Promoter-based silencing

Abstract

The present invention relates to unique strategies and constructs for producing a nucleic acid product that downregulates or prevents expression of a desired target polynucleotide.

1 . A plant transformation construct, comprising at least two copies of a desired polynucleotide operably linked to regulatory elements, wherein the desired polynucleotide comprises a sequence from the promoter of a target gene but not any sequence located downstream of the transcription start of the target gene. 
     
     
         2 . The plant transformation construct of  claim 1 , wherein the desired polynucleotide shares sequence identity to at least 24 contiguous nucleotides of the target gene promoter sequence. 
     
     
         3 . The plant transformation construct of  claim 1 , wherein (A) the regulatory elements are two convergently-oriented, functional plant promoters, wherein the desired polynucleotides are positioned in between and operably linked to the functional promoters; or (B) the regulatory elements are a functional plant promoter and a terminator, wherein the desired polynucleotides are positioned in between and operably linked to the functional promoter and the terminator. 
     
     
         4 . The plant transformation construct of  claim 3 , wherein the functional plant promoter is a tissue-specific promoter. 
     
     
         5 . The plant transformation construct of  claim 4 , wherein the functional plant promoter is specific to a plant tissue selected from the group consisting of in tubers, seeds, leaves, roots, vascular system, flowers, pollen, ovules. 
     
     
         6 . The plant transformation construct of  claim 4 , wherein the functional tissue-specific promoter is selected from the group consisting of a granule-bound starch synthase promoter, and ADP glucose pyrophosphorylase promoter, and a patatin promoter. 
     
     
         7 . The plant transformation construct of  claim 1 , wherein the target gene is selected from the group consisting of an F3′5′H gene involved in flavonoid biosynthesis, a COMT gene involved in lignin biosynthesis, a CCOMT gene involved in lignin biosynthesis, any other gene involved in lignin biosynthesis, an R1 gene involved in starch phosphorylation, a phosphorylase-L gene involved in starch phosphorylation, a PPO gene involved in the oxidation of polyphenols, a polygalacturonase gene involved in pectin degradation, a gene involved in the production of allergens, and a gene involved in fatty acid biosynthesis. 
     
     
         8 . The plant transformation construct of  claim 1 , wherein the desired polynucleotide comprises promoter sequences from multiple target genes. 
     
     
         9 . The plant transformation construct of  claim 8 , wherein the desired polynucleotide comprises promoter sequences from a polyphenol oxidase target gene, a phosphorylase L target gene, and an R1 target gene. 
     
     
         10 . A method for downregulating the expression of a target gene in a plant cell, comprising expressing the plant transformation construct of  claim 1  in a plant cell, wherein the expression level of the target gene is downregulated compared to the expression level of the target gene in an untransformed plant cell. 
     
     
         11 . A plant cell, comprising the plant transformation construct of  claim 1 . 
     
     
         12 . A plant, comprising the plant cell of  claim 11 . 
     
     
         13 . The plant of  claim 12 , wherein the plant is a tuber-bearing plant. 
     
     
         14 . A tuber obtained from the plant of  claim 13 . 
     
     
         15 . A food product made from the tuber of  claim 14 .