Cytoplasmic inhibition of gene expression

Abstract

The invention provides novel genetic constructions for the expression of inhibitory RNA in the cytoplasm of eukaryotic cells. The inhibitory RNA may be an anti-sense RNA or a co-suppressor RNA, and functions to reduce the expression of a gene of interest in the target cell. The genetic constructions of the invention are capable of replicating in the cytoplasm of a eukaryotic cell and comprise a promoter region in functional combination with an encoding polynucleotide. The genetic constructions may be designed so as to replicate in the cytoplasm of plant cells, yeast cells, and mammalian cells. When the eukaryotic cell of interest is a plant cell, the genetic construction is preferably derived from a plant RNA virus. Plant RNA virus derived genetic constructions may employ a plant virus subgenomic promoter, including subgenomic promoters from tobamoviruses in functional combination with the RNA encoding region. In a preferred embodiment of the invention, plant cells are induced to produce elevated levels of the carotenoid phytoene. The elevated levels of phytoene are achieved by inhibiting the expression of the enzyme phytoene desaturase using the vectors of the invention.

1 - 14 . (canceled)  
     
     
         15 . A method of producing a plant cell, which cell has increased production of a gene-product of interest, the method comprising: 
 transfecting a plant cell with a genetic vector capable of replication in the cytoplasm of the plant cell, which vector comprises: a first subgenomic promoter operably linked to a first polynucleotide encoding a target gene inhibitory RNA and a second subgenomic promoter operably linked to a second polynucleotide encoding a viral coat protein; wherein said vector is capable of systemically infecting plants and producing the inhibitory RNA; and, wherein the target gene inhibitory RNA is specific for a target gene comprising a negative regulator of the gene-product of interest.    
     
     
         16 . The method of  claim 15 , wherein the vector is derived from a plant RNA virus.  
     
     
         17 . The method of  claim 16 , wherein the vector is derived from a single stranded RNA plant virus.  
     
     
         18 . The method of  claim 16 , wherein at least one of the subgenomic promoters is derived from a tobamovirus.  
     
     
         19 . The method of  claim 18 , wherein at least one of the subgenomic promoters is derived from a tobacco mosaic virus.  
     
     
         20 . The method of  claim 15 , wherein the subgenomic promoters are heterologous to one another.  
     
     
         21 . The method of  claim 15 , wherein the target gene inhibitory RNA is an anti-sense RNA specific for the target gene.  
     
     
         22 . The method of  claim 15 , wherein the target gene inhibitory RNA is a co-suppressor RNA specific for the target gene.  
     
     
         23 . The method of  claim 15 , wherein the target gene inhibitory RNA is an endogenous plant gene inhibitory RNA specific for the target gene.  
     
     
         24 . The method of  claim 15 , wherein the gene-product of interest is a nucleic acid or a protein.  
     
     
         25 . The method of  claim 15 , wherein the gene-product of interest is phytoene.  
     
     
         26 . The method of  claim 15 , wherein the target gene encodes phytoene desaturase.  
     
     
         27 . The method of  claim 15 , wherein the gene-product of interest is phytoene and the target gene encodes phytoene desaturase  
     
     
         28 . A plant cell produced by the method of  claim 15.