTGGAGATCGAAGTTTCAGCGAGGTTGTCACTGAAGCAAAGAACGCAGGTTTCACTGAGCCTGATCCAAGAGATGATTTATCTGGAACTGATGTTGCAAGGAAGGTGATTATCCTCGCTCGAGAATCTGGACTGAAATTGGACCTCGCTGATCTCCCCATTAGAAGTCTCGTACCAGAACCTCTAAAAGGATGCACTTCTGTTGAAGAATTCATGGAGAAACTCCCACAGTACGATGGAGACCTAGCAAAAGAAAGGCTAGATGCTGAAAACTCTGGGGAAGTTCTGAGATATGTTGGAGTGGTGGACGCTGTTAACCAAAAGGGAACAGTTGAACTTCGAAGATACAAGAAAGAACATCCATTTGCGCAGCTCGCAGGTTCAGACAACATAATAGCCTTCACAACGACAAGGTACAAGGATCATCCACTTATAGTCCGAGGACCTGGAGCTGGTGCTCAAGTCACGGCCGGTGGTATATTCAGCGACATACTAAGGCTTGCATCTTATCTCGGTGCACCGTCTTAA
配列番号3(すなわち、Q524A)では、強調されているGCTはアラニンをコードする突然変異型Gln524に対応し、強調されているCARは野生型Gln443に対応する。ここで、RはGまたはAのいずれであってもよい。
配列番号4
ATGGCGACTCTGAAGCCGTCATTTACTGTTTCTCCGCCGAATAGTAATCCGATTAGATTTGGAAGTTTTCCGCCGCAATGCTTTCTCCGTGTTCCGAAACCGCGGCGACTTATATTGCCTAGGTTTCGGAAGACGACTGGTGGTGGCGGCGGCTTGATTCGATGTGAGCTTCCAGATTTTCATCTATCAGCAACAGCAACTACTGTATCAGGTGTATCGACGGTGAATTTAGTGGATCAAGTTCAGATTCCTAAAGGTGAAATGTGGAGTGTTCACAAGTTTGGTGGGACTTGTGTGGGAAACTCTCAGAGGATCAGAAATGTAGCAGAGGTTATAATCAATGATAATTCCGAAAGAAAACTTGTGGTTGTCTCGGCGATGTCGAAGGTTACGGACATGATGTATGACTTAATCCGCAAGGCACAATCACGAGATGATTCTTATTTATCCGCGTTGGAAGCTGTCTTGGAAAAGCATCGTTTAACAGCTCGTGACCTTCTCGATGGAGATGATCTCGCTAGTTTCTTGTCACATTTGCATAATGATATTAGTAATCTTAAAGCAATGCTTCGTGCTATATACATAGCTGGCCATGCATCAGAGTCGTTTTCAGATTTTGTTGCAGGACATGGGGAGCTTTGGTCTGCTCAGATGCTATCATATGTTGTCAGAAAGACTGGGCTTGAGTGCAAGTGGATGGATACTAGAGACGTGCTCATTGTTAATCCCACCAGCTCTAATCAGGTTGATCCTGATTTTGGTGAATCTGAGAAGAGACTCGATAAATGGTTCTCCTTAAATCCGTCGAAAATTATTATTGCGACTGGGTTTATTGCTAGCACTCCGCAAAATATTCCAACAACTTTGAAAAGAGATGGGAGTGATTTCTCAGCAGCTATTATGGGTGCTTTATTGAGAGCTCGTCAAGTAACCATTTGGACAGATGTTGATGGTGTATACAGTGCGGATCCTCGTAAAGTTAATGAGGCAGTGATACTCCAGACACTTTCTTATCAAGAGGCCTGGGAAATGTCTTATTTTGGAGCAAATGTGTTACATCCTCGCACCATCATTCCTGTGATGCGATATAATATTCCGATTGTGATTAGAAATATTTTCAATCTCTCTGCACCGGGAACAATAATCTGTCAACCTCCTGAAGATGATTATGACCTTAAACTGACAACTCCTGTCAAAGGGTTTGCAACTATTGACAATTTGGCCCTCATAAATGTTGAAGGTACTGGAATGGCTGGTGTACCCGGTACTGCAAGTGACATTTTTGGCTGTGTAAAAGATGTTGGAGCTAATGTGATTATGATATCAGCTGCTAGCAGTGAGCATTCTGTGTGCTTTGCTGTGCCTGAGAAGGAAGTAAACGCAGTCTCTGAGGCATTGCGGTCGAGATTTAGTGAAGCTTTACAAGCGGGACGTCTTTCTCAGATTGAGGTGATACCAAACTGTAGCATCTTAGCTGCAGTCGGCCAGAAAATGGCTAGTACACCTGGAGTTAGTTGTACACTTTTCAGTGCTTTGGCGAAGGCTAATATTAATGTCCGAGCTATATCTGCTGGTTGTTCTGAGTACAATGTTACTGTCGTTATTAAACGTGAAGATAGCGTTAAGGCGTTAAGAGCTGTACACTCGAGGTTTTTCTTGTCAAGAACAACATTAGCAATGGGAATCGTAGGACCGGGCTTGATTGGTGCAACATTACTTGACCAGCTGCGGGATCAGGCTGCTGTTCTCAAACAAGAATTTAACATTGATCTGCGTGTTTTGGGAATCACTGGTTCAAAGAAGATGTTATTGAGTGACATTGGTATTGATTTGTCGAGATGGAGAGAACTTCTAAACGAGAAGGGAACAGAGGCGGATTTGGATAAATTCACTCAACAAGTGCATGGAAATCATTTTATCCCCAACTCTGTAGTGGTTGATTGTACAGCAGACTCTGCTATTGCAAGCCGTTACTATGATTGGTTACGAAAGGGAATTCATGTCATTACCCCAAATAAAAAGGCTAACTCAGGTCCCCTCGATCAGTACTTGAAACTGAGAGATCTTCAAAGGAAATCCTACACTCATTACTTCTACGAAGCCACTGTTGGAGCTGGTCTTCCAATTATCAGCACTTTACGTGGTCTCCTTGAGACAGGAGATAAGATACTACGCATAGAGGGCATTTGCAGTGGAACTTTGAGTTATCTATTCAACAATTTTGTTGGAGATCGAAGTTTCAGCGAGGTTGTCACTGAAGCAAAGAACGCAGGTTTCACTGAGCCTGATCCAAGAGATGATTTATCTGGAACTGATGTTGCAAGGAAGGTGATTATCCTCGCTCGAGAATCTGGACTGAAATTGGACCTCGCTGATCTCCCCATTAGAAGTCTCGTACCAGAACCTCTAAAAGGATGCACTTCTGTTGAAGAATTCATGGAGAAACTCCCACAGTACGATGGAGACCTAGCAAAAGAAAGGCTAGATGCTGAAAACTCTGGGGAAGTTCTGAGATATGTTGGAGTGGTGGACGCTGTTAACCAAAAGGGAACAGTTGAACTTCGAAGATACAAGAAAGAACATCCATTTGCGCAGCTCGCAGGTTCAGACAACATAATAGCCTTCACAACGACAAGGTACAAGGATCATCCACTTATAGTCCGAGGACCTGGAGCTGGTGCTCAAGTCACGGCCGGTGGTATATTCAGCGACATACTAAGGCTTGCATCTTATCTCGGTGCACCGTCTTAA
配列番号4(すなわち、Q443A;Q524A)では、強調されているGCTは、どちらもアラニンをコードする突然変異型Gln443およびGln524に対応する。
配列番号5
MATLKPSFTVSPPNSNPIRFGSFPPQCFLRVPKPRRLILPRFRKTTGGGGGLIRCELPDF
HLSATATTVSGVSTVNLVDQVQIPKGEMWSVHKFGGTCVGNSQRIRNVAEVIINDNSERK
LVVVSAMSKVTDMMYDLIRKAQSRDDSYLSALEAVLEKHRLTARDLLDGDDLASFLSHLH
NDISNLKAMLRAIYIAGHASESFSDFVAGHGELWSAQMLSYVVRKTGLECKWMDTRDVLI
VNPTSSNQVDPDFGESEKRLDKWFSLNPSKIIIATGFIASTPQNIPTTLKRDGSDFSAAI
MGALLRARQVTIWTDVDGVYSADPRKVNEAVILQTLSYQEAWEMSYFGANVLHPRTIIPV
MRYNIPIVIRNIFNLSAPGTIICQPPEDDYDLKLTTPVKGFATIDNLALINVEGTGMAGV
PGTASDIFGCVKDVGANVIMISAASSEHSVCFAVPEKEVNAVSEALRSRFSEALQAGRLS
QIEVIPNCSILAAVGQKMASTPGVSCTLFSALAKANINVRAISQGCSEYNVTVVIKREDS
VKALRAVHSRFFLSRTTLAMGIVGPGLIGATLLDQLRDQAAVLKQEFNIDLRVLGITGSK
