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Artificially synthesized Bt insecticidal gene FLIa as well as preparation method and application thereof

An anti-insect gene, artificial synthesis technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve important economic value, reduce environmental pollution, and broad application prospects.

Active Publication Date: 2016-02-24
JILIN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • 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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  • Artificially synthesized Bt insecticidal gene FLIa as well as preparation method and application thereof
  • Artificially synthesized Bt insecticidal gene FLIa as well as preparation method and application thereof
  • Artificially synthesized Bt insecticidal gene FLIa as well as preparation method and application thereof

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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 the insect-resistant gene Cry1Ab with Domain III of the insect-resistant gene Cry1Ia to obtain the recombinant insect-resistant 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: Transform the codons of 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 FL...

Embodiment 1

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

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

[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-res...

Embodiment 2

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

[0054] Such as figure 2 As shown, digest the plant expression vector pCAMBIA3300 with EcoRI and HindIII, reclaim the fragment ubi-nos between the restriction sites, and connect it into the EcoRI and HindIII 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 SmaI and SacI restriction sites at both ends was connected between the SmaI and SacI sites of the plant expression vector pTF101.1-ubi to construct a plant expression vector for the insect-resistant gene FLIa ( 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, TEV enhancer, bar gene, soybean...

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Abstract

The invention provides an artificially synthesized Bt insecticidal gene FLIa as well as a preparation method and application thereof, belonging to the technical field of insecticidal protein and biosafety. The nucleotide sequence of the Bt insecticidal gene FLIa is shown as SEQ ID NO: 1, and the nucleotide sequence of encoded protein of the Bt insecticidal gene FLIa is shown as SEQ ID NO: 2 in a sequence table. The preparation method of the Bt insecticidal gene FLIa comprises the steps of exchanging and fusing Domain I and Domain II of a insecticidal gene CrylAb and Domian III of a insecticidal gene CrylIa to obtain a recombinant insecticidal gene MCrylI; on the basis of the recombinant insecticidal gene MCrylI, connecting the carbon terminal of a insecticidal gene CrylJal at a 3minute terminal to obtain a recombinant insecticidal gene FLMCrylIa; performing codon reconstruction on the recombinant insecticidal gene FLMCrylIa to obtain the Bt insecticidal gene FLIa. The Bt insecticidal gene FLIa can be stably inherited and expressed in transformation plants, and is high in expression quantity, and obtained transgenic plants are good in insect resistance. The gene is used for transforming crops of corn, cotton, rice, vegetables and the like, and enabling the same to have corresponding insect-resistant activity, so that the using amount of pesticides is reduced and environment pollution and production cost are reduced.

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 Gram-positive bacterium with a very wide distribution, belonging to the Bacillus genus of the Prokaryotic organism (Procaryoticorganism) Bacteria (Bacillaceae) family (Bacillaceae). [0003] During sporulation, Bt produces a parasporal crystal protein with highly specific insecticidal activity, 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 sw...

Claims

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Application Information

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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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