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Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof

A technology of cry1ab-t and insect-resistant genes, which is applied in the fields of application, genetic engineering, and plant gene improvement, can solve the problems of poor insect resistance, unstable expression products, and low expression levels, so as to reduce usage and enhance Expression, the effect of broad application prospects

Active Publication Date: 2012-05-30
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since the Cry1Ab gene is derived from Bacillus thuringiensis, there are problems such as low expression level, unstable expression product, and poor insect resistance effect after it is directly applied to transgenic plants.

Method used

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  • Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof
  • Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof
  • Artificially synthesized Bt insect-resistant gene Cry1Ab-t and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Cry1Ab-t Gene design and artificial synthesis

[0026] Cry1Ab-t Bt gene Cry1Ab On the basis of the original nucleotide sequence of the original nucleotide sequence, a part of the N-terminal 1845bp base sequence SEQ ID No.2 is left, and the preference of the plant gene is adopted while the amino acid composition of the retained sequence remains unchanged. Codon replacement of the corresponding codons of the original Bt, elimination of ATTTA, AATGAA and other AT-rich sequences and ambiguous intron sequences in the Bt gene, and deletion of large inverted repeat sequences and commonly used restriction enzymes in the gene Identify the site sequence, design the coding sequence of the target synthetic Bt gene as shown in the sequence table SEQ ID No.1.

[0027] Blast2 comparison analysis of the original Bt insect-resistant gene Cry1Ab and the synthetic new Bt gene Cry1Ab-t nucleotide coding sequence found that the 1845 bp nucleotide sequence at the 5' end was hom...

Embodiment 2

[0058] Example 2 Expression of Cry1Ab-t gene in Escherichia coli cells and detection of toxicity of expression products

[0059] In order to quickly detect the toxicity of the newly modified Bt insect-resistant protein Cry1Ab-t to corn borer, we constructed a prokaryotic expression vector of the Cry1Ab-t gene to detect the toxicity of the insect-resistant protein Cry1Ab-t in vitro.

[0060] Plasmid pUC1Ab-t was provided by Shanghai Bioengineering Company, containing Cry1Ab-t Gene. According to build Cry1Ab-t The gene prokaryotic expression vector needs to be added at the 5' end of the primer sequence Nd Endonuclease recognition site sequence CATATG, 3-end addition Hind III Endonuclease recognition site sequence AAGCTT. The designed primer sequence is (as shown in SEQ ID NO: 4, 5):

[0061] Upstream primer F1: 5-CATATGGACAACAACCCAAACATC-3

[0062] Downstream primer R1: 5-AAGCTTCTAGTACTCAGCCTCGAATG-3

[0063] The prokaryotic expression vector is pET-28b from Novgen Comp...

Embodiment 3

[0078] Example 3 Plant Transgenic Vector Construction and Agrobacterium Transformation

[0079] The first step: use wxya , SacI Enzyme cut T vector plasmid pUC Cry1Ab-t , recovered with a gel recovery purification kit Cry1Ab-t fragment.

[0080] Step two: use wxya , SacI Digest the plant expression vector CPB, and recover the digested CPB fragments with a gel extraction and purification kit.

[0081] Step 3: Ligate the purified two fragments to construct the plant expression vector pCAMBIA1300- Cry1Ab-t - bar (see figure 2 ), the enzyme was digested and identified, indicating that the vector was constructed correctly (see image 3 ).

[0082] Agrobacterium-mediated genetic transformation (strain LBA4404) was used to transform maize callus and immature embryos. Cry1Ab-t In the gene maize genome, transgenic plants were obtained after screening with the herbicide bialaphos, and transplanted after screening, regeneration, and seedling hardening (see Figure 4 ), ...

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Abstract

The invention provides an insect-resistant gene Cry1Ab-t and an encoding protein thereof. In the gene, by using a plant bias codon and reducing a sequence rich in AT, an inverted repeat sequence and a subsequence-containing undefined eucaryotic deoxyribose nucleic acid (DNA) sequence in the original DNA sequence on the premise of keeping part of amino acid sequences of a Bacillus thuringHansis (Bt) protein Cry1Ab unchanged, a new Cry1Ab-t gene sequence is synthesized by artificial modification, then a prokaryotic expression carrier and a plant expression carrier are constructed for the gene, and a corresponding host cell is converted. In vitro experiments prove that a synthesized Bt toxoprotein has an obvious insecticidal effect on corn borers. The insect-resistant gene Cry1Ab-t and a target protein expressed by the gene can be steadily and efficiently expressed in corns, so that the gene can be used for culturing Cry1Ab-t transgene insect-resistant corns. The artificially synthesizedBt insect-resistant gene Cry1Ab-t can be also used for improving the insect resistance of other crops, fruit trees, vegetables and the like.

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 Cry1Ab-t and its application. Background technique [0002] Corn is not only an important food crop, but also an important feed and industrial raw material. At present, corn pests (mainly corn borer) are serious, causing a large reduction in corn production. Therefore, taking effective measures to control its damage is of great importance to increase corn production and increase farmers' income. meaning. Due to the lack of suitable insect-resistant varieties, the 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. Through transgenic technology, Bt insect-resistant genes can be introduced into corn varieties, thereby ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/32C12N15/70C12N15/74C12N15/82C12N1/21C07K14/325A01H5/00A01N63/02A01P7/04C12R1/01C12R1/19
Inventor 岳润清铁双贵王延召朱卫红齐建双柏松孙静卢彩霞
Owner HENAN ACAD OF AGRI SCI
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