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A new insecticidal protein and its nucleotide sequence

An insecticidal protein and nucleic acid technology, which is applied in the directions of insecticides, biocides, peptides, etc., to enrich insecticidal protein resources, improve insect resistance, and achieve important economic effects.

Active Publication Date: 2020-11-24
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] However, there are few reports that Bt can also produce insecticidal active substances different from the above three proteins, especially insecticidal activity against certain Coleoptera pests, such as the North China black gill beetle (Holotrichia oblita). substance

Method used

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  • A new insecticidal protein and its nucleotide sequence
  • A new insecticidal protein and its nucleotide sequence
  • A new insecticidal protein and its nucleotide sequence

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Experimental program
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Effect test

preparation example Construction

[0050] Table 1 is the preparation table of SDS polyacrylamide gel.

[0051] Table 1 Preparation table of SDS polyacrylamide gel

[0052]

Embodiment 1

[0053] Screening and cloning of embodiment 1 new gene

[0054] Bt genome extraction

[0055] S1: 5mL 1M Tris-HCL, 1mL 0.5M EDTA, 171.15g sucrose, pH7.0, dilute to 500mL, sterilize at 115°C for 20min.

[0056] S2: 10% SDS 4mL+ddH 2 O 6mL, pH4.8-5.2.

[0057] S3: 5M guanidine isothiocyanate, 1M NaAc.

[0058] TE: 10mM Tris-HCL 3mL, 1mM EDTA 600ul, pH 8.0, dilute to 300mL.

[0059] 1) Streak inoculation of Bacillus thuringiensis 261-1 strain on LB medium, and culture overnight at 30°C;

[0060] 2) Scrape all the bacteria on the plate as much as possible, put them in a 1.5ml EP tube, and fully suspend them with 150μl S1;

[0061] 3) Add 100mg of quartz sand, and crush it on the tissue crushing instrument for 1 minute;

[0062] 4) Add 200 μl S2 and mix well;

[0063] 5) Add 400 μl S3, mix thoroughly, and centrifuge at 12000 rpm for 10 minutes;

[0064] 6) Transfer the uppermost supernatant to a 1.5EP tube, add an equal volume of isopropanol, mix well and let stand at -20°C ...

Embodiment 2

[0073] Protein expression and quantitative analysis

[0074] Escherichia coli Rosetta (DE3) strain carrying 261sip01 gene was inoculated in LB liquid medium at an inoculum size of 1%, and cultivated to OD at 37°C 600 When the value reaches between 0.5-1.0, add the inducer 50mM IPTG, induce at 150rpm, 20 ℃ low temperature for 12h. Then centrifuge at 8000 rpm for 3 min at 4°C. The bacterial cells were collected by centrifugation, suspended by adding 50 mM Tris·Cl (pH 8.0); the bacterial cells were disrupted (completely disrupted by ultrasonic routine), and the sonicated bacterial liquid was centrifuged at 12,000 rpm for 15 min at 4°C; then the supernatant was collected. Perform SDS-PAGE quantitative analysis on the supernatant as follows: prepare the following five different concentration gradients of BSA: 0.8 μg / μL, 0.4 μg / μL, 0.2 μg / μL, 0.1 μg / μL, 0.05 μg / μL , and the target protein was serially diluted so that the final concentration was between 0.8-0.05 μg / μL, and the samp...

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Abstract

The invention relates to a novel insecticidal protein and a nucleotide sequence thereof. The insecticidal protein has at least an amino acid sequence in (I) and (II): (I) an amino acid sequence shown as a SEQ ID No.2; (II) an amino acid sequence having the amino acid sequence with consistency of more than 95% shown in the SEQ ID No.2 and having same function with the amino acid sequence shown in the SEQ ID No.2; preferably the amino acid sequence having the amino acid sequence with consistency of more than 98% shown in the SEQ ID No.2 and having same function with the amino acid sequence shown in the SEQ ID No.2.

Description

technical field [0001] The present invention relates to a new insecticidal protein and its nucleotide sequence. Background technique [0002] Bacillus thuringiensis (Bt) is a widely distributed Gram-positive bacterium, an entomopathogenic microorganism with strong toxicity to pests and no toxicity to natural enemies, and no toxicity to higher animals and humans. It is currently the most in-depth study and the most widely used microbial insecticide, and it is active against more than 3000 kinds of pests in 16 orders. [0003] The prior art mainly believes that the reason why Bt can have insecticidal activity is mainly due to its ability to produce various Cry proteins, Cyt proteins and Vip proteins. [0004] However, there are few reports that Bt can also produce insecticidal active substances different from the above three proteins, especially insecticidal activity against certain Coleoptera pests, such as the North China black gill beetle (Holotrichia oblita). substance. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/32A01N47/44A01P7/04
CPCA01N63/10C07K14/325
Inventor 束长龙耿丽丽张杰黄世智宋福平彭琦
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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