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Novel recombinant insect resistant protein as well as preparation method and application thereof

A kind of anti-insect protein and new technology, applied in the field of new recombinant anti-insect protein and its preparation

Active Publication Date: 2015-06-24
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since China launched the "Major Science and Technology Project for the Breeding of New Varieties of Genetically Modified Organisms" in 2008, although there have been some reports on the breeding of transgenic insect-resistant corn varieties, they have not been commercialized. This is mainly due to the insect-resistant effect and transformation of the Bt gene. plant stability

Method used

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  • Novel recombinant insect resistant protein as well as preparation method and application thereof
  • Novel recombinant insect resistant protein as well as preparation method and application thereof
  • Novel recombinant insect resistant protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Determination of the modified region of the Cry protein family

[0062](1) Obtain the amino acid sequences of some members of the Cry protein family from the NCBI database. The 24 selected Cry proteins are Cry1A105 protein, Cry1Aa1 protein, Cry1Ab1 protein, Cry1Ab5 protein, Cry1Ab14 protein, Cry1Ac1 protein, Cry1Ah1 protein, Cry1Ba1 protein, Cry1Bb1 protein protein, Cry1Be1 protein, Cry1Bf1 protein, Cry1Fa1 protein, Cry1Gc protein, Cry1Ia1 protein, Cry1If1 protein, Cry1Ja1 protein, Cry1Jb1 protein, Cry1Jc1 protein, Cry2Ab1 protein, Cry2Ac1 protein, Cry2Ae1 protein, Cry9Aa1 protein, Cry9Ca1 protein, Cry9Ec1 protein.

[0063] (2) Using the pMAL algorithm and perl programming language, the evolution rate analysis method is used to conduct statistical analysis on the above 24 Cry proteins, predict the highly evolutionary activity regions of the above 24 Cry protein amino acid sequences in the molecular evolution process, and determine the evolution rate The fastes...

Embodiment 2

[0066] Example 2 Constructing a Novel Insect-resistant Gene Bank

[0067] (1) Select the genes with anti-lepidoptera activity in the NCBI database to establish a data set, and perform local similarity comparison by BLAST, that is, BLAST comparison.

[0068] Cry genes from different subfamilies were selected according to the difference of quaternary nucleotide homology:

[0069] Cry1Ab1 gene, Cry1Ab5 gene, Cry1Ab14 gene: nucleotide homology greater than 95%;

[0070] Cry1Ab gene, Cry1Ac gene, Cry1Ah gene: nucleotide homology between 78% and 95%;

[0071] Cry1A gene, Cry1B gene, Cry1F gene, Cry1G gene, Cry1I gene, Cry1J gene: nucleotide homology between 45% and 78%;

[0072] Cry1 gene, Cry2 gene, Cry9 gene: the nucleotide homology is less than 45%.

[0073] (2) Since the selected Cry genes are closely related genes, the maximum parsimony method (Maximum parsimony, MP method) was used to establish a phylogenetic tree: firstly, the Clustalx2.0 software was used to perform multi...

Embodiment 3

[0083] Example 3 Construction of recombinant Cry protein

[0084] The 64 novel genes constructed by plasmid pET28 and Example 2 were digested with Nco I endonuclease and BamH I endonuclease respectively; the 64 novel genes digested by double enzymes were respectively connected to plasmid pET28 to complete 64 vectors Construction; the 64 constructed vectors were verified step by step by colony PCR, double-enzyme hydrolysis and sequencing; the verified correct vectors were transferred into the expression host E.coli BL21(DE3)plysS for induced expression, and 64 recombinant Cry proteins were obtained .

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Abstract

The invention discloses a novel recombinant insect resistant protein, as well as a preparation method and an application thereof, relates to the fields of insect resistant protein and biosafety and solves the existing problem of the prior art. The method comprises the following steps: predicting a Cry protein family partial member to determine a directional transformation area from the point of protein natural evolution through a pMAL algorithm; selecting constructed protein by using an MP method to construct 64 recombinant proteins; and synthesizing an insect resistant gene NGc through insect resistant activity test and codon modification, and expressing to obtain the novel recombinant insect resistant protein NGc. The NGc is constructed to a plant expression carrier by taking ubi as a promoter and bar as a selection marker; the insect resistant protein NGc is converted to a corn selfing line through agrobacterium mediated infection, a transgenic offspring is a corn plant with high resistant immune level and with the insect resistant effect better than the trans-CrylAb, CrylGc through an indoor / field insect resistant authentication. Compared with the non-transgenic rice main cultivar Jijing 88, the transgenic rice has an obvious high striped rice borer resistance characteristic; and the recombinant insect resistant protein has high-efficiency expression in a monocotyledon and high lepidopteron resistant biological characteristic.

Description

technical field [0001] The invention relates to the technical field of insect-resistant protein and biosafety, in particular to a novel recombinant anti-insect protein and its preparation method and application. Background technique [0002] Bacillus thuringiensis (Bt for short), the Bt gene is the Bacillus thuringiensis gene, and the Bt poisonous protein encoded by it can be secreted by Bacillus thuringiensis outside the cell. The toxicity of insects such as Pteroptera and Coleoptera comes from the crystal protein produced during spore formation. In 1981, scientists purified this protein and named it Cry, and then they cloned the Cry gene. Bacillus thuringiensis insecticidal crystal protein Cry (hereinafter referred to as Cry protein) is classified according to the difference in amino acid homology: the homology is below 45%, which is the first grade, and is represented by Arabic numerals; the homology is below 45%. Between % and 78%, it is the second grade, represented by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/84C12N15/70C12N15/62A01H5/00
Inventor 郝东云刘相国韩四平尹悦佳刘洋李楠柳青林秀峰高月波郭嘉
Owner JILIN ACAD OF AGRI SCI
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