Use 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 (1178 amino acids) of Cry1Ac insecticidal protein, as shown in SEQ ID NO:3 in the sequence listing; Cry1Ac nucleotide sequence (3537 nucleotides) of encoding corresponding to the amino acid sequence of said Cry1Ac insecticidal protein , as shown in SEQ ID NO:4 in the sequence listing.
[0089] The amino acid sequence (1177 amino acids) of Cry1A.105 insecticidal protein, as shown in SEQ ID NO:5 in the sequence listing; Cry1A.105 nucleotide sequence ( 3534 nucleotides), as shown in SEQ ID NO:6 in the sequence list...
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 Cry2Ab gene
[0094] 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).
[0095] Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 co...
no. 3 example
[0106] The third embodiment, the acquisition of transgenic plants
[0107] 1. Obtaining transgenic soybean plants
[0108] 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 DBN100033, DBN100075 and DBN100076 (including promoter sequence prAtUbi10 of Arabidopsis ubiquitin gene, Cry2Ab nucleotide sequence, Cry1Ac nucleotide sequence, Cry1A.105 nucleotide sequence, PAT gene and tNos termination subsequence) into the soybean genome, obtained the soybean plant transferred to the Cry2Ab nucleotide sequence, the soybean plant transferred to the Cry2Ab-Cry1Ac nucleotide sequence and the soybean transferred to the Cry2Ab-Cry1A.105 nucleotide sequence plants, and wild-type soybean plants were used as controls.
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