Method for controlling pests
A pest and protein technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, angiosperms/flowering plants, etc.
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no. 1 example
[0084] The first embodiment, the acquisition and synthesis of Cry1Fa gene
[0085] 1. Obtain the Cry1Fa nucleotide sequence
[0086] The amino acid sequence (605 amino acids) of the Cry1Fa-01 insecticidal protein, as shown in SEQ ID NO: 1 in the sequence listing; Cry1Fa-01 encoding the amino acid sequence (605 amino acids) corresponding to the Cry1Fa-01 insecticidal protein Nucleotide sequence (1818 nucleotides), as shown in SEQ ID NO:3 in the sequence listing; amino acid sequence (1148 amino acids) of Cry1Fa-02 insecticidal protein, as shown in SEQ ID NO:2 in the sequence listing ; Cry1Fa-02 nucleotide sequence (3447 nucleotides) encoding the amino acid sequence (1148 amino acids) corresponding to the Cry1Fa-02 insecticidal protein, as shown in SEQ ID NO:4 in the sequence listing.
[0087] 2. Obtain the nucleotide sequences of Cry1Ab and Vip3A
[0088] The Cry1Ab nucleotide sequence (1848 nucleotides) encoding the amino acid sequence (615 amino acids) of the Cry1Ab insectic...
no. 2 example
[0091] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0092] 1. Construction of a recombinant cloning vector containing the Cry1F gene
[0093] The synthesized Cry1Fa-01 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 SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; Cry1Fa-01 is the Cry1Fa-01 nucleotide sequence (SEQ ID NO: 3); MCS is the multiple cloning site).
[0094] Then, the recombinant cloning vector DBN01-T was transformed into Escherichia...
no. 3 example
[0106] The third embodiment, the acquisition and verification of corn plants transferred to the Cry1F gene
[0107] 1. Obtaining corn plants transferred to the Cry1F gene
[0108] 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 DBN100014, DBN100012 and DBN100276 (including the promoter sequence of corn Ubiquitin gene, Cry1Fa-01 nucleotide sequence, Cry1Fa-02 nucleotide sequence, Cry1Ab nucleotide sequence, Vip3A nucleotide sequence , PMI gene and Nos terminator sequence) were transferred into the maize genome, and the maize plants transferred to the Cry1Fa-01 nucleotide sequence, the maize plants transferred to the Cry1Fa-01-Cry1Ab nucleotide sequence and the transferred Cry1Fa - the maize plant of 02-Vip3A nucleotide sequence; meanwhile, the wild type maize...
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