Pine wood nematode tra-1 gene and application thereof to development interference
A tra-1, pine xylophilus technology, applied in the fields of application, nematicides, genetic engineering, etc., can solve problems such as interference with the reproductive development of pine xylophilus
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Embodiment 1
[0038] Embodiment 1: the cloning of the gene sequence of pine xylophilus tra-1 gene
[0039] Collection of pine xylophilus
[0040] Prepare fresh botrytis cinerea culture dishes for inoculation and cultivation for 5 days and pine wood nematode plates for 5 days of growth and put them in the ultra-clean bench for ultraviolet sterilization for more than half an hour. After inoculating 1*1cm of nematodes, incubate at 25°C for 5 days at a constant temperature, and filter for 4 hours by Bellman funnel method Finally, collect the nematode liquid at the bottom, centrifuge, rinse with sterile water for 2-3 times, rinse with RNase-free water for 2-3 times, and remove the supernatant for later use.
[0041] Extraction of Total RNA from Pine Xylophilus (Trizol Method)
[0042]The washed nematodes were quickly frozen in liquid nitrogen. After adding 200 μL Trizol dropwise with a pipette gun, grind for 30 seconds with an electric tissue grinder. Then, 800 μL of Trizol was quickly added ...
example 2
[0079] Example 2: Based on the results of Example 1, the cDNA sequence of the tra-1 gene was used to prepare the tra-1 gene dsRNA template
[0080] tra-1 gene dsRNA template primer design
[0081] According to the sequence of the cloned tra-1 gene of B. xylophilus, two pairs of specific primers with T7 promoter were designed and synthesized by using Primer Premier 5.0 software. The designed primers are shown in Table 7:
[0082] Table 7. Pine xylophilus tra-1 gene dsRNA template primer sequence
[0083]
[0084] Synthesis of pine xylophilus tra-1 gene dsRNA template
[0085] The tra-1cDNA sequence template that example 1 is reclaimed obtains, uses the primer that step [0032] obtains as amplification primer, carries out PCR amplification. Among them, BXY_1647700T7F and BXY_1647700T7R were amplified to obtain the tra-1 gene dsRNA template, and the reaction system was as follows:
[0086] Table 8. PCR reaction system of pine xylophilus tra-1dsRNA template
[0087]
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example 3
[0102] Example 3: Based on the results of Example 2, developmental interference was performed using dsRNA of the tra-1 gene
[0103]Filter the plate of pine xylophilus cultured for 5-7 days in an incubator at 25°C for 4-6 hours using the Baermann funnel method, collect the nematode liquid at the bottom of the funnel rubber tube about 4 cm, and pick the 2nd instar larvae of pine wood nematode under a microscope. Rinse with sterile water 2-3 times and set aside;
[0104] dsRNA treatment group: dsRNA solution (final concentration 1.0ug / uL) 120 μL, M9 buffer, 260 μL, constant volume to 500 μL, nematode concentration 4 / μL; the same volume of DEPC-treated water instead of dsRNA solution as the control group, The test was repeated 3 times;
[0105] Place in a shaker at 25°C and incubate with shaking at 120rpm for 24h.
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