Application of insecticidal protein
A protein and pest technology, applied in the field of insecticidal protein
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no. 1 example
[0085] The first embodiment, the acquisition and synthesis of genes
[0086] 1. Obtain the nucleotide sequence
[0087] The amino acid sequence (634 amino acids) of Cry2Ab insecticidal protein, as shown in SEQ ID NO:1 in the sequence listing; The Cry2Ab nucleotide sequence (1905 nucleotides) of encoding corresponding to the amino acid sequence of described Cry2Ab insecticidal protein , as shown in SEQ ID NO:2 in the sequence listing.
[0088] The amino acid sequence (605 amino acids) of Cry1Fa insecticidal protein, as shown in SEQ ID NO:3 in the sequence listing; Cry1Fa nucleotide sequence (1818 nucleotides) of encoding corresponding to the amino acid sequence of said Cry1Fa insecticidal protein , as shown in SEQ ID NO:4 in the sequence listing.
[0089] 2. Synthesize the above nucleotide sequence
[0090] The Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 2 in the sequence listing) and the Cry1Fa nucleotide sequence (as shown in SEQ ID NO: 4 in the sequence listing) we...
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 Cry2Ab gene
[0093] The synthetic Cry2Ab nucleotide sequence was connected into 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, which Build process such as 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; Cry2Ab is the Cry2Ab nucleoside acid sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
[0094] Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 c...
no. 3 example
[0103] The third embodiment, the acquisition of transgenic plants
[0104] 1. Obtaining transgenic corn plants
[0105] 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 The T-DNA in the recombinant expression vectors DBN100033 and DBN100076 (including the promoter sequence of the CaMV35S gene, the Cry2Ab nucleotide sequence, the Cry1Fa nucleotide sequence, the Hpt gene and the Nos terminator sequence) were transferred into the maize genome, and obtained The corn plant transformed with the Cry2Ab nucleotide sequence and the corn plant transformed with the Cry2Ab-Cry1Fa nucleotide sequence; at the same time, the wild-type corn plant was used as a control.
[0106] For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos are isolated from maize and contacted with a susp...
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