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Insecticidal protein, as well as coding gene and application 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, as well as coding gene and application thereof
  • Insecticidal protein, as well as coding gene and application thereof
  • Insecticidal protein, as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

no. 1 example

[0075] The first embodiment, the acquisition and synthesis of PIC9-01 gene sequence

[0076] 1. Obtain the PIC9-01 gene sequence

[0077] The amino acid sequence (699 amino acids) of the PIC9-01 insecticidal protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (699 amino acids) corresponding to the PIC9-01 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.

[0078] 2. Synthesize the above-mentioned PIC9-01 nucleotide sequence

[0079] The PIC9-01 nucleotide sequence (shown as SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Company; the synthesized PIC9-01 nucleotide sequence (SEQ ID NO: 1) The 5' end of the PIC9-01 nucleotide sequence (SEQ ID NO: 1) is also connected with a...

no. 2 example

[0083] The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

[0084] 1. Construction of recombinant cloning vector DBN01-T containing PIC9-01 nucleotide sequence

[0085] The synthesized PIC9-01 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 SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; PIC9-01 It is the nucleotide sequence of PIC9-01 (SEQ ID NO: 1); MCS is the multiple cloning site).

[0086] Then, the recombinant cloning vector DBN01-T was transfor...

no. 3 example

[0102] The third embodiment, the acquisition and verification of corn plants transferred to PIC9-01 nucleotide sequence

[0103] 1. Obtain corn plants transferred to PIC9-01 nucleotide sequence

[0104] 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, PIC9-01 nucleotide sequence, PIC9- 01 substituted nucleotide sequence, PIC9-01 deleted nucleotide sequence, PIC9-01 added nucleotide sequence, known sequence, PMI gene and Nos terminator sequence) were transferred into the maize genome, and the transferred PIC9 Corn plant with -01 nucleotide sequence, corn plant with PIC9-01 substitution nucleotide sequence, corn plant with PIC9-01 deletion nucleotide sequence, corn plant with PIC9-01 addition nucleotid...

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Abstract

The invention provides an insecticidal protein, as well as a coding gene and application thereof. The insecticidal protein comprises (a) protein containing amino acid sequence shown in SEQ ID NO:2; or (b) protein which is an amino acid sequence in (a) substituted, lost or added by one or more amino acids and is derived from (a) with insecticide activity; or (c) protein which is produced through expression of nucleic acid molecules containing a nucleotide sequence shown in SEQ ID NO:1; or (d) protein produced through expression of nucleic acid molecules containing the nucleotide sequence which has a complementary sequence hybridized with the nucleotide sequence shown in SEQ ID NO:1 in a strict condition; or (e) protein produced through expression of nucleic acid molecules containing the nucleotide sequence which has the same codes with the nucleotide sequence of (d). The insecticidal protein has the advantages of high expression capacity and strong toxicity to insects and 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): C07K14/325C12N15/32C12N15/63C12N5/10C12N1/21C12N1/19C12N7/01A01K67/033C12P21/02C12N15/82C12N15/84A01H5/00A01N47/44A01P7/04
Inventor 张爱红庞洁杨进孝牛瑞琪董雷庞雪兵杨秋妹傅学乾
Owner BEIJING DABEINONG BIOTECHNOLOGY CO LTD
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