DNA sequences for ammonium transporter, plasmids, bacteria, yeasts, plant cells and plants containing the transporter

Abstract

There are described DNA sequences, that contain the coding region of ammonium transporters, which after introduction in suitable vectors are introduced into the plant genome and lead to the formation of mRNA which makes possible either the formation of new ammonium transporters, or prevents the formation of endogenous ammonium transporters in transgenic plants. There are further described plasmids, bacteria, yeast strains, plant cells and transgenic plants, which contain the sequences of ammonium transporters as a constituent of a recombinant DNA, and also a process for the identification and isolation of DNA sequences, coding for ammonium transporters.

1 . DNA sequences that contain the coding region of a plant ammonium transporter, characterised in that the information contained in the nucleotide sequence when integrated in a plant genome makes possible under control of a promoter in sense orientation, the expression of a translatable mRNA, which leads to the synthesis of an ammonium transporter in transgenic plants.  
     
     
         2 . DNA sequences, that contain the coding region of a plant ammonium transporter, characterised in that the information contained in the nucleotide sequence when integrated in a plant genome makes possible under control of a promoter in anti-sense orientation, the expression of a non-translatable mRNA, which prevents the synthesis of an endogenous ammonium transporter in transgenic plants.  
     
     
         3 . A DNA sequence according to  claim 1  or  2 , characterised in that, it contains the following nucleotide sequence (Seq-ID No 1):  
       
         
           
                 
                 
                 
               
                     
                 
                                           TTGTTCTGTAAACTCTCAAC 
                   20 
                     
                 
                     
                 
                   ATG TCT TGC TGG GCC ACC GAT CTC GCC GTC CTG TTG GGT CCT AAT 
                   65 
                 
                   Net Ser Gys Ser Ala Thr Asp Leu Ala Val Leu Leu Gly Pro Asn 
                 
                     
                 
                   GCC ACG GCG GCG GCC AAC TAC ATA TGT GGC CAG CTA GGC GAC GTC 
                   110 
                 
                   Ala Thr Ala Ala Ala Asn Tyr Ile Cys Gly Gln Leu Gly Asp Val 
                 
                     
                 
                   AAC AAC AAA TTC ATC GAC ACC GCT TTC GCT ATA GAC AAC ACT TAC 
                   155 
                 
                   Asn Asn Lys Phe Ile Asp Thr Ala Phe Ala Ile Asp Asn Thr Tyr 
                 
                     
                 
                   CTC CTC TTC TCC GCC TAC CTT GTC TTC TCT ATG CAG CTT GGC TTC 
                   200 
                 
                   Leu Leu Phe Ser Ala Tyr Leu Val Phe Ser Met GIn Leu Gly Phe 
                 
                     
                 
                   GCT ATG CTC TGT GCC CGT TCC GTG AGA GCC AAG AAT ACT ATG AAC 
                   245 
                 
                   Ala Met Leu Cys Ala Gly Ser Val Arg Ala Lys Asn Thr Met Asn 
                 
                     
                 
                   ATC ATG CTT ACC AAC GTC CTT GAC GCT GCA GCC GGT GGT CTC TTC 
                   290 
                 
                   Ile Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly Gly Leu Phe 
                 
                     
                 
                   TAT TAT CTG TTT GGC TAC GCC TTT GCC TTT GGA TCT CCG TCC AAT 
                   335 
                 
                   Tyr Tyr Leu Phe Gly Tyr Ala Phe Ala Phe Gly Ser Pro Ser Asn 
                 
                     
                 
                   GGT TTC ATC GGT AAA CAC TAC TTT GGT CTC AAA GAC ATC CCC ACG 
                   380 
                 
                   Gly Phe Ile Gly Lys His Tyr Phe Gly Leu Lys Asp Ile Pro Thr 
                 
                     
                 
                   GCC TCT GCT GAC TAC TCC AAC TTT CTC TAC CAA TGG GCC TTT GCA 
                   425 
                 
                   Ala Ser Ala Asp Tyr Ser Asn Phe Leu Tyr Gln Trp Ala Phe Ala 
                 
                     
                 
                   ATC GCT GCG GCT GGA ATC ACA AGT GGC TCG ATC GCT GAA CGG ACA 
                   470 
                 
                   Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr 
                 
                     
                 
                   CAG TTC GTG GCT TAC CTA ATC TAT TCC TCT TTC TTA ACC GGG TTT 
                   515 
                 
                   Gln Phe Val Ala Tyr Leu Ile Tyr Ser Ser Phe Leu Thr Gly Phe 
                 
                     
                 
                   GTT TAC CCG GTC GTC TCT CAC TGG TTC TGG TCA GTT GAT GGA TGG 
                   560 
                 
                   Val Tyr Pro Val Val Ser His Trp Phe Trp Ser Val Asp Gly Trp 
                 
                     
                 
                   GCC AGC CCG TTC CGT ACC GAT GGA GAT TTG CTT TTC AGC ACC GGA 
                   605 
                 
                   Ala Ser Pro Phe Arg Thr Asp Gly Asp Leu Leu Phe Ser Thr Gly 
                 
                     
                 
