DOMINANT-NEGATIVE GENETIC MANIPULATION TO MAKE LOW-NICOTINE TOBACCO PRODUCTS

1 . A chimeric nucleic acid construct comprising at least one transcription repressor sequence in translational fusion with at least one transcription factor sequence encoding for a transcription factor or fragment thereof that positively regulates nicotine biosynthesis, wherein the chimeric nucleic acid construct is operably linked to a heterologous nucleic acid.

2 . The chimeric nucleic acid of claim 1 , wherein the transcription repressor sequence encodes for a transcription repressor selected from the group consisting of: Engrailed (En 298 ) (SEQ ID NO: 11), SRDX (SEQ ID NO: 12), AtERF4 (SEQ ID NO: 13), NtERF3 (SEQ ID NO: 15), SUPERMAN (SEQ ID NO: 17), LEUNIG (SEQ ID NO: 18), and SEUSS (SEQ ID NO: 19).

3 . The chimeric nucleic acid of claim 1 , wherein the transcription factor sequence encoding for a transcription factor or fragment thereof that positively regulates nicotine biosynthesis is selected from the group consisting of:

(a) the nucleic acid sequence of SEQ ID NO: 1 (NtERF1), SEQ ID NO: 2 (NtERF5), SEQ ID NO: 3 (NtERF10), SEQ ID NO: 4 (NtERF32), SEQ ID NO: 5 (NtERF221), SEQ ID NO: 6 (NtERF241), SEQ ID NO: 7 (NtMYC1a), SEQ ID NO: 8 (NtMYC1b), SEQ ID NO: 9 (NtMYC2a), or SEQ ID NO: 10 (NtMYC2b); and

(b) a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence of (a), and which encodes for a protein comprising at least one DNA binding domain that positively regulates nicotine biosynthesis.

4 . An expression vector comprising the chimeric nucleic acid of claim 1 , wherein the operably linked heterologous nucleic acid comprises one or more control sequences suitable for directing expression in a Nicotiana host cell.

5 . A Nicotiana host cell transformed with the chimeric nucleic acid construct of claim 1 .

6 . A genetically engineered nicotinic alkaloid-producing Nicotiana plant comprising a cell comprising the chimeric nucleic acid construct of claim 1 .

7 . The engineered Nicotiana plant of claim 6 , wherein the plant is a Nicotiana tabacum plant.

8 . Seeds from the genetically engineered Nicotiana plant of claim 6 , wherein the seeds comprise the chimeric nucleic acid construct.

9 . A tobacco product comprising the engineered Nicotiana plant of claim 6 .

10 . A method for reducing nicotine in a Nicotiana plant, comprising:

(a) introducing into a Nicotiana plant an expression vector comprising a chimeric nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter suitable for directing expression in a Nicotiana plant cell operably linked to at least one transcription repressor sequence in translational fusion with at least one transcription factor sequence encoding for a transcription factor or fragment thereof that positively regulates nicotine biosynthesis; and

(b) growing the plant under conditions which allow for the expression of the chimeric nucleic acid;

wherein expression of the chimeric nucleic acid results in production of a dominant negative form of the transcription factor and the plant having a reduced nicotine content as compared to a non-transformed control plant grown under similar conditions.

11 . The method of claim 10 , wherein the transcription repressor sequence encodes for transcription repressor selected from the group consisting of: Engrailed (En 298 ) (SEQ ID NO: 11), SRDX (SEQ ID NO: 12), AtERF4 (SEQ ID NO: 13), NtERF3 (SEQ ID NO: 15), SUPERMAN (SEQ ID NO: 17), LEUNIG (SEQ ID NO: 18), and SEUSS (SEQ ID NO: 19).

12 . The method of claim 10 , wherein the transcription factor that positively regulates nicotine biosynthesis is selected from the group consisting of:

(a) the nucleic acid sequence of SEQ ID NO: 1 (NtERF1), SEQ ID NO: 2 (NtERF5), SEQ ID NO: 3 (NtERF10), SEQ ID NO: 4 (NtERF32), SEQ ID NO: 5 (NtERF221), SEQ ID NO: 6 (NtERF241), SEQ ID NO: 7 (NtMYC1a), SEQ ID NO: 8 (NtMYC1b), SEQ ID NO: 9 (NtMYC2a), or SEQ ID NO: 10 (NtMYC2b); and

(b) a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of (a), and which encodes for a protein comprising at least one DNA binding domain that positively regulates nicotine biosynthesis.

13 . A genetically engineered Nicotiana plant produced by the method of claim 10 , wherein the plant has reduced expression of nicotine as compared to a control plant.

14 . A product comprising the genetically engineered plant of claim 13 or portions thereof, wherein the product has a reduced nicotine content as compared to a product produced from a control plant.

15 . The product of claim 14 , wherein the product is a reduced-nicotine tobacco product selected from the group consisting of cigarette tobacco, reconstituted tobacco, cigar tobacco, pipe tobacco, cigarettes, cigars, chewing tobacco, snuff, snus, and lozenges.

16 . Seeds from the genetically engineered Nicotiana plant of claim 13 , wherein the seeds comprise the chimeric nucleic acid construct.

17 . A genetically engineered Nicotiana plant having reduced nicotine content as compared to a non-transformed control plant, wherein the plant comprises plant cells comprising:

a chimeric nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter suitable for directing expression in the Nicotiana plant cell operably linked to at least one transcription repressor sequence in translational fusion with at least one transcription factor sequence encoding for a transcription factor or fragment thereof that positively regulates nicotine biosynthesis,

wherein expression of the chimeric nucleic acid results in production of a dominant negative form of the transcription factor and the plant having a reduced nicotine content as compared to a non-transformed control plant grown under similar conditions.

