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Insecticidal proteins and methods for their use

A technology for insects and pests, applied in the field of molecular biology

Active Publication Date: 2017-08-29
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

[0401] Example 1 - Identification of insecticidal protein activity from Selaginella kraussiana

[0402] BLAST (Basic Local Alignment Search Tool; Altschul et al., (1993) J. Mol. Biol. 215:403-410; see also ncbi.nlm.nih.gov / BLAST / , which can be accessed using the www prefix) identified the amino acid sequence of SEQ ID NO:9. Primers were designed using the transcriptome sequence to clone the PtIP-96Aa cDNA sequence. This clone uses KOD Hot Start DNA A PCR kit (Novagen, Merck KGaA, Darmstadt, Germany) was generated by polymerase chain reaction using total RNA from Paleopsis praecox (sample Id. PS-8780) as a template. Cloned PCR products were confirmed by sequencing. Based on DNA sequencing, the PtIP-96Aa polynucleotide sequence is shown as SEQ ID NO:4 and the encoded polypeptide sequence is shown as SEQ ID NO:9.

[0403] In a 96-well plate format, the three pests were tested using plant tissue protein extracts obtained from Lepidoptera pachyrhiza sample PS-8780 overlaid o...

Embodiment 2

[0406] Example 2 - Transcriptome sequencing of Cuiyuncao

[0407] The Transcriptome of C. paliscens from sample Id. PS-8780 was prepared as follows. used for total RNA isolation Kit for the isolation of total RNA from frozen tissues. used from Inc.'s TruSeq TM The mRNA-Seq Kit and Protocol (San Diego, CA) prepared sequencing libraries from the resulting total RNA. Briefly, mRNA was isolated by ligation to oligo(dT) magnetic beads, fragmented to an average size of 180nt, reverse-transcribed into cDNA by random hexamer primers, end-repaired, A-tailed to 3', and bound to Have Indexed TruSeq TM Joint connection. use TruSeq TM The ligated cDNA fragments were amplified by PCR with primers, and the Agilent The quality and quantity of purified PCR products were checked on a DNA7500 chip. After quality and quantity evaluation, by using double-strand specific nuclease (Duplex Specific Nuclease, DSN) ( Moscow, Russia) treatment normalized 100 ng of the transcript libr...

Embodiment 3

[0409] Identification of embodiment 3-PtIP-96 polypeptide homologues

[0410] Homologs of the PtIP-96Aa polypeptide (SEQ ID NO: 4) were identified using BLAST gene identities expressed in the internal DUPONT PIONEER transcriptome database of ferns and other native plants. Table 2 shows the PtIP-96Aa polypeptide homologues and organisms identified therefrom. In some cases, congeners were identified from pooled samples of fern isolates and / or species indicated in Table 2 as "Mix 1", "Mix 3" and "Mix 4". The ferns in the pooled samples are shown in Table 3.

[0411] Table 2

[0412]

[0413]

[0414] table 3

[0415]

[0416]

[0417] cDNA is generated from the original organism with identified homologues by reverse transcription from total RNA or synthesized based on sequences assembled from transcriptomes. The cDNA-derived genes encoding PtIP-96 homologues were PCR amplified from their corresponding cDNAs using primers designed to encode the sequences of t...

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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] 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 "6584WOPCT_sequence_listing", the date of creation is September 14, 2015, the file size is 255 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 in the preparation of insecticidal formulations and in the production of transgenic insect-resistant plants. Background technique [0004] Biological control of insect pests of agricultural interest using microbial agents such as fungi, bact...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/82A01N37/46A01N65/00A01N65/04A01P7/04
CPCA01N37/46A01N65/00A01N65/04C07K14/415C12N15/8286Y02A40/146C07K14/145C12N15/82
Inventor J.K.巴里D.W.克拉克J.J.恩格里斯A.安格E.J.谢佩斯J.祁J.A.里塞
Owner PIONEER HI BRED INT INC
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