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Pseudomonas aeruginosa flagellin for improving disease resistance of plants as well as coding gene and application of pseudomonas aeruginosa flagellin

A Pseudomonas aeruginosa and flagellin technology, which is applied in the field of agricultural biology, can solve the problems of unclear, difficult to use, and low sensitivity of the activator protein and Harpin protein receptors of Alternaria vermin, and reduce agricultural production. Cost, low concentration of use, the effect of improving resistance to disease

Active Publication Date: 2019-12-20
CHENGDU LUSYNO BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the receptors of the activating protein and Harpin protein of Alternaria ultrafines are not yet clear, which means that it is difficult to make scientific and effective guidance on their use in theory, and their scope of application can only be judged by experimental experience. Use concentration, which requires a long-term and continuous exploration process; second, plants are less sensitive to these plant immune pesticides, the final use concentration is 100μmol, the amount used per mu is 100g, and the cost is more than 40 yuan / mu

Method used

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  • Pseudomonas aeruginosa flagellin for improving disease resistance of plants as well as coding gene and application of pseudomonas aeruginosa flagellin
  • Pseudomonas aeruginosa flagellin for improving disease resistance of plants as well as coding gene and application of pseudomonas aeruginosa flagellin
  • Pseudomonas aeruginosa flagellin for improving disease resistance of plants as well as coding gene and application of pseudomonas aeruginosa flagellin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The construction of embodiment 1flagellin expression vector

[0037] Using the bioinformatics software Geneious R9 to analyze the gene sequence of flagellin gene flagellin of Pseudomonas aeruginosa, combined with the restriction endonuclease cutting site of pET-28b prokaryotic expression vector, according to the principle of open reading frame code ORF, design The upstream primer with NdeI restriction site and the downstream primer with XholI restriction site.

[0038] Forward primer: 5'-GAC CATATG ATGGCTCTTACTGTTAAT-3' (SEQ ID NO: 3), wherein the underline is the enzyme cutting sequence of NdeI;

[0039] Reverse primer: 5'-TCA CTCGAG TTATCTAAGCAAAGACAACACAGA-3' (SEQ ID NO: 4), where the underline is the restriction sequence of XholI.

[0040] The full-length flagellin gene was amplified by PCR, and the amplification program was as follows: 98°C for 30s; 34 cycles of 98°C for 10s, 55°C for 10s, and 72°C for 30s; 72°C for 2min; and storage at 4°C.

[0041] The PCR ...

Embodiment 2

[0043] Example 2 Protein expression condition optimization

[0044] The recombinant plasmid pET-28b-flagellin expression vector was transformed into Escherichia coli BL21(DE3) by the heat shock method to obtain a recombinant Escherichia coli containing the recombinant plasmid pET-28b-flagellin, and the recombinant Escherichia coli was named BL21(DE3) / pET-28b-flagellin. The method of extracting the plasmid and transforming the expression host strain is the same as that in Example 1. After the verification is correct, the expression is carried out. The correct expression strain will be verified to be activated overnight, and the strain containing pET-28b empty plasmid will be used as a control. Add 1mL of overnight culture solution to 100mL LB liquid medium containing 100μg / mL kanamycin (1% inoculum size), culture at 37°C, shake at 200r / min for 2-3h until OD 600 It is 0.6-0.8. At this time, set the concentration gradient of the inducer IPTG (add IPTG with final concentration...

Embodiment 3

[0046] Expression and purification of the protein of embodiment 3

[0047] 1. Inoculate BL21(DE3) / pET-28b-flagellin in LB liquid medium, and culture overnight at 37°C and 200rpm / min with shaking.

[0048] 2. Induce protein expression according to the method and conditions shown in Example 2, that is: transfer the overnight bacterial solution to LB liquid medium containing 100 μg / mL kanamycin at a volume ratio of 1:100, 37°C, 200rpm / min shaking culture to OD 600 The value is 0.6, then add the final concentration of 0.1mmol / LIPTG, shake culture at 30°C and 200rpm / min for 12h, and collect the bacteria.

[0049] 3. Take the bacterial cells obtained in step 2, resuspend them with PBS buffer, and perform ultrasonic crushing (parameters for ultrasonic crushing: total ultrasonic time 30 minutes, every 5 seconds for 5 seconds, power 200W), then centrifuge at 12000rpm for 10 minutes, and collect the supernatants respectively liquid and sediment.

[0050] 4. Perform SDS-PAGE on the s...

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Abstract

The invention discloses pseudomonas aeruginosa flagellin for improving disease resistance of plants as well as a coding gene and application of the pseudomonas aeruginosa flagellin. An amino acid sequence of the protein is shown in SEQ ID NO:1 or is an amino acid sequence which is obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence shown in the SEQ ID NO:1 and can express the same functional protein; and a nucleotide sequence of the gene coding the protein is shown in SEQ ID NO:2 or is a nucleotide sequence which is obtained by substituting, deleting and / or adding one or more nucleotides to the nucleotide sequence shown in the SEQ ID NO:2 and can code the same functional protein. As an immunity elicitor, the protein can remarkably improve disease resistance of plants, has low use concentration, takes effects quickly and can effectively reduce the agricultural production cost.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a Pseudomonas aeruginosa flagellin for improving plant disease resistance, its coding gene and application. Background technique [0002] At present, many biopesticide products have been widely used in the world, the most widely used are the bacterial biopesticide Bacillus thuringiensis (Bt) and agricultural antibiotics such as Jinggangmycin, Zhongshengmycin, and Brassin. These products achieve the purpose of insect and disease resistance through antibacterial and insecticidal substances of biological origin. Compared with chemical insecticides and antibacterial agents, these products have a shorter degradation cycle and greater environmental compatibility. However, the occurrence of pests and diseases is a process of interaction between plants and pathogens. In the past, the research and development of biopesticides often ignored the role of plant immunity ...

Claims

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Application Information

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IPC IPC(8): C07K14/21C12N15/31A01N47/44A01P21/00
CPCA01N47/44C07K14/21
Inventor 蔡易李琦郭晋雅李雍周鑫琼张丽梅
Owner CHENGDU LUSYNO BIOTECHNOLOGY CO LTD
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