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Insecticidal protein, and coding gene and purposes thereof

A technology of insecticidal protein and insecticidal gene, applied in the field of insecticidal protein

Active Publication Date: 2013-12-18
BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002]Pest pests are the main factor leading to crop loss, causing significant economic losses to farmers, and even affecting the survival of the local population
In order to control plant pests, people usually use broad-spectrum chemical insecticides and biological insecticides, but both have limitations in practical application: chemical insecticides will cause environmental pollution problems and lead to the emergence of resistant insects. However, biopesticides are easily degraded in the environment and need to be repeatedly applied in production, which greatly increases the production cost
There is no report on the expression level and toxicity of PIC9 insecticidal protein in plants

Method used

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  • Insecticidal protein, and coding gene and purposes thereof
  • Insecticidal protein, and coding gene and purposes thereof
  • Insecticidal protein, and coding gene and purposes thereof

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0080] The first embodiment, the acquisition and synthesis of PIC9 gene sequence

[0081] 1. Obtain the PIC9 gene sequence

[0082] The amino acid sequence (650 amino acids) of the PIC9 insecticidal protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (650 amino acids) corresponding to the PIC9 insecticidal protein encoding is obtained according to the corn preference codon Nucleotide sequence (1953 nucleotides), as shown in SEQ ID NO:1 in the sequence listing. For maize codon usage bias, please refer to http: / / www.kazusa.or.jp / codon / cgi-bin / showcodon.cgi?species=381124.

[0083] 2. Synthesis of the above PIC9 nucleotide sequence

[0084] The PIC9 nucleotide sequence (shown as SEQ ID NO:1 in the sequence listing) was synthesized by Nanjing KingScript Biotechnology Co., Ltd.; the 5' end of the synthesized PIC9 nucleotide sequence (SEQ ID NO:1) An AscI restriction site is also connected, and a SpeI restriction site is also connected to the 3' end ...

no. 2 example

[0089] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0090] 1. Construction of recombinant cloning vector DBN01-T containing PIC9 nucleotide sequence

[0091] The synthesized PIC9 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, 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 start codon; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; PIC9 is the promoter of PIC9 Nucleotide sequence (SEQ ID NO: 1); MCS is multiple cloning site).

[0092] Then, the recombinant cloning vector DBN01-T was transformed into Escheri...

no. 3 example

[0110] The third embodiment, the acquisition and verification of corn plants transferred to PIC9 nucleotide sequence

[0111] 1. Obtaining corn plants transferred to the PIC9 nucleotide sequence

[0112] According to the routinely used Agrobacterium infection method, the immature embryos of the aseptically cultivated corn variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 4 in the second example, so as to infect 2 and 3 in the second example. T-DNA (including the promoter sequence of corn Ubiquitin gene, PIC9 nucleotide sequence, PIC9 substitution nucleotide sequence, PIC9 deletion nucleotide sequence, PIC9 addition nucleotide sequence, PIC9-Ab nucleotide sequence, known sequence, PMI gene and Nos terminator sequence) were transferred into the maize genome to obtain Corn plants transferred to PIC9 nucleotide sequence, corn plants transferred to PIC9 substitution nucleotide sequence, corn plants transferred to PIC9 deletion nucleotide sequence, corn pla...

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PUM

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Abstract

The invention relates to an insecticidal protein, its gene encoding and the uses thereof. The protein comprises: (a) a protein consisting of an amino acid sequence shown by SEQ ID NO:2 or a sequence of amino acid residuses at positions 1 to 640 thereof; or (b) a protein derived from (a) by substitution, deletion, or addition of one or more amino acid residuses of the amino acid sequences in (a), and having insecticidal activity; or (c) a protein generated by the expression of nucleic acid molecules containing a sequence of amino acid residuses at the positions 1 to 1920 of SEQ ID NO:1; or (d) a protein generated by the expression of nucleic acid molecules containing a complementary sequence that hybridized with the sequence of nucleotides at the positions 1 to 1920 of SEQ ID NO:1 under stringent conditions. The protein has high expression level and strong toxicity against pests.

Description

technical field [0001] The present invention relates to an insecticidal protein, its encoding gene and application, in particular to a transformed PIC9 insecticidal protein, its encoding gene and application. Background technique [0002] Plant pests are the main factor leading to the loss of crops, causing significant economic losses to farmers, and even affecting the living conditions of the local population. In order to control plant pests, people usually use broad-spectrum chemical insecticides and biological insecticides, but both have limitations in practical application: chemical insecticides will cause environmental pollution problems and lead to the emergence of resistant insects. However, biopesticides are easily degraded in the environment and require repeated application in production, which greatly increases production costs. [0003] In order to solve the limitations in the practical application of chemical insecticides and biological insecticides, scientists...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/63C12N5/10C12N1/21C12N1/19C12N7/01A01K67/033C12P21/02C12N15/82C12N15/84A01H5/00C07K19/00A01N47/44A01P7/04A01N63/50
CPCC12N15/09C07K14/325A01N63/00A01N37/44A01N63/02C12N15/8286A01N37/46Y02A40/146A01N63/50
Inventor 张爱红庞洁牛瑞琪牛丽红高超苗猛猛杨东海
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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