The use of insecticidal protein
A technology of insecticidal protein and protein, applied in the field of insecticidal protein
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no. 1 example
[0086] The first embodiment, the acquisition and synthesis of genes
[0087] 1. Obtain the nucleotide sequence
[0088] The amino acid sequence (789 amino acids) of Vip3A-01 insecticidal protein, as shown in SEQ ID NO: 1 in the sequence listing; The Vip3A-01 nucleus of encoding corresponding to the amino acid sequence (789 amino acids) of described Vip3A-01 insecticidal protein Nucleotide sequence (2370 nucleotides), as shown in SEQ ID NO:2 in the sequence listing. Amino acid sequence (789 amino acids) of Vip3A-02 insecticidal protein, as shown in SEQ ID NO: 3 in the sequence listing; coding corresponds to the Vip3A-02 of the amino acid sequence (789 amino acids) of said Vip3A-02 insecticidal protein Nucleotide sequence (2370 nucleotides), as shown in SEQ ID NO:4 in the sequence listing.
[0089] The amino acid sequence (615 amino acids) of Cry1Ab insecticidal protein, as shown in SEQ ID NO:5 in the sequence listing; The Cry1Ab nucleotide sequence (1848 nucleotides), as sho...
no. 2 example
[0092] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0093] 1. Construction of a recombinant cloning vector containing the Vip3A gene
[0094] The synthetic Vip3A-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 initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; Vip3A-01 is the Vip3A -01 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
[0095]Then, the recombinant cloning vector DBN01-T was transformed into Escherichia...
no. 3 example
[0106] The third embodiment, the acquisition of transgenic plants
[0107] 1. Obtaining transgenic corn plants
[0108] According to the commonly used Agrobacterium infection method, the immature embryos of the aseptically cultured corn variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the second embodiment, so that the T-DNA of recombinant expression vectors DBN100002, DBN100003 and DBN100740 (including the promoter sequence of corn Ubiquitin gene, Vip3A-01 nucleotide sequence, Vip3A-02 nucleotide sequence, Cry1Ab nucleotide sequence, Hpt gene and Nos terminator Sequence) was transferred into the corn genome, and the corn plant transferred to the Vip3A-01 nucleotide sequence, the corn plant transferred to the Vip3A-02 nucleotide sequence, and the corn plant transferred to the Cry1Ab+Vip3A-01 nucleotide sequence were obtained ; At the same time, wild-type maize plants were used as controls.
[0109] For Agrobacterium-mediated transformation of m...
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