Fusion insecticidal protein Cry1Am, and coding gene and application thereof

An insecticidal protein and gene technology, applied in the field of genetic engineering, can solve the problems of accelerating pest resistance, restricting the application period of Cry1A insect-resistant transgenic crops, etc., and achieve the effect of improving insecticidal ability and high insecticidal activity

Active Publication Date: 2015-08-26
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since 1996, a variety of Bt genetically modified insecticidal crops have been approved for commercial production, but most of these insecticidal genetically modified crops use Cry1A insecticidal protein genes. The emergence of resistance restricts the application period of Cry1A-type insect-resistant transgenic crops

Method used

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  • Fusion insecticidal protein Cry1Am, and coding gene and application thereof
  • Fusion insecticidal protein Cry1Am, and coding gene and application thereof
  • Fusion insecticidal protein Cry1Am, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of fusion insecticidal protein coding gene Cry1Am inducible expression vector

[0024] The gene encoding the fusion insecticidal protein Cry1Am, including the nucleotide sequence encoding the upstream 658 amino acids of Cry1Ab, the nucleotide sequence encoding the fusion peptide of 30 amino acids, the nucleotide sequence encoding the domain III of the Cry1Ia protein, and the encoding Cry1Ie protein Nucleotide sequences of domains I and II. The amino acid sequence of the fusion insecticidal protein Cry1Am is shown in Seq ID No.1. According to the maize codon preference, the gene encoding the fusion insecticidal protein Cry1Am was modified and synthesized. The nucleotide sequence of the gene Cry1Am is shown in Seq ID No.2. The gene sequence was synthesized by Shanghai Sangong and constructed on the pUC57 vector , and the ampicillin-resistant plasmid pUC57-Cry1Am was constructed.

[0025] Add 20 ng of plasmid pUC57-Cry1Am and plasmid pET30a (purcha...

Embodiment 2

[0028] Example 2 Induction and purification of fusion insecticidal protein Cry1Am

[0029] The correct plasmid pET30a-Cry1Am verified by sequencing and digestion was transferred into the strain Transetta (DE3) purchased from Quanshijin Company, a single clone was picked, and positive plaques were verified by PCR amplification. Inoculate the positive bacterial plaques into 10 mL of LB liquid medium (containing appropriate antibiotics), and culture with shaking overnight at 37°C. Escherichia coli strain Transetta (DE3) containing the pET30a-Cry1Am plasmid was inoculated into 10 mL LB liquid medium (containing appropriate antibiotics) at a ratio of 1:100, and cultured overnight at 37°C with shaking.

[0030] The next day, inoculate into 200mL LB liquid medium (containing appropriate antibiotics) at a ratio of 1:200, culture at 37°C with shaking at 200rpm until OD 600 If it is 0.4-0.6, add IPTG to a final concentration of 0.5mM, shake at 16°C, 160rpm, and shake for about 20hrs to...

Embodiment 3

[0039] Example 3 Indoor Corn Borer Insecticide Test Fused with Insecticidal Protein Cry1Am

[0040] The corn borers tested were sensitive corn borers and corn borers resistant to Cry1Ac protein. Under the conditions of indoor temperature of 28±1°C, photoperiod (L:D) of 16:8h, and relative humidity of 70-80%, the artificial feed mixing method was used to carry out the indoor corn borer test, and the purified fusion protein Cry1Am and other proteins The amount of 25 μg / g was added to the artificial feed to make feeding feed, and the feed was evenly divided into three 48-well cell culture plates, and each hole was inoculated with one newly hatched larvae of Corn borer, and a total of 144 larvae were inoculated at each concentration. After 7 days, the worm mortality was counted. The results showed that the insecticidal effect of fusion insecticidal protein Cry1Am on sensitive corn borer was significantly higher than that of Cry1Ab, Cry1Ie or Cry1Ia1, and the insecticidal effect o...

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Abstract

The invention discloses a novel fusion insecticidal protein Cry1Am, and a coding gene and an application thereof. The fusion insecticidal protein has high insecticidal activity against wild type and Cry1Ab/Cry1Ac-resistant insects, and can be used for killing lepidopteran pests such as Asiatic corn borer, and for improving the insecticidal capacity of transgenic crops. The fusion insecticidal protein can effectively kill corn borers with Cry1Ac protein resistance, and can be used in insect resistance control.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a fusion insecticidal protein Cry1Am, its coding gene and application. Background technique [0002] Insect pests are a major factor causing severe crop yield reduction. The most common means of controlling agricultural pests is to use broad-spectrum chemical insecticides and some biological insecticides. Most of the chemical insecticides have the characteristics of broad spectrum and high toxicity. When killing the target pests, they often kill many beneficial insects together, seriously destroying the ecological balance and causing serious pollution to the environment; on the other hand, pesticide residues are harmful to humans, Livestock health also poses a serious threat. Biopesticides are easy to degrade and highly compatible with the environment, but they need to be applied repeatedly in production, which greatly increases production costs. In order to make up for the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/82A01H5/00
CPCC07K14/325C07K2319/00C12N15/8286
Inventor 刘允军王国英张煜文刘艳
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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