Use of insecticidal protein
A protein and pest technology, applied in the field of insecticidal protein
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no. 1 example
[0084] The first embodiment, the acquisition and synthesis of genes
[0085] 1. Obtain the nucleotide sequence
[0086] The amino acid sequence (789 amino acids) of Vip3Aa-01 insecticidal protein, as shown in SEQ ID NO: 1 in the sequence listing; The Vip3Aa nucleotide sequence (2370 nucleosides) of encoding corresponding to the amino acid sequence of said Vip3Aa insecticidal protein acid), as shown in SEQ ID NO:2 in the sequence listing.
[0087] Amino acid sequence (789 amino acids) of Vip3Aa-02 insecticidal protein, as shown in SEQ ID NO:3 in the sequence listing; Vip3Aa-02 nucleotide sequence ( 2370 nucleotides), as shown in SEQ ID NO:4 in the sequence listing.
[0088] The amino acid sequence (615 amino acids) of Cry1Ab insecticidal protein, as shown in SEQ ID NO:5 in the sequence listing; Cry1Ab nucleotide sequence (1848 nucleotides) of encoding corresponding to the amino acid sequence of said Cry1Ab insecticidal protein , as shown in SEQ ID NO:6 in the sequence listin...
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 Vip3Aa gene
[0094]The synthetic Vip3Aa-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, whose construction process is as follows figure 1 Shown (among them, Amp represents the ampicillin resistance gene; f1ori represents the replication origin of phage f1; LacZ is the actual codon of LacZ; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; Vip3Aa-01 is the promoter of Vip3Aa- 01 nucleotide sequence (SEQ ID NO: 2); MCS is a multiple cloning site).
[0095] Then, the recombinant cloning vector DBN01-T was t...
no. 3 example
[0108] The third embodiment, the acquisition of transgenic plants
[0109] 1. Obtaining transgenic soybean plants
[0110] According to the routinely used Agrobacterium infection method, the cotyledon node tissue of the aseptically cultivated soybean variety Zhonghuang 13 was co-cultured with the Agrobacterium described in 3 in the second example, so that the recombinant gene constructed in 2 in the second example T-DNA of expression vectors DBN100002, DBN100741, DBN100003 and DBN100370 (including the promoter sequence of Arabidopsis ubiquitin gene, Vip3Aa-01 nucleotide sequence, Vip3Aa-02 nucleotide sequence, Vip3Aa-02-Cry1Ab nucleotide sequence sequence, Vip3Aa-02-Cry2Ab nucleotide sequence, PAT gene and tNos terminator sequence) were transferred into the soybean genome, and the soybean plants transferred to the Vip3Aa-01 nucleotide sequence and the Vip3Aa-02 nucleoside Acid sequence soybean plants, soybean plants transformed with Vip3Aa-02-Cry1Ab nucleotide sequence, and s...
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