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Gall wasp control agents

A nucleic acid and nucleotide technology, applied in the field of gene silencing, which can solve the problems of chemical pesticides that are unfavorable to the environment, harmful to non-target crops and fauna, etc.

Active Publication Date: 2014-02-19
FUTURAGENE ISRAEL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical pesticides may be environmentally unfriendly, are not selective, and may be harmful to non-target crops and fauna

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] gall bee transcriptome sequencing

[0164] Gall wasp-infected leaves were collected from infected red gum from Emek, Israel. Gall wasp larvae were removed from galls found in leaves and / or petioles by cutting and opening the galls with a sharp knife under a binocular microscope. Use a mixture of larvae from various larval developmental stages. Place batches of 100 larvae in microtubes on ice. The tubes were then sealed and immediately frozen in liquid nitrogen and stored at -80°C until further processing. Total RNA was isolated using the MasterPure RNA purification kit and manual (MRC85102 - Epicentere Biotechnologies). The total RNA volume was 50 μl. Total RNA was then treated with DNAse to remove any remaining residual DNA, followed by isolation of poly A mRNA (MicroPoly(A) Purist, Small Scale mRNA Purification Kit, AM1919Ambion). The final volume of mRNA was 20 μl. Purified mRNA was stored at -80°C until 454 sequencing. 454 sequencing was performed according...

Embodiment 2

[0166] Identification of Li and Om target genes and sequences

[0167]Unique viable Li and Om genes that are critical for cellular processes or proper developmental progression in specific tissues or whole organisms are selected as targets for gene silencing. First, standard procedures based on degenerate primers of known homologous Hymenopteran sequences will be used to identify fragments of the target gene. Thereafter, the respective transcriptomes of Li and Om gall wasp larvae were sequenced using the 454 Sequencer deep sequencing platform (454 Life Sciences; Branford, CT, USA; now Roche, Basel). Using the Roche software package and annotated using the Blast2Go program (available at http: / / www.blast2go.org / ), sequences were assembled and the results annotated based on sequence alignments with known published Hymenopteran transcriptomes.

[0168] Table 1 gives SEQ ID NO: Complete or partial gene sequences of the identified genes 1-9 from Li and Om.

[0169] Table 1. Li...

Embodiment 3

[0184] Identification of Li and Om target genes and sequences

[0185] A BLAST (NCBI) comparison (15,16) using 141 genes that are lethal when expressed as RNAi in Drosophila was used to identify 127 direct to homologous sequences. The identified Nv sequences were further used to screen the lethal genes of the Om and Li transcriptome libraries prepared in Example 1. The screen identified 39 potential target sequences from the Om transcriptome library and 48 potential targets from the Li transcriptome library comprising a contiguous reading frame of at least 500 nucleotides, and 1 sequence comprising a 309 nucleotide Potential Li-derived target genes (SEQ ID NO:76) of contiguous open reading frames, or at least 50% of each predicted full-length gene. In one example, a BLAST (NCBI) comparison using the Drosophila melanogaster gene ADV37321 (CG18740) (15), which has been identified as an RNAi-lethal factor, was used to identify the Nv homolog gene XP_001605573. The Nv homolog...

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Abstract

The present invention relates to the field of RNA-mediated gene silencing in insect species. The present invention is based, in part, on the inventors' sequencing of genes from eucalyptus invasive species gall wasp pests Leptocybe invasa (Li) and Ophelimus maskelli (Om). In certain aspects, the invention provides Li and Om nucleic acids, derivatives thereof and the use of such nucleic acids and derivatives as gall wasp control agents.

Description

[0001] sequence listing [0002] This application contains a Sequence Listing submitted via EFS-Web in ASCII format, the entire contents of which are hereby incorporated by reference. Said ASCII copy made on March 30, 2012 is named 34702WO1.txt and is 312,664 bytes in size. [0003] Cross References to Related Applications [0004] This application claims priority to US Provisional Application 61 / 469,469, filed March 30, 2011 and US Provisional Application 61 / 592,175, filed January 30, 2012. The entire contents of each of the foregoing provisional applications are hereby incorporated by reference. technical field [0005] The present invention relates to the field of double stranded RNA (dsRNA) mediated gene silencing in insect species. Background technique [0006] Gall wasp infestation of eucalyptus occurs in both the northern and southern hemispheres and threatens commercial eucalyptus plantations in China, Australia, Israel, and Brazil. Efforts to control eucalyptus-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/713
CPCC12N15/8218C12N15/8286C07K14/43568Y02A90/40Y02A40/146C12N15/113
Inventor D·阿维萨尔H·斯坦因Z·萨尼D·西格尔
Owner FUTURAGENE ISRAEL
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