Method of identifying a nucleic acid sequence in a plant

Abstract

The present invention provides a method of compiling a plant functional gene profile, a method of changing the phenotype or biochemistry of a plant, a method of determining a change in phenotype or biochemistry of a plant, and a method of determining the presence of a trait in plant. The methods comprise expressing transiently a nucleic acid sequence of a plant into a host plant to affect phenotypic or biochemical changes in the host plant. A viral vector functional genomic screen has been developed to identify nucleotide sequences in transfected plants by systemically knocking out endogenous gene expression in an antisense mechanism. Once the presence of a trait in a plant is identified by phenotypic or biochemical changes in the host plant, the nucleic acid insert in the cDNA clone or in the vector that results in the changes is then sequenced. The present invention exemplifies that genes encoding GTP binding proteins in one plant can silence endogenous gene expression in a different plant.

What is claimed is:  
     
         1 . A method for identifying a nucleic acid sequence in a donor plant having the same function as that in a host plant that belongs to a different genus, family, order, class, subdivision, or division from said donor plant, said method comprising the steps of: 
 (a) preparing a library of cDNAs, genomic DNAs, or a pool of RNAs of said donor plant;    (b) constructing recombinant viral nucleic acids obtained from a tobamo virus comprising an unidentified nucleic acid insert obtained from said library in an antisense orientation relative to said DNA or RNA sequence of said donor plant;    (c) infecting a host plant with one or more said recombinant viral nucleic acids, and transiently expressing said unidentified nucleic acid in the host plant;    (d) detecting one or more changes in said host plant;    (e) identifying said recombinant viral nucleic acid that results in changes in said host plant;    (f) determining the sequence of said nucleic acid insert in said recombinant viral nucleic acid; and    (g) determining the sequence of an entire open reading frame of a gene in said donor from which said nucleic acid insert is derived.    
     
     
         2 . The method according to  claim 1 , wherein said nucleic acid sequence comprising a GTP binding protein open reading frame.  
     
     
         3 . A method for identifying a nucleic acid sequence in a host plant having the same function as that in a donor plant that belongs to a different genus, family, order, class, subdivision, or division from said donor plant, said method comprising the steps of: 
 (a) preparing a cDNA library, a genomic DNA library, or a pool of RNA of said donor plant;    (b) constructing recombinant viral nucleic acids obtained from a tobamo virus comprising an unidentified nucleic acid insert obtained from said library in an antisense orientation relative to said DNA or RNA sequence of said donor plant;    (c) infecting a host plant with one or more said recombinant viral nucleic acids, and transiently expressing said unidentified nucleic acid in the host plant;    (d) detecting one or more changes in said host plant;    (e) identifying said recombinant viral nucleic acid that results in changes in said host plant; and    (f) determining the sequence of said nucleic acid insert in said recombinant viral nucleic acid; and    (g) determining the sequence of an entire open reading frame of a gene in said host plant, the expression of which gene is affected by said insert.    
     
     
         4 . The method according to  claim 3 , wherein said nucleic acid sequence comprising a GTP binding protein open reading frame.  
     
     
         5 . A method for detecting the presence of a nucleic acid sequence that has homology in a donor plant and in a host plant, wherein said donor plant and said host plant belong to a different genus, family, order, class, subdivision, or division from said donor plant, said method comprising the steps of: 
 (a) preparing a cDNA library, a genomic DNA library, or a pool of RNA of said donor plant;    (b) constructing recombinant viral nucleic acids obtained from a tobamo virus comprising an unidentified nucleic acid insert obtained from said library in an antisense orientation relative to said DNA or RNA sequence of said donor plant;    (c) infecting a host plant with one or more said recombinant viral nucleic acids, and transiently expressing said unidentified nucleic acid in the host plant; and    (d) detecting one or more changes in said host plant.    
     
     
         6 . The method according to  claim 5 , further comprising the steps of: 
 (e) identifying said recombinant viral nucleic acid that results in changes in said host plant;    (f) determining the sequence of said nucleic acid insert in said recombinant viral nucleic acid; and    (g) determining the sequence of an entire open reading frame of said donor from which said nucleic acid insert is derived.    
     
     
         7 . The method according to  claim 5 , wherein said nucleic acid sequence comprising a GTP binding protein open reading frame having an antisense orientation.