18 . The plant of claim 17 , wherein the transcription repressor sequence encodes for a transcription repressor selected from the group consisting of: Engrailed (En 298 ) (SEQ ID NO: 11), SRDX (SEQ ID NO: 12), AtERF4 (SEQ ID NO: 13), NtERF3 (SEQ ID NO: 15), SUPERMAN (SEQ ID NO: 17), LEUNIG (SEQ ID NO: 18), and SEUSS (SEQ ID NO: 19).

19 . The plant of claim 17 , wherein the transcription factor that positively regulates nicotine biosynthesis is selected from the group consisting of:

(a) the nucleic acid sequence of SEQ ID NO: 1 (NtERF1), SEQ ID NO: 2 (NtERF5), SEQ ID NO: 3 (NtERF10), SEQ ID NO: 4 (NtERF32), SEQ ID NO: 5 (NtERF221), SEQ ID NO: 6 (NtERF241), SEQ ID NO: 7 (NtMYC1a), SEQ ID NO: 8 (NtMYC1b), SEQ ID NO: 9 (NtMYC2a), or SEQ ID NO: 10 (NtMYC2b); and

(b) a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence of (a), and which encodes for a protein comprising at least one DNA binding domain that positively regulates nicotine biosynthesis.

20 . A product comprising the genetically engineered plant of claim 17 or portions thereof, wherein the product has a reduced nicotine content as compared to a product produced from a control plant.

21 . The product of claim 20 , wherein the product is a reduced-nicotine tobacco product selected from the group consisting of cigarette tobacco, reconstituted tobacco, cigar tobacco, pipe tobacco, cigarettes, cigars, chewing tobacco, snuff, snus, and lozenges.

22 . Seeds from the genetically engineered Nicotiana plant of claim 17 , wherein the seeds comprise the chimeric nucleic acid construct.

23 . A method of making a genetically engineered Nicotiana plant cell having reduced nicotine content, the method comprising:

introducing into a Nicotiana plant cell an expression vector comprising a chimeric nucleic acid construct comprising, in the 5′ to 3′ direction, a promoter suitable for directing expression in a Nicotiana plant cell operably linked to at least one transcription repressor sequence in translational fusion with at least one transcription factor sequence encoding for a transcription factor or fragment thereof that positively regulates nicotine biosynthesis;

wherein expression of the chimeric nucleic acid results in production of a dominant negative form of the transcription factor and the plant cell having a reduced nicotine content as compared to a non-transformed control plant cell.

24 . The method of claim 23 , wherein the transcription repressor sequence encodes for transcription repressor selected from the group consisting of: Engrailed (En 298 ) (SEQ ID NO: 11), SRDX (SEQ ID NO: 12), AtERF4 (SEQ ID NO: 13), NtERF3 (SEQ ID NO: 15), SUPERMAN (SEQ ID NO: 17), LEUNIG (SEQ ID NO: 18), and SEUSS (SEQ ID NO: 19).

25 . The method of claim 23 , wherein the transcription factor that positively regulates nicotine biosynthesis is selected from the group comprising of:

(a) the nucleic acid sequence of SEQ ID NO: 1 (NtERF1), SEQ ID NO: 2 (NtERF5), SEQ ID NO: 3 (NtERF10), SEQ ID NO: 4 (NtERF32), SEQ ID NO: 5 (NtERF221), SEQ ID NO: 6 (NtERF241), SEQ ID NO: 7 (NtMYC1a), SEQ ID NO: 8 (NtMYC1b), SEQ ID NO: 9 (NtMYC2a), or SEQ ID NO: 10 (NtMYC2b); and

(b) a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence of (a), and which encodes for a protein comprising at least one DNA binding domain that positively regulates nicotine biosynthesis.

26 . The method of claim 23 , wherein the Nicotiana plant cell is Nicotiana tabacum .

SEQUENCE LISTING

SEQ ID NO: 1 (711 bp)

NtERF1 ORF (GenBank Accession No.: D38123.1)

1

atgaatcaac caatttatac agagttgccg ccggcgaatt ttccgggaga atttccggtg

61

taccgccgga attcaagctt cagtcgtcta atcccatgtt taactgaaac atggggcgac

121

ttaccactaa aagtcgacga ttctgaagat atggtaattt atactctctt aaaagacgct

181

cttaacgtcg gatggtcgcc gtttaatttc agcgccggcg aagtaaaatc ggagcagagg

241

gaggaagaaa ttgtggtttc tccggcggag acgacggccg cgccggcggc tgagttacct

301

aggggaaggc attacagagg tgttagacga cggccgtggg ggaaatttgc ggcggagatt

361

agggatccgg cgaagaatgg agctagggtt tggcttggaa catacgaaac agatgaagaa

421

gctgcaattg cttatgataa agcggcttat agaatgcgcg gttcaaaggc tcatttaaat

481

tttccacata gaatcggttt aaatgaaccg gaaccggttc gagttacggc gaaaagacga

541

gcatcgcctg aaccggctag ttcgtcggaa aatagttcac ctaaacggag aagaaaggct

601

gttgcaactg agaaatctga agcagtagaa gtggagagta aatcaaatgt tttgcaaact

661

ggatgtcaag ttgaactatt gacacgtcga catcaattat tagtcagtta a

SEQ ID NO: 2 (705 bp)

