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Axmi115 variant insecticidal gene and methods for its use

Inactive Publication Date: 2013-04-18
ATHENIX
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent is about using genetic engineering to create organisms that are resistant or tolerant to pests. These organisms can be used for agricultural purposes. The patent also describes how to create proteins that have pesticidal activity, or detect the presence of pesticidal proteins or nucleic acids in products or organisms.

Problems solved by technology

This toxin binds to apical brush border receptors in the midgut of the target larvae and inserts into the apical membrane creating ion channels or pores, resulting in larval death.

Method used

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  • Axmi115 variant insecticidal gene and methods for its use
  • Axmi115 variant insecticidal gene and methods for its use

Examples

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experimental examples

Example 1

Design and Testing of Axmi115 Fusion Proteins

[0097]Axmi115 is described in U.S. Patent Publication 20100004176 (the amino acid sequence is set forth herein as SEQ ID NO:43). This gene shares 70% sequence homology with Vip3Aa. A codon optimized version of Axmi115 (also referred to herein as Axmi115v01 and set forth in SEQ ID NO:42) was cloned and expressed using the E. coli expression vector. The protein produced was shown in in vitro bioassay to have pesticidal activity against various insect pests including European corn borer (ECB), corn earworm (CEW), fall armyworm (FAW) and black cutworm (BCW).

[0098]Axmi005 is also described in U.S. Patent Publication 20100004176. This gene shares 94% sequence homology with Vip3Aa. A codon optimized version of Axmi005 (optAxmi005, which is set forth herein as SEQ ID NO:44) was cloned and expressed using the E. coli expression vector. The protein produced was shown in in vitro bioassay to have pesticidal activity against various insect p...

example 2

Directed Evolution of Axmi115v02

[0126]Directed evolution has been used to improve the potency and activity profile of Axmi115 against ECB, Hz, FAW, BCW, and VBC. To identify regions of Axmi115 involved in insect toxicity, a number of Axmi115 / Axmi005 sequence swap variants in the C-terminal part of Axmi115 were created. Twenty-one blocks of sequence divergence between Axmi115 and Axmi005 were designated (see U.S. Patent Publication No. 20100004176 which is herein incorporated by reference in its entirety) and these sequence blocks in Axmi115 were replaced with the corresponding Axmi005 sequence blocks. Bioassays of hybrid proteins showed that substitutions in blocks 2, 3, 10 and 18 are linked to increased insect toxicity.

[0127]Point mutant libraries were created that targeted positions in blocks 2, 3, 10 and 18. These point mutant libraries were assayed against ECB, Hz, FAW, BCW and VBC at 1.5× coverage at the 4 replicate level. Re-assays were carried out at the 4 replicate level, an...

example 3

Additional Assays for Pesticidal Activity

[0136]The nucleotide sequences of the invention can be tested for their ability to produce pesticidal proteins. The ability of a pesticidal protein to act as a pesticide upon a pest is often assessed in a number of ways. One way well known in the art is to perform a feeding assay. In such a feeding assay, one exposes the pest to a sample containing either compounds to be tested or control samples. Often this is performed by placing the material to be tested, or a suitable dilution of such material, onto a material that the pest will ingest, such as an artificial diet. The material to be tested may be composed of a liquid, solid, or slurry. The material to be tested may be placed upon the surface and then allowed to dry. Alternatively, the material to be tested may be mixed with a molten artificial diet, and then dispensed into the assay chamber. The assay chamber may be, for example, a cup, a dish, or a well of a microtiter plate.

[0137]Assays...

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Abstract

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. The toxin coding sequences can be used in DNA constructs or expression cassettes for expression in plants and bacteria. Compositions include transformed bacteria, plants, plant cells, tissues, and seeds. In particular, polynucleotide sequences and the toxin proteins encoded thereby are provided. Also provided are antibodies specifically binding to those amino acid sequences. In particular, the invention encompasses nucleotide sequences encoding fusion proteins, as well as biologically active variants and fragments thereof, wherein the fusion protein contains the C-terminal portion of SEQ ID NO:43. The fusion protein may also contain the N-terminal portion of SEQ ID NO:45. The invention also includes the nucleotide sequence of SEQ ID NO:47 and 1-14, or a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:48 and 15-31, including biologically active variants and fragments thereof.

Description

CROSS REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 61 / 471,848, filed Apr. 5, 2011, the contents of which are herein incorporated by reference in their entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY[0002]The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named “APA116021USSEQLIST.txt”, created on Mar. 28, 2012, and having a size of 241 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.FIELD OF THE INVENTION[0003]This invention relates to the field of molecular biology. Provided are novel genes that encode pesticidal proteins. These proteins and the nucleic acid sequences that encode them are useful in preparing pesticidal formulations and in the production of tr...

Claims

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Application Information

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IPC IPC(8): C12N15/82
CPCC07K2319/00C12N15/8286C07K2319/24C07K14/325C12N15/8285A01H5/00C12N15/82Y02A40/146
Inventor HEINRICHS, VOLKERLEHTINEN, DUANE
Owner ATHENIX
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