Oedaleus asiaticus protein tyrosine kinase PTK as well as encoding gene and application thereof
A technology of tyrosine kinase and coding gene, which is applied in the field of protein tyrosine kinase PTK and its coding gene and its application, can solve the problems of lagging disease and insect pest control, and achieve the effect of reducing overall expressiveness and weight
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Embodiment 1
[0072] Example 1. Acquisition of protein tyrosine kinase PTK and its coding gene in P. asiaticus
[0073] 1. Extraction of total RNA from P. asiaticus
[0074] use The RNA isolation reagent was used to extract the RNA from the tissue samples of P. trocarpus. Specific steps are as follows:
[0075] 1) Put a 2mL homogenizer in an oven at 160°C for 3 hours, then cool to room temperature for later use.
[0076] 2) Put the homogenizer on ice, add 1mL RNA isolation reagent and 100-200 mg of P. asiaticus tissue, ground.
[0077] 3) Transfer the homogenate to a 1.5mL centrifuge tube and let stand at room temperature for 5min. Centrifuge at 13000r for 5min at 4°C.
[0078] 4) Transfer the supernatant to a clean 1.5mL centrifuge tube, add 200μL chloroform, and vortex for 15s. Place at room temperature for 5 minutes. Centrifuge at 13000r for 10min at 4°C.
[0079] 5) Transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube, add 200 μL of chloroform, and vortex for 30 sec...
Embodiment 2
[0112] Embodiment 2, the dsRNA of the protein tyrosine kinase gene PTK of the Asian locust and its application in pest control
[0113] 1. Synthesis of dsRNA
[0114] Using T7RiboMAX TM The Express RNAi System Kit synthesizes dsRNA. Specific steps are as follows:
[0115] 1) Synthesis of dsRNA primers
[0116] Design primers based on the cloned gene fragments, amplify the target fragment to about 600bp, and introduce T7 promoter at the 5' end of the primers. The primer sequences are as follows: PTK RNAi F: 5'-GGATCCTAATACGACTCACTATAGGGCACGAACTGATGAGGACCT-3'; PTK RNAi R: 5'-GGATCCTAATACGACTCACTATAGGAACTTGCGGACGAACTTGAG-3'.
[0117] 2) Preparation of DNA template
[0118] The bacterial liquid plasmid was extracted with a kit, and the plasmid containing the gene fragment (recombinant vector in Example 1) was used as a template, and PTK RNAi F and PTK RNAi R were used for PCR amplification to obtain the target fragment containing the T7 promoter sequence.
[0119] The PCR r...
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