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Insecticidal protein as well as coded gene and application of insecticidal protein

A technology of insecticidal protein and coding gene, which is applied in the field of genetic engineering to achieve the effect of improving insecticidal ability

Active Publication Date: 2015-01-28
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

However, the toxicity of Cry1Ie protein is lower than that of insecticidal protein Cry1Ac, which limits its application to some extent.

Method used

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  • Insecticidal protein as well as coded gene and application of insecticidal protein
  • Insecticidal protein as well as coded gene and application of insecticidal protein
  • Insecticidal protein as well as coded gene and application of insecticidal protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Construction of Cry1m7 inducible expression vector.

[0033] The gene sequence of Cry1m7 was commissioned by Shanghai Shenggong to be synthesized and constructed on the pUC57 vector, which constitutes the ampicillin-resistant plasmid pUC57-Cry1m7.

[0034] Add 20ng of plasmid pUC57-Cry1m7 and plasmid pET30a (purchased from Novagen, USA) to 50μL of E. coli competent cells Trans5α, ice bath for 30min; heat shock at 42°C for 90sec, ice bath for 2min; add 300μL LB liquid medium, After resuming culture at 37°C with low-speed shaking for 1hr, spread the bacterial solution on a plate containing the corresponding antibiotics, and dry; incubate in the 37°C incubator upside down for 15hr. Pick out the monoclonal plaque in 10ml of LB liquid medium containing ampicillin, and cultivate overnight at 37℃ with shaking. The next day, the plasmid was extracted according to the operation steps of Tiangen Plasmid Small Extraction Kit, and NanoDrop 2000C Micro UV A spectrophotometer quantified ...

Embodiment 2

[0048] Induction and purification of Cry1m7 and Cry1Ie proteins.

[0049] The plasmid pET30a-Cry1m7, which was sequenced and verified by restriction enzyme digestion, was transferred into the strain Transetta (DE3) purchased from Quanshijin Company, a single clone was picked, and the positive plaque was verified by PCR amplification. The positive plaque was inoculated into 10 mL of LB liquid medium (containing appropriate antibiotics), and cultured with shaking at 37°C overnight. The Escherichia coli strain Transetta (DE3) containing pET30a-Cry1Ie plasmid was stored at -70℃ in the laboratory, and was inoculated into 10mL LB liquid medium (with appropriate antibiotics) at a ratio of 1:100, and cultured with shaking at 37℃ overnight.

[0050] The next day, inoculate 200mL LB liquid medium (containing appropriate antibiotics) at 1:200, culture at 37°C, 200rpm shaking to an OD600 of 0.4-0.6, add IPTG to a final concentration of 0.5mM, 16°C shaker, 160rpm, shaking Culture for about 20 ...

Embodiment 3

[0060] Indoor corn borer test of Cry1Ie purified protein.

[0061] Under the conditions of indoor temperature of 28±1℃, photoperiod (L:D) 16:8h, and relative humidity of 70% to 80%, the corn borer test was carried out with artificial feed mixing method, namely, the purification of Cry1m7 and Cry1Ie respectively Protein is added to artificial feed to make 0μg / g, 0.025μg / g, 0.05μg / g, 0.25μg / g, 0.5μg / g, 2.5μg / g, 5μg / g, 25μg / g, 50μg / g total 9 Feeding feeds with a concentration gradient, the feed prepared with the corresponding concentration of each protein is divided into three 48-well cell culture plates, and each hole of the corn borer newly hatched larvae is inoculated with a total of 144 insects at each concentration, and the statistics are counted after 7 days Insect mortality and average insect weight Cry1m7 and Cry1Ie purified protein indoor bioassay data analysis LC50 results are shown in Table 1. From the data in Table 1, it can be seen that the insecticidal effect of Cry1m...

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Abstract

The invention relates to an insecticidal protein Cry1m7, which is 1) a protein composed of an amino acid sequence shown by SEQ ID NO:1, or 2) a protein which is obtained by substituting, deleting or inserting one or more amino acid sequences in the amino acid sequence shown in SEQ ID NO:1 and has an equivalent activity. The invention further provides a coded gene, an expression vector and an engineering bacterium of the insecticidal protein, an application of the protein to insecticides and an application of the coded gene of the protein to plant conversion. The invention provides the high-toxicity insecticidal protein Cry1m7 and the coded gene thereof; and the insecticidal protein provided by the invention can be used for killing Asiatic corn borers and other lepidoptera pests to improve the insecticidal capability of genetically modified crops.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to an insecticidal egg and its coding gene and application. Background technique [0002] Insect pests are a major factor that causes serious crop yield reduction. The most common means to prevent agricultural pests is to use broad-spectrum chemical pesticides and some biological pesticides. Most chemical pesticides have the characteristics of broad-spectrum and high toxicity. While killing the target pests, they often kill many beneficial insects together, seriously disrupting the ecological balance and causing serious pollution to the environment. On the other hand, pesticide residues are harmful to humans, The health of livestock also poses a serious threat. Biological insecticides are easily degradable and highly compatible with the environment, but they need to be repeatedly applied in production, which greatly increases production costs. In order to make up for the shortcomings...

Claims

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Application Information

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IPC IPC(8): C07K14/325C12N15/32C12N15/82A01H5/00A01N47/44A01P7/04
CPCA01N63/10C07K14/325C12N15/82C12N15/8286
Inventor 刘允军张煜文王国英何康来
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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