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Use of vip3ab in combination with cry1ca for management of resistant insects

A technology of insects and mixtures, applied in the field of combined use of Vip3Ab protein and Cry1Ca protein

Active Publication Date: 2012-10-24
CORTEVA AGRISCIENCE LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] That said, some of the qualities of insect-resistant transgenic plants, while enabling the rapid and widespread adoption of the technology, also raise concerns that pest populations may become resistant to the insecticidal proteins produced by these plants

Method used

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  • Use of vip3ab in combination with cry1ca for management of resistant insects
  • Use of vip3ab in combination with cry1ca for management of resistant insects
  • Use of vip3ab in combination with cry1ca for management of resistant insects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-V

[0092] Example 1 - Production and Trypsin Treatment of Vip3Ab and Cry1Ca Proteins

[0093] Genes encoding the Cry1Ca and Vip3Ab1 protoxins were expressed in a Pseudomonas fluorescens expression strain and the full-length proteins were isolated as insoluble inclusion bodies. Washed inclusion bodies were solubilized by stirring at 37°C for 2 hr in a buffer containing 20 mM CAPS buffer, pH 11, +10 mM DDT, +0.1% 2-mercaptoethanol. The solution was centrifuged at 27,000 xg for 10 min at 37°C, and the supernatant was treated with 0.5% (w / v) TCPK-treated trypsin (Sigma). The solution was incubated with mixing for an additional 1 h at room temperature, filtered and then loaded onto a Pharmacia Mono Q 1010 column equilibrated with 20 mM CAPS pH 10.5. After washing the loaded column with 2 column volumes of buffer, the truncated toxin was eluted with a linear gradient of 0-0.5 M NaCl in 20 mM CAPS at a flow rate of 1.0 ml / min over 15 column volumes. Purified tryptic truncated Cry pr...

Embodiment 2-C

[0095] Example 2-Iodination of Cry1Ca core toxin protein

[0096] Although in a few selected examples Cry1Ca can be radiolabeled and functions well in receptor binding assays, previous work has shown that Cry1Ca is very difficult to radiolabel with traditional methods. we decided to use 125 Cry1Ca was radiolabeled with radiolabeled fluorescein-5-maleimide, a method already used for active radiolabeling of Cry1Fa (Prov. 69919). Iodination of fluorescein-5-maleimide and subsequent conjugation of this radiolabeled chemical to CrylCa results in specific radiolabeling of cysteines of the protein. Thus, this labeling procedure is highly specific at the residues labeled. The CrylCa core toxin segment (residues 29-619) contains two cysteine ​​amino acid residues, at positions 210 and 438. Palmer et al. (1997) demonstrated that the phenyl ring of fluorescein-5-maleimide can be radioiodinated and then reacted with proteins containing sulfhydryl groups (e.g. as donated by free cyste...

Embodiment 3

[0099] Example 3-Cry1Ca and Vip3Ab core toxin protein from Fall Armyworm (S.frugiperda) Competitive binding assay of BBMVs

[0100] Homologous and heterologous competition binding assays were performed with 150 μg / mL BBMV protein and 2 nM of 125I-radiolabeled Cry1Ca core toxin protein. Concentrations of 0.1, 1, 10, 100 and 1000 nM of cognate competing non-radiolabeled Cry1Ca core toxin protein added to the reaction mixture. Heterologous trypsin-truncated Vip3Ab protein was tested at 10 and 1,000 nM, which was added simultaneously with radioactive CrylCa core toxin protein to ensure true binding competition. Incubation was performed at 28° for 1 hr. The amount of 125I-labeled CrylCa core toxin protein not bound to BBMV's (i.e., not bound to insect receptor protein) was compared to bound protein by centrifuging the BBMV mixture at 16,000 x g for 8 min and removing the supernatant from the resulting pellet. separate. Precipitate with ice-cold binding buffer (PBS; 11.9 mM N...

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Abstract

The subject invention includes methods and plants for controlling fall army worm lepidopteran insects, said plants comprising a V1p3Ab insecticidal protein and a Cry1Ca insecticidal protein, and various combinations of other proteins comprising this pair of proteins, to delay or prevent development of resistance by the insects.

Description

Background of the invention [0001] Humans grow grains for food and energy applications. Humans also grow many other crops, including soybeans and cotton. Insects undermine human efforts by eating and destroying plants. Pests cost billions of dollars each year to control, and they cause another billions of dollars in damage. Synthetic organic chemical insecticides are the main tool used to control pests, but in some areas biopesticides, such as insecticidal proteins from Bacillus thuringiensis (Bt), play an important role. The ability to produce insect-resistant plants by transforming Bt insecticidal protein genes has revolutionized modern agriculture and has increased the importance and value of insecticidal proteins and their genes. [0002] Several Bt proteins have been used to generate insect-resistant transgenic plants and they have now been successfully registered and commercialized. These include Cry1Ab, Cry1Ac, Cry1F and Cry3Bb in corn / corn, Cry1Ac and Cry2Ab in cot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00A61K36/899A01N63/50
CPCA01N37/46C12N15/8286A01G1/001Y02A40/146A01N63/50A01N63/23A01N65/00A01G22/40A01G22/50
Inventor T.米德K.纳瓦N.P.斯托尔J.J.希茨A.T.伍斯利S.L.伯顿
Owner CORTEVA AGRISCIENCE LLC
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