COMBINATIONS INCLUDING Cry34Ab/35Ab AND Cry6Aa PROTEINS TO PREVENT DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.)

a technology of corn rootworms and proteins, which is applied in the field of conjugations including cry34ab/35ab and cry6aa proteins to prevent the development of resistance in corn rootworms (diabrotica spp.), can solve the problems of affecting the survival of corn rootworms, and affecting the growth of corn rootworms. , to achieve the effect of preventing the development of resistan

Inactive Publication Date: 2013-06-27
DOW AGROSCIENCES LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0020]The subject invention relates in part to Cry34Ab / 35Ab in combination with Cry6Aa. The subject invention relates in part to the surprising discovery that Cry34Ab / Cry35Ab and Cry6Aa are useful for preventing development of resistance (to either insecticidal protein system alone) by a corn rootworm (Diabrotica spp.) population. As one skilled in the art will recognize with the benefit of this disclosure, plants producing these insecticidal Cry proteins will be useful to mitigate concern that a corn rootworm population could develop that would be resistant to either of these insecticidal protein systems alone.

Problems solved by technology

Insects eat and damage plants and thereby undermine these human efforts.
Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict.
Damage caused by insect pests is a major factor in the loss of the world's corn crops, despite the use of protective measures such as chemical pesticides.
Lodging eventually reduces corn yield and often results in death of the plant.
By feeding on cornsilks, the adult beetles reduce pollination and, therefore, detrimentally affect the yield of corn per plant.
However, if two toxins bind to two different receptors, this could be an indication that the insect would not be simultaneously resistant to those two toxins.
Predicting competitive binding for the Cry34 / 35 system is also further complicated by the fact that two proteins are involved in the Cry34 / 35 binary system.

Method used

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  • COMBINATIONS INCLUDING Cry34Ab/35Ab AND Cry6Aa PROTEINS TO PREVENT DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.)
  • COMBINATIONS INCLUDING Cry34Ab/35Ab AND Cry6Aa PROTEINS TO PREVENT DEVELOPMENT OF RESISTANCE IN CORN ROOTWORMS (DIABROTICA SPP.)

Examples

Experimental program
Comparison scheme
Effect test

example 1

Construction of Expression Plasmids Encoding Cry34Ab1, Cry35Ab1, and Cry6Aa1 Full-Length Toxins

[0089]Standard cloning methods were used in the construction of Pseudomonas fluorescens (Pf) expression plasmids engineered to produce a full-length Cry34Ab1, Cry35Ab1, and Cry6Aa1 Cry proteins, respectively. Restriction endonucleases from New England BioLabs (NEB; Ipswich, Mass.) were used for DNA digestion and T4 DNA Ligase from Invitrogen was used for DNA ligation. Plasmid preparations were performed using the Plasmid Mini kit (Qiagen, Valencia, Calif.), following the instructions of the supplier. DNA fragments were purified using the Millipore Ultrafree®-DA cartridge (Billerica, Mass.) after agarose Tris-acetate gel electrophoresis. The basic cloning strategy entailed subcloning the coding sequences (CDS) of a full-length of these Cry proteins into pMYC1803 for Cry34Ab1 and Cry35Ab1, and into pDOW1169 for Cry6Aa1 at SpeI and XhoI (or SalI that is compatible with XhoI) restriction sites...

example 2

Growth and Expression

[0091]Growth and expression analysis in shake flasks production of Cry34Ab1, Cry35Ab1, and Cry6Aa1 toxins for characterization including Bt receptor binding and insect bioassay was accomplished by shake-flask-grown P. fluorescens strains harboring expression constructs (e.g. clone pMYC2593 for Cry34Ab1, pMYC3122 for Cry35Ab1, and pDAB102018 for Cry6Aa1). Seed cultures for Cry34Ab1 and Cry35Ab1 grown in P. fluorescens medium overnight supplemented with 20 μg / ml tetracycline were used to inoculate 200 mL of the same medium with 20 μg / ml tetracycline. However, the seed culture for Cry6Aa1 was grown in M9 minimal broth overnight and was used to inoculate 200 mL of the P. fluorescens medium without antibiotic. Expressions of Cry34Ab1, Cry35Ab1, and Cry6Aa1 toxins via the Ptac promoter were induced by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubation of 24 hours at 28-30° C. with shaking at 300 rpm. Cultures were sampled at the time ...

example 3

Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples

[0092]At each sampling time, the cell density of the samples was adjusted to OD600=20 and 1-mL aliquots are centrifuged at 14,000×g for five minutes. The cell pellets were frozen at −80° C. Soluble and insoluble fractions from frozen cell pellet samples were generated using EasyLyse™ Bacterial Protein Extraction Solution (EPICENTRE® Biotechnologies, Madison, Wis.). Each cell pellet was resuspended in 1 mL EasyLyse™ solution and further diluted 1:4 in lysis buffer and incubated with shaking at room temperature for 30 minutes. The lysate was centrifuged at 14,000 rpm for 20 minutes at 4° C. and the supernatant was recovered as the soluble fraction. The pellet (insoluble fraction) was then resuspended in an equal volume of phosphate buffered saline (PBS; 11.9 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, pH7.4). Samples were mixed at 3:1 with 4× Laemmli sample buffer containing β-mercaptoethanol and boiled for 5 minutes prior to loa...

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Abstract

The subject invention relates in part to Cry34Ab/35Ab in combination with Cry6Aa. The subject invention relates in part to the surprising discovery that combinations of Cry34Ab/Cry35Ab and Cry6Aa are useful for preventing development of resistance (to either insecticidal protein system alone) by a corn rootworm (Diabrotica spp.) population. Included within the subject invention are plants producing these insecticidal Cry proteins, which are useful to mitigate concern that a corn rootworm population could develop that would be resistant to either of these insecticidal protein systems alone. Plants (and acreage planted with such plants) that produce these two insecticidal protein systems are included within the scope of the subject invention. The subject invention also relates in part to combinations of Cry34Ab/35Ab and Cry3Aa proteins “triple stacked” with a Cry6Aa protein. Transgenic plants, including corn, comprising a cry6Aa gene, cry34Ab/35Ab genes, and a cry3Aa gene are included within the scope of the subject invention. Thus, such embodiments target rootworms with three modes of action.

Description

BACKGROUND[0001]Humans grow corn for food and energy applications. Corn is an important crop. It is an important source of food, food products, and animal feed in many areas of the world. Insects eat and damage plants and thereby undermine these human efforts. Billions of dollars are spent each year to control insect pests and additional billions are lost to the damage they inflict.[0002]Damage caused by insect pests is a major factor in the loss of the world's corn crops, despite the use of protective measures such as chemical pesticides. In view of this, insect resistance has been genetically engineered into crops such as corn in order to control insect damage and to reduce the need for traditional chemical pesticides.[0003]Over 10 million acres of U.S. corn are infested with corn rootworm species complex each year. The corn rootworm species complex includes the northern corn rootworm (Diabrotica barberi), the southern corn rootworm (D. undecimpunctata howardi), and the western co...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/82A01G1/00
CPCC07K14/325C12N15/8286B32B1/02A01N37/18Y10T428/13A01G1/001A01H5/10A01N43/50B65D85/00A01H5/00C12N5/04Y02A40/146C12N15/8251A01N65/44A01G7/06
Inventor NARVA, KENNETHFENCIL, KRISTIN J.HEY, TIMOTHY D.MEADE, THOMASLI, HUARONGWOOSLEY, AARON T.OLSON, MONICA B.
Owner DOW AGROSCIENCES LLC
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