DsRNA as insect control agent

A technology for insects and insecticides, applied in the control of insects, genetic constructs of dsRNA, preventing insects from invading the field, and can solve problems such as unknown gene expression

Inactive Publication Date: 2009-02-18
DEVGEN PTE LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] Although RNAi technology in plants, C. elegans, and mammalian cells has been generally known in the art for several years, the use of RNAi to downregulate gene expression in insects has hitherto been virtually unknown

Method used

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  • DsRNA as insect control agent
  • DsRNA as insect control agent
  • DsRNA as insect control agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0264] Example 1: Silencing of Target Genes in C. elegans in High Throughput Screening

[0265] A C. elegans genome-wide library was made between the same two T7 promoters and terminators in the pGN9A vector (WO 01 / 88121), which in the presence of T7 polymerase expression induced by IPTG sense and reverse expression of direction.

[0266] This library was transformed into the AB301-105(DE3) bacterial strain in a 96-well plate format. For genome-wide screening, these bacterial cells were fed a nuclease-deficient C. elegans nuc-1 (e1392) strain.

[0267] C. elegans nuc-1(e1392) was fed in 96-well plates with dsRNA produced in the AB301-105(DE3) bacterial strain as follows: Nuc-1 eggs were transferred to separate plates and The L1 generation was synchronized by incubating them simultaneously at 20°C. Add 100 μL of liquid growth medium containing IPTG and OD containing the C. elegans dsRNA library. 600 10 μL of AB301-105 (DE3) bacterial cell culture was added to a 96-well plat...

Embodiment 2

[0281] Example 2: Identification of orthologs of Drosophila melanogaster

[0282] As described in Example 1, a number of C. elegans lethal sequences were identified and can be used to identify orthologues in other species. For example, the C. elegans lethal sequence can be used to identify orthologous sequences in Drosophila melanogaster. That is, each C. elegans sequence can be used to search public databases such as GenBank for D. melanogaster orthologs. Select possible Drosophila melanogaster orthologs with a high degree of sequence homology (E value preferably less than or equal to 1E-30) that are the best hits from a mutual blast, the latter referring to those from different organisms ( For example, the sequences of Caenorhabditis elegans and Drosophila melanogaster) are mutually the best blast hit results. For example, sequence C from C. elegans was aligned to sequence D from Drosophila melanogaster using BLAST. If D. melanogaster sequence D is the best hit for sequ...

Embodiment 3

[0283] Embodiment 3: Potato beetle (Colorado potato beetle, CPB)

[0284] A. Cloning the partial gene sequence from the potato leaf beetle

[0285] Potato leaf beetle (Colorado beetle; source: Jeroen van Schaik, Entocare CV Biologische Gewasbescherming, Postbus 162, 6700 AD Wageningen, the Netherlands) isolate high quality intact RNA. Genomic DNA was removed from this RNA preparation by DNase (Cat. Nr. 1700, Promega) treatment following the manufacturer's instructions. According to the manufacturer's instructions, using a commercially available kit (SuperScript TM III Reverse Transcriptase, Cat. Nr. 18080044, Invitrogen, Rockville, Maryland, USA) to obtain cDNA.

[0286] To isolate cDNA sequences comprising part of the LD001, LD002, LD003, LD006, LD007, LD010, LD011, LD014, LD015, LD016, LC018, and LD027 genes, Amplitaq Gold (Cat. Nr. N8080240, Applied Biosystems) for a series of PCR reactions.

[0287] The degenerate primer sequences used to amplify each gene are give...

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Abstract

The present invention concerns methods for controlling insect infestation via RNAi- mediated gene silencing, whereby the intact insect cell(s) are contacted with a double-stranded RNA from outside the insect cell(s) and whereby the double-stranded RNA is taken up by the intact insect cell(s). In one particular embodiment, the methods of the invention are used to alleviate plants from insect pests. Alternatively, the methods are used for treating and/or preventing insect infestation on a substrate or a subject in need of such treatment and/or prevention. Suitable insect target genes and fragments thereof, dsRNA constructs, recombinant constructs and compositions are disclosed.

Description

technical field [0001] The present invention relates to the field of double stranded RNA (dsRNA) mediated gene silencing in insect species. More specifically, the present invention relates to genetic constructs designed to express dsRNAs corresponding to novel target genes. These constructs are especially useful in RNAi-mediated pest control. The present invention also relates to methods for controlling insects, methods for preventing insect infestation, and methods for down-regulating gene expression in insects using RNAi. Background technique [0002] Insects and other pests can cause injury and even death through their bites or stings. Also, many pests transmit bacteria and other pathogens that cause disease. For example, mosquitoes transmit pathogens that cause malaria, yellow fever, encephalitis, and other diseases. The bubonic plague (or Black Death) is caused by bacteria that infect rats and some other rodents. Compositions for controlling microscopic pest infest...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00C12N5/10C12N15/12C12N15/11C12N15/113
CPCC12N15/8286Y02A40/146
Inventor 罗曼·雷马克斯劳伦特·库布勒格特·普莱廷克埃尔丝·万布勒
Owner DEVGEN PTE LTD
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