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Phi-4 polypeptides and methods for their use

A PHI-4, AXMI-205 technology, applied in the field of molecular biology

Inactive Publication Date: 2016-04-06
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While these genetically engineered insect-resistant crops have proven to be very commercially successful, they are only resistant to a narrow range of economically important insect pests

Method used

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  • Phi-4 polypeptides and methods for their use
  • Phi-4 polypeptides and methods for their use
  • Phi-4 polypeptides and methods for their use

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0588] Example 1: Generation of the PHI-4 gene

[0589] A polynucleotide having a single codon substitution compared to the PHI-4 polypeptide of SEQ ID NO: 1 was generated. As described in the Examples below, the corresponding PHI-4 polypeptides were expressed, purified and assayed for WCRW insecticidal activity to evaluate the corresponding activity diversity. A reverse mutagenesis primer and a complementary forward mutagenesis primer were designed to produce the desired amino acid substitution at the site of interest. Typically, the mutagenic primers are between 30 and 45 bases in length and flank the site of interest by two or more bases, usually 10 to 15 bases. For saturation mutagenesis, degenerate primers covering all possible amino acid residues were used. Unless otherwise stated, mutagenesis reactions described were performed using the Agilent QuikChange TM Lightening site-directed mutagenesis kit (Agilent'sQuikChange TM LighteningSite-DirectedMutagenesiskit). A...

example 2

[0591] Example 2: Purification of MBP::PHI-4 Fusion Polypeptides

[0592] The polynucleotide encoding the PHI-4 polypeptide was used as a fusion with MBP (maltose binding protein) in an improved pMAL vector (New England BioLab) (i.e. MBP::PHI-4; SEQ ID NO:6 )Express. The pMAL vector was modified to attach a 6X histidine tag to the N-terminus of MBP. To clone the polynucleotide encoding the MBP::PHI-4 fusion protein (SEQ ID NO: 6), Sph1 and BamH1 sites were engineered into the vector at the cloning site. The polynucleotide (SEQ ID NO: 1) encoding the PHI-4 polypeptide (SEQ ID NO: 2) was amplified using a forward primer (SEQ ID NO: 32) covering the Sph1 site and a reverse primer (SEQ ID NO: 33) covering the BamH1 site. This PCR product was digested with Sph1 and BamH1 and cloned into pMAL previously cut with the same enzymes. The forward primer was designed such that the polynucleotides encoding both MBP and the PHI-4 polypeptide (SEQ ID NO: 2) within the MBP::PHI-4 gene (S...

example 3

[0594] Example 3: MBP::PHI-4 polypeptide (SEQ ID NO:6) and MBP::PHI-4-SFR12-004 polypeptide (SEQ ID NO:6) NO: 31) Determination of WCRW Insecticidal Activity

[0595] Substantially as in Cong, R. et al. (Proceedings of the 4th Pacific Rim Conference on Biotechnology of Bacillus thuringiensis and its environmental impact), pp. 118-123, edited by R.J. Akhurst, C.E. Beard and P. Hughes , 2002 publication, described in Canberra, Australia (Canberra, Australia)), measure MBP::PHI-4 polypeptide (SEQIDNO:6) and MBP::PHI-4-SFR12-004 polypeptide (SEQIDNO :31; Example 8) Activity against WCRW (Western Corn Rootworm Diabroticavirgiferavirgifera). The assay was performed with artificial baits containing dilutions of these polypeptides. MBP::PHI-4-SFR12-004 polypeptide fusion (SEQ ID NO:31) and MBP::PHI-4 fusion (SEQ ID NO:6) were prepared as above, and 10 μL protein sample was mixed with 50 μL molten (40-50°C) Artificial insect bait mix prepared especially for Diabrotica sp. with lo...

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Abstract

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest, as probes for the isolation of other homologous (or partially homologous) genes. The insecticidal proteins find use in controlling, inhibiting growth or killing lepidopteran, coleopteran, dipteran, fungal, hemipteran, and nematode pest populations and for producing compositions with insecticidal activity.

Description

[0001] References to Sequence Listings Submitted Electronically [0002] Submit the official text of the sequence listing electronically via EFS-Web as a sequence listing in ASCII format, the file name is "5414WOPCT_Sequence_Listing", the date of creation is March 4, 2014, the file size is 5,935 kilobytes, and the file submitted at the same time as this manual. The Sequence Listing contained in this document in ASCII format is part of this specification and is hereby incorporated by reference in its entirety. technical field [0003] The present invention relates to the field of molecular biology. Novel genes encoding pesticidal proteins are provided. These insecticidal proteins and nucleic acid sequences encoding them can be used to prepare insecticidal preparations and to produce transgenic insect-resistant plants. Background technique [0004] The biological control of insect pests of agricultural interest using microbial agents such as fungi, bacteria, or another inse...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/82A01N63/02A01P7/04A01H5/00C07K19/00A01N63/50
CPCC07K14/195C12N15/8205C12N15/8286C07K2319/00A01N63/50A01N63/25A01N63/23A01N63/22A01N63/20A01N63/28A01N63/27Y02A40/146
Inventor R.丛J.侯Z.侯P.A.帕特坦T.亚马莫托
Owner PIONEER HI BRED INT INC
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