KMLLSDIGIDLSRWRELLNEKGTEADLDKFTQQVHGNHFIPNSVVVDCTADSAIASRYYD
WLRKGIHVITPNKKANSGPLDQYLKLRDLQRKSYTHYFYEATVGAGLPIISTLRGLLETG
DKILRIEGICSGTLSYLFNNFVGDRSFSEVVTEAKNAGFTEPDPRDDLSGTDVARKVIIL
ARESGLKLDLADLPIRSLVPEPLKGCTSVEEFMEKLPQYDGDLAKERLDAENSGEVLRYV
GVVDAVNQKGTVELRRYKKEHPFAQLAGSDNIIAFTTTRYKDHPLIVRGPGAGAQVTAGG
IFSDILRLASYLGAPS 配列番号5では、位置443にある突然変異型アラニン(A)、および位置524にある野生型グルタミン(Q)が強調されている。
配列番号6
MATLKPSFTVSPPNSNPIRFGSFPPQCFLRVPKPRRLILPRFRKTTGGGGGLIRCELPDF
HLSATATTVSGVSTVNLVDQVQIPKGEMWSVHKFGGTCVGNSQRIRNVAEVIINDNSERK
LVVVSAMSKVTDMMYDLIRKAQSRDDSYLSALEAVLEKHRLTARDLLDGDDLASFLSHLH
NDISNLKAMLRAIYIAGHASESFSDFVAGHGELWSAQMLSYVVRKTGLECKWMDTRDVLI
VNPTSSNQVDPDFGESEKRLDKWFSLNPSKIIIATGFIASTPQNIPTTLKRDGSDFSAAI
MGALLRARQVTIWTDVDGVYSADPRKVNEAVILQTLSYQEAWEMSYFGANVLHPRTIIPV
MRYNIPIVIRNIFNLSAPGTIICQPPEDDYDLKLTTPVKGFATIDNLALINVEGTGMAGV
PGTASDIFGCVKDVGANVIMISQASSEHSVCFAVPEKEVNAVSEALRSRFSEALQAGRLS
QIEVIPNCSILAAVGQKMASTPGVSCTLFSALAKANINVRAISAGCSEYNVTVVIKREDS
VKALRAVHSRFFLSRTTLAMGIVGPGLIGATLLDQLRDQAAVLKQEFNIDLRVLGITGSK
KMLLSDIGIDLSRWRELLNEKGTEADLDKFTQQVHGNHFIPNSVVVDCTADSAIASRYYD
WLRKGIHVITPNKKANSGPLDQYLKLRDLQRKSYTHYFYEATVGAGLPIISTLRGLLETG
DKILRIEGICSGTLSYLFNNFVGDRSFSEVVTEAKNAGFTEPDPRDDLSGTDVARKVIIL
ARESGLKLDLADLPIRSLVPEPLKGCTSVEEFMEKLPQYDGDLAKERLDAENSGEVLRYV
GVVDAVNQKGTVELRRYKKEHPFAQLAGSDNIIAFTTTRYKDHPLIVRGPGAGAQVTAGGIFSDILRLASYLGAPS
配列番号6では、位置524にある突然変異型アラニン(A)および位置443にある野生型グルタミン(Q)が強調されている。
配列番号7
MATLKPSFTVSPPNSNPIRFGSFPPQCFLRVPKPRRLILPRFRKTTGGGGGLIRCELPDF
HLSATATTVSGVSTVNLVDQVQIPKGEMWSVHKFGGTCVGNSQRIRNVAEVIINDNSERK
LVVVSAMSKVTDMMYDLIRKAQSRDDSYLSALEAVLEKHRLTARDLLDGDDLASFLSHLH
NDISNLKAMLRAIYIAGHASESFSDFVAGHGELWSAQMLSYVVRKTGLECKWMDTRDVLI
VNPTSSNQVDPDFGESEKRLDKWFSLNPSKIIIATGFIASTPQNIPTTLKRDGSDFSAAI
MGALLRARQVTIWTDVDGVYSADPRKVNEAVILQTLSYQEAWEMSYFGANVLHPRTIIPV
MRYNIPIVIRNIFNLSAPGTIICQPPEDDYDLKLTTPVKGFATIDNLALINVEGTGMAGV
PGTASDIFGCVKDVGANVIMISAASSEHSVCFAVPEKEVNAVSEALRSRFSEALQAGRLS
QIEVIPNCSILAAVGQKMASTPGVSCTLFSALAKANINVRAISAGCSEYNVTVVIKREDS
VKALRAVHSRFFLSRTTLAMGIVGPGLIGATLLDQLRDQAAVLKQEFNIDLRVLGITGSK
KMLLSDIGIDLSRWRELLNEKGTEADLDKFTQQVHGNHFIPNSVVVDCTADSAIASRYYD