NtERF5/NtERF121 ORF (GenBank Accession No.: AY655738.1)

1

atgtcaagta actcaagccc actagaaata gacacttcat tttcacattc caacttcttc

61

tttctccaag atcaatcacc aattttacaa tgggatgatg atcttttctt caatgatcca

121

tggtttgatg atgatcaatc accaattata ccatgtaact cagagaaaga tgaaaatcat

181

caagtatttg aagaatcctc agacaataca atcatgtcaa aaggaagtag ccatggtcaa

241

gaattagaag aggtaacatc ccaagaagaa aaagaaaaag aagaagaaga aaaacactat

301

ataggagtta gaaaaaggcc atggggtaaa tatgcagcag aaataagaga ttcaacaaga

361

aatggaatta gggtttggtt agggacattt gatacagctg aagaagctgc tttagcttat

421

gatcaagctg cattatcgat gagaggtcct tggtctcttc ttaattttcc attggagaaa

481

gtcaagaaat cacttgaaaa aattgagtat tcttgtaaag atggattgtc tcctgctgct

541

gttctaaaag ctactcataa aacaaggaga gtgaagcata aaagaagtag tagaaagaaa

601

aagaataaag aaactcataa tgttattgtt tttgaggact tgggtgctga gttattagaa

661

gagcttttaa tgacttcatc acaacattcg tgtcgaaggg actga

SEQ ID NO: 3 (858 bp)

NtERF10/JAP1 gene (GenBank Accession No.: CQ808845.1)

1

acgggggggg gggggggggg ggacttgaag actgggaagc tccattaacg agctccgaca

61

actcaacagc ctctgattta agccgaagca atagcattga gtccaacatg tttcctaatt

121

gcttgcccaa tgaatataat tatacagctg atatgttttt taacgatatc tttaatgaag

181

gcattgttgg ctatggattt gagccagctt ctgaatttac actccccagt atcaaattgg

241

agccagaaat gactgtacaa tcacctgcaa tatggaattt accggagttt gtggcgccgc

301

cggagacggc ggcggaggtg aaactggaac caccggcgcc gcaaaaggca aagcattata

361

ggggagtgag agtgaggccg tgggggaagt ttgcagcgga aattagggat ccggcaaaga

421

atggggcaag ggtgtggctg ggtacgtatg agacggcaga ggacgcagcg tttgcttatg

481

acaaggcggc gtttcgcatg cgggggtcac gtgcattgct taatttcccg ttaaggatta

541

attctggtga gcctgatccc attagagttg gttctaaaag gtcatcaatg tcgccggagt

601

attcttcttc ttcatcgtcg tcggcgtcgt cgccgaagag gaggaagaag gtatctcaag

661

ggacggagct aacggtgtta taggtcccaa ctgggttctg tgtagtgatt aagaaaaata

721

gaattagtcg agggaatttg ttttttactt ggctgaagta atgaatttgt tatttattta

781

ttttttgact gtggttgaaa ttgaatcaaa aaaaaaaaaa aaaaagtact agtcgacgcg

841

tggcctagta gtagtaga

SEQ ID NO: 4 (702 bp)

NtERF32/EREBP2/ERF2 ORF (GenBank Accession No.: D38126.1)

1

atgtatcaac caatttcgac cgagctacct ccgacgagtt tcagtagtct catgccatgt

61

ttgacggata catggggtga cttgccgtta aaagttgatg attccgaaga tatggtaatt

121

tatgggctct taagtgacgc tttaactgcc ggatggacgc cgtttaattt aacgtccacc

181

gaaataaaag ccgagccgag ggaggagatt gagccagcta cgattcctgt tccttcagtg

241

gctccacctg cggagactac gacggctcaa gccgttgttc ccaaggggag gcattatagg

301

ggcgttaggc aaaggccgtg ggggaaattt gcggcggaaa taagggaccc agctaaaaac

361

ggcgcacggg tttggctagg gacttatgag acggctgaag aagccgcgct cgcttatgat

421

aaagcagctt acaggatgcg cggctccaag gctctattga attttccgca taggatcggc

481

ttaaatgagc ctgaaccggt tagactaacc gctaagagac gatcacctga accggctagc

541

tcgtcaatat catcggcttt ggaaaatggc tcgccgaaac ggaggagaaa agctgtagcg

601

gctaagaagg ctgaattaga agtgcaaagc cgatcaaatg ctatgcaagt tgggtgccag

661

atggaacaat ttccagttgg cgagcagcta ttagtcagtt aa

SEQ ID NO: 5 (924 bp)

NtERF221/ORC1 gene (GenBank Accession No.: CQ808982.1)

