Parental rnai suppression of kruppel gene to control hemipteran pests
a technology of hemipteran pests and rnai suppression, which is applied in the field of gene suppression of kruppel gene to control hemipteran pests, can solve the problems of reducing the number of viable progeny produced, reducing the growth and/or reproduction of hemipteran pests, etc., and achieves the effect of reducing the capacity of an existing generation and reducing the number of viable progeny
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example 1
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Template Preparation by PCR and dsRNA Synthesis
[0210]The strategies used to provide specific templates for kruppel dsRNA production are shown in FIG. 1A and FIG. 1B. Template DNAs intended for use in kruppel dsRNA synthesis are prepared by PCR using specific primers and first-strand cDNA prepared from total RNA. For kruppel selected target gene regions, two separate PCR amplifications are performed. FIG. 1. The first PCR amplification introduces a T7 promoter sequence at the 5′ end of the amplified sense strands. The second reaction incorporates the T7 promoter sequence at the 5′ ends of the antisense strands. The two PCR amplified fragments for each region of the target genes are then mixed in approximately equal amounts, and the mixture is used as transcription template for dsRNA production. FIG. 1.
[0211]For the YFP negative control, a single PCR amplification was performed. FIG. 1B. The PCR amplification introduced a T7 promoter sequence at the 5′ ends of the amplified sens...
example 2
[0212]Quantitative Real-Time PCR: Relative expression levels of transcripts are analyzed by probe hydrolysis quantitative Real-Time PCR (qPCR) using Roche LightCycler480. The PCR primers and hydrolysis probes are designed using LightCycler Probe Design Software 2.0 (Roche). dsRNA-injected insects or eggs are frozen on dry ice. Tissue disruption is performed with a Klecko™ tissue pulverizer (Garcia Manufacturing, Visalia, Calif.) in the kit RLT lysis buffer with one stainless steel bead. Following tissue maceration, the total RNA is isolated in high throughput format using the RNeasy96® kit (Qiagen, #74182) following the kit protocol with the optional on column DNaseI treatment step at ½× suggested concentration for 30 minutes and additional DNaseI digest using Turbo™ DNase (AM2238, Life Technologies, Carlsbad Calif.) for 1 hour at room temperature on the elutant.
[0213]First-strand cDNA synthesis for High-Throughput qPCR assay: High capacity cDNA RT kit (part#4368813. Lif...
example 3
ion of Plant Transformation Vectors
[0215]An entry vector harboring a target gene construct for dsRNA hairpin formation comprising segments of kruppel (SEQ ID NO:1) is assembled using a combination of chemically synthesized fragments (DNA2.0, Menlo Park, Calif.) and standard molecular cloning methods. Intramolecular hairpin formation by RNA primary transcripts is facilitated by arranging (within a single transcription unit) two copies of a target gene segment in opposite orientation to one another, the two segments being separated by a linker sequence (e.g. ST-LS1 intron, SEQ ID NO:7; Vancanneyt et al. (1990) Mol. Gen. Genet. 220:245-250). Thus, the primary mRNA transcript contains the two kruppel gene segment sequences as large inverted repeats of one another, separated by the linker sequence. A copy of a promoter (e.g. maize ubiquitin 1, U.S. Pat. No. 5,510,474; 35S from Cauliflower Mosaic Virus (CaMV); promoters from rice actin genes; ubiquitin promoters; pEMU; MAS; maize H3 histo...
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