Characterisation of gene function using double stranded RNA inhibition

Abstract

There is provided a method of identifying DNA responsible for conferring a particular phenotype in a cell which method comprises a) constructing a cDNA or genomic library of the DNA of said cell in a suitable vector in an orientation relative to a promoter(s) capable of initiating transcription of said cDNA or DNA to double stranded (ds) RNA upon binding of an appropriate transcription factor to said promoter(s), b) introducing said library into one or more of said cells comprising said transcription factor, and c) identifying and isolating a particular phenotype of said cell comprising said library and identifying the DNA or cDNA fragment from said library responsible for conferring said phenotype. Using this technique it is also possible to assign function to a known DNA sequence by a) identifying a homologue(s) of said DNA sequence in a cell, b) isolating the relevant DNA homologue(s) or a fragment thereof from said cell, c) cloning said homologue or fragment thereof into an appropriate vector in an orientation relative to a suitable promoter(s) capable of initiating transcription of dsRNA from said DNA homologue or fragment upon binding of an appropriate transcription factor to said promoter(s) and d) introducing said vector into said cell from step a) comprising said transcription factor.

1 . A method of alleviating infestation of plant pests, which method comprises 
 a) identifying a DNA sequence from said pest which is critical for its survival, growth, proliferation,    b) cloning said sequence from step a) or a fragment thereof in a suitable vector in an orientation relative to promoter(s) such that said promoter(s), is capable of initiating transcription of said DNA sequence to RNA or dsRNA upon binding of an appropriate transcription factor to said promoter(s), and    c) introducing said vector into the plant.    
     
     
         2 . A method according to  claim 1 , wherein said DNA sequence is provided between two promoters such that binding of the transcription factor to the promoters results in transcription of the DNA to dsRNA.  
     
     
         3 . A method according to  claim 1 , wherein said DNA sequence is provided in a sense and an antisense orientation relative to said promoter such that binding of the transcription factor to the promoter results in transcription of the DNA to dsRNA.  
     
     
         4 . A method according to  claim 1 , wherein said pest is a nematode worm.  
     
     
         5 . A method according to  claim 4 , wherein said nematode comprises any of Tylenchulus ssp., Radopholus ssp., Rhadinaphelenchus ssp., Heterodera ssp., Rotylenchulus ssp., Pratylenchus ssp., Belonolaimus ssp., Canjanus ssp., Meloidogyne ssp., Globodera ssp., Nacobbus ssp., Ditylenchus ssp., Aphelenchoides ssp., Hirschmenniella ssp., Anguina ssp., Hoplolaimus ssp., Heliotylenchus ssp., Criconemella ssp, Xiphinema ssp., Longidorus ssp., Trichodorus ssp., Paratrichodorus ssp., or Aphelenchs ssp.  
     
     
         6 . A method according to  claim 2 , wherein said DNA sequence or fragment thereof is cloned between two tissue specific promoters.  
     
     
         7 . A method according to  claim 6 , wherein said tissue specific promoters are root specific promoters.