1

atccagaatt aataaaccct agtaagtgaa agtgaaagaa actactcatc caaatatcta

61

tagaaaagta aatgaatccc gctaatgcaa ccttctcttt ctctgagctt gatttccttc

121

aatcaataga aaaccatctt ctgaattatg attccgattt ttctgaaatt ttttcgccga

181

tgagttcaag taacgcattg cctaatagtc ctagctcaag ttttggcagc ttcccttcag

241

cagaaaatag cttggatacc tctctttggg atgaaaactt tgaggaaaca atacaaaatc

301

tcgaagaaaa gtccgagtcc gaggaggaaa caaaggggca tgtcgtggcg cgtgagaaaa

361

acgcgacaca agattggaga cggtacatag gagttaaacg gcggccgtgg gggacgtttt

421

cggcggagat aagggacccg gagagaagag gcgcgagatt atggctagga acttacgaga

481

ccccagagga cgcagcattg gcttacgatc aagccgcttt caaaatccgc ggctcgagag

541

ctcggctcaa ttttcctcac ttaattggat caaacattcc taagccggct agagttacag

601

cgagacgtag ccgtacgcgc tcaccccagc catcgtcttc ttcatgtacc tcatcatcag

661

aaaatgggac aagaaaaagg aaaatagatt tgataaattc catagccaaa gcaaaattta

721

ttcgtcatag ctggaaccta caaatgttgc tataactgta tttaatttgg aaggaattaa

781

ttaaggttat tctatgtctt tgtattagaa tttagaataa ttccctaaag ctcctgaaga

841

acgaaacttg taaacatctc tctgtctccg tatcatgttc taatttaaca tgaaattaca

901

tgagcgcaaa aaaaaaaaaa aaaa

SEQ ID NO: 6 (741 bp)

NtERF241 ORF

ATGTATCAACCCATTTCTACAGAATTCCCAGTATATCACCGGACTTCAAGTTTCAGTAGTCTCAT

GCCATGTTTGACGGATACTTGGGGTGACTTGCCGTTAAAAGTTGATGATTCCGAAGATATGGTAA

TTTATGGGCTCTTAAGTGACGCTTTAACTACCGGATGGACGCCGTTTAATTTAACGTCCACCGAA

ATAAAAGCCGAGCCGAGGGAAGAGATTGAGCCAGCTACGAGTCCTGTTCCTTCAGTGGCTCCACC

CGCGGAGACTACGACGGCTCAAGCCGTCGTGCCCAAGGGAAGGCATTATAGGGGCGTCAGGCAAA

GGCCGTGGGGGAAATTTGCGGCGGAAATAAGGGACCCAGCTAAAAATGGCGCACGGGTTTGGCTA

GGGACTTATGAGACGGCTGAAGAAGCCGCGCTCGCTTATGATAAAGCAGCTTACAGGATGCGCGG

CTCCAAGGCTCTATTGAATTTTCCGCATAGGATCGGCTTAAATGAGCCTGAACCGGTTAGGCTGA

CCGTTAAGAGACGATCACCTGAACCGGCCGTTAAGAGACGATCACCTGAACCGGCTAGCTCGTCA

ATATCACCGGCTTCGGAAAATAGCTTGCCGAAGCGGAGGAGAAAAGCTGTAGCGGCTAAGCAAGC

TGAATTAGAAGTGCAGAGCCGATCAAATGTAATGCAAGTTGGGTGCCAAATGGAACAATTTCCAG

TTGGCGAGCAGCTATTAGTTAGTTAA

SEQ ID NO: 7 (2046 bp)

NtMYC1a ORF (GenBank Accession No.: GQ859158.1)