                   GCG ATA GAT TTC GCT GGG TCC GGT GTT GTT CAT ATG GTC GGA GGT 
                   650 
                 
                   Ala Ile Asp Phe Ala Gly Ser Gly Val Val His Met Val Gly Gly 
                 
                     
                 
                   ATC GCT GGA CTC TGG GGT GCG CTC ATC GAA GGT CCA CGA CTT GGC 
                   695 
                 
                   Ile Ala Gly Leu Trp Gly Ala Leu Ile Glu Gly Pro Arg Leu Gly 
                 
                     
                 
                   CGG TTC GAT AAC GGA GGC CGT GCC ATC GCT CTT CGT GGC CAC TCG 
                   740 
                 
                   Arg Phe Asp Asn Gly Gly Arg Ala Ile Ala Leu Arg Gly His Ser 
                 
                     
                 
                   GCG TCA CTT GTT GTC CTT GGA ACA TTC CTC CTC TGG TTT GGA TGG 
                   785 
                 
                   Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu Trp Phe Gly Trp 
                 
                     
                 
                   TAC GGA TTT AAC CCC GGT TCC TTC AAC AAG ATC CTA GTC ACG TAC 
                   830 
                 
                   Tyr Gly Phe Asn Pro Gly Ser Phe Asn Lys Ile Leu Val Thr Tyr 
                 
                     
                 
                   GAG ACA GGC ACA TAC AAC GGC CAG TGG AGC GCG GTC GGA CGG ACA 
                   875 
                 
                   Glu Thr Gly Thr Tyr Asn Gly Gln Trp Ser Ala Val Gly Arg Thr 
                 
                     
                 
                   GCT GTC ACA ACA ACG TTA GCT GGC TGC ACC GCG GCG CTG ACA ACC 
                   920 
                 
                   Ala Val Thr Thr Thr Leu Ala Gly Cys Thr Ala Ala Leu Thr Thr 
                 
                     
                 
                   CTA TTT GGG AAA CGT CTA CTC TCG GGA CAT TGG AAC GTC ACT GAT 
                   965 
                 
                   Leu Phe Gly Lys Arg Leu Leu Ser Gly His Trp Asn Val Thr Asp 
                 
                     
                 
                   GTA TGC AAC GGC CTC CTC GGA GGG TTT GCA GCC ATA ACT GGT GGC 
                   1010 
                 
                   Val Cys Asn Gly Leu Leu Gly Gly Phe Ala Ala Ile Thr Gly Gly 
                 
                     
                 
                   TGC TCT GTC GTT GAG CCA TGG GCT GCG ATC ATC TGC GGG TTC GTG 
                   1055 
                 
                   Cys Ser Val Val Glu Pro Trp Ala Ala Ile Ile Cys Gly Phe Val 
                 
                     
                 
                   GCG GCC CTA GTC CTC CTC GGA TGC AAC AAG CTC GCT GAG AAG CTC 
                   1100 
                 
                   Ala Ala Leu Val Leu Leu Gly Cys Asn Lys Leu Ala Glu Lys Leu 
                 
                     
                 
                   AAA TAC GAC GAC CCT CTT GAG GCA GCA CAA CTA CAC GGT GGT TGC 
                   1145 
                 
                   Lys Tyr Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly Gly Cys 
                 
                     
                 
                   GCT GCG TGG GGA CTA ATA TTC ACG GCT CTC TTC GCT CAA GAA AAG 
                   1190 
                 
                   Gly Ala Trp Gly Leu Ile Phe Thr Ala Leu Phe Ala Gln Glu Lys 
                 
                     
                 
                   TAC TTG AAC CAG ATT TAC GGC AAC AAA CCC GGA AGG CCA CAC GGT 1235 
                 
                   Tyr Leu Asn Gln Ile Tyr Gly Asn Lys Pro Gly Arg Pro His Gly 
                 
                     
                 
                   TTG TTT ATG GGC GGT GGA GGA AAA CTA CTT GGA GCT CAG CTG ATT 
                   1280 
                 
                   Leu Phe Met Gly Gly Gly Gly Lys Leu Leu Gly Ala Gln Leu Ile 
                 
                     
                 
                   CAG ATC ATT GTG ATC ACG GGT TGG GTA ACT GCG ACC ATG GGG ACA 
                   1325 
                 
                   Gln Ile Ile Val Ile Thr Gly Trp Val Ser Ala Thr Met Gly Thr 
                 
                     
                 
                   CTT TTC TTC ATC CTC AAG AAA ATG AAA TTG TTG CGG ATA TCG TCC 
                   1370 
                 
                   Leu Phe Phe Ile Leu Lys Lys Met Lys Leu Leu Arg Ile Ser Ser 
                 
                     
                 
                   GAG GAT GAG ATG GCC GGT ATG GAT ATG ACC AGG CAC GGT GGT TTT 
                   1415 
                 
                   Glu Asp Glu Met Ala Gly Met Asp Met Thr Arg His Gly Gly Phe 
                 
                     
                 
