Nucleotide sequences and methods for controlling insect infestation
A nucleotide sequence and polynucleotide technology, applied in the field of controlling the two-spot firefly beetle, can solve the problems of antisense sequence instability and antisense sequence instability
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no. 1 example
[0101] The first embodiment, the determination of the target sequence of the two-spot firefly beetle
[0102] 1. Extraction of total RNA from the two-spot firefly beetle
[0103] Using the newly hatched instars of the two-spotted firefly beetle as materials, the RNA was extracted by the conventional Trizol method, purified by conventional methods, and treated with DNase to obtain a concentration of ≥300ng / μL, a total amount of ≥6μg, and OD 260 / 280 1.8-2.2 for total RNA samples.
[0104] 2. Isolation of mRNA and synthesis of cDNA
[0105] The mRNA with polyA was isolated with oligo-dT magnetic beads, and then the first-strand cDNA was synthesized with random hexamers and Invitrogen's Superscript II reverse transcriptase kit.
[0106] 3. Screen out a target gene
[0107] From the analysis of the larval library, the expression level is moderate, and it may be involved in the screening of important metabolic pathways. A target gene rsbsnf7 of the two-spotted firefly beetle was ...
no. 2 example
[0108] The second embodiment, the construction of plant expression vector
[0109] Entrusted Nanjing KingScript Biotechnology Co., Ltd. to synthesize two expression cassettes according to the sequence of p35S-R-tNos-p35S-Hpt-tNos, and connected them to the plant expression vector through EcoRI and HindIII, and named it DBN110048. The schematic diagram of the vector is as follows figure 1 Shown (Kan: kanamycin gene; RB: right border; pr35S: cauliflower mosaic virus 35S (SEQ ID NO: 3); R (SEQ ID NO: 2): the reverse complementary nucleotide sequence of r ( r is the target sequence of the target gene rsbsnf7, the nucleotide sequence of SEQ ID NO:1)+spacer sequence+r; tNos: the terminator of the nopaline synthase gene (SEQ ID NO:4); Hpt: hygromycin phosphotransfer Enzyme gene (SEQ ID NO:5); LB: left border).
[0110] The recombinant expression vector DBN110048 was transformed into Escherichia coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 μL E...
no. 3 example
[0111] The third embodiment, transformation of recombinant expression vector into Agrobacterium
[0112] The recombinant expression vector DBN110048 that has been constructed correctly was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression Carrier); placed in liquid nitrogen for 10 minutes, in a warm water bath at 37°C for 10 minutes; the transformed Agrobacterium LBA4404 was inoculated in an LB test tube and cultivated for 2 hours at a temperature of 28°C and a rotation speed of 200rpm, and then applied to a medium containing 50mg / L Rifampicin (Rifampicin) and 100mg / L Kanamycin (Kanamycin) on the LB plate until a positive single clone grows, pick the single clone culture and extract its plasmid, and use restriction endonucleases EcoRI and HindIII to Recombinant expression vector DBN110048 was digested and verif...
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