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Insect-resistant fusion gene, fused protein and application of fused protein

A fusion gene and fusion protein technology, applied in the fields of application, hybrid peptides, angiosperms/flowering plants, etc., can solve the problems of narrow insecticidal spectrum, insecticidal protein pest resistance, low insecticidal activity, etc., and achieve insecticidal Increased activity, slowing down of pest resistance, and broad-spectrum insecticidal effects

Active Publication Date: 2013-05-01
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 low insecticidal activity.
At the same time, the long-term use of a single insecticidal protein in large quantities may also cause the development of pest resistance

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1, construction of Cry1Ab-Cry9A and Cry1Ac-Cry9A fusion genes:

[0021] The genes encoding the insecticidal proteins of Cry9Aa, Cry1Ab, Cry1Ac, and Cry1Ca were all synthesized by Shanghai Sangong, and their DNA sequences are SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 . These genes were respectively cloned between the sites of restriction endonucleases BamHI and SacI in the vector pET28a expression vector, and the obtained vectors were named pET-9Aa, pET-1Ab, pET-1Ac and pET-1Ca respectively.

[0022] Cry1Ab-Cry9Aa construction process: The Cry1Ab fragment was obtained by digesting pET-1Ab with BamHI and XmaI, and the Cry9Aa fragment was obtained by digesting pET-9Aa with XmaI and SacI. The vector pET28a was digested with BamHI and SacI and connected with Cry1Ab and Cry9Aa to obtain the vector pET-Cry1Ab-Cry9Aa. This vector contains a fusion gene, Cry1Ab-Cry9Aa (eg, SEQ ID NO: 1), which encodes a fusion protein whose amino acid sequence is SEQ ID...

Embodiment 2

[0026] Embodiment 2, the preparation of insecticidal protein:

[0027] The vectors pET-9Aa, pET-1Ab, pET-1Ac, pET-1Ca, Cry1Ab-Cry9Aa, CryAc-Cry9Aa and Cry1Ab-Cry1Ca containing insecticidal genes were introduced into BL21Star cell line (E. Single clones were selected on LB solid medium with Namycin. 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 0.5 mM, 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

[0028] Embodiment 3, the insecticidal activity assay of the insecticidal protein expressed in Escherichia coli:

[0029] 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 the same as in 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:

[0030] Table 1. Insecticidal rate of Cry1Ab-Cry9Aa

[0031] insecticidal protein

[0032] Table 2. Insecticidal rate of Cry1Ab-Cry1Ca

[0033] insecticidal protein

[0034]The results showed that the insecticidal activity of the fusion protein Cry1Ab-Cry9Aa was significantly higher than that of Cry1Ab or Cry9Aa alone, and also significantly higher...

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PUM

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Abstract

The invention discloses an insect-resistant fusion gene, comprising a nucleotide sequence for coding BT crystal toxin cry1 and a nucleotide sequence for coding Cry9Aa toxin from 5'-3'sequentially; and the two nucleotide sequences are arranged in the same open reading frame. The insect-resistant fusion gene comprises Cry1Aa, Cry1Ab, Cry1Ac, a modifier gene of the Cry1Aa, a modifier gene of the Cry1Ab or a modifier gene of the Cry1Ac, and further comprises Cry9Aa or a modifier gene of the Cry9Aa. The invention further discloses a fused protein coded by the insect-resistant fusion gene, and the Cry1 crystal toxin and Cry9Aa crystal toxin are sequentially arranged from N end o C end of the fused protein. The fused protein in the invention is used for preparing transgenic insect-resistant crops.

Description

technical field [0001] 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 specific applications of the fusion protein. Background technique [0002] Pests cost global agricultural production an estimated $8 billion per year. The control of pests currently mainly relies on the use of chemical pesticides, but pesticide residues will cause harm to human health. Therefore, people have successfully selected transgenic crops using insecticidal toxins 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, and there are many kinds of insecticidal proteins. More commonly used are Bt crystal proteins, such as Cry1Ab, Cry1C, etc., which have been ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C07K19/00A01H5/00
Inventor 高建华沈志成
Owner ZHEJIANG UNIV