Insecticidal proteins and methods for their use

A technology of insecticidal activity and sequence, applied in pesticides, chemical instruments and methods, botanical equipment and methods, etc.

Active Publication Date: 2016-12-14
PIONEER HI BRED INT INC +1
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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 commercially successful, they are only resistant to a narrow range of economically important insect pests

Method used

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  • Insecticidal proteins and methods for their use
  • Insecticidal proteins and methods for their use
  • Insecticidal proteins and methods for their use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0707] Example 1-Insecticide from the fern Asplenium australasicum (PS-8566) Identification of protein activity

[0708] The insecticidal proteins PtIP-50Aa and PtIP-65Aa from the pteridophyte Nanyangshan Suhua PS-8566 were identified by protein purification, mass spectrometry (MS) and PCR cloning as follows.

[0709] Nanyangshan Suhua was collected by co-investigators and assigned the identification number PS-8566. Send the evidence specimens of Nanyangshan Suhua to the Herbarium of Delaware State University (Delaware State University Herbarium) where the identity is verified. Collect PS-8566, quickly freeze in liquid nitrogen and store at -80°C. After storage, it was ground into a fine powder with a Geno ball mill (SPEX, Metuchen, NJ) at liquid nitrogen temperature. To extract protein, add 20 mL of 50 mM Tris buffer (pH 8.0), 150 mM KCl, 2.5 mM EDTA, 1.5% polyvinyl polypyrrolidone (PVPP) and protease inhibitor mixture (Roche Diagnostics, Germany) to every 5 g of fresh tissue ...

Embodiment 2

[0716] Example 2-Transcriptome sequencing of PtIP-50 and PtIP-65

[0717] The transcript set of Nanyang Shansuhua PS-8566 was prepared as follows. Use for total DNA separation The kit isolates total RNA from frozen tissue. Used from TruSeq of Inc. (San Diego, CA) TM The mRNA-Seq kit and protocol prepare a sequencing library from the total RNA obtained. In short, mRNA is separated by ligation to oligo (dT) magnetic beads, fragmented into an average size of 180 nt, reverse transcribed into cDNA by random hexamer primers, end repaired, 3'A tail is added, and the band Have Indexed TruSeq TM Connector connection. use TruSeq TM The primers are used for PCR amplification of the ligated cDNA fragments, and the Check the quality and quantity of the purified PCR products on the DNA7500 chip. After the quality and quantity evaluation, by using Duplex Specific Nuclease (DSN) ( Moscow, Russia) processing standardized a 100 ng transcript library. Standardization was achieved by a...

Embodiment 3

[0723] Example 3:-Identification of PtIP-50 and PtIP-65 homologs

[0724] The BLAST (basic partial alignment search tool; Altschul et al., (1993) J.Mol.Biol.215:403-410; see also ncbi.nlm.nih.gov / BLAST / can be used www prefix to access it) search to determine gene identity based on sequence similarity. The polynucleotide sequences of PtIP-50Aa (SEQ ID NO: 43) and PtIP-65Aa (SEQ ID NO: 1) were analyzed. Through BLAST gene identification in the DUPONT PIONEER internal transcriptome database of ferns and other primitive plants, it was identified that PtIP-50Aa (SEQ ID NO: 71) and PtIP-65Aa (SEQ ID NO: 22) are in the same Multiple homologues in the source range. Table 8 shows the plant sample ID# of PtIP-65 polypeptide homologues and polynucleotides encoding PtIP-50 homologues, ferns or other original plant species, internal clone names and corresponding sequence identifiers. Table 9 shows the plant sample ID# of PtIP-65 polypeptide homologues and polynucleotides encoding PtIP-50...

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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 including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal 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] cross reference [0002] This application claims the benefits of U.S. Provisional Application 61 / 937,288 filed on February 7, 2014, which is incorporated herein by reference in its entirety. [0003] Reference to sequence listing submitted electronically [0004] Electronically submit the official text of the sequence list as a sequence list in ASCII format through EFS-Web, the file name is "20150203_5295PCT_SeqListing", the creation date is February 3, 2015, the file size is 1,281 kilobytes, and the file Submit at the same time as this manual. The sequence listing contained in the ASCII format file is a part of this specification, and the full text is incorporated herein by reference. Technical field [0005] The present disclosure relates to the field of molecular biology. A novel gene encoding insecticidal protein is provided. These insecticidal proteins and the nucleic acid sequences encoding them can be used to prepare insecticidal preparations and can be used to produ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29A01N65/04A01P7/04C12N15/82C12N5/10A01H5/00A01N63/50
CPCA01N65/04C07K14/415C12N15/8286A01N63/50Y02A40/146A01N63/10A01N47/44A01N65/00C07K2319/00
Inventor J.巴里K.哈耶斯刘璐E.舍佩斯N.亚帕尼
Owner PIONEER HI BRED INT INC
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