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Recombinant anti-insect protein and its preparation method and application
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A kind of anti-insect protein and protein technology, applied in the field of recombinant anti-insect protein and its preparation
Active Publication Date: 2017-08-29
JILIN ACAD OF AGRI SCI
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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
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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.
[0066] Example 2 Constructing an 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 quaternarynucleotide homology:
[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 multiple c...
Embodiment 3
[0083] Example 3 Construction of recombinant Cry protein
[0084] The plasmid pET28 and the 64 genes constructed in Example 2 were digested with NcoI endonuclease and BamHIendonuclease respectively; the 64 genes digested with double enzymes were respectively connected to the plasmid pET28 to complete the construction of 64 vectors; The 64 constructed vectors were verified step by step by PCR, double-enzymehydrolysis and sequencing; the verified correct vectors were transferred into the expression host E.coliBL21(DE3)plysS for induced expression, and 64 recombinant Cry proteins were obtained.
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Abstract
The recombinant anti-insectprotein and its preparation method and application relate to the fields of anti-insectprotein and biosafety, and solve the problems existing in the prior art. Using the pMAL algorithm to predict some members of the Cry protein family from the perspective of protein natural evolution to determine the directional transformation region; use the MP method to build a tree to select the proteins to be transformed to construct 64 recombinant proteins; identify insect-resistant activity tests, and synthesize insect-resistant codons The gene NGc is expressed to obtain the recombinant insect-resistant protein NGc. Construct NGc on a plantexpression vector with ubi as a promoter and bar as a selection marker; Agrobacterium-mediated infection transfers the insect-resistant gene NGc to maize inbred lines, and the transgenic offspring are identified as high-immunity by indoor / field insect resistance level, the insect resistance effect was significantly higher than that of Cry1Ab and Cry1Gc-transformed corn plants; compared with the non-transgenic rice main variety Jijing 88, the transgenic rice has obvious high resistance to Chipotle borer; the recombinant insect-resistant protein has the function of monocot plants Biological characteristics of medium high expression and high resistance to Lepidoptera insects.
Description
technical field [0001] The invention relates to the technical field of insect-resistant protein and biosafety, in particular to a recombinant anti-insect protein and its preparation method and application. Background technique [0002] Bacillus thuringiensis (Bacillus thuringiensis, referred to as Bt), the Bt gene is the Bacillus thuringiensis gene, and the Bt poisonous protein encoded by it can be secreted by Bacillus thuringiensis outside the bacterial body. The toxicity of insects such as Coleoptera 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...
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