Patent EA-003835-B1 (EA)
Title: Isolated purified xanthosine n<7> methyl transferase from coffee plant, coding nucleic acid thereof, coffee plant transformed cells, caffeine free transformed coffee plants and coffee beans, and methods for inhibiting production of caffeine by coffee plant cells
Inventor: STILES JOHN I
IPC: C12N15/82
Filed: 1997-03-24
Abstract: 1. An isolated xanthosine N<7> methyl transferase from a coffee plant Coffea arabica, consisting essentially of the amino acid sequence: 1Met Ala Phe Val Ala Arg Gln Trp Phe Leu Leu Ser Ile 14Ile Asn Val Val Val Val Cys Phe Leu Lys Pro Phe Ala 27Leu Gly Glu Gln Gln Val Pro Cys Tyr Phe Ile Phe Gly 40Asp Ser Gln Asp Asp Asn Gly Asn Asn Asn His Leu Asn 53Thr Thr Ala Arg Ala Asn Tyr Pro Pro Tyr Gly Ile Asp 66Phe Pro Glu Gly Pro Thr Gly Arg Phe Thr Asn Gly Arg 79Asn His Ala Asp Phe Ile Gly Glu Leu Leu Gly Phe Asp 92Ser Tyr Ile Pro Pro Phe Ala Asn Thr Lys Gly Arg Asp 105Ile Thr Lys Gly Ile Asn Tyr Ala Ser Gly Ala Ser Gly 118Ile Leu Asp Gln Thr Gly Arg His Leu Gly Asp Leu Phe 131Ser Phe Asn Glu Gln Leu His Asn His Glu Arg Ala Ile 144Ser Arg Ile Val Arg Leu Ile Gly Asn Arg Ser Ala Thr 157Lys Glu Tyr Leu Ala Lys Cys Leu Tyr Thr Val Ala Leu 170Gly Asn Asn Asp Tyr Ile Asn Asn Tyr Leu Leu Pro Glu 183Tyr Tyr Pro Thr Ser His Leu Tyr Thr Pro Arg Glu Phe 196Ala Ser Leu Leu Ile Arg His Tyr Ser Gln Gln Leu Arg 209Thr Leu Tyr Arg Leu Gly Ala Arg Lys Ile Ala Val Phe 222Gly Leu Gly Trp Leu Gly Cys Ile Pro Ala Glu Leu Ser 235Thr Asp Gly Asn Cys Val Asp Ser Ile Asn Glu Glu Val 248Leu Leu Phe Asn Asp Lys Leu Lys Pro Leu Val Asp Glu 261Leu Asn Thr Glu Leu Ser Gly Ala Gln Phe Leu Tyr Val 274Asp Val Ile Ala Ile Asn Leu Asn Asn Leu Ser Thr Pro 287Ala Glu Ile Thr Ile Gly Asn Ala Pro Cys Cys Asn Val 300Ser Ala Ala Val Ala Gly Gly Gln Cys Ile Pro Gly Gln 313Ile Pro Cys Ser Asn Arg Asn Gln Tyr Tyr Phe Trp Asp 326Asp Phe His Pro Ser Glu Val Val Asn Glu Ala Tyr Ser 339Arg Leu Ala Tyr Ser Ala Leu Ser Ser Leu Leu Asp Ala 352Asp Pro Leu Ala Ile Gly Gly Leu Thr Gly Lys Asn Cys 365His Asp Lys Val Lys Ile Gln. comprising tryptic fragments with the amino acid sequences (i) Ile, Asn, Tyr, Ala, Ser, Gly, Ala, Ser, Gly, Ile, Leu, Asp, Gln-Gly, or (ii) Ile, Asn, Tyr, Ala, Ser, Gly, Ala, Ser, Gly, Ile, Leu, Asp, Gln-Gly, Thr; or (iii) Gln, Tyr, Val, Pro, Cys, Tur, Phe, (Thr/Asp) Phe, Ile, Asp, (Asp), (Gln), Asp. 2. An isolated nudeic acid sequence from a coffee plant Coffea arabica that codes on expression for a xanthosine N<7> methyl transferase having the following structure: 1 CCTCTGACTT GCTAAACCTA CCATTACCTT TTTCTTCTTG TCATCTGCAT 51 TCATGGCTTT TGTAGCCAGG CAATGGTTTC TCCTATCCAT CATTAATGTA 101 GTGGTTGTCT GTTTCTTGAA ACCATTTGCC CTAGGCGAAC AACAGGTCCC 151 TTGCTACTTC ATTTTTGGAG ACTCACAAGA TGACAATGGC AACAATAATC 201 ACCTGAACAC CACTGCCAGG GCAAATTATC CACCTTACGG CATTGATTTC 251 CCAGAAGGTC CAACTGGTCG CTTCACCAAT GGTCGAAATC ATGCAGACTT 301 CATTGGTGAG CTCCTTGGAT TTGACAGCTA CATACCTCCA TTTGCAAATA 51 CAAAAGGCCG GGATATCACT AAAGGCATTA ATTATGCTTC GGGAGCATCT 401 GGAATTCTTG ATCAGACCGG TCGTCACCTG GGCGATCTCT TCAGCTTCAA 451 CGAACAATTG CACAATCACG AGAGAGCAAT TTCGCGCATC GTGCGGTTGA 501 TTGGAAACAG ATCTGCAACA AAAGAATATC TAGCCAAATG