Down-regulation and silencing of allergen genes in transgenic peanut plants

Abstract

An allergen-free transgenic peanut seed is produced by recombinant methods. Peanut plants are transformed with multiple copies of each of the allergen genes, or fragments thereof, to suppress gene expression and allergen protein production. Alternatively, peanut plants are transformed with peanut allergen antisense genes introduced into the peanut genome as antisense fragments, sense fragments, or combinations of both antisense and sense fragments. Peanut transgenes are under the control of the 35S promoter, or the promoter of the Ara h2 gene to produce antisense RNAs, sense RNAs, and double-stranded RNAs for suppressing allergen protein production in peanut plants. A full length genomic clone for allergen Ara h2 is isolated and sequenced. The ORF is 622 nucleotides long. The predicted encoded protein is 207 amino acids long and includes a putative transit peptide of 21 residues. One polyadenilation signal is identified at position 951. Six additional stop codons are observed. A promoter region was revealed containing a putative TATA box located at position−72. Homologous regions were identified between Ara h2, h6, and h7, and between Ara h3 and h4, and between Ara h1P41B and Ara h1P17. The homologous regions will be used for the screening of peanut genomic library to isolate all peanut allergen genes and for down-regulation and silencing of multiple peanut allergen genes.

1 . A method for producing a transgenic peanut plant with reduced or undetectable allergen protein content in the seed, comprising the steps of: 
 (a) transforming a recipient peanut plant cell with a DNA construct comprising a peanut allergen antisense gene, or a peanut allergen sense gene, or a combination thereof, or fragments thereof;    (b) regenerating a peanut plant from the recipient cell which has been transformed with the DNA construct; and    (c) identifying a fertile transgenic peanut that produces seeds having reduced or undetectable allergen protein content.    
     
     
         2 . The method of  claim 1 , wherein the peanut allergen gene is selected from the group consisting of Ara h1, Ara h2, Ara h3, Ara h4, Ara h5, Ara h6, and Ara h7.  
     
     
         3 . The method of  claim 1 , wherein the recipient cell is transformed by the  Agrobacterium -mediated method.  
     
     
         4 . The method of  claim 1 , wherein the recipient cell is transformed by the biolistic method.  
     
     
         5 . The method according to  claim 1 , wherein the peanut allergen sense or antisense gene, or a fragment thereof, comprises at least a portion of the nucleotide sequence shown in  FIG. 2  (SEQ ID NO: 1).  
     
     
         6 . The method according to  claim 1 , wherein the peanut allergen sense or antisense gene, or fragment thereof, comprises at least a portion of the nucleotide sequence shown in  FIG. 3  (SEQ ID NO: 3).  
     
     
         7 . The method according to  claim 1 , wherein the peanut allergen sense or antisense gene or fragment thereof, comprises at least a portion of the nucleotide sequence shown in  FIG. 4  (SEQ ID NO: 4).  
     
     
         8 . The method according to  claim 1 , wherein the peanut allergen sense or antisense gene, or fragment thereof, comprises at least a portion of the nucleotide sequence shown in  FIG. 5  (SEQ ID NO: 5).  
     
     
         9 . The method according to  claim 1 , wherein the peanut allergen sense or antisense gene, or fragment thereof, comprises at least a portion of the nucleotide sequence shown in  FIG. 7  (SEQ ID NO: 6).