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Bollworm insect resistance management in transgenic plants

An insecticidal, cotton boll technology, applied in plant products, genetic engineering, botany equipment and methods, etc.

Inactive Publication Date: 2011-05-18
BAYER BIOSCIENCE N V
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] There are no reports demonstrating saturable binding of Cry2A proteins based on direct saturability assays using a fixed concentration of the binding site (i.e. BBMV) to which increasing concentrations of the labeled protein are added

Method used

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  • Bollworm insect resistance management in transgenic plants
  • Bollworm insect resistance management in transgenic plants
  • Bollworm insect resistance management in transgenic plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0135] 1.1. Materials and methods

[0136] Toxin purification and toxin activation

[0137] Cry1Ac-expressing Bacillus thuringiensis strain HD73 from the Bacillus Genetic Stock Collection (Columbus, OH) was grown in CCY medium (Stewart et al., 1981) for 48 hours at 28.5°C with continuous shaking and supplemental air. The precipitated insoluble fraction was washed twice with 1M NaCl, 10 mM EDTA and once with 10 mM KCl. Cry1Ac crystals were dissolved in freshly prepared carbonate buffer (50 mM Na2CO3 / NaHCO3, 10 mM DTT; pH 10.5) and incubated at room temperature for 2.5 hours with shaking at 150 rpm. The insoluble residue was discarded by centrifugation at 25000 xg for 10 minutes at 4°C. Solubilized Cry1Ac protoxin was activated by incubation with trypsin (SigmaT-8642) at a trypsin:protein ratio of 1:10 (w:w) for 2 hours at 37°C. After centrifugation at 25000 x g for 10 minutes at 4°C, the supernatant was dialyzed against buffer A (20 mM Tris-HCl, pH 8.65), filtered and used...

Embodiment 2

[0178] Several methods are contemplated for obtaining the combined expression of at least two insecticidal protein genes such as the Cry2Ae and Cry1Ab genes in transgenic plants such as maize or cotton plants.

[0179] The first method is based on sequential transformation steps in which plants transformed with the first chimeric gene are retransformed to introduce the second gene. This sequential transformation preferably uses two different selectable marker genes, such as a kanamycin resistance gene and a phosphinothricin acetyltransferase gene that confers resistance to the glufosinate herbicide (e.g., the well-known pat or bar genes ). The use of these two selectable markers has been described in De Block et al. (1987).

[0180] The second method is based on the co-transformation of two chimeric genes encoding different insecticidal proteins on different plasmids in a single step. Integration of two genes can be selected for by use of selectable markers linked to the res...

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Abstract

This invention relates to the use of a combination of different proteins insecticidal to Helicoverpa zeaor Helicoverpa armigeran an insect resistance management process, wherein such proteins are: a) a Cry2A protein such as Cry2Aa, Cry2Ab, or Cry2Ae and b) a Cry1A, Cry1F or VIP3A protein, particularly wherein such proteins binds saturably to the insect midgut membrane of Helicoverpa zeaor Helicoverpa armigera, as well as plants and seeds expressing such combination of proteins, which are used to delay or prevent the development of resistance in populations of such insect species.

Description

field of invention [0001] The invention relates to the field of control of plant diseases and insect pests, in particular to the control of insects. The present invention relates to the use of transgenic plant cells and plants in insect resistance management programs, wherein the genome of said cells and plants (or more typically, predecessor plant cells or plants) has been provided with at least two genes, each The genes encode different proteins that are insecticidal against Helicoverpa zea or Helicoverpa armigera, where these proteins saturably bind the brush border membrane of these insect species, the proteins are : a) Cry2A protein and b) Cry1A, Cry1F or VIP3A protein, eg VIP3A, Cry1Ac, Cry1Ab or Cry1A.105 protein. In one embodiment, such plants are used to delay or prevent the emergence of resistance to crop plants in cotton bollworm populations. [0002] In addition, the present invention provides simultaneous or sequential use of Cry2A protein and VIP3A, Cry1A or Cr...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H5/10
CPCC12N15/8286Y02A40/146
Inventor C·S·赫尔南德茨A·范 弗里埃特J·范 里J·F·曼扎纳罗
Owner BAYER BIOSCIENCE N V
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