Pest control method
A technology of pests, two-spotted mites, applied in the field of pest control
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no. 1 example
[0083] The first embodiment, the acquisition and synthesis of Cry1A.105 gene
[0084] 1. Obtain the nucleotide sequence of Cry1A.105
[0085] The amino acid sequence (1177 amino acids) of the Cry1A.105 insecticidal protein, as shown in SEQ ID NO: 1 in the sequence listing; Cry1A.105 encoding the amino acid sequence (1177 amino acids) corresponding to the Cry1A.105 insecticidal protein Nucleotide sequence (3534 nucleotides), as shown in SEQ ID NO:2 in the sequence listing.
[0086] 2. Obtain the nucleotide sequences of Cry2Ab and Vip3A
[0087] The Cry2Ab nucleotide sequence (1905 nucleotides) encoding the amino acid sequence (634 amino acids) of the Cry2Ab insecticidal protein, as shown in SEQ ID NO: 3 in the sequence listing; the amino acid sequence (789 nucleotides) encoding the Vip3A insecticidal protein amino acid) Vip3A nucleotide sequence (2370 nucleotides), as shown in SEQ ID NO:4 in the sequence listing.
[0088] 3. Synthesize the above nucleotide sequence
[0089]...
no. 2 example
[0090] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0091] 1. Construction of a recombinant cloning vector containing the Cry1A.105 gene
[0092] The synthesized Cry1A.105 nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T , its construction process is as follows figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; Cry1A.105 is the promoter of Cry1A .105 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
[0093] Then, the recombinant cloning vector DBN01-T was trans...
no. 3 example
[0104] The third embodiment, the acquisition and verification of corn plants transferred to the Cry1A.105 gene
[0105] 1. Obtaining corn plants transferred to the Cry1A.105 gene
[0106] According to the commonly used Agrobacterium infection method, the immature embryos of the aseptically cultured maize variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the second example, so that the T-DNA in recombinant expression vectors DBN100032, DBN100076 and DBN100029 (including the promoter sequence of maize Ubiquitin gene, Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence, Vip3A nucleotide sequence, PMI gene and Nos terminator sequence ) into the maize genome, and obtained the maize plant with the Cry1A.105 nucleotide sequence, the maize plant with the Cry1A.105-Cry2Ab nucleotide sequence and the Cry1A.105-Vip3A nucleotide sequence maize plants; meanwhile, wild-type maize plants were used as controls.
[0107] For Agrobacterium-mediated transfor...
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