WLRKGIHVITPNKKANSGPLDQYLKLRDLQRKSYTHYFYEATVGAGLPIISTLRGLLETG
DKILRIEGICSGTLSYLFNNFVGDRSFSEVVTEAKNAGFTEPDPRDDLSGTDVARKVIIL
ARESGLKLDLADLPIRSLVPEPLKGCTSVEEFMEKLPQYDGDLAKERLDAENSGEVLRYV
GVVDAVNQKGTVELRRYKKEHPFAQLAGSDNIIAFTTTRYKDHPLIVRGPGAGAQVTAGGIFSDILRLASYLGAPS
配列番号7では、位置443および524にある突然変異型アラニン(A)が強調されている。
(i)植物細胞を、第1の態様の遺伝子構築物または第2の態様のベクターで形質転換することと、
(ii)形質転換された細胞から植物を再生することと、
を含む。
緑色の葉または黄変した葉に対してトレオニン濃度に関する試験を行なった。葉片を採取し、分析に使用し、測定は、葉片当たりのトレオニンの量に基づくか(すなわち、thrの量/葉の面積)、上澄み中のタンパク質の量と関連づけられた(すなわち、トレオニンの量/1mgのタンパク質)。葉片を規定の体積の水と一緒にすり潰し、遠心分離して不溶な葉のくずを沈降させた。次いで、このプロセスで得られた上澄みをPhenomenex EZfaast Kitを用いて処理した。これは、標準的なLC/MS装置を用いて定量化することができるように抽出物中のアミノ酸を誘導体化するために使用される専売キットである。
本発明者らは、シロイヌナズナ(At4g19710)由来の二機能性AK:HSDH野生型配列に対して部位特異的突然変異誘発を行なった。従って、最初に下記プライマーを用いるPCRによってシロイヌナズナ由来の葉特異的cDNAライブラリーから野生型配列を単離した:
正方向At4g19710 ATGGCGACTCTGAAGCCGTCATTTAC(配列番号8);
逆方向At4g19710 TTAAGACGGTGCACCGAGATAAGATGC(配列番号9)。
正方向Gln443 GTGATTATGATATCAGCTGCTAGCAGTGAGCATTCTG(配列番号10);および
逆方向Gln443 CAGAATGCTCACTGCTAGCAGCTGATATCATAATTCAC(配列番号11)。
正方向Gln524 GTCCGAGCTATATCTGCTGGTTGTTCTGAGTACAATG(配列番号12);および
逆方向Gln524 CATTGATCTCAGAACAACCAGCAGATATAGCTCGGAC(配列番号13)。
タバコ(Nicotiana tabacum)植物(品種K326)を使用して葉片を得、これを老化特異的プロモーターSAG12(その配列は図6、すなわち配列番号1に示されている)の制御下で、目的の遺伝子(すなわち、突然変異型AK:HSDH、図7(すなわち配列番号2、3および4)に示されているAK:HSDH配列)を保持するバイナリーベクターで(電気穿孔法によって)予め形質転換されていたアグロバクテリウム・ツメファシエンスと共培養した。バイナリーベクターがプロモーターを含むがAK:HSDH遺伝子を含まない対照個体群を同時に育てた。次いで、これらの葉片を組織培養プロトコルによって処理して小植物(Horsch et al. Science 227: 1229-1231, 1985)を得た。各小植物は、細菌からDNAを抽出し、植物のゲノムDNAに組み込む単一の形質転換イベントから得られたものであった。これらの植物を温室に移し、成熟するまで育てた。成熟した葉を植物から切り離し、ポリテン袋に入れて35℃で72時暗所に置いた。この時間の後、この葉を使用して葉片を得、上記実施例1に記載したように、トレオニン含有量を評価した。
SAG12:アスパラギン酸キナーゼ(pBNP 138-0253-001)の実験個体群から選択された植物細胞株をノースカロライナで2008年の間現場で育てた。この葉を火力乾燥し、選択した遊離アミノ酸の存在を分析した。この分析は、内部および外部標準較正によって確認されているLC/MSによるものであった。分析物が較正に使用される範囲を超える場合は、表2では「>S」として表中に示し、図では、関連する棒の上に星で示す。
Claims (25)
- トレオニン非感受性アスパラギン酸キナーゼ活性を有するポリペプチドをコードするコード配列に作動可能に連結された老化特異的プロモーターを含む遺伝子構築物。
- 前記老化特異的プロモーターが、シロイヌナズナの老化関連遺伝子から単離されたものである、請求項1に記載の遺伝子構築物。
- 前記老化特異的プロモーターが、SAG12、SAG13、SAG101、SAG21およびSAG18からなる群またはその機能的な変異体もしくは断片から選択される、請求項1または2に記載の遺伝子構築物。