1

atgactgatt acagcttacc caccatgaat ttgtggaata ctagtggtac taccgatgac

61

aacgttacta tgatggaagc ttttatgtct tctgatctca cttcattttg ggctacttct

121

aattctactg ctgttgctgc tgttacctct aattctaatc atattccagt taatacccca

181

acggttcttc ttccgtcttc ttgtgcctct actgtcacag ctgtggctgt cgatgcttca

241

aaatccatgt cttttttcaa ccaagaaacc cttcaacagc gtcttcaaac gctcattgat

301

ggtgctcgtg agacgtggac ctatgccatc ttttggcagt catccgccgt tgatttaacg

361

agtccgtttg tgttgggctg gggagatggt tactacaaag gtgaagaaga taaagccaat

421

aggaaattag ctgtttcttc tcctgcttat atagctgagc aagaacaccg gaaaaaggtt

481

ctccgggagc tgaattcgtt gatttccggc acgcaaaccg gcactgatga tgccgtcgat

541

gaagaagtta ccgacactga atggttcttc cttatttcca tgacccagtc gtttgttaac

601

ggaagtgggc ttccgggtca ggccttatac aattccagcc ctatttgggt cgccggagca

661

gagaaattgg cagcttccca ctgcgaacgg gctcggcagg cccagggatt cgggcttcag

721

acgatggttt gtattccttc agcaaacggc gtggttgaat tgggctccac ggagttgatt

781

attcagagtt ctgatctcat gaacaaggtt agagtattgt ttaacttcaa taatgatttg

841

ggctctggtt cgtgggctgt gcaacccgag agcgatccgt ccgctctttg gctcactgat

901

ccatcgtctg cagctgtaca agtcaaagat ttaaatacag ttgaggcaaa ttcagttcca

961

tcaagtaata gtagtaagca agttgtattt gataatgaga ataatggtca cagttgtgat

1021

aatcagcaac agcaccattc tcggcaacaa acacaaggat tttttacaag ggagttgaac

1081

ttttcagaat tcgggtttga tggaagtagt aataatagga atgggaattc atcactttct

1141

tgcaagccag agtcggggga aatcttgaat tttggtgata gcactaagaa aagtgcaaat

1201

gggaacttat tttccggtca gtcccatttt ggtgcagggg aggagaataa gaagaagaaa

1261

aggtcacctg cttccagagg aagcaatgaa gaaggaatgc tttcatttgt ttcaggtaca

1321

atcttgcctg cagcttctgg tgcgatgaag tcaagtggat gtgtcggtga agactcctct

1381

gatcattcgg atcttgaggc ctcagtggtg aaagaagctg aaagtagtag agttgtagaa

1441

cccgaaaaga ggccaaagaa gcgaggaagg aagccagcaa atggacgtga ggaacctttg

1501

aatcacgtcg aagcagagag gcaaaggaga gagaaattaa accaaaggtt ctacgcttta

1561

agagctgttg ttccgaatgt gtccaagatg gacaaggcat cactgcttgg agatgcaatt

1621

tcatatatta atgagctgaa gttgaagctt caaactacag aaacagatag agaagacttg

1681

aagagccaaa tagaagattt gaagaaagaa ttagatagta aagactcaag gcgccctggt

1741

cctccaccac caaatcaaga tcacaagatg tctagccata ctggaagcaa gattgtagat

1801

gtggatatag atgttaagat aattggatgg gatgcgatga ttcgtataca atgtaataaa

1861

aagaaccatc cagctgcaag gttaatggta gccctcaagg agttagatct agatgtgcac

1921

catgccagtg tttcagtggt gaatgatttg atgatccaac aagccacagt gaaaatgggt

1981

agcagacttt acacggaaga gcaacttagg atagcattga catccagagt tgctgaaaca

2041

cgctaa

SEQ ID NO: 8 (2040 bp)

NtMYC1b ORF (GenBank Accession No.: GQ859159.1)

1

cgcagacccc tcttttcacc catttctctc tctctctctc tctctctctc tatatatata

61

tatatctttc acgccaccat atccaactgt ttgtgctggg tttatggaat gactgattac

121

agcttaccca ccatgaattt gtggaatact agtggtacta ccgatgacaa cgtttctatg

181

atggaatctt ttatgtcttc tgatctcact tcattttggg ctacttctaa ttctactact

241

gctgctgtta cctctaattc taatcttatt ccagttaata ccctaactgt tcttcttccg

301

tcttcttgtg cttctactgt cacagctgtg gctgtcgatg cttcaaaatc catgtctttt

361

ttcaaccaag aaactcttca gcagcgtctt caaaccctca ttgatggtgc tcgtgagacg

421

tggacctatg ccatcttttg gcagtcatcc gtcgttgatt tatcgagtcc gtttgtgttg

481

ggctggggag atggttacta caaaggtgaa gaagataaag ccaataggaa attagctgtt

541

tcttctcctg cttatattgc tgagcaagaa caccgaaaaa aggttctccg ggagctgaat

601

tcgttgatct ccggcacgca aaccggcact gatgatgccg tcgatgaaga agttaccgac

661

actgaatggt tcttccttat ttccatgacc caatcgtttg ttaacggaag tgggcttccg

721

ggtcaggcct tatacaattc cagccctatt tgggtcgccg gagcagagaa attggcagct

781

tcccactgcg aacgggctcg gcaggcccag ggattcgggc ttcagacgat ggtttgtatt

841

ccttcagcaa acggcgtggt tgaattgggc tccacggagt tgataatcca gagttgtgat

901

ctcatgaaca aggttagagt attgtttaac ttcaataatg atttgggctc tggttcgtgg

961

gctgtgcagc ccgagagcga tccgtccgct ctttggctca ctgatccatc gtctgcagct

1021

gtagaagtcc aagatttaaa tacagttaag gcaaattcag ttccatcaag taatagtagt

1081

aagcaagttg tgtttgataa tgagaataat ggtcacagtt ctgataatca gcaacagcag

1141

cattctaagc atgaaacaca aggatttttc acaagggagt tgaatttttc agaatttggg

1201

tttgatggaa gtagtaataa taggaatggg aattcatcac tttcttgcaa gccagagtcg

1261

ggggaaatct tgaattttgg tgatagtact aagaaaagtg caaatgggaa cttattttcg

1321

ggtcagtccc attttggggc aggggaggag aataagaaca agaaaaggtc acctgcttcc

1381

agaggaagca atgaagaagg aatgctttca tttgtttcgg gtacaatctt gcctgcagct

1441

tctggtgcga tgaagtcaag tggaggtgta ggtgaagact ctgatcattc ggatcttgag

1501

gcctcagtgg tgaaagaagc tgaaagtagt agagttgtag aacccgaaaa gaggccaaag

1561

aagcgaggaa ggaagccagc aaatggacgg gaggaacctt tgaatcacgt cgaagcagag

1621

aggcaaagga gagagaaatt aaaccaaagg ttctacgcat taagagctgt tgttccgaat

1681

gtgtccaaga tggacaaggc atcactgctt ggagatgcaa tttcatatat taatgagctg

1741

aagttgaagc ttcaaaatac agaaacagat agagaagaat tgaagagcca aatagaagat

1801

ttaaagaaag aattagttag taaagactca aggcgccctg gtcctccacc atcaaatcat

1861

gatcacaaga tgtctagcca tactggaagc aagattgtag acgtggatat agatgttaag

1921

ataattggat gggatgcgat gattcgtata caatgtaata aaaagaatca tccagctgca

1981

aggttaatgg tagccctcaa ggagttagat ctagatgtgc accatgccag tgtttcagtg

2041

gtgaacgatt tgatgatcca acaagccact gtgaaaatgg gtagcagact ttacacggaa

2101

gagcaactta ggatagcatt gacatccaga gttgctgaaa cacgctaa

SEQ ID NO: 9 (1980 bp)

NtMYC2a ORF (GenBank Accession No.: GQ859160.1)

