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Insecticidal protein and coding gene and application thereof

A technology of insecticidal protein and insecticidal gene, applied in the field of insecticidal protein, can solve the problems of environmental pollution, increase of production cost, need for repeated application and the like

Active Publication Date: 2013-03-27
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 PIC6 insecticidal protein in plants

Method used

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  • Insecticidal protein and coding gene and application thereof
  • Insecticidal protein and coding gene and application thereof
  • Insecticidal protein and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0083] The first embodiment, the acquisition and synthesis of PIC6-02t gene sequence

[0084] 1. Obtain the PIC6-02t gene sequence

[0085] The amino acid sequence (660 amino acids) of the PIC6-02t insecticidal protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (660 amino acids) corresponding to the PIC6-02t insecticidal protein is obtained according to the maize preference codon ) nucleotide sequence (1983 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.

[0086] 2. Synthesize the above-mentioned PIC6-02t nucleotide sequence

[0087] The PIC6-02t nucleotide sequence (shown as SEQIDNO:1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the 5' end of the synthesized PIC6-02t nucleotide sequence (SEQIDNO:1) A SpeI restriction site is also connected, and the 3' end of the PIC6-02t nucl...

no. 2 example

[0091]The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0092] 1. Construction of recombinant cloning vector DBN01-T containing PIC6-02t nucleotide sequence

[0093] The synthesized PIC6-02t 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; PIC6-02t is the promoter of PIC6 -02t nucleotide sequence (SEQ ID NO: 1); MCS is a multiple cloning site).

[0094] Then, the recombinant cloning vector DBN01-T...

no. 3 example

[0110]The third embodiment, the acquisition and verification of corn plants transferred to PIC6-02t nucleotide sequence

[0111] 1. Obtaining corn plants transferred to the PIC6-02t 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, PIC6-02t nucleotide sequence, PIC6-02t Substitution nucleotide sequence, PIC6-02t deletion nucleotide sequence, PIC6-Ib nucleotide sequence, natural sequence, PMI gene and Nos terminator sequence) were transferred into the maize genome, and the PIC6-02t nuclear The corn plant of the nucleotide sequence, the corn plant of the PIC6-02t substitution nucleotide sequence, the corn plant of the PIC6-02t deletion nucleotide sequence, the corn plant of ...

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Abstract

The invention relates to an insecticidal protein and a coding gene and an application thereof. The insecticidal protein contains a protein (a) formed by an amino acid sequence from 1st to 650th sites shown as SEQ ID NO:2, or a protein (b) formed by the amino acid sequence shown as the SEQ ID NO:2, or a protein (c) with insecticidal activity, which is derived from the protein (a) or (b), and is formed by replacing and / or deleting and / or adding one or more amino acid in the amino acid sequence of the protein (a) or (b), or a protein (d) formed by an amino acid sequence with at least 90% of sequence identity with the amino acid sequence of the protein (a) or (b). The insecticidal protein has the advantages of high expression quantity, good stability, and high toxicity to insect pests.

Description

technical field [0001] The present invention relates to an insecticidal protein, its encoding gene and application, in particular to a transformed PIC6-02t 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, scienti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/63C12N5/10C12N1/21C12N1/19C12N7/01A01K67/033C12P21/02C12N15/82A01H5/00A01N47/44A01P7/04C07K19/00C12N15/62
Inventor 张爱红程鹏杨旭韩超张云珠王飞鹏杨秋妹王小艺李涛
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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