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Insect-resistant fusion gene, protein encoded by insect-resistant fusion gene, as well as expression vector and application of insect-resistant fusion gene

A technology of fusing genes and insect-resistant genes, applied in the field of genetic engineering, can solve the problems of low insect resistance, low expression of toxic proteins, and unstable expression products of transgenic plants, achieve broad application space and market prospects, and overcome gene silencing. Phenomenon, effect of excellent insecticidal activity

Pending Publication Date: 2020-05-01
广州哈维种业有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the potential problems in the application of the insect-resistant plants with the above-mentioned insect-resistant genes are increasingly revealed, which affects their sustainable use.
For example, the transgenic plants obtained by directly applying the original insect-resistant genes to transgenic plants have very low insect resistance, poor insect resistance effects, low and low expression levels of toxic proteins, and unstable expression products, which cannot meet the needs of agricultural production. Most of the insect pest control needs are difficult to apply; with the wide application of insect-resistant genes, insects will also develop resistance to insecticidal proteins; the insect-resistant spectrum of insect-resistant genes is narrow; the expression of foreign genes in plants has gene " silence, etc.

Method used

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  • Insect-resistant fusion gene, protein encoded by insect-resistant fusion gene, as well as expression vector and application of insect-resistant fusion gene
  • Insect-resistant fusion gene, protein encoded by insect-resistant fusion gene, as well as expression vector and application of insect-resistant fusion gene
  • Insect-resistant fusion gene, protein encoded by insect-resistant fusion gene, as well as expression vector and application of insect-resistant fusion gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Anti-insect gene Cry1Ab and VIP3A(a) transformation

[0047] The inventor based on a large number of long-term experimental research and practical experience, in the original Cry1Ab (GenBank serial number is AY847289.1) A substantial improvement was made on the basis of the insect-resistant gene, mainly including removing part of the redundant base sequence, leaving only the nucleotide sequence containing the functional domain, codon modification, confirmation And exclude multiple AT-enriched regions such as ATTTA and AATGAA in the original insect-resistant gene DNA sequence and inverted repeat sequences in the gene sequence, and remove some commonly used restriction endonuclease recognition site sequences ( Xba I. Sac I), the sequence of the intron of the undefined eukaryotic DNA sequence and the sequence that may cause the premature termination of the gene transcription or the instability of the mRNA are reduced, and the stop codon at the 3' end is ...

Embodiment 2

[0054] Example 2: Anti-insect fusion gene CryAb-VIP3A build

[0055] Optimized insect-resistant fusion gene CryAb-VIP3A ( CryAb-VIP3A-MR ), transformed from Example 1 CryAb Gene sequence, connecting sequence L and embodiment one transformation VIP3A(a) Gene sequence composition. Cry1Ab After the stop codon at the 3 end of the gene is removed, the reading frame of the gene can be continuously expressed until the optimally modified vip3A(a) Gene, formed after optimized transformation Cry1Ab gene and vip3A fusion gene. The nucleotide sequence of the connecting sequence L is shown in SEQ ID NO.4; the nucleotide sequence of the anti-insect fusion gene is shown in SEQ ID NO.5, and the encoded amino acid sequence is shown in SEQ ID NO.6.

[0056] To detect modified CryAb-VIP3A The prokaryotic expression vector of the fusion gene was constructed according to the in vitro expression of the gene and the toxicity to corn borer and other pests. According to the needs ...

Embodiment 3

[0062] Example 3: Anti-insect fusion gene CryAb-VIP3A Expressed in E. coli

[0063] 1. Construction of recombinant E. coli

[0064] Transform the recombinant plasmid pET-CryAb-VIP3A into Escherichia coli E. coli BL21(DE3), positive transformants were screened out. After extracting the plasmid and verifying digestion, select positive transformants and inoculate them into resistant medium, culture overnight at 37°C, transfer with 2% inoculum, and culture to OD 600 The value is about 0.5-0.6, and it is stored at 4°C for later use.

[0065] 2. Detection of recombinant bacterial expression products Indoor insecticidal effect test

[0066] (1) The experimental treatments were divided into 4 groups:

[0067] Escherichia coli containing recombinant pET-CryAb-VIP3A was induced by IPTG, then ultrasonically disrupted, and the collected supernatant was used as the test group; cultured under the same conditions containing the mCryAb Gene sequence of recombinant pET28b vector E....

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Abstract

The invention discloses an insect-resistant fusion gene, a protein encoded by the insect-resistant fusion gene, as well as an expression vector and application of the insect-resistant fusion gene; andthe purpose of the invention is to solve the technical problem of irresistible trend of drug resistance of insects to pesticides. According to the invention, an insect-resistant fusion gene and an insect-resistant fusion gene CryAb-VIP3A are designed. The invention also provides a protein encoded by the insect-resistant fusion gene CryAb-VIP3A, and designs an expression vector constructed by using the insect-resistant fusion gene CryAb-VIP3A as well as a recombinant bacterium constructed by using the expression vector. The invention further discloses application of the insect-resistant fusiongene CryAb-VIP3A in preparation of insect-resistant plant cells. The invention still further discloses application of the protein encoded by the insect-resistant fusion gene CryAb-VIP3A in preparation of pesticides. The insect-resistant fusion gene CryAb-VIP3A disclosed by the invention can be stably expressed in corn so as to avoid biological toxicity caused by transferring of an original VIP3A(a) gene into the plant alone and overcome the phenomenon of gene silencing in foreign gene expression, so that more choices are provided for plant pest control; and thus, a new method is provided forkeeping down the trend of the drug resistance of the insects to the pesticides.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to an insect-resistant fusion gene, its encoded protein, its expression vector and its application. Background technique [0002] There are as many as one million species of pests that feed on crops, causing huge economic losses and seriously affecting crop yield and quality. At present, the control of crop pests mainly depends on chemical pesticides, and the long-term use of chemical pesticides in large quantities will cause great harm to the environment and health. In order to reduce environmental pollution and potential harm to human health, the use of biotechnology to develop new agents has become a research hotspot. [0003] With the development of molecular biology technology and the emergence of technologies such as gene cloning and DNA manipulation, humans began to clone insect-resistant genes into engineered bacteria, and further obtained resistant transgenic p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C12N15/62C07K19/00C12N15/82C12N15/66A01H5/00A01H6/46A01N37/46A01P7/04
CPCC07K14/325C12N15/8205C12N15/66C12N15/8286C07K2319/00
Inventor 铁健雄
Owner 广州哈维种业有限公司
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