Structure specific recognition protein 1 (SSRP1) nucleic acid molecules to control insect pests
A nucleic acid molecule and molecular technology, applied in the field of genetic control, can solve problems such as the inability to accurately identify RNAi targets a priori
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Embodiment 1
[0214] Example 1: Pollen beetle transcriptome
[0215] insect. Larval and adult pollen beetles were collected from fields of flowering rape plants (Giessen, Germany). Juvenile adults (each Treatment group: n=20; 3 repetitions) for challenge. Bacterial cultures were grown at 37°C with agitation and optical density was monitored at 600 nm (OD600). Cells were harvested by centrifugation at OD600-1 and resuspended in phosphate buffered saline. Using a dissecting needle immersed in an aqueous solution of 10 mg / ml of LPS (purified Escherichia coli (E. coli) endotoxin; SIGMA, Taufkirchen, Germany) and bacterial and yeast cultures, the abdomen of adult pollen beetles was introduced ventrolaterally. mixture. At the same point in time, collect primordial Beetles and larvae (n=20 per group, 3 replicates per group).
[0216] RNA isolation. Eight hours after immunization, total RNA was extracted from frozen beetles and larvae using TriReagent (Molecular Research Centre, Cincinna...
Embodiment 2
[0218] Example 2: Mortality of pollen beetles treated with ssrp1 iRNA
[0219] A PCR product (SEQ ID NO: 4) of approximately 332 bp was generated by PCR using gene-specific primers containing the T7 polymerase promoter sequence at the 5' end. The PCR fragment was cloned into the pGEM T easy vector according to the manufacturer's protocol and sent to a sequencing company for sequence verification. Then according to the manufacturer's protocol, T7 RNA polymerase ( RNAi Kit, Applied Biosystems), dsRNA was generated from PCR constructs generated from sequenced plasmids.
[0220] injection bioassay . Under a dissecting stereomicroscope, inject approximately 100 nL dsRNA (1 μg / uL) into adult beetles using a micromanipulator. Animals were anesthetized on ice and then taped to double-sided tape. The control group received the same volume of water. Due to lack of animals, it was not possible to test all controls at all stages. Controls were performed on different days due to l...
Embodiment 3
[0228] Example 3: Agrobacterium-mediated transformation of canola hypocotyls
[0229] 10-20 transgenic Brassica napus plants containing an RNAi construct encoding a hairpin dsRNA targeting ssrpl were generated for pollen beetle challenge. The polynucleotide encoding the hairpin dsRNA comprising the contiguous nucleotide sequence of PB ssrp1 (eg, SEQ ID NO:4) is SEQ ID NO:11.
[0230] Agrobacterium preparation. Agrobacterium strains containing binary plasmids were grown in YEP medium (Bacto Peptone) containing streptomycin (100 mg / mL) and spectinomycin (50 mg / mL) TM 20.0 gm / L and yeast extract 10.0 gm / L) plates were streaked and incubated at 28°C for 2 days. Scrape the propagated Agrobacterium strains containing the binary plasmid from the 2-day-old streak plate using a sterile inoculating loop. Then, the scraped Agrobacterium strain containing the binary plasmid was inoculated into 150 mL of modified YEP liquid containing streptomycin (100 mg / mL) and spectinomycin (50 mg / m...
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