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Protein having phytopathogen killing activity, and coding gene and application thereof

A plant pathogen and protein technology, applied in the direction of plant gene improvement, application, plant products, etc., can solve the problems of lack of application, lack of insect resistance genes, etc.

Active Publication Date: 2013-01-30
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These toxin proteins have been widely used in plant transgenic insect resistance. However, these genes are generally protected by foreign patents, and my country lacks insect resistance genes with independent intellectual property rights.
On the other hand, due to the widespread use of a limited number of highly active insecticidal protein genes, the problem of pest resistance to insecticidal proteins has emerged

Method used

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  • Protein having phytopathogen killing activity, and coding gene and application thereof
  • Protein having phytopathogen killing activity, and coding gene and application thereof
  • Protein having phytopathogen killing activity, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Embodiment 1, Serratia entomophila insecticidal protein gene spro

[0120] The present inventor clones an insecticidal protein gene spro from Serratia insectophilus DSM12358 bacterial strain, the Spro protein encoded by this gene is a metalloprotease, consists of 487 amino acid residues, and it is different from the proteins reported in the past The highest sequence similarity is only 53%, so it belongs to a new protein.

[0121] The nucleotide sequence of the Serratia entomophila insecticidal protein gene spro of the present invention is as follows (SEQ ID NO: 1, figure 2 ):

[0122] atgcaatcta ctaaaaaggc aattgaagtt actgaatcca gtcttgcggc tgcaaccacc

[0123] ggctacgatg cagtagacga tctgttgcat taccacgagc gcggtaatgg gatccaggtt

[0124] aatggcaagg actcattttc taccgaacaa gctgggctgt ttattacccg tgagaaccaa

[0125] acctggaatg gttataaagt ttttggccag ccggtcaaat taacgttttc cttcccggac

[0126] tataagttct cttccaccaa cgtcgccggc gacaccgggc tgagcaagtt cagcgcggaa

[0127] cagcagcagc...

Embodiment 2

[0157] Embodiment 2, bacterial culture and the extraction of genomic DNA

[0158] 1) Culture of Serratia entomophila

[0159] The frozen Serratia entomophila DSM12358 (purchased from German Collection of Microorganisms and Cell Cultures) was picked, inoculated on LB agar plates, and cultured at 37°C for 16 hours to activate the strains. Then pick a well-separated colony, inoculate it in LB liquid medium, shake it at 37° C. (200 r / min) and cultivate it for 12 hours before use.

[0160] 2) Extraction of Serratia entomophila DSM12358 total DNA

[0161] Take 1.5 mL of the cultured Serratia entomophila bacteria liquid, centrifuge at 12000 g for 2 min to collect the cells, resuspend the cells with 567 μL TE buffer, add 30 μL of 10% (w / v) SDS and 3 μL of 20 mg / mL proteinase K, After thorough mixing, incubate at 37°C for 1 hour; add 100 μL of 5M NaCl, mix well, add 80 μL of CTAB / NaCl (10% (w / v) CTAB, 0.7mol / L NaCl)) solution, and bathe in water at 65°C for 10 minutes; Add an equal ...

Embodiment 3

[0162] Embodiment 3, the cloning of Serratia entomophila insecticidal protein gene spro

[0163] Design and synthesize the following two primers,

[0164] 5' GAGCTC ATG CAA TCT ACT AAA AAG GCAA 3' (SEQ ID NO: 3);

[0165] 5' AAGCTT TTA CAC GAT AAA GTC CGT GGC 3' (SEQ ID NO: 4);

[0166] Sac I and HindIII restriction sites (underlined parts) were respectively added at the 5' ends of the two primers. Using the Serratia entomophila genome DNA as a template, the above two primers are used to amplify the Serratia entomophila insecticidal protein gene by PCR method. The Taq DNA polymerase was selected from the high-fidelity rTaq DNA polymerase of Bao Biological Engineering Co., Ltd. The PCR amplification program was: 94°C for 4min; 94°C for 1min, 58°C for 1min, and 72°C for 1min, a total of 30 cycles; 72°C for 10min to drop to 10°C. PCR products were detected by agarose gel electrophoresis. Under UV irradiation, the target DNA band is excised. Then, Axygen Gel Extraction...

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Abstract

The invention relates to a protein having a phytopathogen killing activity, and a coding gene and an application thereof. The protein can widely and strongly position lepidoptera insects comprising cotton bollworm and the like, and can be used for the insect pest control of plants (crops).

Description

technical field [0001] The invention belongs to the technical field of microbial genetic engineering or biological pesticide genetic engineering. Specifically, it relates to a protein with the activity of killing plant pathogens, its coding gene and application. Background technique [0002] In recent decades, extensive research has been carried out on microbial insecticides with the theme of entomopathogenic microorganisms. Due to the different pathogenic microorganisms used, the insecticidal principles and mechanisms of microbial insecticides are also different. It has been confirmed that the insecticidal mechanism of bacterial insecticides is usually closely related to bacterial toxin proteins (Yu Ziniu, Production and Application of Bacillus thuringiensis [M]. Beijing: Agricultural Press 1993). Since the discovery of the first toxin protein diphtheria toxin, hundreds of toxin proteins have been found in various bacteria, and detailed research work has been carried out o...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/57C12N15/63C12N1/21C12N1/19C12N5/10A01N63/00A01P7/04C12N15/84A01H5/00C12R1/425
Inventor 周志华孙琴严兴王钱福钱昌丽王成树
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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