RNAi expression vector of Plutella xylostella tor gene and its construction method and application
A technology of expression vectors and plant expression vectors, applied in application, genetic engineering, plant genetic improvement, etc., to achieve the effect of delaying pest resistance and reducing the use of pesticides
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Embodiment 1
[0031] The construction of embodiment 1 Plutella xylostella TOR gene RNAi vector
[0032] 1. Acquisition and synthesis of TOR gene RNAi fragment of diamondback moth
[0033] Through the Plutella xylostella genome database ( http: / / iae.fafu.edu.cn / DBM / ), find out the full-length CDS sequence of the TOR gene of Plutella xylostella: TOR1: 7140bp; TOR2: 6882bp. Using the CDS of Plutella xylostella TOR to perform homologous alignment in NCBI, find out the three most conserved sequences, and fuse the three sequences. Add an Asc I-Not I restriction site at the 5' end of the fused sequence, add a SbfI-BbvC I restriction site at the 3' end, and name the sequence TOR-RNAi (658bp), and the sequence is as follows: As shown in the list Seq ID No: 1, the sequence was synthesized.
[0034] 2. Construction of Plutella xylostella TOR gene RNAi vector
[0035] Follow the steps below:
[0036] (1) Synthesize the sequence shown in the sequence table Seq ID No: 2, the sequence is named 35S-IN...
Embodiment 2
[0055] The acquisition of embodiment 2 transgenic Arabidopsis plants
[0056] Follow the steps below:
[0057] (1) The pE35S-TORRI vector obtained in Example 1 is transformed into the Agrobacterium LBA4404 bacterial strain, and the transformation steps are: 1) adding the pE35S-TORRI vector plasmid DNA (using plasmid Extracted by DNA extraction kit, refer to the kit manual for operation method) 5 μL, mix gently and then ice-bath for 30 minutes; 2) Put it in liquid nitrogen for 1 minute, then immediately put it in a 37°C water bath for 5 minutes; 3) Take it out and centrifuge put the tube on ice for 3 minutes; 4) add the liquid in the centrifuge tube to 500 μL LB liquid medium in the ultra-clean workbench, shake and culture at 28 ° C and 200 rpm for 4 h; 5) take out the bacterial solution in the Ping and Kanamycin 50 mg / L LB solid medium were plated, and after drying, they were cultured upside down in a 28°C incubator. Colonies were visible in about 2 days.
[0058] A single co...
Embodiment 3
[0061] Example 3 Research on insect resistance of transgenic Arabidopsis
[0062] 1. Group rearing of diamondback moth
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