                   GCT TAT ATG TAC TTT GAT GAT CAT GAG TCT CAC AAA GCC ATT CAG 
                   1460 
                 
                   Ala Tyr Met Tyr Phe Asp Asp Asp Glu Ser His Lys Ala Ile Gln 
                 
                     
                 
                   CTT AGG AGA GTT GAG CCA CGA TCT CCT TCT CCT TCT GGT GCT AAT 
                   1505 
                 
                   Leu Arg Arg Val Glu Pro Arg Ser Pro Ser Pro Ser Gly Ala Asn 
                 
                     
                 
                   ACT ACA CCT ACT CCG GTT TGATTTGGAT TTTTACTTTT ATTCTCTATT 
                   1553 
                 
                   Thr Thr Pro Thr Pro Val 
                 
                     
                 
                   TTCTAGAGTA TTATTTTAAA TGATGTTTTG TGATACTTAA ATATTGTTTT 
                   1603 
                 
                   GGATATTTTT TTGGCATTTC AGTAATGTTT TAGATGTACA GTTTCATGGG 
                   1653 
                 
                     
                 
                   GTTGTGATGA TAATATCTAT GTGGTCATTT GTGTTCTCTT TGGAGTTAAA 
                   1703 
                 
                   AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 
                   1748 
                 
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         4 . A process for the identification and isolation of DNA sequences from plants that code for ammonium transporters, characterised in that it includes the following steps: 
 a) transformation of a yeast strain, which cannot grow in media which contain ammonium as the only nitrogen source, with a plant cDNA-library using suitable expression vectors,    b) selection and propagation of transformands, which after expression of plant cDNA-sequences, can grow in media which contain ammonium as the only nitrogen source, and    c) isolation of the expression vectors, which carry a plant cDNA insert from the selected transformand.    
     
     
         5 . DNA sequences from plants, that code for a protein with the biological activity of an ammonium transporter, obtainable by a process according to  claim 4 .  
     
     
         6 . Plasmids, containing DNA sequences according to any one of claims  1 - 3 .  
     
     
         7 . Plasmid p35S-MEP-a (DSM 8651).  
     
     
         8 . Plasmid p35S-a-MEP-a (DSM 8652).  
     
     
         9 . Use of the plasmid according to any one of claims  6 - 8  or derivatives or parts thereof for the transformation of pro- and eukaryotic cells.  
     
     
         10 . Transgenic plants, in which the DNA sequences of the invention are introduced as a constituent of a recombinant DNA molecule, in which this recombinant DNA molecule is stably integrated into the genome, and in whose cells based on the presence of these sequences there is achieved either a synthesis of an additional plant ammonium transporter whereby these cells can take up larger amounts of ammonium in comparison with untransformed plants or there is achieved an inhibition of the synthesis of endogenous ammonium transporters whereby such cells show a reduced uptake of ammonium in comparison with untransformed cells  
     
     
         11 . Yeasts, containing DNA sequences according to any one of claims  1 - 3 .  
     
     
         12 . Bacteria, containing DNA sequences according to any one of claims  1 - 3 .  
     
     
         13 . Plant cells, in which the DNA sequences of the invention are introduced as a constituent of a recombinant DNA molecule, in which this recombinant DNA molecule is stably integrated into the genome, and in whose cells based on the presence of these sequences there is achieved either a synthesis of an additional plant ammonium transporter whereby these cells can take up larger amounts of ammonium in comparison with untransformed plants or there is achieved an inhibition of the synthesis of endogenous ammonium transporters whereby such cells show a reduced uptake of ammonium in comparison with untransformed cells.  
     
     
         14 . Use of plant cells according to  claim 13  for the regeneration of whole plants.  
     
     
         15 . Use of the DNA sequences according to any one of claims  1 - 3 , for the preparation of derivatives with changed specificity of the ammonium transporter.  
     
     
         16 . Use of the DNA sequences according to any one of claims  1 - 3 , for the isolation of homologous sequences from the genome of bacteria, fungi and transgenic plants.  
     
     
         17 . Use of the DNA sequences according to claims  1 ,  3  or  5 , in combination with a transcription promoter in sense orientation, for the expression of a translatable mRNA, which makes possible the synthesis of an ammonium transporter in pro- and eukaryotic cells.  
     
     
         18 . Use of the DNA sequences according to claims  2 ,  3  or  5 , in combination with a transcription promoter in anti-sense orientation, for the expression of a non-translatable mRNA, which prevents the synthesis of an endogenous ammonium transporter in pro- and eukaryotic cells.  
     
     
         19 . Use of the DNA sequences according to any one of claims  1 - 3  or  5  for the preparation of transgenic plants with changed nitrogen metabolism.  
     
     
         20 . Use of the DNA sequences according to any one of claims  1 ,  3  or  5  for the preparation of transgenic crop plants, such as tobacco, potatoes, beets and maize with increased yield.  
     
     
         21 . Use of the DNA sequences according to any one of claims  2 ,  3  or  5  for the preparation of transgenic crop plants, such as tobacco, potatoes, beets and maize under low input conditions.  
     
     
         22 . Use of the DNA sequences according to any one of claims  2 ,  3  or  5  for the preparation of transgenic crop plants, which also grow on acid soils.