TCTGTACACT 551 GTTGCATTGG GGAATAATGA TTACATCAAC AACTACTTGT TGCCAGAATA 601 TTATCCTACC AGCCACCTAT ATACTCCAAG AGAATTTGCC AGCTTGTTAA 651 TTAGGCATTA TTCTCAGCAA CTACGGACTT TGTACAGATT GGGGGCAAGA 701 AAAATAGCCG TTTTTGGGCT TGGTTGGCTT GGCTGCATAC CTGCTGAGTT 751 ATCTACAGAT GGTAACTGTG TGGATTCTAT TAACGAGGAA GTTCTGTTAT 801 TCAATGACAA GCTCAAGCCA CTGGTTGATG AACTGAATAC CGAGTTAAGC 851 GGTGCACAAT TTCTTTATGT AGATGTGATA GCAATCAATT TGAACAATTT 901 ATCCACCCCT GCAGAAATTA CAATTGGCAA TGCACCATGC TGCAACGTGT 95l CTGCAGCAGT TGCTGGTGGA CAGTGTATTC CTGGGCAAAT TCCCTGCAGC 1001 AACAGGAACC AATATTATTT TTGGGATGAT TTCCATCCCA GTGAAGTAGT 1051 CAATGAAGCA TATTCAAGAT TAGCATATTC TGCGTTATCC TCATTACTTG 1101 ATGCTGATCC TCTTGCCATT GGCGGCCTAA CAGGCAAAAA CTGTCATGAT 1151 AAAGTGAAGA TACAATAGAC TGTATCTATG TGTCCCATGA TATTTCTATA 1201 TTCCAAGTTT CCGACAAGTC AAACTCAATG TAATAAAACT TGAGAGTCCG 1251 AATGTGCTAG TGTGATGTTA TCTCCTCAAT GGAAACAATA TGTTATCATT 1301 AATCTCAGAC TATTTATAAT TACTATTAAA AAAAAAAAAA AAAAAAA; and also variants of said nudeic acid sequence that are degenerate with respect to the foregoing DNA sequences. 3. A method for producing caffeine free coffee beans comprising: transforming coffee plants with a DNA sequence that is antisense to the DNA sequence or to a nudeic acid sequence or variants thereof are degenerate characterized in claim 2, and harvesting the fruit from the transformed coffee plants. 4. A coffee plant transformed with a nudeic acid sequence of claim 2 that codes on transcription for an RNA that is antisense to the mRNA that codes on expression for a xanthosine-N<7> methyltransferase of claim 1, wherein the RNA has a length sufficient to interfere with the expression of xanthosine-N<7>-methyltransferase. 5. A coffee plant transformed with a nudeic add sequence of claim 2 that codes on transcription for an RNA that is sense to the mRNA that codes on expression for a xanthosine-N<7> methyltransferase of claim 1, wherein the RNA has a length sufficient to interfere with the expression of xanthosine-N<7>-methyltransf erase. 6. The coffee plant of claim 4, wherein the nudeic acid sequence is linked to a transcription promoter in an antisense orientation. 7. The coffee plant of claim 5, wherein the nucleic acid sequence is linked to a transcription promoter in a sense orientation. 8. A coffee bean from the coffee plant set forth in any of claims 4 to 7. 9. A transforming vector comprising a transcription promoter operably linked to the nudeic add sequence as set forth in claim 2. 10. The transforming vector of claim 9, wherein the nucleic acid sequence is operably linked to the transcription promoter in a sense orientation. 11. The transforming vector of claim 9, wherein the nucleic acid sequence is operably linked to the transcription promoter in an antisense orientation. 12. The transforming vector of claim 9, wherein the promoter is a cauliflower mosaic virus 35S promoter. 13. The transforming vector of claim 9, wherein the vector is a modified plasmid pBI-121. 14. A coffee plant cell transformed with a nucleic acid sequence of claim 2 that codes on transcription for an RNA that is antisense to the mRNA that codes on expression for a xanthosine-N<7> methyltransferase of claim 1, wherein the RNA has a length sufficient to interfere with the expression of xanthosine-N<7>-methyltransferase. 