1

atgacggatt atagaatacc aacgatgact aatatatgga gcaatactac atccgatgat

61

aatatgatgg aagctttttt atcttctgat ccgtcgtcgt tttggcccgg aacaactact

121

acaccaactc cccggagttc agtttctcca gcgccggcgc cggtgacggg gattgccgga

181

gacccattaa agtctatgcc atatttcaac caagagtcac tgcaacagcg actccagact

241

ttaatcgatg gggctcgcaa agggtggacg tatgccatat tttggcaatc gtctgttgtg

301

gatttcgcga gcccctcggt tttggggtgg ggagatgggt attataaagg tgaagaagat

361

aaaaataagc gtaaaacggc gtcgttttcg cctgacttta tcacggaaca agcacaccgg

421

aaaaaggttc tccgggagct gaattcttta atttccggca cacaaaccgg tggtgaaaat

481

gatgctgtag atgaagaagt aactgatact gaatggtttt ttctgatttc catgacacaa

541

tcgtttgtta acggaagcgg gcttccgggc ctggcgatgt atagttcaag cccgatttgg

601

gttactggaa cagagagatt agctgtttct cactgtgaac gggcccgaca ggcccaaggt

661

ttcgggcttc agactattgt ttgtattcct tcagctaatg gtgttgttga gctcgggtca

721

actgagttga tattccagac tgctgattta atgaacaagg ttaaagtttt gtttaatttt

781

aatattgata tgggtgcgac tacgggctca ggatcgggct catgtgctat tcaggccgag

841

cccgatcctt cagccctttg gctgactgat ccggcttctt cagttgtgga agtcaaggat

901

tcgtcgaata cagttccttc aaggaatacc agtaagcaac ttgtgtttgg aaatgagaat

961

tctgaaaatg ttaatcaaaa ttctcagcaa acacaaggat ttttcactag ggagttgaat

1021

ttttccgaat atggatttga tggaagtaat actcggtatg gaaatgggaa tgcgaattct

1081

tcgcgttctt gcaagcctga gtctggtgaa atcttgaatt ttggtgatag tactaagagg

1141

agtgcttgca gtgcaaatgg gagcttgttt tcgggccaat cacagttcgg gcccgggcct

1201

gcggaggaga acaagaacaa gaacaagaaa aggtcacctg catcaagagg aagcaacgat

1261

gaaggaatcc tttcatttgt ttcgggtgtg attttgccaa gttcaaacac ggggaagtcc

1321

ggtggaggtg gcgattcgga tcaatcagat ctcgaggctt cggtggtgaa ggaggcggat

1381

agtagtagag ttgtagaccc cgagaagaag ccgaggaaac gagggaggaa accggctaac

1441

gggagagagg agccattgaa tcatgtggag gcagagagac aaaggaggga gaaattgaat

1501

caaagattct atgcacttag agctgttgta ccaaatgtgt caaaaatgga taaagcatca

1561

cttcttggtg atgcaattgc atttatcaat gagttgaaat caaaggttca gaattctgac

1621

tcagataaag aggacttgag gaaccaaatc gaatctttaa ggaatgaatt agccaacaag

1681

ggatcaaact ataccggtcc tcccccgtca aatcaagaac tcaagattgt agatatggac

1741

atcgacgtta aggtgatcgg atgggatgct atgattcgta tacaatctaa taaaaagaac

1801

catccagccg cgaggttaat gaccgctctc atggaattgg acttagatgt gcaccatgct

1861

agtgtttcag ttgtcaacga gttgatgatc caacaagcga ctgtgaaaat gggaagccgg

1921

ctttacacgc aagaacaact tcggatatca ttgacatcca gaattgctga atcgcgatga

SEQ ID NO: 10 (1977 bp)

NtMYC2b ORF (GenBank Accession No.: GQ859161)

