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Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof

A technology of cry1f-t and insect-resistant genes, which is applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of lack of insect-resistant varieties, ecological imbalance, environmental pollution, etc., to reduce usage, reduce environmental pollution, The effect of significant economic value

Active Publication Date: 2012-09-12
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of suitable insect-resistant varieties, the current main method to solve insect pests is to spray chemical insecticides during the growth process; however, chemical insecticides kill pests and their natural enemies at the same time, causing ecological imbalance and environmental pollution

Method used

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  • Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof
  • Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof
  • Artificially synthesized Bt insect-resistant gene Cry1F-t and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Design and artificial synthesis of embodiment 1Cry1F-t gene

[0025] The Cry1F-t gene is based on the original nucleotide sequence of the Bt gene Cry1F, leaving a partially deleted N-terminal 1809bp base sequence SEQ ID No.5, and the amino acid composition of the retained sequence remains unchanged overall Under normal circumstances, the corresponding codons of the original Bt are replaced with the preferred codons of the plant genes, the AT-rich sequences and unclear intron sequences such as ATTTA and AATGAA in the Bt gene are eliminated, and the large reverse in the gene is eliminated. The repetitive sequence and the commonly used restriction endonuclease recognition site sequence are designed to design the coding sequence of the target synthetic Bt gene as shown in the sequence table SEQ ID No.1.

[0026] The newly modified Bt insect-resistant gene AdhI-Cry1F-t containing the enhancer AdhI (as shown in the sequence listing SEQ.ID NO: 4) was commissioned by Shanghai B...

Embodiment 2

[0046] Example 2 Expression of the newly modified Bt insect-resistant gene AdhI-Cry1F-t containing the enhancer AdhI in Escherichia coli cells and the toxicity detection of the expression product

[0047] In order to quickly detect the toxicity of the newly modified Bt insect-resistant protein Cry1F-t to corn borer, we constructed a prokaryotic expression vector of the AdhI-Cry1F-t gene and tested the toxicity of the insect-resistant protein Cry1F-t in vitro.

[0048] Plasmid pUCAdhI-Cry1F-t is provided by Shanghai Bioengineering Company and contains the Cry1F-t gene. According to the needs of constructing the prokaryotic expression vector of Cry1F-t gene, the NdeI endonuclease recognition site sequence CATATG was added to the 5' end of the primer sequence, and the HindIII endonuclease recognition site sequence AAGCTT was added to the 3' end. The primer sequences were designed as:

[0049] Upstream primer F1: 5-CATATGGGAAGGTGCAAGGATTGCTC-3

[0050] Downstream primer R1: 5-AA...

Embodiment 3

[0066] Embodiment 3 Plant transgenic vector construction and Agrobacterium transformation

[0067] Step 1: Digest the T vector plasmid pUCAdhI-Cry1F-t with XbaI and SacI, and recover the AdhI-Cry1F-t fragment with a gel extraction and purification kit.

[0068] Step 2: Digest the plant expression vector CPB with XbaI and SacI, and recover the digested CPB fragments with a gel extraction and purification kit.

[0069] The 3rd step: two fragments after the purification carry out ligation reaction, construct the plant expression vector pCAMBIA1300-AdhI-Cry1F-t-Bar, enzyme carries out enzyme digestion identification, shows that vector construction is correct (see image 3 ).

[0070] Agrobacterium-mediated genetic transformation (strain LBA4404) was used to transform corn callus and immature embryos, and the Cry1F-t gene maize genome was screened by the herbicide bialaphos to obtain transgenic plants. After screening, Carry out the transplanting of transgenic corn after regenerati...

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Abstract

The invention provides an insect-resistant gene Cry1F-t and application thereof. A Cry1F gene is completely modified to obtain the Cry1F-t insect-resistant gene as shown by SEQ ID NO: 1; an AdhI enhancer is added in the front of the Cry1F-t insect-resistant gene; the Cry1F-t insect-resistant gene is put on an expression vector including a strong promoter 35S; and the Cry1F-t gene is transferred into a corn genome through an agrobacterium-mediated genetic transformation method to obtain a transgenic material including the Cry1F-t. Proved by an experiment, the Bt toxalbumin modified and synthesized by the invention has an obvious insect killing effect on corn borers; and the Cry1F-t and a target protein expressed by the Cry1F-t can be stably and efficiently expressed in a corn, so that the Cry1F-t and the target protein are used for culturing a Cry1F-t transgenic insect-resistant corn. The Bt insect-resistant gene Cry1F-t can be also applied in the improvement of the insect resistance of other farm crops, fruit trees or vegetables and the like, and an effective approach is provided for solving insect pests in the production of plants such as the corn.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biological control, in particular to an artificially synthesized Bt insect-resistant gene Cry1F-t and its application. Background technique [0002] The Bt gene encodes an insecticidal crystal protein, which is derived from Bacillus thuringHansis, a Gram-positive Agrobacterium. It produces insecticidal parasporal crystal proteins called delta-endotoxins during sporulation (the gene controlling the synthesis of this protein is on the plasmid), and these proteins have high insecticidal activity. The principle of action is that this insect-resistant protein can be dissolved by alkaline intestinal fluid and hydrolyzed into smaller active toxin fragments - core fragments (Hofte and Whiteley, 1989). The active fragment is resistant to further proteolysis by proteases, and the activated protein binds to the brush vesicles in the insect gut, causing perforation and affecting osmotic balance, cells...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C07K14/325C12N15/70C12N15/113A01N47/44A01P7/04C12N15/84C12N1/21A01H5/00
Inventor 铁双贵岳润清卢彩霞齐建双王延召柏松陈娜
Owner HENAN ACAD OF AGRI SCI
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