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Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application

An insect-resistant protein and fusion protein technology, applied in the field of insect-resistant genes and insect-resistant proteins, can solve the problem of not being able to obtain efficient insecticidal ability of fusion proteins, and achieve the improvement of insecticidal activity, reduction of insect resistance, and insecticidal spectrum. wide effect

Inactive Publication Date: 2015-11-04
HANGZHOU RUIFENG BIOTECH LIMITED
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

In addition, even if the combination of two individual anti-insect proteins has a synergistic effect, it cannot be concluded that their fusion protein has efficient insecticidal ability; this is because: in protein biochemistry, the physical mixing of two individual proteins Different from fusion, the synergistic effect obtained after fusion cannot be confirmed according to the synergistic effect of physical mixing; moreover, the activities of fusion proteins obtained by fusion at different positions are quite different

Method used

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  • Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application
  • Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application
  • Insect-resistant protein, insect-resistant fusion protein, coding gene, carrier and application

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Experimental program
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Effect test

Embodiment 1

[0024] Embodiment 1, the acquisition of Cry2Aj gene and the construction of expression vector

[0025] The gene encoding the Cry2Aj insecticidal protein was synthesized by Shanghai Sangong, and its DNA sequence is SEQ ID NO:2. The Cry2Aj gene was cloned into the expression vector pET28a (Novagen, Cambridge, UK) between the sites of restriction enzymes BamHI and SacI, and the resulting vector was named pET-2Aj, and the amino acid sequence encoded by the Cry2Aj gene was SEQ ID NO: 1 for the proteins indicated.

[0026] Description: The expression frame of the nucleic acid sequence of SEQ ID NO: 2 starts from the first start codon (ATG) and ends before the last stop codon (TAA), with a length of 1902bp. The nucleic acid sequence GGATCC before the first ATG is a BamHI restriction site, the subsequent ACC is a ribosome binding site, and finally GAGCTC is a SacI restriction site.

Embodiment 2

[0027] Example 2, the acquisition of Cry1Ab-Cry2Aj fusion gene and the construction of expression vector

[0028] The fusion gene encoding the insecticidal protein of Cry1Ab-Cry2Aj was synthesized by Shanghai Sangong, and there is a connecting peptide between Cry1Ab and Cry2Aj, and its amino acid sequence is PGKGGG. The DNA sequence of the Cry1Ab-Cry2Aj fusion gene is SEQ ID NO:4. The Cry1Ab-Cry2Aj fusion gene is cloned between the restriction endonuclease BamHI and SacI sites in the expression vector pET28a, and the resulting vector is named pET-1Ab-2Aj, and the Cry1Ab-Cry2Aj fusion gene encodes an amino acid sequence as SEQ ID No :3 fusion proteins.

[0029] Due to the needs of the experiment, Shanghai Sangong was commissioned to synthesize the Cry1Ab gene, whose DNA sequence is SEQ ID NO:6. The Cry1Ab gene was cloned between the restriction endonuclease BamHI and SacI sites in the expression vector pET28a, and the obtained vector was named pET-1Ab. The Cry1Ab gene encoded...

Embodiment 3

[0032] Embodiment 3, the preparation of insecticidal protein

[0033] The vectors pET-1Ab, pET-2Aj and pET-1Ab-2Aj containing insecticidal genes were respectively introduced into BL21Star (Escherichia coli) cell line, and cultured on LB solid medium containing 50mg / L kanamycin at 37°C. clone. Inoculate a single colony into 100ml of LB bacterial culture medium, shake and culture at 37°C until OD600=0.6, then add IPTG (Isopropyl-β-D-thiogalactoside) to a concentration of 0.5mM, and continue to cultivate 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 pH 7.0, 20 mM Tris-HCl buffer solution to the precipitate, and ultrasonically break to obtain insecticidal protein Cry1Ab, insecticidal protein Cry 2Aj and insecticidal protein Cry 1Ab-Cry 2Aj respectively. so get

[0034] The recombinant protein was used to measure the insecti...

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Abstract

The invention discloses an insect-resistant protein, an insect-resistant fusion protein, a coding gene, a carrier and application. According to the invention, the insect-resistant protein has an amino acid sequence with 90% of homology compared with SEQ ID NO: 1; and the insect-resistant fusion protein, from an N end to a C end, successively contains a BT crystal toxin Cry1 protein and an insect-resistant protein Cry2Aj. The invention first discloses the BT crystal toxin Cry2Aj protein with high insecticidal ability, and designs an artificial protein molecule which is prepared by fusing a Cry1Ab crystal toxin and the crystal toxin Cry2Aj protein; and compared with an original physical mixture of Cry1 and Cry2 crystal toxins, the artificial protein molecule has the following advantages: insecticidal activity is increased by more than 10 times, and insecticidal spectrum is wider; the insecticidal protein provided by the invention can kill main lepidopterous pests like cotton bollworms, beet armyworms, corn borer and Chilo suppressalis of rice, corn and cotton; moreover, the fusion protein disclosed in the invention can effectively retard occurrence of insect resistance.

Description

(1) Technical field [0001] The invention relates to an insect-resistant gene and an insect-resistant protein with high insecticidal ability, an insect-resistant fusion gene and a fusion protein with high insecticidal ability, and the construction of insect-resistant crops with the insect-resistant protein or insect-resistant fusion protein specific applications. (2) Background technology [0002] Pests cost global agricultural production an estimated $8 billion per year. There are complex types of rice pests. From sowing, seedling stage to maturity stage, the seeds, seedlings, roots, stems, leaves, flower spikes, and grains can all be harmed by different pests. There are more than 385 species of known rice pests in China, of which more than 40 species are the most important, such as the three stem borers, the two stem borers and the large borer, which are widely distributed and cause serious damage. At present, the control of pests mainly relies on chemical pesticides, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/325C12N15/32C12N5/10C07K19/00C12N15/62A01N47/44A01P7/04
CPCC07K14/325A01N47/44C07K2319/00
Inventor 沈志成张先文林朝阳王东芳
Owner HANGZHOU RUIFENG BIOTECH LIMITED
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