Insect-resistant fusion gene M2CryAb-VIP3A as well as expression vector, product and application thereof

A fusion gene and expression carrier technology, applied in antibody mimics/scaffolds, use of vectors to introduce foreign genetic material, application, etc., can solve the problem that Bt fusion genes are difficult to balance biological safety and efficient insecticide, and achieve excellent insecticide Effect, important economic value, effect of broad application prospects

Active Publication Date: 2022-03-01
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The object of the present invention is to provide a kind of anti-insect fusion gene M2CryAb-VIP3A , its expression vector, product and its ap...

Method used

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  • Insect-resistant fusion gene M2CryAb-VIP3A as well as expression vector, product and application thereof
  • Insect-resistant fusion gene M2CryAb-VIP3A as well as expression vector, product and application thereof
  • Insect-resistant fusion gene M2CryAb-VIP3A as well as expression vector, product and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Anti-insect fusion gene M2CryAb-VIP3A the acquisition

[0035] choose to Cry1Ab Based on the original DNA sequence (GenBank: AY847289.1) with a length of 3468 bp of the gene, the functional domain and core region (protein activity region) of the original sequence of the Cry1Ab gene were analyzed in depth. Cry1Ab The 1845bp functional domain and core region at the N-terminal of the original sequence, the 1620bp base sequence at the C-terminal of the original sequence is removed; and based on long-term practical experience, the retained Cry1Ab Nucleotide sequences in the core region are subjected to structural optimization, codon optimization, and other directional transformations, for example, to exclude multiple AT-enriched regions such as AATGAA and ATTTA in the original insect-resistant gene DNA sequence and inverted repeat sequences that exist in the gene sequence , to remove or reduce the sequence of introns of unclear eukaryotic DNA sequences and se...

Embodiment 2

[0038] Example 2: Anti-insect fusion gene M2CryAb-VIP3A Construction of prokaryotic expression system

[0039] The modified anti-insect fusion gene was cut and ligated with the high-efficiency prokaryotic expression vector pET28b+ to construct a prokaryotic expression vector with the fusion target gene, and the protein expression and biological function of the target gene were determined. According to the needs of cloning the insect-resistant fusion gene, the NdeI endonuclease recognition site sequence CATATG was added to the 5' end of the primer sequence, and the HindIII endonuclease recognition site sequence AAGCTT was added to the 3' end.

[0040] Cloning the insect-resistant gene based on the synthetic sequence, constructing the insect-resistant gene in the pET28b+ prokaryotic expression system, and then transforming BL21(DE3) competent cells with the constructed pET to determine the gene protein expression level, and the fusion gene was tested indoors The biological func...

Embodiment 3

[0041] Example 3: Preparation of insecticidal protein M2CryAb-VIP3A

[0042] The constructed recombinant plasmid pET- and its corresponding control pET-mCryAb, pET-mVIP3A and pET28b+ empty vector were respectively introduced into BL21(DE3) cell line (Escherichia coli), and single bacteria were picked and inoculated in LB liquid containing kanamycin Shake culture overnight at 37°C in culture medium; inoculate the bacterial solution into LB liquid medium containing kanamycin at a ratio of 1:100 and shake culture until the OD600 value is about 0.5-0.6, and add IPTG to a final concentration of 0.5 mM continued shaking induction for 4 hours; collected the induced bacterial liquid, centrifuged at 4000rpm for 10 minutes, discarded the supernatant, and collected the bacterial cells. Add 20mM Tris-HCl lysis buffer to the precipitate to resuspend, add lysozyme to a final concentration of 1mg / ml, place on ice for 30 minutes; ultrasonically disrupt the bacteria, centrifuge at 4000rpm for ...

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Abstract

The invention relates to an insect-resistant fusion gene M2CryAb-VIP3A as well as an expression vector, a product and application of the insect-resistant fusion gene M2CryAb-VIP3A, and aims to solve the technical problem that the existing Bt fusion gene is difficult to give consideration to both biological safety and efficient insect killing. The nucleotide sequence of the insect-resistant fusion gene M2CryAb-VIP3A is as shown in SEQ ID NO. 4, and the corresponding protein sequence of the insect-resistant fusion gene M2CryAb-VIP3A is as shown in SEQ ID NO. 5. The fusion gene does not contain a sensitizer sequence and can be efficiently expressed, the biological safety and the insecticidal effect are both considered, and an expression product of the fusion gene has excellent resistance to pests such as ostrinia nubilalis and spodoptera frugiperda and is wider in insecticidal spectrum. After the M2CryAb-VIP3A gene is introduced into corn, a new crop variety which is resistant to stem borers and also resistant to other lepidoptera pests or coleoptera pests (such as spodoptera exigua and the like) is obtained, and the service life of the resistant variety is effectively prolonged through fusion or polymerization of the gene; the insect-resistant fusion gene expression protein M2CryAb-VIP3A disclosed by the invention has an excellent insecticidal effect, can be applied to preparation of insect-resistant preparations, and is green, environment-friendly and pollution-free.

Description

technical field [0001] The invention relates to the technical field of biological genetic engineering, in particular to an insect-resistant fusion gene M2CryAb-VIP3A , its expression vector, product and application thereof. Background technique [0002] Pest is a general term for insects that are harmful to humans. There are as many as one million kinds of pests that have been discovered and confirmed that can harm crops, and these large numbers of pests will bring incalculable economic losses to agricultural production every year. However, the current control of crop pests still relies on chemically synthesized pesticides; however, long-term and large-scale use of chemical pesticides will bring serious harm to the ecological environment. With the rapid development of bio-genetic engineering technology, researchers have gradually begun to use insect-resistant genes to transform plants, and then cultivate new varieties of transgenic crops with resistance. [0003] At prese...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/70C12N15/84C12N1/21A01H5/00A01H6/46A01N63/50A01N37/46A01P7/04C12R1/19
CPCC07K14/325C12N15/70C12N15/8286A01N63/50A01N37/46C07K2319/00Y02A40/146
Inventor 岳润清孟昭东孙琦鲁守平李文兰李文才于艳丽赵勐曾廷儒张庆伟
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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