15. A coffee plant cell transformed with a nucleic acid sequence of claim 2 that codes on transcription for an RNA that is sense to the mRNA that codes on expression for a xanthosine-N<7> methyltransferase, wherein the RNA has a length sufficient to interfere with the expression of xanthosine-N<7>-methyltransferase. 16. The coffee plant cell of claim 14, wherein the nudeic add sequence is operably linked to a transcription promoter in n sense orientation. 17. The coffee plant cell of claim 14, wherein the nucleic acid sequence is operably linked to a transcription promoter in an antisense orientation. 18. A transformed coffee plant ceil produced by the process of inserting a transforming vector of claim 9 into a coffee plant cell. 19. The transformed coffee plant cell of claim 88, wherein the nucleic add sequence is operably linked to the transcription promoter in a sense orientation. 20. The transformed coffee plant cell of claim 28, wherein the nucleic acid sequence is operably linked to the transcription promoter in an antisense orientation. 21. The transformed coffee plant cell of any of claims 14 to 20, wherein the cell exhibits reduced caffeine production compared to a coffee plant cell that is not transformed with the nucleic acid sequence. 22. A coffee plant regenerated from the transformed coffee plant cell of any of claims 14 to 21. 23. A coffee bean from a regenerated coffee plant according to claim 22. 24. A method for inhibiting production of caffeine by a coffee plant cell, comprising the steps of: providing a transforming vector comprising a nucleic add sequence of claim 2 that codes on transcription for an RNA that has a length sufficient to interfere with the expression of a xanthosine-N<7> methyltransferase of claim 1, wherein the nucleic add sequence is operably linked to a transcription promoter in an antisense orientation; and inserting the transforming vector into a coffee plant cell, wherein the nucleic acid sequence thereafter becomes inserted into the genome of the coffee plant cell to form a transformed cell, and wherein the transformed cell exhibits a reduced caffeine production compared to a coffee plant cell that has not been transformed with the nucleic acid sequence. 25. A method for inhibiting production of caffeine by a coffee plant cell, comprising the steps of: providing a transforming vector comprising a nucleic acid sequence of claim 2 that codes on transcription for an RNA that has a length sufficient to interfere with the expression of a xanthosine-N<7> methyltransferase of claim 1, wherein the nudeic acid sequence is operably linked to a transcription promoter in a sense orientation; and inserting the transforming vector into a coffee plant cell, wherein the nucleic acid sequence thereafter becomes inserted into the genome of the coffee plant cell to form a transformed cell, and wherein the transformed cell exhibits a reduced caffeine production compared to a coffee plant cell that has not been transformed with the nudeic acid sequence.