Insecticidal proteins from plants and methods for their use

A technique for killing insects, insects, applied in the field of molecular biology

Pending Publication Date: 2019-05-21
PIONEER HI BRED INT INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although they have proven to be very commercially successful, these genetically engineered insect-resi

Method used

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

Examples

Experimental program
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example 1

[0412] EXAMPLE 1 - Efficacy from the fern Japonica "Red Beauty" against ear moth, European corn borer, Identification of Insecticidal Proteins Active in Fall Spodoptera, Soybean Spodoptera, and Lyme Spodoptera

[0413] The insecticidal protein IPD103Aa (SEQ ID NO: 2) was identified from the commercial cultivar Japonicus "Hongyan" (designated NY15 herein) by protein purification, mass spectrometry (MS) and PCR cloning. Insecticidal activity against lepidopteran pests was observed from protein extracts from the Japanese cover fern "Red Face" using an artificial diet-based assay.

[0414] NY15 plant material was snap frozen in liquid nitrogen and stored at -80°C. Remove frozen samples from storage and at liquid nitrogen temperature, use 2010 (SPEX Metuchen, NJ) ground it into a fine powder. For protein extraction, add extraction buffer (50 mM Tris, pH 8.0, 150 mM KCl, 2.5 mM EDTA, 1.5% polyvinylpyrrolidone and "Complete, EDTA-free" protease inhibitor cocktail (Roche Com...

example 2

[0418] Example 2 - Transcriptome sequencing and cloning of IPD103Aa of Japanese scorpion fern "Hongyan"

[0419] The transcriptome of J. japonica 'Hongyan' (NY15) was prepared as follows. use Reagent test kit Total RNA was isolated from frozen tissues. Using TruSeq TM mRNA-Seq kit and from Inc. (San Diego, CA [San Diego, CA]) protocol for preparation of sequencing libraries from the resulting total RNA. Briefly, mRNA was isolated via attachment to oligo(dT) beads, fragmented to an average size of 180 nt, initial reverse transcription into cDNA by random hexamers, end repaired, 3'A-tailed, and Indexed TruSeq TM Adapter connection. use TruSeq TM The ligated cDNA fragments were amplified by PCR with primers, and the Agilent Quality and quantity of purified PCR products were checked on a DNA 7500 chip. After qualitative and quantitative assessment, by using dual specificity nuclease (DSN) ( Moscow, Russia) process to normalize the 100 ng transcript library. N...

example 3

[0422] Example 3 - Purification of IPD103Aa expressed in E. coli

[0423] SEQ ID NO: 2 encoding IPD103Aa (SEQ ID NO: 2) was encoded using an in-frame NdeI / XhoI restriction site (with coding sequence for the N-terminal 6x His tag followed by a thrombin cleavage site (SEQ ID NO: 40)). The polynucleotide of ID NO: 1 was subcloned into pET14b vector middle. Transformation of chemically competent cells with pET plasmid DNA containing the IPD103Aa gene C41(DE3)SOLOs cells for recombinant protein expression. Transformed E. coli cells were grown overnight at 37°C with ampicillin selection, then inoculated into fresh 2xYT medium (1:25), and further grown to an optical density of approximately 0.8. Protein expression was induced by adding 0.3 mM IPTG, and the cells were further grown at 16°C for 16 hours. According to the manufacturer's protocol, by using HisPur TM E. coli expressed proteins were purified by immobilized metal ion chromatography on cobalt resin (Clonetech, Mou...

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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 areinsecticidal 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 References to Related Applications [0002] This application claims priority to US Provisional Patent Application Serial No. 62 / 357,501, filed July 01, 2016, which is hereby incorporated by reference in its entirety. [0003] References to Sequence Listings Submitted Electronically [0004] An official copy of this Sequence Listing is submitted electronically via EFS-Web as a Sequence Listing in ASCII format under the file name "6762WOPCT_SequenceListing", created on June 1, 2017, and having a size of 546 kilobytes, and at the same time as this specification submit. The Sequence Listing included in this file in ASCII format is part of this specification and is hereby incorporated by reference in its entirety. technical field [0005] The present disclosure relates to the field of molecular biology. Novel genes encoding pesticidal proteins are provided. These pesticidal proteins and nucleic acid sequences encoding them can be used in the preparation of pesticid...

Claims

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Application Information

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IPC IPC(8): A01H1/00A01H5/00A01N63/00C12N15/09C12N15/31A01N63/50
CPCA01N37/46C07K14/415C12N15/8286A01N63/50Y02A40/146
Inventor J.巴瑞C.克拉克R.格贝A.林J.马西斯A.安B.佩特森-布克T.C.沃菲谢卫平N.雅帕尼钟晓红
Owner PIONEER HI BRED INT INC
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