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
[0084] The first embodiment, the acquisition and synthesis of genes
[0085] 1. Obtain the Cry1Fa nucleotide sequence
[0086] The amino acid sequence (605 amino acids) of Cry1Fa-01 insecticidal protein, as shown in SEQ ID NO:1 in the sequence listing; Cry1Fa-01 nucleotide sequence ( 1818 nucleotides), as shown in SEQ ID NO: 3 in the sequence listing; the amino acid sequence (1148 amino acids) of Cry1Fa-02 insecticidal protein, as shown in SEQ ID NO: 2 in the sequence listing; the encoding corresponds to the The Cry1Fa-02 nucleotide sequence (3447 nucleotides) of the amino acid sequence of the Cry1Fa-02 insecticidal protein is shown in SEQ ID NO:4 in the sequence listing.
[0087] 2. Obtain the Cry1Ac nucleotide sequence
[0088] The amino acid sequence (1156 amino acids) of Cry1Ac insecticidal protein, as shown in SEQ ID NO:5 in the sequence listing; Cry1Ac nucleotide sequence (3471 nucleotides) of encoding corresponding to the amino acid sequence of said Cry1Ac insecticida...
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 Cry1Fa gene
[0093] The synthetic 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 promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; Cry1Fa-01 is the promoter of Cry1Fa -01 nucleotide sequence (SEQ ID NO: 3); MCS is the multiple cloning site).
[0094]Then, the recombinant cloning vector DBN01-T was transformed...
no. 3 example
[0105] The third embodiment, the acquisition of transgenic plants
[0106] 1. Obtaining transgenic soybean plants
[0107] 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 DBN100014, DBN100013 and DBN100756 (including the promoter sequence of Arabidopsis ubiquitin gene, Cry1Fa-01 nucleotide sequence, Cry1Fa-02 nucleotide sequence, Cry1Ac nucleotide sequence, PAT gene and tNos terminator sequence) into the soybean genome, obtained the soybean plant transferred to the Cry1Fa-01 nucleotide sequence, the soybean plant transferred to the Cry1Fa-02 nucleotide sequence and the Cry1Fa-02-Cry1Ac nucleotide sequence sequenced soybean plants, and wild-type soybean plants were used as controls.
[0108] For Agrobacter...
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