Compositions and methods to control insect pests

Inactive Publication Date: 2011-03-03
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0006]In another embodiment, a method for controlling a pest, such as a Coleopteran plant pest or a Diabrotica plant pest, is provided. The method comprises feeding to a pest a composition comprising a silencing element, wherein the silencing element, when ingested by the pest, reduces the level of a target sequence in the pest and

Problems solved by technology

Insect pests are a serious problem in agriculture.
They destroy millions of acres of staple crops such as corn, soybeans, peas, and cotton.
Yearly, these pests cause over $100 billion dollars in crop damage in the U.S. alone.
However, these

Method used

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  • Compositions and methods to control insect pests
  • Compositions and methods to control insect pests

Examples

Experimental program
Comparison scheme
Effect test

example 1

In Vitro Transcript dsRNA Screening Method

[0119]A cDNA library was produced from neonate western corn rootworm larvae by standard methods. A selected cDNA clone containing an expressed sequence tag is amplified in a PCR using universal primers to the plasmid backbone and flanking the EST insert. The universal primers also contain T7 RNA polymerase sites. 1 ul of the PCR reaction is used as the template for an in vitro transcription (IVT) reaction to produce long double stranded RNAs. Following enzymatic digestion and removal of the DNA template and single stranded RNA, the IVT reaction products are incorporated into artificial insect diet as described below.

Insect Bioassays

[0120]2.5 ul of the IVT reaction are added to a given well of a 96 well microtiter plate. 25 ul of molten lowmelt Western corn rootworm diet are added to the sample and shaken on an orbital shaker to mix the sample and diet. Once the diet has solidified, neonate rootworms are added to the well. An average of 5 neo...

example 2

Sequences Having Insecticidal Activity

[0122]DNA sequences which encode double stranded RNAs which were shown to have insecticidal activity against corn rootworms using the assay described in Example 1 are set forth below. Non-limiting examples of target polynucleotides are set forth below in Table 1.

TABLE 1SEQ ID NO: 1>iwm2c.pk005.e1.fis1SEQ ID NO: 2>iwm2c.pk004.b13.fis1SEQ ID NO: 3>iwm2s.pk003.o11.fis1SEQ ID NO: 4>iwm2c.pk002.e24.fis1SEQ ID NO: 5>iwm2c.pk002.e24.fis1SEQ ID NO: 6>iwm2c.pk011.n17.fis1SEQ ID NO: 7>idv1c.pk001.d14.f.fis1SEQ ID NO: 8>idv1c.pk001.e9.f.fis1SEQ ID NO: 9>idv1c.pk001.m5.f.fis1SEQ ID NO: 10>idv1c.pk001.n1.f.fis1SEQ ID NO: 11>idv1c.pk002.c5.f.fis1SEQ ID NO: 12>idv1c.pk002.f20.f.fis1SEQ ID NO: 13>idv1c.pk002.j17.f.fis1SEQ ID NO: 14>idv1c.pk002.n13.f.fis1SEQ ID NO: 15>idv1c.pk003.d6.f.fis1SEQ ID NO: 16>idv1c.pk003.f8.f.fis1SEQ ID NO: 17>idv1c.pk003.f9.f.fis1SEQ ID NO: 18>idv1c.pk003.j4.f.fis1SEQ ID NO: 19>idv1c.pk003.j6.f.fis1SEQ ID NO: 20>idv1c.pk003.j20.f.fis1...

example 3

Transformation of Maize

[0123]Immature maize embryos from greenhouse donor plants are bombarded with a plasmid containing the silencing element of the invention operably linked to either a tissue specific, tissue selective, or constitutive promoter and the selectable marker gene PAT (Wohlleben et al. (1988) Gene 70:25-37), which confers resistance to the herbicide Bialaphos. In one embodiment, the constructs will express a long double stranded RNA of the target sequence set forth in table 1. Such a construct can be linked to the dMMB promoter. Alternatively, the selectable marker gene is provided on a separate plasmid. Transformation is performed as follows. Media recipes follow below.

Preparation of Target Tissue

[0124]The ears are husked and surface sterilized in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two times with sterile water. The immature embryos are excised and placed embryo axis side down (scutellum side up), 25 embryos per plate, on 560Y medium...

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Abstract

Methods and compositions are provided which employ a silencing element that, when ingested by a pest, such as a Coleopteran plant pest or a Diabrotica plant pest, decrease the expression of a target sequence in the pest. In specific embodiments, the decrease in expression of the target sequence controls the pest and thereby the methods and compositions are capable of limiting damage to a plant. The present invention provides various target polynucleotides set forth in any one of SEQ ID NOS: 1-236 or active variants and fragments thereof, wherein a decrease in expression of one or more the sequences in the target pest controls the pest (i.e., has insecticidal activity). Further provided are silencing elements which when ingested by the pest decrease the level of the target polypeptide and thereby control the pest. In specific embodiment, the pest is D. virgifera virgifera, D. barberi, D. speciosa, or D. undecimpunctata howardi. Plants, plant part, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Application No. 61 / 330,484, filed on May 3, 2010 and U.S. Provisional Application No. 61 / 237,880, filed Aug. 28, 2009, the contents of which are herein incorporated by reference in their entirety.FIELD OF THE INVENTION[0002]The present invention relates generally to methods of molecular biology and gene silencing to control pests.REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB[0003]The official copy of the sequence listing is submitted concurrently with the specification as a text file via EFS-Web, in compliance with the American Standard Code for Information Interchange (ASCII), with a file name of 391924SEQLIST.txt, a creation date of Aug. 25, 2010 and a size of 306 Kb. The sequence listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.BACKGROUND OF THE INVENTION[0004]Insect pests are a serious pro...

Claims

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Application Information

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IPC IPC(8): A01N57/16C07H21/04C07H21/02A01H5/00A01H5/10A01P7/04C12N5/10C12N15/63A01N63/60
CPCA01N63/00C12N15/8218C12N15/8286C12N15/113A01N57/16A01N65/20A01N65/44C12N2310/531A01N63/60Y02A40/146C12N2310/11
Inventor BROGLIE, KAREN E.KRISS, KEVINLU, ALBERT LAURENCEMUTHALAGI, MANIPRESNAIL, JAMES K.
Owner EI DU PONT DE NEMOURS & CO
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