Methods and vectors for site-specific recombination in plant cell plastids

Abstract

Novel compositions and methods useful for genetic engineering of plant cells are provided. In particular, plastid constructs comprising recombining sites are provided. Such constructs find use in methods for site specific recombination in plant cell plastids. Plant cells and plants comprising the constructs described herein, as well as plant cells and plants produced by the methods of the present invention are of interest.

1 . A recombinant nucleic acid sequence comprising: 
 a plastid construct comprising,    at least one DNA sequence, and    at least two recombining sites.    
     
     
         2 . The recombinant nucleic acid sequence according to  claim 1 , 
 wherein said at least one DNA sequence is a first DNA sequence and a second DNA sequence, and    wherein said recombining sites are positioned between said first and said second DNA sequences.    
     
     
         3 . The recombinant nucleic acid sequence according to  claim 1 , 
 wherein said at least two recombining sites is a first recombining site and a second recombining site, and    wherein said at least one DNA sequence is positioned between said first and said second recombining sites.    
     
     
         4 . The recombinant nucleic acid construct according to  claim 1 , wherein each of said recombining sites are selected from the group consisting of Lox, FRT, and R.  
     
     
         5 . The recombinant nucleic acid construct according to  claim 1 , further comprising regions of homology for integration into the plastid genome.  
     
     
         6 . A plant cell comprising the construct according to  claim 1 .  
     
     
         7 . A plant comprising the plant cell according to  claim 6 .  
     
     
         8 . A recombinant nucleic acid construct comprising, in the 5′ to 3′ direction of transcription: 
 a transcriptional initiation region functional in a plant cell, an organelle targeting sequence, and a nucleic acid sequence encoding recombinase.    
     
     
         9 . The recombinant nucleic acid construct according to  claim 8 , wherein said transcriptional initiation region is selected from the group consisting of a transcriptional initiation region functional during zygote formation, and a transcriptional initiation region functional during seed germination.  
     
     
         10 . The recombinant nucleic acid construct according to  claim 8 , wherein said targeting sequence directs the recombinase to the plant cell plastid.  
     
     
         11 . The recombinant nucleic acid construct according to  claim 8 , wherein said recombinase is a bacteriophage P1 Cre recombinase.  
     
     
         12 . A method for the production of a plant having transformed plastids, comprising: 
 introducing into a plant cell a first recombinant DNA sequence comprising a plastid construct comprising at least one DNA sequence, and at least two recombining sites,    providing a recombinase to said plant cells, and    regenerating a plant having at least one plant cell containing said first DNA construct.    
     
     
         13 . The method according to  claim 12 , wherein said recombinase is provided to said plant cells by introducing a second recombinant comprising: 
 a transcriptional initiation region, an organelle targeting region, and a nucleic acid sequence encoding recombinase.    
     
     
         14 . The method according to  claim 12 , 
 wherein said at least one DNA sequence is a first DNA sequence and a second DNA sequence, and    wherein said recombining sites are positioned between said first and said second DNA sequences.    
     
     
         15 . The method according to  claim 12 , 
 wherein said at least two recombining sites is a first recombining site and a second recombining site, and    wherein said at least one DNA sequence is positioned between said first and said second recombining sites.    
     
     
         16 . The method according to  claim 15 , wherein said first and said second recombining sites are parallel, i.e., in directly repeated orientation.  
     
     
         17 . The method according to  claim 16 , whereby excision of the DNA sequence located between said first and said second recombining sites occurs.  
     
     
         18 . The method according to  claim 17 , wherein said DNA sequence encodes a selectable marker.  
     
     
         19 . The method according to  claim 18 , further comprising introducing a third construct into a plant cell obtained from said regenerated plant.  
     
     
         20 . The method according to  claim 12 , 
 wherein each of said recombining sites are selected from the group consisting of Lox, FRT, and R.    
     
     
         21 . The recombinant nucleic acid construct according to  claim 12 , further comprising regions of homology for integration into the plastid genome.  
     
     
         22 . A plant cell produced according to method of  claim 12.