- 前記老化特異的プロモーターが、SAG12またはその機能的な変異体もしくは断片である、請求項3に記載の遺伝子構築物。
- 前記プロモーターが、実質的に配列番号1に示されているようなヌクレオチド配列またはその機能的な変異体もしくは断片を含む、請求項3または4に記載の遺伝子構築物。
- 前記ポリペプチドが、トレオニン非感受性アスパラギン酸キナーゼ(AK)またはその機能的な変異体もしくは断片である、前記請求項のいずれかに記載の遺伝子構築物。
- 前記ポリペプチドが、トレオニン非感受性二機能性アスパラギン酸キナーゼ−ホモセリン脱水素酵素(AK−HSDH)である、前記請求項に記載の遺伝子構築物。
- トレオニン非感受性アスパラギン酸キナーゼ活性を有する前記ポリペプチドをコードする前記コード配列が、シロイヌナズナ属、トウモロコシ属、フラベリア属またはクレオメ属に由来する、前記請求項のいずれかに記載の遺伝子構築物。
- トレオニン非感受性アスパラギン酸キナーゼ活性を有する前記ポリペプチドをコードする前記コード配列が、シロイヌナズナ属、好ましくはシロイヌナズナ(A.thaliana)に由来する、前記請求項のいずれかに記載の遺伝子構築物。
- 前記AK−HSDHの少なくとも1つのトレオニン結合部位が突然変異されている、請求項7〜9のいずれか1項に記載の遺伝子構築物。
- 前記シロイヌナズナAK−HSDHがGln443および/またはGln524で突然変異されている、請求項7〜10のいずれか1項に記載の遺伝子構築物。
- 前記AK−HSDHがGln443およびGln524で突然変異されている、請求項7〜11のいずれか1項に記載の遺伝子構築物。
- 請求項1〜12のいずれか1項に記載の遺伝子構築物を含むベクター。
- 同じ条件下で栽培された対応する野生型植物よりも高濃度のトレオニンをその葉に蓄積させる形質転換植物の産生方法であって、
i)植物細胞を請求項1〜12のいずれか1項に記載の遺伝子構築物または請求項13に記載のベクターで形質転換することと、
ii)前記形質転換された細胞から植物を再生することと、
を含む方法。 - 請求項1〜13のいずれか1項に記載の遺伝子構築物または請求項13に記載のベクターを含む細胞。
- 請求項1〜9のいずれか1項に記載の遺伝子構築物または請求項13に記載のベクターを含む形質転換植物。
- 前記植物がアブラナ科、イネ科またはナス科由来である、請求項16に記載の形質転換植物。
- 前記植物がタバコ植物である、請求項16または17に記載の形質転換植物。
- 請求項16〜18のいずれか1項に記載の形質転換植物から得ることができる植物繁殖用製品。
- 前記植物繁殖用製品が種子である、請求項19に記載の植物繁殖用製品。
- 同じ条件下で栽培された野生型植物から採取した収穫された葉の中の対応するトレオニン濃度よりも高濃度のトレオニンを含有する収穫された葉であって、前記葉が、請求項16〜18のいずれか1項に記載の形質転換植物から収穫されたものまたは請求項14に記載の方法によって生産されたものである葉。
- 突然変異型タバコ植物から得られたトレオニン豊富なタバコを含む喫煙物品であって、前記突然変異体が老化した葉の中にトレオニンを過剰産出することができる喫煙物品。
- 前記突然変異型タバコ植物が、請求項1〜12のいずれか1項に記載の遺伝子構築物または請求項13に記載のベクターで形質転換されている、請求項22に記載の喫煙物品。
- 植物の適応度を損なうことなく対応する野生型の濃度よりも植物の葉の中のトレオニン濃度を上昇させる方法であって、植物代謝を変化させて葉の老化開始後にトレオニンの産生の上昇を達成することを含む方法。
- 前記方法が、前記植物を請求項1〜12のいずれか1項に記載の構築物または請求項13に記載のベクターで形質転換することを含む、請求項24に記載の方法。
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US5773691A (en) * | 1992-03-19 | 1998-06-30 | E. I. Du Pont De Nemours And Company | Chimeric genes and methods for increasing the lysine and threonine content of the seeds of plants |
US6969782B2 (en) * | 1993-10-06 | 2005-11-29 | Ajinomoto Co., Inc. | Method of producing transgenic plants having improved amino acid composition |
