Emblycoris indica synaptic fusion protein binding protein RpSDP and application thereof
A technology of syntaxin and binding protein, which is applied in the field of genetic engineering, can solve the problems of poor applicability and lack of pest control technology for the stink bug, and achieve the effect of reducing the survival rate
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Embodiment 1
[0038] Example 1 Cloning of the RpSDP gene fragment of the stinkbug RpSDP
[0039] 1. Amplification of the RpSDP gene fragment of the stink bug
[0040] (1) Take some adults or nymphs of the stink bug, put them into 1ml Trizol (Takara) and grind them thoroughly; then add 400μl chloroform to mix vigorously, then centrifuge at 12,000rpm 4°C for 15 minutes, carefully absorb the top aqueous phase liquid, and transfer to a new centrifuge tube; add an equal volume of isopropanol, mix well and let stand at room temperature for 10 minutes; then centrifuge at 12,000rpm for 10 minutes, discard the supernatant and add 1ml of 75% ethanol to the pellet; centrifuge at 9600rpm for 5 minutes at 4°C Discard the supernatant, and add an appropriate amount of RNase-free water to the pellet; measure the RNA concentration with NanoDrop;
[0041] (2) Use the ReverTra Ace q PCR RT Master with gDNA remover kit to reverse-transcribe the extracted total RNA of the stink bug: First, denature the total...
Embodiment 2
[0053] Embodiment 2: the dsRNA synthesis of RpSDP gene
[0054] 1. T7 primer PCR amplification and purification
[0055] (1) Using the recombinant plasmid or bacterial liquid containing the RpSDP gene as a template, use the primers (shown in Table 2) with the T7 promoter sequence to amplify the target gene. The primers were synthesized by Nanjing KingScript Biotechnology Co., Ltd., and the amplification system 2× Max Buffer 100μl, dNTP Mix (10mM each) 4μl, Max Super-Fidelity DNA Polymerase 4μl, upstream and downstream (SEQ ID NO.5 and SEQ ID NO.6) 4μl each, recombinant plasmid or bacterial solution 4μl, and finally use ddH 2 O make up to 200 μl. The PCR amplification conditions are: 95°C for 3 minutes; 95°C for 15 seconds, 60°C for 1 minute, 35 cycles; 72°C for 10 minutes.
[0056] Table 2
[0057]
[0058] (2) The amplified product is separated by agarose gel electrophoresis, and recovered by a DNA agarose gel recovery kit, and finally a large number of single R...
Embodiment 3
[0066] Example 3: The effect of the dsRNA introduction point of RpSDP gene on the survival rate of insects
[0067] The dsRNA of the RpSDP gene is introduced into the body of the stink bug by microinjection, and the specific method is as follows:
[0068] (1) Take some nymphs of stinkbug. use CO 2 Anesthetize for 10s;
[0069] (2) Use a capillary puller (P 97, Sutter Instrument) to pull the glass capillary (Wuhan Weitan Scientific Instrument Co., Ltd.) to a suitable size, and the program parameters used are set as: heat=800pull=150vel=150time=80;
[0070] (3) inject the dsRNA of GFP and RpSDP genes into the glass capillary respectively with a micro-pippet head (Eppendorf);
[0071] (4) Install the glass capillary that has added the sample into a microinjector (Eppendorf), and introduce the dsRNA into the stink bug body under a stereoscope. The parameters of the microinjector are set as: injection pressure 1300 pah, injection time 0.3 s, compensation pressure 10pah;
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