1

atgacggact atagaatacc aacgatgact aatatatgga gcaatacaac atccgacgat

61

aacatgatgg aagctttttt atcttctgat ccgtcgtcgt tttgggccgg aacaaataca

121

ccaactccac ggagttcagt ttctccggcg ccggcgccgg tgacggggat tgccggagac

181

ccattaaagt cgatgccgta tttcaaccaa gagtcgctgc aacagcgact ccagacgtta

241

atcgacgggg ctcgcgaagc gtggacttac gccatattct ggcaatcgtc tgttgtggat

301

ttcgtgagcc cctcggtgtt ggggtgggga gatggatatt ataaaggaga agaagacaag

361

aataagcgta aaacggcggc gttttcgcct gattttatta cggagcaaga acaccggaaa

421

aaagttctcc gggagctgaa ttctttaatt tccggcacac aaactggtgg tgaaaatgat

481

gctgtagatg aagaagtaac ggatactgaa tggttttttc tgatttcaat gactcaatcg

541

tttgttaacg gaagcgggct tccgggcctg gctatgtaca gctcaagccc gatttgggtt

601

actggaagag aaagattagc tgcttctcac tgtgaacggg cccgacaggc ccaaggtttc

661

gggcttcaga ctatggtttg tattccttca gctaatggtg ttgttgagct cgggtcaact

721

gagttgatat tccagagcgc tgatttaatg aacaaggtta aaatcttgtt tgattttaat

781

attgatatgg gcgcgactac gggctcaggt tcgggctcat gtgctattca ggctgagccc

841

gatccttcaa ccctttggct tacggatcca ccttcctcag ttgtggaagt caaggattcg

901

tcgaatacag ttccttcaag taatagtagt aagcaacttg tgtttggaaa tgagaattct

961

gaaaatgtta atcaaaattc tcagcaaaca caaggatttt tcactaggga gttgaatttt

1021

tccgaatatg gatttgatgg aagtaatact aggagtggaa atgggaatgt gaattcttcg

1081

cgttcttgca agcctgagtc tggcgaaatc ttgaattttg gtgatagtac taagagaaat

1141

gcttcaagtg caaatgggag cttgttttcg ggccaatcgc agttcggtcc cgggcctgcg

1201

gaggagaaca agaacaagaa caagaaaagg tcacctgcat caagaggaag caatgaagaa

1261

ggaatgcttt catttgtttc gggtgtgatc ttgccaagtt caaacacggg gaagtccggt

1321

ggaggtggcg attcggatca ttcagatctc gaggcttcgg tggtgaagga ggcggatagt

1381

agtagagttg tagaccccga gaagaggccg aggaaacgag gaaggaaacc ggctaacggg

1441

agagaggagc cattgaatca tgtggaggca gagaggcaaa ggagggagaa attgaatcaa

1501

agattctatg cacttagagc tgttgtacca aatgtgtcaa aaatggataa agcatcactt

1561

cttggtgatg caattgcatt tatcaatgag ttgaaatcaa aggttcagaa ttctgactca

1621

gataaagatg agttgaggaa ccaaattgaa tctttaagga atgaattagc caacaaggga

1681

tcaaactata ccggtcctcc accgccaaat caagatctca agattgtaga tatggatatc

1741

gacgttaaag tcatcggatg ggatgctatg attcgtatac aatctaataa aaagaaccat

1801

ccagccgcga ggttaatggc cgctctcatg gaattggact tagatgtgca ccatgctagt

1861

gtttcagttg tcaacgagtt gatgatccaa caagcgacag tgaaaatggg gagccggctt

1921

tacacgcaag agcagcttcg gatatcattg acatccagaa ttgctgaatc gcgatga

SEQ ID NO: 11 (298 AA)

Engrailed (En 298 ) protein from  Drosophila  (the first 298 amino acids encoded by the

Engrailed gene from  Drosophila )

Met Met Met Asn Ala Phe Ile Glu Pro Ala Gln His His Leu Ala Ser

1               5                   10                  15

Tyr Gly Leu Arg Met Ser Pro Asn Thr Thr Ala Ser Asn Ser Asn Ala

            20                  25                  30

Gln Gln Gln Gln Gln Gln Gln Leu Glu Met Thr Gln Gln Gln Gln Gln

        35                  40                  45

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Asp Gln Glu Ser Ala

    50                  55                  60

Ala Ala Thr Ala Ala Ala Tyr Gln Asn Ser Gly Tyr Gly His Phe Asn

65                  70                  75                  80

Ser Tyr Ala Ser Arg Asp Phe Leu Leu Gly Arg Arg Glu Ala Glu Tyr

                85                  90                  95

Gly Val Ala Gly Ser Ala Gly Gln Ala Ser Ala Ala Ala Asp Ser Met

            100                 105                 110

Leu Phe Ser Gly Phe Pro Ala Gln Ala Ala Glu Leu Gly Ser Gly Phe

        115                 120                 125

Gly Gln His Pro Phe His Ser His His His His His Gln Met Arg Met

    130                 135                 140

Gly Met Ala Asp Ala Tyr Ala Ala Gly His Pro Tyr Asn His His Gly

145                 150                 155                 160

Asn Phe Pro Thr Ala Ala Val His His Pro Val Val His His Pro Ser

                165                 170                 175

His His Ala Met Ser Ala Met His Pro Ala Gly Ala Gly Ala Phe Leu

            180                 185                 190

Arg Tyr Met Arg His Gln Pro Ala Ser Ser Ala Ser Ser Val Lys Gln

        195                 200                 205

Glu Met Gln Cys Leu Trp Ile Asp Pro Asp Gln Pro Gly Leu Val Pro

    210                 215                 220

Pro Gly Gly Arg Lys Thr Cys Asn Lys Val Phe His Ser Met His Glu

225                 230                 235                 240

Ile Val Thr His Leu Thr Val Glu His Val Gly Gly Pro Glu Cys Thr

                245                 250                 255

Thr His Ala Cys Phe Trp Val Gly Cys Ser Arg Asn Gly Arg Pro Phe

            260                 265                 270

Lys Ala Lys Tyr Lys Leu Val Asn His Ile Arg Val His Thr Gly Glu

        275                 280                 285

Lys Pro Phe Ala Cys Pro His Pro Gly Cys

    290                 295

SEQ ID NO: 12 (12 AA)

EAR repressor domain protein also known as SRDX (A 12-amino acid sequence of the

Arabidopsis  Ethylene-responsive transcription factor 4 (ERF4))

LDLDLELRLGFA

SEQ ID NO: 13 (24 AA)

EAR repressor domain protein also known as AtERF4 (A 24-amino acid sequence of the

Arabidopsis  Ethylene-responsive transcription factor 4 (ERF4))

EGGMEKRSQLLDLDLNLPPPSEQA

SEQ ID NO: 14 (225 AA)

