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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 farmers' economic loss, need for repeated application, increase of production cost, etc.

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

[0078] The first embodiment, the acquisition and synthesis of PIC6-01 gene sequence

[0079] 1. Obtain the PIC6-01 gene sequence

[0080] The amino acid sequence (699 amino acids) of the PIC6-01 insecticidal protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (699 amino acids) corresponding to the PIC6-01 insecticidal protein is obtained according to the maize preference codon amino acids) nucleotide sequence (2100 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.

[0081] 2. Synthesize the above-mentioned PIC6-01 nucleotide sequence

[0082] The PIC6-01 nucleotide sequence (shown as SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the synthesized PIC6-01 nucleotide sequence (SEQ ID NO: 1) The 5' end of the PIC6-01 nucleotide sequence (SEQ ID NO: 1) is also connected with...

no. 2 example

[0086] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0087] 1. Construction of recombinant cloning vector DBN01-T containing PIC6-01 nucleotide sequence

[0088] The synthesized PIC6-01 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 , 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 SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; PIC6-01 is the nucleotide sequence of PIC6-01 (SEQ ID NO: 1); MCS is the multiple cloning site).

[0089]Then, the recombinant cloning vector DBN01-T was transformed i...

no. 3 example

[0105] The third embodiment, the acquisition and verification of corn plants transferred to PIC6-01 nucleotide sequence

[0106] 1. Obtaining corn plants transferred to the PIC6-01 nucleotide sequence

[0107]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, PIC6-01 nucleotide sequence, PIC6-01 The substituted nucleotide sequence, PIC6-01 truncated nucleotide sequence, PIC6-01 added nucleotide sequence, natural sequence, PMI gene and Nos terminator sequence) were transferred into the maize genome, and the transferred PIC6- Corn plant with 01 nucleotide sequence, corn plant with PIC6-01 substituted nucleotide sequence, corn plant with PIC6-01 truncated nucleotide sequence, corn plant with PIC6-01 added...

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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 shown as SEQ ID NO:2, or a protein (b) with insecticidal activity, which is derived from the protein (a), 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 a protein (c) formed by an amino acid sequence with at least 90% of sequence identity with the SEQ ID NO:2. 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 modified PIC6-01 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 Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/63C12N5/10C12N1/13C12N1/19C12N1/21C12N7/01C12P21/02A01H5/00A01N63/02A01P7/04
Inventor 张爱红贾志伟李胜兵杨旭王利君王登元王志新牛丽红黄金存于福宽
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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