Compositions and methods to control insect pests

A technology for expression cassettes and polynucleotides, applied in the field of compositions and methods for controlling insect pests, and capable of solving problems such as environmental impact

Inactive Publication Date: 2016-02-17
PIONEER HI BRED INT INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, these modes of insecticidal activity offer varying levels of spe

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0197] Example 1: In vitro transcript dsRNA screening method

[0198] cDNA libraries were generated from newly hatched western corn rootworm larvae by standard methods. Selected cDNA clones containing expressed sequence tags were amplified in PCR using universal primers from the plasmid backbone flanking the EST insert. This universal primer also contains a T7 RNA polymerase site. The product of this PCR reaction is used as a template for an in vitro transcription (IVT) reaction that produces long double-stranded RNA. Following enzymatic digestion and removal of DNA template and single-stranded RNA, the IVT reaction products were incorporated into artificial insect baits as described below.

[0199] Different target selection strategies were used in the present invention to identify RNAi-active targets with insecticidal activity in a corn rootworm bait-based assay. cDNA libraries were generated by standard methods from the midgut of newly hatched or 3rd instar western cor...

example 2

[0204] Example 2. Sequences having insecticidal activity

[0205] The DNA sequence encoding the double-stranded RNA demonstrated to have insecticidal activity against corn rootworm using the assay described in Example 1 is shown below. Non-limiting examples of target polynucleotides are shown in Tables 1A and B below and the following sequences: SEQ ID NO: 1, 4, 5, 8, 9, 12, 13, 16, 17, 20, 21, 24, 25, 28 , 29, 32, 33, 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 54, 55, 56, 57, 60, 61, 64, 65, 68, 69, 72, 73 ,76,77,80,81,84,85,88,89,92,93,96,97,100,101,104,105,108,109,112,113,116,117,120,121,124 ,125,128,129,132,133,136,137,140,141,144,145,148,149,152,153,156,157,160,161,164,165,168,169,172,173 ,176,177,180,181,184,185,188,189,192,193,196,197,200,201,204,205,208,209,212,213,216,217,220,221,224 ,225,228,229,232,233,236,237,240,241,244,245,248,249,252,253,256,257,260,261,264,265,268,269,272,273 ,276,277,280,281,284,285,288,289,292,293,296,297,300,301,304,305,308,309,312,313,31...

example 3

[0209] Example 3. Identification of RNAi active targets from other insects

[0210]To identify RNAi-active genes from other important corn pests or non-target insects, samples from northern corn rootworm (Diabrotica barberi), southern corn rootworm (Diabrotica undecimpunctata), Mexican bean lady beetle (Epilachnavarivestis), Colorado potato beetle (Leptinotarsadecemlineata), Transcriptome experiments were performed on the 3rd instar larvae of Orius insidiosus and Coleomegillamaculata, [CMAC]. Homologous transcripts of RNAi active leaders are listed in Table 3 (SeqNo. 693 to 723). This sequence data is important for designing fragments that suppress target pest genes and avoid down-regulation of the same genes in non-target insects.

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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. The present invention provides various target polynucleotides set forth in any one of SEQ ID NOS: disclosed herein, (but not including the forward and reverse primers.) or active variants and fragments thereof, or complements thereof, wherein a decrease in expression of one or more of the sequences in the target pest controls the pest (i.e., has insecticidal activity). Plants, plant parts, bacteria and other host cells comprising the silencing elements or an active variant or fragment thereof of the invention are also provided.

Description

[0001] Cross references to related patent applications [0002] This application claims the benefit of U.S. Provisional Application No. 61 / 785,680, filed March 14, 2013, and U.S. Utility Application No. 13 / 831,230, filed March 14, 2013, both of which are incorporated by reference in their entirety way incorporated into this article. technical field [0003] The present invention generally relates to methods of molecular biology and gene silencing for controlling pests. [0004] For submissions via EFS-WEB as text files [0005] References to Sequence Listings [0006] Pursuant to 37C.F.R. §1.52(e)(5), the sequence listing filed on March 14, 2014 is hereby created on March 14, 2014 as a text of 697,429 bytes in size and entitled "36446_0007P1_ST25.txt" This document is incorporated by reference. Background technique [0007] Insect pests are a serious problem in agriculture. They destroy millions of acres of staple crops such as corn, soybeans, peas, and cotton. In t...

Claims

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Application Information

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IPC IPC(8): C07H21/02C07H21/04C12N5/04C12N5/10C12N15/00
CPCC07H21/02C07H21/04C12N5/04C12N15/10C12N15/8218C12N15/8286Y02A40/146
Inventor X.胡J.普雷斯奈尔N.里奇特曼S.迪伊恩M.范阿伦L.普罗克
Owner PIONEER HI BRED INT INC
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