Nicotiana tabacum  ethylene responsive element binding factor 3 (NtERF3) protein

1

mavknkvsng nlkggnvktd gvkevhyrgv rkrpwgryaa eirdpgkksr vwlgtfdtae

61

eaakaydtaa refrgpkakt nfpsptenqs pshsstvess sgengvhapp hapleldltr

121

rlgsvaadgg dncrrsgevg ypifhqqptv avlpngqpvl lfdslwragv vnrpqpyhvt

181

pmgfngvnag vgptvsdsss aveenqydgk rgidldlnla ppmef

SEQ ID NO: 15 (35 AA)

EAR repressor domain of NtERF3 protein (amino acids 191-225)

VGPTVSDSSSAVEENQYDGKRGIDLDLNLAPPMEF

SEQ ID NO: 16 (204 AA)

Arabidopsis thaliana  SUPERMAN (SUP) protein

1

mersnsielr nsfygrarts pwsygdydnc qqdhdyllgf swpprsytcs fckrefrsaq

61

algghmnvhr rdrarlrlqq spsssstpsp pypnpnysys tmansppphh spltlfptls

121

ppsspryrag lirslspksk htpenacktk kssllveage atrftskdac kilrndeiis

181

leleigline seqdldlelr lgfa

SEQ ID NO: 17 (30 AA)

EAR repressor domain of SUPERMAN (SUP) protein (amino acids 175-204)

NDEIISLELEIGLINESEQDLDLELRLGFA

SEQ ID NO: 18 (969 AA)

LEUNIG transcriptional co-repressor protein ( Arabidopsis thaliana )

1

msqtnweadk mldvyihdyl vkrdlkataq afqaegkvss dpvaidapgg flfewwsvfw

61

difiartnek hsevaasyie tqmikareqq lqqsqhpqvs qqqqqqqqqq iqmqqlllqr

121

aqqqqqqqqq qhhhhqqqqq qqqqqqqqqq qqqqqhqnqp psqqqqqqst pqhqqqptpq

181

qqpqrrdgsh langsanglv gnnsepvmrq npgsgsslas kayeervkmp tqresldeaa

241

mkrfgdnvgq lldpshasil ksaaasgqpa gqvlhstsgg mspqvqtrnq qlpgsavdik

301

seinpvltpr tavpegslig ipgrfavlsv sfqlvkrvkc sistsahknl lrindwfpvs

361

gsnqgsnnlt lkgwpltgfd qlrsgllqqq kpfmqsqsfh qlnmltpqhq qqlmlaqqnl

421

nsqsvseenr rlkmllnnrs mtlgkdglgs svgdvlpnvg sslqpggsll prgdtdmllk

481

lkmallqqqq qnqqqgggnp pqpqpqpqpl nqlaltnpqp qssnhsihqq eklggggsit

541

mdgsisnsfr gneqvlknqs grkrkqpvss sgpanssgta ntagpspssa pstpsthtpg

601

dvismpnlph sggssksmmm fgtegtgtlt spsnqladmd rfvedgsldd nvesflsqed

661

gdqrdavtrc mdvskgftft evnsvrastt kvtcchfssd gkmlasaghd kkavlwytdt

721

mkpkttleeh tamitdirfs psqlrlatss fdktvrvwda dnkgyslrtf mghssmvtsl

781

dfhpikddli cscdndneir ywsinngsct rvykggstqi rfqprvgkyl aassanlvnv

841

ldvetqairh slqghanpin svcwdpsgdf lasvsedmvk vwtlgtgseg ecvhelscng

901

nkfqscvfhp aypsllvigc yqslelwnms enktmtlpah eglitslavs tatglvasas

961

hdklvklwk

SEQ ID NO: 19 (877 AA)

SEUSS transcriptional co-repressor protein ( Arabidopsis thaliana )

1

mvpseppnpv gggenvppsi lggqggaplp sqpafpslvs prtqfgnnms msmlgnapni

61

ssllnnqsfv ngipgsmism dtsgaesdpm snvgfsglss fnassmvspr ssgqvqgqqf

121

snvsanqlla eqqrnkkmet qsfqhgqqqs mqqqfstvrg gglagvgpvk mepgqvsndq

181

qhgqvqqqqg kmlrnlgsvk lepqqiqamr nlaqvkmepq hseqslflqg qqrqqqqqqq

241

qqflqmpgqs pqaqmnifqq qrlmqlqqqq llksmpqqrp qlpqqfqqqn lplrpplkpv

301

yepgmgaqrl tqymyrqqhr pednniefwr kfvaeyfapn akkrwcvsmy gsgrqttgvf

361

pqdvwhceic nrkpgrgfea taevlprlfk ikyesgtlee llyvdmpres qnssgqivle

421

yakatqesvf ehlrvvrdgq lrivfspdlk ifswefcarr heeliprrll ipqvsqlgsa

481

aqkyqqaaqn attdsalpel qnncnmfvas arqlakalev plvndlgytk ryvrclqise

541

vvnsmkdlid ysretrtgpi eslakfprrt gpssalpgps pqqasdqlrq qqqqqqqqqq

601

qqqqqqqqqq qqqtvsqntn sdqssrqval mqgnpsngvn yafnaasast stssiaglih

661

qnsmkgrhqn aaynppnspy ggnsvqmqsp sssgtmvpss sqqqhnlptf qsptsssnnn

721

npsqngipsv nhmgstnspa mqqagevdgn esssvqkiln eilmnnqahn nssggsmvgh

781

gsfgndgkgq anvnssgvll mngqvnnnnn tniggaggfg ggigqsmaan ginningnns

841

lmngrvgmmv rdpngqqdlg nqllgavngf nnfdwna