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A kind of artificially synthesized bt insect-resistant gene flia and its preparation method and application

An anti-insect gene, artificial synthesis technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve broad application prospects, the method is simple and effective, and the effect of reducing the amount of use

Active Publication Date: 2018-02-13
JILIN ACAD OF AGRI SCI
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  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • A kind of artificially synthesized bt insect-resistant gene flia and its preparation method and application
  • A kind of artificially synthesized bt insect-resistant gene flia and its preparation method and application
  • A kind of artificially synthesized bt insect-resistant gene flia and its preparation method and application

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preparation example Construction

[0040] The invention provides a method for preparing the above-mentioned artificially synthesized Bt insect-resistant gene FLIa, comprising the following steps:

[0041] Step 1: Exchange and fuse Domain I and Domain II of insect resistance gene Cry1Ab with Domain III of insect resistance gene Cry1Ia to obtain recombinant insect resistance gene MCry1I.

[0042] Step 2. On the basis of the recombined insect-resistant gene MCry1I, connect the carbon terminus of the insect-resistant gene Cry1Ja1 at the 3' end to obtain the recombined insect-resistant gene FLMCry1Ia; the recombined insect-resistant gene FLMCry1Ia has the greatest homology with the amino acid sequence of the Cry gene was 88%.

[0043] Step 3: Perform codon modification on the recombinant insect-resistant gene FLMCry1Ia to obtain the Bt insect-resistant gene FLIa, the nucleotide sequence of which is shown in SEQ ID NO: 1 in the sequence listing. The codon transformation of the recombinant insect-resistant gene FLMCr...

Embodiment 1

[0049] Example 1 The acquisition of novel insect-resistant gene FLIa

[0050] Domain III is the recognition region between Cry protein and receptor protein, and is the main functional region to determine the target. The insecticidal spectrum of insecticidal proteins can be increased by exchanging Domain III domains between Cry proteins with different insecticidal spectrum.

[0051] Such as figure 1 As shown, in the present invention, Domain I and Domain II of insect-resistant gene Cry1Ab are exchanged and fused with Domain III of insect-resistant gene Cry1Ia to obtain recombinant insect-resistant gene MCry1I. In order to increase the stability of gene expression, on the basis of the recombinant insect-resistant gene MCry1I, the carbon terminus of the insect-resistant gene Cry1Ja1 was connected at the 3' end to obtain the recombinant insect-resistant gene FLMCry1Ia. The maximum homology of amino acid sequence of Cry gene is 88%.

[0052] In order to further improve the insect...

Embodiment 2

[0053] Embodiment 2 Construction of novel insect-resistant gene FLIa plant expression vector

[0054] Such as figure 2 As shown, the plant expression vector pCAMBIA3300 was digested with EcoR I and Hind III, and the fragment ubi-nos between the enzyme cutting sites was reclaimed, which was connected between the EcoR I and Hind III sites of the plant expression vector pTF101.1 (such as image 3 shown), construct the plant expression vector pTF101.1-ubi (such as Figure 4 shown). The artificially synthesized Bt insect-resistant gene FLIa with Sma I and Sac I restriction sites at both ends was connected into the plant expression vector pTF101.1-ubi between the Sma I and Sac I sites to construct the insect-resistant gene FLIa. Plant expression vectors (such as Figure 5 shown), the plant expression vector was named pTF101.1-ubi-FLIa, and its nucleotide sequence is shown in SEQ ID NO:3.

[0055] The constructed plant expression vector pTF101.1-ubi-FLIa includes: P35S promoter,...

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Abstract

An artificially synthesized Bt insect-resistant gene FLIa and its preparation method and application belong to the technical field of insect-resistant protein and biosafety. The nucleotide sequence of the Bt insect-resistant gene FLIa is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2 in the sequence listing. The preparation method of the Bt insect-resistant gene FLIa is as follows: Domain I and Domain II of the insect-resistant gene Cry1Ab are exchanged and fused with Domain III of the insect-resistant gene Cry1Ia to obtain a recombinant insect-resistant gene MCry1I; on the basis of the recombinant insect-resistant gene MCry1I, 3 The ' end was connected to the carbon terminus of the insect-resistant gene Cry1Ja1 to obtain the recombinant insect-resistant gene FLMCry1Ia; the codon modification of the recombinant insect-resistant gene FLMCry1Ia was carried out to obtain the Bt insect-resistant gene FLIa. The Bt insect-resistant gene FLIa can be stably inherited and expressed in transformed plants, and the expression level is high, and the obtained transgenic plants have good insect resistance. The gene transforms crops such as corn, cotton, rice, and vegetables to have corresponding insect-resistant activities, thereby reducing the use of pesticides and reducing environmental pollution and production costs.

Description

technical field [0001] The invention belongs to the technical field of insect-resistant protein and biosafety, and in particular relates to an artificially synthesized Bt insect-resistant gene FLIa and its preparation method and application. Background technique [0002] Bacillus thuringiensis (Bt) is a widely distributed Gram-positive bacterium belonging to the genus Bacillus of the class Bacteria and the family Bacillus. [0003] Bt produces a parasporal crystal protein with highly specific insecticidal activity during sporulation, called δ-endotoxin or insecticidal crystal protein (ICP). In the midgut of insect larvae, the protoxin is transformed into a polypeptide molecule by protease hydrolysis, and the activated toxin can interact with specific receptors on the surface of sensitive insect intestinal epithelial cells, induce cells to produce some pores, and disrupt cell permeability Balance, and cause cell swelling and even lysis, along with the above process, the larv...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/32C12N15/82C12N15/10C07K14/325A01H5/00
CPCC07K14/325C12N15/8286
Inventor 郝东云刘相国李楠韩四平尹悦佳柳青刘洋康领生王阳闫玮玉
Owner JILIN ACAD OF AGRI SCI
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