Nucleotide sequences and methods for controlling insect infestation
A nucleotide sequence and polynucleotide technology, which is applied in the field of control of Sterna chinensis, can solve the problems of antisense sequence instability, antisense sequence instability, etc.
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no. 1 example
[0109] The first embodiment, the determination of the target sequence
[0110] Through whole-transcriptome sequencing, the transcriptomes of the larvae and adults of S. brownfoot were obtained, and seven target sequences from five candidate genes of S. brownfoot were screened from each metabolic pathway. The specific information is shown in Table 1. Shown:
[0111] Table 1. Specific information table of 5 candidate genes
[0112]
no. 2 example
[0113] The second embodiment, the construction of plant expression vector
[0114] According to cauliflower mosaic virus 35S promoter-target sequence sense strand-spacer sequence-target sequence antisense strand are connected together, and form expression vector with marker gene Hpt which can confer hygromycin selection.
[0115] The two ends of the sense primer have EcoR I and Hind III restriction sites, the antisense primers have Xho I and Sac I restriction sites at both ends, and the spacer primers have Hind III and Sac I restriction sites at both ends .
[0116] The recombinant cloning vector containing the sense strand was digested by EcoR I and Hind III, and the sense strand fragment was recovered. The recombinant expression vector DBNBC-01 was subjected to the same double digestion to recover the linearized plasmid, and ligated with the target fragment r1 to obtain the recombinant expression vector DBNBC-01-r1. The recombinant expression vector DBNBC-01-r1 was double ...
no. 3 example
[0119] The third embodiment, transformation of recombinant expression vector into Agrobacterium
[0120]The correct recombinant expression vectors DBN110017, DBN110018, DBN100999, DBN110000, DBN110013, DBN110016 and DBN110041 were transformed into Agrobacterium LBA4404 (Invitrogen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were as follows: : 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, 37 ° C warm water bath for 10 minutes; inoculate the transformed Agrobacterium LBA4404 in LB test tubes at a temperature of 28 ° C and a rotation speed of 200 rpm Cultivate under conditions for 2 hours, spread on LB plates containing 50 mg / L Rifampicin and 100 mg / L Kanamycin until positive single clones grow, pick single clones for culture and extract their Plasmids, the recombinant expression vectors DBN110017, DBN110018, DBN100999, DBN110000, DBN110013, DBN110016 and DB...
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