A kind of anti-insect protein mvip3an18, coding gene and application

A technology of mvip3an18, encoding gene, applied in the field of genetic engineering and molecular biology, to achieve the effect of enhanced structural stability and high expression

Active Publication Date: 2022-03-18
九圣禾种业股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Transformation of Vip3A protein, by improving its stability in transgenic plants, and then obtaining transgenic plants with high expression of Vip3A protein to make it suitable for agricultural production is rarely rewarded

Method used

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  • A kind of anti-insect protein mvip3an18, coding gene and application
  • A kind of anti-insect protein mvip3an18, coding gene and application
  • A kind of anti-insect protein mvip3an18, coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Artificial design of insect-resistant protein mVIP3An18 and synthesis of its coding gene

[0034]1) First, train according to the collected information on the expression levels of various proteins expressed naturally or heterologously in maize cells, and establish a vector machine algorithm model, and use this model to analyze the stability of the simulated natural VIP3A protein in maize leaf cells. The relationship of N-terminal amino acid composition;

[0035] 2) According to the analysis results, firstly, we made three amino acid sequences at the N-terminus of the published natural VIP3A protein on Genbank, which may lead to the removal of the amino acids recognized by ubiquitinase in maize cells, so as to preliminarily reduce its activity in maize cells. The risk of poor stability and low content of expression products in heterologous expression

[0036] 3) According to the simulation results, new parameter settings were made, and the exhaustive method w...

Embodiment 2

[0039] Example 2: Construction of plant expression vectors for two insect-resistant proteins and preparation of genetically transformed engineering bacteria

[0040] 1) Using the commercialized plant expression vector pCAMBIA3301 as the backbone of the plant expression vector, the effects of artificial design and modification on the heterologous expression of insecticidal proteins in maize cells were verified. First, the plant expression vector is digested with restriction enzymes to remove the reporter gene GUS gene contained therein. Next, by adding a restriction endonuclease recognition site at the 5' end of the amplification primer, the same endonuclease recognition site was added to both ends of the coding gene of the natural Vip3A protein and the modified mVIP3An18 protein, respectively Restriction endonuclease digestion was performed, and the resulting cohesive ends were ligated into the commercialized plant expression vector pCAMBIA3301 with the GUS gene removed, and t...

Embodiment 3

[0042] Embodiment 3: Obtaining of corn transgenic plants of two insect-resistant proteins

[0043] The Agrobacterium carrying different vectors prepared in Example 2 is used as the engineered bacterium of genetic transformation, and the young corn embryos 12 days after pollination as the plant receptor material are co-cultured for 3 days in a dark environment, after induced culture and 35mg / After L glufosinate-ammonium resistance screening to generate resistant callus, the callus was differentiated, and the transgenic maize plants with two insect-resistant proteins before and after transformation were finally obtained by PCR detection of the obtained resistant plants.

[0044] In order to study whether the two insect-resistant genes will affect the plant genetic transformation operation, and then increase or reduce the operational difficulty of obtaining its transgenic plants for insect-resistant breeding applications, we repeated the above experiment in three different batche...

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Abstract

The invention provides an anti-insect protein mVIP3An18, an encoding gene and an application. The amino acid sequence of the insect-resistant protein mVIP3An18 is shown in SEQ ID No. 1, and the base sequence of the encoding gene is shown in SEQ ID No. 2. The applications are the application of the encoding gene in the cultivation of insect-resistant transgenic corn plants, the application of the recombinant insect-resistant protein mVIP3An18, and the application of the insect-resistant protein mVIP3An18 in the preparation of insecticides. The present invention increases the expression amount of the anti-insect protein in the transgenic material through the artificial transformation of the anti-insect protein and the optimization of the plant expression vector without affecting its original anti-insect function and other agronomic traits of the transgenic maize, so it can more easily obtain the anti-insect protein Effect.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and molecular biology, in particular to an insect-resistant protein mVIP3An18, a coding gene and its application. Background technique [0002] Although the large-scale use of pesticides is currently the main means of preventing and controlling pests and diseases, it not only increases the labor input of crop production and increases production costs, but also poses a threat to the health of users due to pesticide residues. The strategy of transgenic crops with various insect-resistant genes derived from Bacillus thuringiensis to control pests has brought significant economic benefits to global agricultural production and greatly reduced the dependence on chemical pesticides in food production. Therefore, in recent years, the planting area of ​​transgenic insect-resistant corn, rice, cotton and other crops has increased year by year, and the mining and development of new safe and reli...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/32C12N15/82C12N5/10A01H5/00A01H6/46A01N47/44A01P7/04
CPCC07K14/325C12N15/8286A01N47/44C12N2800/22
Inventor 舍亚涛高雪飞高翔舍强陈微林郭建强张宇赵海菊马玉龙孙志军
Owner 九圣禾种业股份有限公司
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