Insect-resistant fusion gene as well as insect-resistant fusion protein and application of insect-resistant fusion gene and insect-resistant fusion protein

A fusion gene and fusion protein technology, applied in the field of plant genetic engineering, can solve problems such as low insecticidal activity, narrow insecticidal spectrum, and insecticidal protein pest resistance, and achieve broad insecticidal spectrum, improved insecticidal activity, and slow down The effect of pest resistance

Inactive Publication Date: 2013-07-24
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, a single insecticidal toxin often has a narrow insecticidal spectrum and relatively low insecticidal activity
At the same time, long-term heavy use of a single insecticidal protein may also lead to the development of pest resistance[2,3]

Method used

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  • Insect-resistant fusion gene as well as insect-resistant fusion protein and application of insect-resistant fusion gene and insect-resistant fusion protein
  • Insect-resistant fusion gene as well as insect-resistant fusion protein and application of insect-resistant fusion gene and insect-resistant fusion protein
  • Insect-resistant fusion gene as well as insect-resistant fusion protein and application of insect-resistant fusion gene and insect-resistant fusion protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, construction of cry1Ac-cry1Ie fusion gene:

[0026] The genes encoding Cry1Ac and Cry1Ie insecticidal proteins were commissioned by Shanghai Sangong to synthesize, and their DNA sequences are SEQ ID NO: 4 and SEQ ID NO: 6, respectively. The cry1Ac gene was cloned between the sites of restriction enzymes BamHI and SacI in the vector pET28a expression vector, and the resulting vector was named pET-1Ac, which encoded a protein whose amino acid sequence was SEQ ID NO: 3; the cry1Ie gene was Cloned into the vector pET28a expression vector between the sites of restriction enzymes BamHI and SacI, the obtained vector was named pET-1Ie, which encoded a protein whose amino acid sequence was SEQ ID NO:5.

[0027] Construction process of pET-1Ac-1Ie: the cry1Ac fragment was obtained by digesting pET-1Ac with BamHI and XmaI, and the cry1Ie fragment was obtained by digesting pET-1Ie with XmaI and SacI. The vector pET28a was digested with BamHI and SacI and ligated with...

Embodiment 2

[0028] Embodiment 2, the preparation of insecticidal protein:

[0029] Vectors pET-1Ac, pET-1Ie and pET-1Ac-1Ie containing insecticidal genes were respectively introduced into BL21 Star (E.coli) cell line, and single clones were selected on LB solid medium containing 50 mg / L kanamycin. Inoculate a single colony into 100 ml of LB bacterial culture medium, shake culture at 37°C to OD 600 =0.6, then add IPTG (Isopropyl-β-D-thiogalactoside) to a concentration of 0.5mM, and continue culturing under the same conditions for 4 hours. The culture solution was centrifuged at 5000 g for 10 minutes to pellet E. coli cells, and then the supernatant was discarded to collect the pellet. Add 30 ml of 20 mM Tris-HCl buffer solution to the precipitate, and ultrasonically crush it. The recombinant protein thus obtained was used for the determination of insecticidal activity.

Embodiment 3

[0030] Embodiment 3, the insecticidal activity assay of the insecticidal protein expressed in Escherichia coli:

[0031] 100 microliters of the insecticidal proteins obtained in Example 2 were applied alone or mixed on the surface of 0.5 square centimeters of insect artificial feed, and fed to newborn first-instar larvae for insecticidal activity determination. The preparation method of the negative control was similar to Example 2, but the plasmid was the pET28a vector itself without any inserted DNA. After raising for 7 days, the insecticidal rate was counted, and the results are shown in Tables 1 and 2:

[0032] Table 1. Insecticidal rate of Cry1Ac-Cry1I

[0033]

[0034] The results showed that the insecticidal activity of the fusion protein Cry1Ac-Cry1I was significantly higher than that of Cry1Ac or Cry1I alone, and also significantly higher than that of the mixture of Cry1Ac and Cry1I.

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Abstract

The invention belongs to the field of plant gene engineering, relates to a method for obtaining an insect-resistant fusion gene with high insect killing capacity, an insect-resistant fusion gene and an insect-resistant fusion protein with high insect killing capacity and an application of constructing insect-resistant crops by using the fusion protein. Specifically, the invention discloses an insect-resistant fusion gene. The insect-resistant fusion gene comprises a nucleotide sequence for coding bacillus thuringiensis insecticidal crystal Cry1Ac and a nucleotide sequence for coding Cry1Ie toxin from 5' to 3'; the Cry1Ac and the Cry1Ie nucleotide are connected by an XmaI digestion site; and the two nucleotide sequences are in the same open reading frame. The invention further discloses an insect-resistant fusion protein coded by the insect-resistant fusion gene. The invention further discloses applications of the insect-resistant fusion gene and the insect-resistant fusion protein in transgenic insect-resistant crops.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering. The invention relates to a method for obtaining an insect-resistant fusion gene with high insecticidal ability, an insect-resistant fusion gene and a fusion protein with high insecticidal ability, and the specific application of using the fusion protein to construct insect-resistant crops. Background technique [0002] Pests cost global agricultural production an estimated $8 billion per year. At present, the control of pests mainly relies on the use of chemical pesticides, but pesticide residues will cause harm to human health. Therefore, people have successfully selected insecticidal toxin transgenic crops for insect resistance. At present, transgenic insect-resistant corn and cotton have been planted in large quantities. [0003] The key technology for transgenic crops to resist insects is to obtain insecticidal proteins with excellent performance. There are many kinds of insectici...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00A01P7/04C12N15/82A01H5/00
Inventor 方军沈志成张薇秦毅
Owner ZHEJIANG UNIV
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