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Method for suppressing Bt (Bacillus thuringiensis) CRY toxin resistance of insects by using toxins needing no cadherin receptors

A toxin and resistance technology, applied in the field of using toxins that do not require cadherin receptors to curb insect resistance to Bacillus thuringiensis CRY toxin, can solve problems that have not yet been implemented in large-scale field trials

Active Publication Date: 2015-07-01
UNIV NAT AUTONOMA DE MEXICO +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this strategy appears to be useful for retrograde resistance, careful large-scale field trials have not been performed (Tabashnik et al., 2005)

Method used

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  • Method for suppressing Bt (Bacillus thuringiensis) CRY toxin resistance of insects by using toxins needing no cadherin receptors
  • Method for suppressing Bt (Bacillus thuringiensis) CRY toxin resistance of insects by using toxins needing no cadherin receptors
  • Method for suppressing Bt (Bacillus thuringiensis) CRY toxin resistance of insects by using toxins needing no cadherin receptors

Examples

Experimental program
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Effect test

Embodiment 1

[0096] Example 1. Example of a DNA construct encoding a modified 3-domain Cry toxin lacking an alpha-1 helix obtained using the method of the invention.

[0097] To illustrate the method of the invention, two 3-domain Cry toxins were chosen: Cry1Ab and Cry1Ac. In both, the promoter region, the coding region of the complete protein and the alpha-1 helix were identified as follows:

[0098]

[0099] pb, base pair

[0100] To remove the coding region of the α-1 helix, genetic constructs of modified 3-domain Crv toxins called Cry1AbMod and Cry1AcMod were amplified as follows:

[0101] In these cases, perform Figure 4 The 3 PCR steps shown in . The total DNA of the Bacillus thuringiensis strain Bt407 containing the pHT315Cry1Ab plasmid and the total DNA of the BtHD73 strain (which contain the Cry1Ab and Cry1Ac genes, respectively; according to reports in Gene Bank, accession numbers X98793 and ML 1068, respectively) were used as templates. For PCR1, the forward oligo contains...

Embodiment 2

[0105] Example 2. Obtaining vectors and host cells containing DNA constructs encoding modified 3-domain Cry toxins and their use in recombinant methods for the production of modified 3-domain Cry toxins.

[0106] A vector containing the gene constructs Cry1AbMod and Cry1AcMod cloned in the double origin of replication vector pHT-315 (Lereclus et al., 1989) was obtained. By transforming the decrystallized (acristalífera) strain Bt407Cry with the above vector - (serotype H1, Lereclus et al., 1989) to obtain genetically engineered host cells containing the construct.

[0107] To produce the modified and unmodified (wild-type (silvestre)) 3-domain Cry toxins of this example, transformed and wild-type (silvestre) strains were supplemented with 10 μg / ml of erythromycin in vegetative sporulation medium (Lereclus et al., 1995) for 3 days. Modified and unmodified 3-domain Cry toxin crystals (Thomas and Ellar, 1983) were recovered and purified by sucrose gradient and dissolved in 10 ...

Embodiment 3

[0108] Example 3. Formation of modified 3-domain Cry toxin oligomers used as examples (Crv1AbMod and Cry1AcMod).

[0109] Bt-R containing toxin-binding domain 1 Oligomer formation was promoted in vitro by protease activation of the Cry1A protoxin in the presence of protein fragments or in the presence of scFv73 antibodies mimicking these toxin binding regions of the CADR receptor (Gómez et al., 2003). When incubated with CADR fragments containing the 7-12, 11-12, or 12 repeats of CADR, the Cry1Ab protoxin produces 250 kDa oligomers ( Image 6 ).

[0110] When Cry1AbMod and Cry1AcMod protoxins were trypsinized in the absence of CADR receptors, they produced 250 kDa oligomers, but under these conditions the wild-type (silvestre) protoxins Cry1Ab and Cry1Ac did not form oligomers, but only Produces a 60kDa monomer ( Figure 7 ). These results indicate that when the modified 3-domain Cry toxins are proteolytically activated in the absence of primary receptors, they generate 25...

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Abstract

The invention relates to a method for suppressing the Bt (Bacillus thuringiensis) CRY toxin resistance of insects by using toxins not needing cadherin receptors. The invention relates to a method for obtaining DNA constmcciones coding a Cry toxin (also known as Cry toxin, Bt toxin or delta-endotoxin) which has three structural domains lacking alpha-1 helixes. The DNA constmcciones is modified to code protein which can kill insects with resistance to a corresponding unmodified Cry toxin. The invention relates to DNA constmcciones of a modified 3-structural domain Cry toxin gene, modified 3-structural domain Cry protein, methods, a molecular carrier containing the DNA constmcciones, a host cell containing the DNA constmcciones and recombination methods for generating modified 3-structural domain Cry toxins. Besides, the invention relates to a preparation containing the modified 3-structural domain Cry toxins. The insect resistance to unmodified Cry toxins is caused by the reduction of the combination of the toxins and receptors in the intestines of the insects. More concretely, the invention relates to expression of modified 3-structural domain Cry toxins, methods for expressing the modified 3-structural domain Cry toxins, transgenic microbes and plants containing the DNA constmcciones for expressing the modified 3-structural domain Cry toxins, and a method for suppressing the unmodified CRY toxin resistance of insects.

Description

[0001] This application is an international application PCT / MX2007 / 000068 entered into China with an international filing date of June 8, 2007, and the application number is 200780053274.5, entitled "Using a toxin that does not require cadherin receptors to curb insects' response to Bacillus thuringiensis CRY toxin The divisional application of the invention patent application. technical field [0002] The Cry toxins of Bacillus thuringiensis (Bt) are of great value for their ability to control pests in different cultivars, forest trees and insects that transmit diseases in humans. The development of resistance of insect infestation to Bt toxin is a major obstacle to the continued successful use of this environmentally friendly insecticide. The main mechanism of resistance to Cry toxins in insects involves reduced binding of Cry toxins to specific receptors located in the insect gut (Ferré and Van Rie, 2002). [0003] The Cry1A proteins are a group of Bt toxins that kill some...

Claims

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Application Information

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IPC IPC(8): C12N15/32C12N15/82C12N1/21C12N5/10C07K14/325C12P21/02A01N47/44A01P7/04
Inventor L.帕多-罗佩斯E.B.塔巴什尼克M.索维伦-查韦斯M.A.布拉沃-德拉帕拉
Owner UNIV NAT AUTONOMA DE MEXICO
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