Expression cassette and plasmids for a guard cell specific expression and their use for the introduction of transgenic plant cells and plants

Abstract

There is described an expression cassette and plasmids containing an expression cassette, which contains the transcriptional regulatory starter region for a guard cell specific gene expression. In the expression cassette is contained the guard cell promoter having the sequence Seq. ID No: 1. The starter region in the expression cassette ensures a guard cell specific gene expression and causes no expression in mesophyllic cells or epidermal cells of the leaves of plants. There is also described the preparation of the expression cassette and plasmids containing this expression cassette. Also described is the use of the expression cassette and plasmids containing this expression cassette for preparing transgenic plant cells and plants.

1 . An expression cassette characterised in that it mediates a DNA sequence with a transcriptional regulatory starter region that ensures a guard cell specific gene expression in the closed cells of leaves of plants and no expression in mesophyllic cells or epidermal cells of the leaves.  
     
     
         2 . An expression cassette according to  claim 1  characterised in that there is contained on the DNA sequence an approximately 3.2 kb size HincII/BglII promoter fragment of the ADP glucose pyrophosphorylase gene GS6-11 or a part thereof.  
     
     
         3 . An expression cassette according to  claim 2  characterised in that there is contained on the DNA sequence of the guard cell specific promoter the sequence: Seq ID No:1  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   HindIII 
                 
                     
                   AAGCTTCGTA AAGAATATTT TATCATAGTA AAACATGATT  
                 
                 
                 
                 
               
                     
                   ATCAAGTAAA 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   AGTGAACAAA GGGAGTAATA TGAAGATTTA TCATGTATTT  
                 
                 
                 
                 
               
                     
                   AAAAGCTCAA 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   TAGTGATTAT AATTTGAGGG ACTAAATAAA TTTAAGGAGT  
                 
                 
                 
                 
               
                     
                   TGTTAATATA 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   TTCCGAGAAA ATAAAATATT GTTTAAGTAG AAAAGTTATG  
                 
                 
                 
                 
               
                     
                   GGGTGTATAA 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   GTTAAATAAT AATATTTTGT AAATAGGGAT ATCGAAATGA  
                 
                 
                 
                 
               
                     
                   GTATAAATAG 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   AAAGATAGCA AGGTTTCTCG TGAGAGTTCA CAAGCCAATA  
                 
                 
                 
                 
               
                     
                   AAGCTGATCA 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   CACTCCCCTT TGTATGTCCA CTCAACAACA CAACTTCTTG  
                 
                 
                 
                 
               
                     
                   TGATTCACTT 
                     
                 
                     
                     
                 
                 
                 
               
                     
                   TCAATTCTAG ATCGGGGATC C 
                 
                     
                     
                 
                     
                   BamHI. 
                 
                     
                     
                 
             
                
                
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
               
            
             
                
                
               
            
             
                
                
                
                
               
            
           
         
       
     
     
         4 . An expression cassette according to any one of the preceding claims characterised in that the DNA sequence contained therein, which is fused to suitable coding DNA sequences, changes the plant guard cells therein so that a permanent opening or closing or lengthening or shortening of the opening period of these cells is made possible.  
     
     
         5 . An expression cassette according to any one of the preceding claims characterised in that the transcriptional regulatory starter region for a guard cell specific gene expression can be inserted after a DNA sequence, that contains the information for the formation of endogenous products or the formation of heterologous expression products in crops.  
     
     
         6 . Plasmids containing an expression cassette according to any one of  claims 1  to  5 .  
     
     
         7 . Plasmid pAS (DSM 6906) consisting of an approximately 13.2 kb size DNA sequence in which there is contained an approximately 2.0 kb size kanamycin resistance gene, and an approximately 3.2 kb size HincII/BglII promoter fragment of the regulatory starter area of the ADP glucose pyrophosphorylase gene GS6-11 of  Solanum tuberosum,  a polylinker and the transcription terminator of the nopaline synthase gene.  
     
     
         8 . Plasmid pAS-GUS (DSM 6907), consisting of an approximately 15.2 kb size DNA sequence in which there is contained an approximately 2.0 kb size kanamycin resistance gene, an approximately 3.2 kb size HincII/BglII promoter fragment of the regulatory starter area of the ADP glucose pyrophosphorylase gene GS6-11 from  Solanum tuberosum,  the approximately 2 kb size coding region β-glucuronidase and the transcription terminator of the nopaline synthase gene.  
     
     
         9 . Plasmid pS1-D4GUS or derivatives thereof containing the DNA sequence Seq ID No:1, according to  claim 3  or parts thereof.  
     
     
         10 . Process of genetic manipulation of plant cells containing an expression cassette with a DNA sequence for the closed cell specific gene expression that consists of the following steps, 
 a. Isolating clone GS6-11,    b. preparing plasmid pSF-6, using the approximately 12 kb size SalI-fragment of the clone GS6-11,    c. preparing the plasmid pH6-1 using an approximately 5.3 kb size HincII-fragment from the plasmid pSF-6,    d. preparing the plasmids pAS (DSM 6906), PAS-GUS (DSM 6907) and/or pS1-D4GUS using the approximately 3.2 kb size HincII/BglII-fragment of the regulatory starter area of the ADP glucose pyrophosphorylase gene of  Solanum tuberosum  from the plasmid pH 6-1, and    e. transferring the plasmid pAS (DSM6906), pAS-GUS (DSM6907) and/or pS1-D4GUS into a plant cell.    
     
     
         11 . A plant cell prepared according to the process of  claim 10 .  
     
     
         12 . A plant or plant tissue regenerated from a plant cell according to  claim 11 .  
     
     
         13 . A plant cell containing an expression cassette according to any one of  claims 1  to  5 .  
     
     
         14 . Use of an expression cassette according to any one of  claims 1  to  5  for the expression of genes in guard cells that change the regulation of gas exchange and transpiration.  
     
     
         15 . Use of expression cassette to any one of  claims 1  to  5  for the expression of homologous or heterologous genes in guard cells of transformed plants.  
     
     
         16 . Use of an expression cassette to any one of  claims 1  to  5  for the expression of genes in guard cells that cause a local change of the phytohormone level.  
     
     
         17 . Use of the plasmid pAS, pAS-GUS or pS1-D4GUS or derivatives thereof for the transformation of crops.  
     
     
         18 . Use of a plasmid pAS, pAS-GUS or pS1-D4GUS pAS or derivatives thereof for the regulation of endogenous processes or for the preparation of heterogenous products in crops.  
     
     
         19 . A plant according to  claim 12  characterised in that it is a tobacco plant.  
     
     
         20 . A plant according to  claim 12  characterised in that it is a potato plant.  
     
     
         21 . A plant according to  claim 12  characterised in that it is a tomato plant.  
     
     
         22 . A plant according to  claim 12  characterised in that it is a sugar beet plant.