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Fusion protein capable of efficiently resisting spodoptera frugiperda and application thereof

A technology of Spodoptera frugiperda and fusion protein, which is applied in the field of anti-insect fusion protein and its application, can solve the problems of low sensitivity of Cry1A protein and difficult control effect, so as to achieve beneficial biological activity, improve insecticidal activity, and slow down risk effect

Active Publication Date: 2021-02-23
HANGZHOU RUIFENG BIOTECH LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The insect has high fecundity, strong migratory ability, and a wide range of hosts. It is especially harmful to corn. Its boring and feeding characteristics make it difficult for traditional chemical pesticides to obtain the expected control effect.
Spodoptera frugiperda is sensitive to Cry1F and Vip3A proteins, but not to Cry1A protein (Frankenhuyzen2009)

Method used

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  • Fusion protein capable of efficiently resisting spodoptera frugiperda and application thereof
  • Fusion protein capable of efficiently resisting spodoptera frugiperda and application thereof
  • Fusion protein capable of efficiently resisting spodoptera frugiperda and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, vector construction of insect-resistant fusion gene, preparation of recombinant protein

[0030] The coding genes involved in the present invention are all artificially synthesized by Shanghai Sangong Company, wherein the anti-insect fusion protein Cry1Da-Vip3 is composed of BT insecticidal crystal protein Cry1Da and BT vegetative phase insecticidal protein Vip3 through fusion connection, and the amino acid sequence is SEQ ID NO:1, the nucleotide sequence of the coding gene cry1da-vip3 is SEQ ID NO:5. The anti-insect fusion protein Cry1Fa-Vip3 is composed of BT insecticidal crystal protein Cry1Fa and BT vegetative phase insecticidal protein Vip3 through fusion connection, the amino acid sequence is SEQ ID NO: 2, and the nucleotide sequence of the coding gene cry1fa-vip3 is SEQ ID NO :6. The amino acid sequence of BT insecticidal crystal protein Cry1Da is SEQ ID NO: 9, the nucleotide sequence of the coding gene cry1da is SEQ ID NO: 7, the amino acid sequen...

Embodiment 2

[0035] Embodiment 2, the biological activity assay of recombinant protein to Lepidoptera insects

[0036] Experimental objects: Select the newly hatched larvae (within 2 hours after birth) of common Lepidoptera pests (Spodoptera frugiperda, Corn borer) as the tested insects.

[0037] Experimental group: the recombinant protein prepared by the method of embodiment 1 carries out the pre-experiment of biological activity (setting 1ng / cm 2 , 10ng / cm 2 , 100ng / cm 2 , 1000ng / cm 2 Four groups of concentrations); According to the results of the preliminary experiment, 8 groups of concentration gradients (concentration ranges as shown in Table 1 below, diluted with PBS solution) are set to measure the concentration interval that has 50% lethality to insects. The number of insects tested at each concentration was 80.

[0038] Table 1 Recombinant protein concentration gradient table (ng / cm2) for measuring the biological activity of Spodoptera frugiperda 2 )

[0039]

[0040] ...

Embodiment 3

[0055] Example 3, Determination of Insecticidal Activity of Recombinant Fusion Proteins to BT-resistant Spodoptera frugiperda

[0056] Experimental objects: Select the resistant Spodoptera frugiperda which is insensitive to Cry1Fa protein and the resistant Spodoptera frugiperda which is insensitive to Vip3A protein as the research objects, and measure the insecticidal biological activity of the recombinant protein. within) as the tested insects.

[0057] Experimental group: the specific implementation method, preparation of experimental materials and negative control group are the same as those described in Example 2; the only difference is that the tested insects are different, here is BT-resistant Spodoptera frugiperda.

[0058] Determination method: Determination of lethal concentration LC of recombinant protein to insects by surface tiling method 50 . The specific implementation is consistent with the method described in Embodiment 2.

[0059] The results of the determi...

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Abstract

The invention discloses a fusion protein capable of efficiently resisting spodoptera frugiperda. The fusion protein is formed by fusion connection of BT insecticidal crystal protein and BT vegetativeinsecticidal protein Vip3, and the BT insecticidal crystal protein is BT insecticidal crystal protein Cry1Da or BT insecticidal crystal protein Cry1Fa. The fusion protein has higher insecticidal activity on noctuidae pests, significantly improves the insecticidal activity on lepidoptera noctuidae pests such as the spodoptera frugiperda, is beneficial to the insect-resistant protein to exert the biological activity, and alleviates the risk of pest resistance. The fusion protein can be used for simultaneously and efficiently killing main lepidoptera pests on rice, corn and soybeans such as spodoptera frugiperda, armyworms, corn borers, cotton bollworms, beet armyworms, cutworms, chilo suppressalis, pink rice borers and cnaphalocrocis medinalis.

Description

[0001] (1) Technical field [0002] The invention relates to an insect-resistant fusion protein and its application, in particular to an insect-resistant fusion protein highly resistant to Lepidoptera pests such as Spodoptera frugiperda and its application. [0003] (2) Background technology [0004] Pests cost billions of dollars annually in global agricultural production. The control of pests currently mainly relies on the use of chemical pesticides and the promotion of genetically modified crops. Residues of chemical pesticides can have adverse effects on human health and the environment. The use of biotechnology to cultivate transgenic insect-resistant crops containing insect-resistant genes can greatly reduce the use of chemical pesticides and effectively protect crops from pests. There are many kinds of insecticidal proteins, and the insecticidal crystal proteins from Bacillus thuringiensis (BT) used most at present, such as Cry1A, Cry1F, etc.; and BT vegetative period ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/82A01H5/00A01H6/46A01H6/54
CPCC07K14/325C12N15/8286C07K2319/00
Inventor 许超沈志成
Owner HANGZHOU RUIFENG BIOTECH LIMITED
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