Insecticidal protein, and encoding gene and use thereof
A technology of insecticidal protein and insecticidal gene, applied in the field of insecticidal protein, can solve problems such as environmental pollution, affecting the living conditions of the population, and farmers' economic losses.
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no. 1 example
[0073] The first embodiment, the acquisition and synthesis of PIC10-01 gene sequence
[0074] The amino acid sequence (630 amino acids) of the PIC10-01 insecticidal protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (630 amino acids) encoding the insecticidal protein corresponding to the PIC10-01 is obtained according to rice preference codons amino acid) nucleotide sequence (1896 nucleotides), as shown in SEQID NO:1 in the sequence listing. The PIC10-01 nucleotide sequence (shown as SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the synthesized PIC10-01 nucleotide sequence (SEQ ID NO: 1) The 5' end of the PIC10-01 nucleotide sequence (SEQ ID NO: 1) is also connected with a PvuI restriction site at the 3' end.
no. 2 example
[0075] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0076] 1. Construction of recombinant cloning vector DBN01-T containing PIC10-01 nucleotide sequence
[0077] The synthesized PIC10-01 nucleotide sequence was ligated 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 , 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; PIC10-01 is the promoter of PIC10 -01 nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).
[0078] Then, the recombinant cloning vector DBN01-...
no. 3 example
[0088] The third embodiment, the acquisition and verification of rice plants transferred to the PIC10-01 nucleotide sequence
[0089] 1. Obtain rice plants transferred to the PIC10-01 nucleotide sequence
[0090] According to the routinely used Agrobacterium infection method, the callus of the aseptically cultured japonica rice variety Nipponbare was co-cultured with the Agrobacterium described in 4 in the second embodiment, so that the recombinant strains constructed in 2 and 3 in the second embodiment The T-DNA (including the promoter sequence of maize Ubiquitin gene, PIC10-01 nucleotide sequence, known sequence, PMI gene and Nos terminator sequence) in the expression vectors DBN100323 and DBN100323N (known sequence) was transferred into rice chromosome In the group, the rice plants transformed with the PIC10-01 nucleotide sequence and the rice plants transformed with the known sequence were obtained; at the same time, wild-type rice plants were used as controls.
[0091] F...
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