Insecticidal proteins from plants and methods for their use
An insect protein, insecticidal technology, applied in the field of molecular biology
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example 1
[0441] EXAMPLE 1 - ISOLATION AND IDENTIFICATION OF INSECTICIDAL PROTEINS ACTIVE AGAINST Lepidoptera Species from the Pteridophyte Pteris
[0442]Targeting of soybean armyworm ((SBL)( Insecticidal activity of soybean spider moth)) and corn earworm ((CEW)(corn earworm)). This insecticidal activity exhibits heat sensitivity and protease sensitivity indicative of protein properties.
[0443] Pteris miltiorrhiza (PS930) and Pteris genus (PS995) had similar activity profiles, anion-exchange separated fractions, and similar SDS-PAGE protein profiles at the crude level to crude samples. Due to the limited amount of material, the samples were pooled into one sample for purification steps.
[0444] Combined plant material of PS930 and PS995 was removed from storage at -80°C and kept at liquid nitrogen temperature with Geno / 2010 ball mill (SPEX Sample Metuchen, NJ) grinds it into a fine powder. By adding extraction buffer (50 mM Tris, pH 8.0, 150 mM KCl, 2.5 mM EDTA, 1.5% polyv...
example 2
[0447] Example 2 - Transcriptome sequencing of Pteris albicans and cloning of IPD113Aa
[0448] The transcriptome of a synonym of Pteris albolineata, Pteris albolineata (ID#PS930), was prepared as follows. For total RNA isolation by using Kit for the isolation of total RNA from frozen tissues. Use from Inc. (San Diego, CA) TruSeq TM mRNA-Seq Kits and Protocols Prepare 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, reverse-transcribed into cDNA by random hexamer priming, 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 ...
example 3-I
[0451] Example 3 - Expression of IPD113Aa on transient leaf tissue and insect bioassays
[0452] In order to confirm the activity of the IPD113Aa polypeptide (SEQ ID NO: 1), the corresponding gene (SEQ ID NO: 127) was cloned into the viral promoter dMMV (Dey et al., (1999) PlantMol. Biol. [Plant Molecular Biology] 40 :771-782) in a transient expression system under control. Agrobacterium strains containing the IPD113Aa expression construct were infiltrated into leaves. The Agroinfiltration method of introducing a suspension of Agrobacterium cells into intact plant cells so that superinfection and subsequent plant-derived transgene expression can be measured or studied is well known in the art (Kapila et al., (1997) Plant Science [Plant Science] 122:101-108). Briefly, single leaves of bush bean (common bean (Phaseolus vulgaris)) were agro-infiltrated with standardized bacterial cell cultures of test and control strains. Leaf discs were excised from each plantlet and treate...
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