Down-regulating gene expression in insect plants

Abstract

The present invention relates to the prevention and/or control of infestation by insect pest species. In particular, the invention relates to down-regulation of expression of target genes in insect pests using interfering ribonucleic acid (RNA) molecules. Also described are transgenic plants that (i) express or are capable of expressing interfering RNAs of the invention and (ii) are resistant to infestation by insect pest species. Compositions containing the interfering RNAs of the invention are also provided.

What is claimed: 
     
         1 . An isolated polynucleotide encoding a dsRNA, wherein said dsRNA comprises annealed complementary strands, one strand of which comprises or consists of a sequence of at least 21 contiguous nucleotides that is 100% identical to a target nucleotide sequence within the target gene, wherein said target gene:
 (i) has a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, or a nucleotide sequence that, when the two sequences are optimally aligned and compared, is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof; or   (ii) is an insect pest ortholog of a gene having a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, wherein the two orthologous genes are similar in sequence to such a degree that when the two genes are optimally aligned and compared, the ortholog has a sequence that is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iii) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence encoded by the nucleotide sequence represented by at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iv) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence represented in any of SEQ ID NOs 216 and 238.   
     
     
         2 . A DNA construct comprising the polynucleotide of  claim 1 . 
     
     
         3 . The DNA construct of  claim 2 , wherein the DNA construct is an expression construct and wherein the polynucleotide sequence is operably linked to at least one regulatory sequence capable of driving expression of the polynucleotide sequence. 
     
     
         4 . A host cell comprising the polynucleotide of  claim 1 . 
     
     
         5 . A host cell comprising the DNA construct of  claim 3 . 
     
     
         6 . The host cell of  claim 4 , wherein the host cell is a prokaryotic or a eukaryotic cell. 
     
     
         7 . The host cell of  claim 6 , wherein the host cell is a bacterial cell or a plant cell. 
     
     
         8 . The host cell of  claim 5 , wherein the host cell is a prokaryotic or a eukaryotic cell. 
     
     
         9 . The host cell of  claim 8 , wherein the host cell is a bacterial cell or a plant cell. 
     
     
         10 . A composition comprising a dsRNA comprising annealed complementary strands, one strand of which comprises or consists of a sequence of at least 21 contiguous nucleotides that is 100% identical to a target nucleotide sequence within the target gene, wherein said target gene:
 (i) has a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, or a nucleotide sequence that, when the two sequences are optimally aligned and compared, is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof; or   (ii) is an insect pest ortholog of a gene having a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, wherein the two orthologous genes are similar in sequence to such a degree that when the two genes are optimally aligned and compared, the ortholog has a sequence that is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iii) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence encoded by the nucleotide sequence represented by at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iv) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence represented in any of SEQ ID NOs 216 and 238.   
     
     
         11 . The composition of  claim 10 , further comprising at least one suitable carrier, excipient or diluent. 
     
     
         12 . The composition of  claim 10 , wherein the dsRNA is expressed inside a host cell. 
     
     
         13 . A method for generating a transgenic plant comprising:
 (a) transforming a plant cell with the DNA construct of  claim 1 ;   (b) regenerating a plant from the transformed plant cell; and   (c) growing the transformed plant under conditions suitable for the expression of the double-stranded RNA from the recombinant DNA construct   
     
     
         14 . A transgenic plant produced by the method of  claim 13 . 
     
     
         15 . A transgenic plant, reproductive or propagation material for a transgenic plant, or a cultured transgenic plant cell, which expresses or is capable of expressing at least one dsRNA, wherein said dsRNA comprises annealed complementary strands, one strand of which comprises or consists of a sequence of at least 21 contiguous nucleotides that 100% identical to a target nucleotide sequence within the target gene, wherein said target gene:
 (i) has a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, or a nucleotide sequence that, when the two sequences are optimally aligned and compared, is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof; or   (ii) is an insect pest ortholog of a gene having a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, wherein the two orthologous genes are similar in sequence to such a degree that when the two genes are optimally aligned and compared, the ortholog has a sequence that is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iii) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence encoded by the nucleotide sequence represented by at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iv) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence represented in any of SEQ ID NOs 216 and 238.   
     
     
         16 . A seed produced from the transgenic plant of  claim 15 . 
     
     
         17 . A method to down regulate expression of a target gene in an insect pest species, comprising contacting said insect pest species with an effective amount of at least one double-stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises annealed complementary strands, one strand of which comprises or consists of a sequence of at least 21 contiguous nucleotides that is 100% identical to a target nucleotide sequence within the target gene, wherein said target gene:
 (i) has a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, or a nucleotide sequence that, when the two sequences are optimally aligned and compared, is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof; or   (ii) is an insect pest ortholog of a gene having a nucleotide sequence comprising at least one of SEQ ID NOs 11, 31, 134, and 148, or the complement thereof, wherein the two orthologous genes are similar in sequence to such a degree that when the two genes are optimally aligned and compared, the ortholog has a sequence that is at least 85% identical to at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iii) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence encoded by the nucleotide sequence represented by at least one of SEQ ID NOs 11, 31, 134, and 148; or   (iv) has a nucleotide sequence encoding an amino acid sequence that, when the two amino acid sequences are optimally aligned and compared, is at least 70% preferably at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence represented in any of SEQ ID NOs 216 and 238.   
     
     
         18 . The method of  claim 17 , wherein the insect pest species is a plant pest. 
     
     
         19 . The method of  claim 18 , wherein the plant pest is selected from the  Leptinotarsa  genus or  Lygus  genus. 
     
     
         20 . The method of  claim 19 , wherein the plant pest is  Leptinotarsa decemlineata  or  Lygus hesperus.