Application of insecticidal protein
A protein and pest technology, applied in the field of insecticidal protein
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no. 1 example
[0083] The first embodiment, the acquisition and synthesis of genes
[0084] 1. Obtain the nucleotide sequence
[0085] 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.
[0086] The amino acid sequence (1177 amino acids) of Cry1A.105 insecticidal protein, as shown in SEQ ID NO:3 in the sequence listing; Cry1A.105 nucleotide sequence ( 3534 nucleotides), as shown in SEQ ID NO:4 in the sequence listing.
[0087] 2. Synthesize the above nucleotide sequence
[0088] The Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 2 in the sequence listing) and the Cry1A.105 nucleotide sequence (as shown in SEQ ID NO: 4 in the sequence listing) were provided by Nanjing GenScript Co., Ltd.; the 5' end of the synthetic Cry2Ab...
no. 2 example
[0089] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0090] 1. Construction of a recombinant cloning vector containing the Cry2Ab gene
[0091] 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).
[0092]Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 co...
no. 3 example
[0102] The third embodiment, the acquisition of transgenic plants
[0103] 1. Obtaining transgenic corn plants
[0104] 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 recombinant expression vectors DBN100744, DBN100745 and DBN100029 (including the promoter sequence of maize Ubiquitin gene, Cry2Ab nucleotide sequence, Cry1A.105 nucleotide sequence, Hpt gene and Nos terminator sequence) were transferred to maize chromosome In the group, the corn plants transferred to Cry2Ab nucleotide sequence, the corn plant transferred to Cry1A.105 nucleotide sequence and the corn plant transferred to Cry1A.105-Cry2Ab nucleotide sequence were obtained; as comparison.
[0105] For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos were isolated from maize and contact...
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