BR9510174A (pt) * | 1994-07-08 | 1997-11-04 | Du Pont | Gene quimérico planta sementes método para aumentar o teor de treonina das sementes de plantas |
WO1996038574A1 (en) * | 1995-05-31 | 1996-12-05 | Pioneer Hi-Bred International, Inc. | Methods of increasing accumulation of essential amino acids in seeds |
US5959179A (en) * | 1996-03-13 | 1999-09-28 | Monsanto Company | Method for transforming soybeans |
CN1242050A (zh) * | 1996-11-18 | 2000-01-19 | 阿韦贝公司 | 超量产生至少两个天冬氨酸家族氨基酸的具有天然高含水量的转基因植物 |
US20020148003A1 (en) * | 1999-06-07 | 2002-10-10 | Avebe | Transgenic plant or plants with a naturally high water content overproducing at least two amino acids of the aspartate family |
DE10014546A1 (de) * | 2000-03-23 | 2001-09-27 | Degussa | Für das dapC-Gen kodierende Nukleotidsequenzen und Verfahren zur Herstellung von L-Lysin |
US6730832B1 (en) * | 2001-09-10 | 2004-05-04 | Luis Mayan Dominguez | High threonine producing lines of Nicotiana tobacum and methods for producing |
CN1820073A (zh) * | 2003-05-07 | 2006-08-16 | 雷尼森有限责任公司 | 一种或多种氨基酸的水平增加的植物 |
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CL2011000302A1 (es) | 2011-06-17 |
GB0814927D0 (en) | 2008-09-24 |
EP2310496A1 (en) | 2011-04-20 |
BRPI0917658B1 (pt) | 2019-01-02 |
AU2009281139A1 (en) | 2010-02-18 |
EP2310496B1 (en) | 2016-11-02 |
AP2011005612A0 (en) | 2011-04-30 |
NZ590889A (en) | 2012-08-31 |
KR20110041575A (ko) | 2011-04-21 |
CN102203243B (zh) | 2015-05-20 |
MX2011001649A (es) | 2011-05-02 |
CA2732800A1 (en) | 2010-02-18 |
HK1162585A1 (en) | 2012-08-31 |
SG193797A1 (en) | 2013-10-30 |
GB2462645A (en) | 2010-02-17 |
RU2011109341A (ru) | 2012-09-20 |
ZA201100905B (en) | 2012-07-25 |
JP2015128439A (ja) | 2015-07-16 |
US20110226267A1 (en) | 2011-09-22 |
CN102203243A (zh) | 2011-09-28 |
BRPI0917658A2 (pt) | 2014-11-18 |
JP5795959B2 (ja) | 2015-10-14 |
WO2010018234A1 (en) | 2010-02-18 |
US20120199144A2 (en) | 2012-08-09 |
RU2515927C2 (ru) | 2014-05-20 |
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