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Screening method of pseudomonas protegens mutant strain and application thereof to biological control

A technology of Pseudomonas fluorescens and mutant strains, applied in pest control, microbial-based methods, chemicals for biological control, etc., can solve the problems of insufficient secondary metabolites and weak antibacterial ability

Active Publication Date: 2017-09-01
SHANDONG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the genome sequencing results show that the secondary metabolites of this bacterium are not abundant, and the antibacterial ability is weak

Method used

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  • Screening method of pseudomonas protegens mutant strain and application thereof to biological control
  • Screening method of pseudomonas protegens mutant strain and application thereof to biological control
  • Screening method of pseudomonas protegens mutant strain and application thereof to biological control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] The screening method of Pseudomonas fluorescens mutant strain Pf5-△retS, the specific operation steps are as follows:

[0084] (1) The plasmid pBBR1-Rha-TEGpsy-kan (this plasmid can express recombinase in Pseudomonas) is introduced into wild-type Pseudomonas protegens Pf5 (Pseudomonas protegens Pf5) by electroporation, and electroporation The final bacteria were spread on the plate of LB medium + kanamycin (km, 30 μg / mL), and 12 single colonies were randomly selected to extract plasmids for enzyme digestion and identification, and the correct transformant Pf5::pBBR1-Rha was screened -TEGpsy-kan;

[0085] (2) Knock out the retS gene on the genome of Pseudomonas protegens Pf5. 将线性DNA片段loxM-genta(此片段通过PCR的方法得到,所用的一对引物为RetS-Genta-loxM-5'GCACACGCCCTTGCCGTGCGGTCATTA CGCCGCGCATAGTTATAATCAGGCATCAACCAACGAAGGGATTTCGCCAGCTGAATTA CATTCCCAACCG / RetS-Genta-loxM-3'TGGAGCATGGTG GGAGCTCACGACTAAAGGAGGGCGAGCGAGAGTTTAACAGGCGCCGCAGAGCCTGT CGGCTCACAACTTAAATGTGAAAGTGGGTC,分别如SEQ ID NO.15 and S...

Embodiment 2

[0091] Embodiment 2: The screening method of Pseudomonas fluorescens mutant strain Pf5-NiF, the specific operation steps are as follows:

[0092](1) Using the method of Red / ET direct cloning, the genomic DNA of Pseudomonas stutzeri DSM4166 (Pseudomonas stutzeri DSM4166) was first digested with restriction endonucleases Afl II and Ssp I, and the obtained 69kb NiF Nitrogen fixation gene island ( figure 2 ) After the gel electrophoresis of the DNA fragment is verified correctly, it is then connected to the corresponding expression vector, and the primers used are:

[0093] Primer 1: AGTGAATTGTAATACGACTCACTATAGGGCGAATTCGAGCTCGGTACCCGCTTAAGTACGGCTACCTGGAGCTCGCG CCAGTG, as shown in SEQ ID NO.1;

[0094] Primer 2: TACGGCTACCTGGAGCTCGCGCCAGTGCTTGCCGACATCGAATCACGGCCGCTGCTGCAGCACGTGGTGGTCACCGGCCGGGATCCGTTTAAACACAAATGGCAAGGGCTAATG, as shown in SEQ ID NO.2;

[0095] Primer 3: ATTGATGTTTTCCTTGGCCAGCGCCTCGAACATCCGGCTGGCGACGCCTGCGTGCGAACGCATACCGACACCGACGATAGGGATCCGTTTAAACGGTG TGGTAGCTCGCG...

Embodiment 3

[0112] Example 3: The screening method of Pseudomonas fluorescens mutant strain Pf5-△retS-NiF, the specific operation steps are as follows:

[0113] The plasmid pBeloBAC11-oriT-TnpA-genta-NiF from Escherichia coli ET12567 was introduced into the mutant Pseudomonas protegens Pf5-△retS by conjugative transfer, and then the NiF gene could be randomly inserted into Pf5-△retS by transposition. In the genomic DNA of △retS. The detailed operation of conjugative transfer is as follows: the mutant Pseudomonas protegens Pf5-△retS (LB medium, 30°C) and Escherichia coli ET12567 (LB+genta 2μg / mL medium, 37°C) were cultured overnight respectively. The next day, wash the mutated Pseudomonas protegens Pf5-△retS and Escherichia coli ET12567 twice with fresh LB medium respectively, dissolve them in 500 μL LB respectively, and mix them together to make a total of 1 mL. After centrifuging at 9000rpm for 1 minute, discard most of the supernatant, leave 100 μL of bacterial solution and resuspend t...

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Abstract

The invention discloses a pseudomonas protegens mutant strain Pf5-NiF, Pf5-delatretS or Pf5-delatretS-NiF, a screening method and application thereof. By a Red / ET recombining and direct cloning technology, an NiF nitrogen fix gene island in a genome of pseudomonas stutzeri DSM4166 is integrally cloned into a genome of the pseudomonas protegens Pf5 to obtain a genetic engineering strain Pf5-NiF through smooth heterogeneous expression, so that the pseudomonas protegens Pf5 without a biological nitrogen fixing function itself generates the biological nitrogen fixing function; in addition, a retS gene in the genome of the pseudomonas protegens Pf5 is knocked out through directional unmarked gene knockout to obtain a genetic engineering strain Pf5-delatretS, so that the expression amount of antibiotic 2, 4-diacetyl phloroglucinol and haematochrome is increased, and thus the pseudomonas protegens Pf5 mutant strain with stronger bactericidal activity is obtained.

Description

technical field [0001] The invention relates to a mutant strain of Pseudomonas fluorescens, in particular to a method for screening the mutant strain of Pseudomonas fluorescens and its application in biological control. The invention belongs to the field of biotechnology. Background technique [0002] Pseudomonas protegens (Pseudomonas protegens) is a Gram-negative rod-shaped bacterium commonly found in plant rhizosphere and soil, and is one of the most studied plant growth-promoting rhizobacteria (PGPR). Due to its fast reproduction speed, strong adaptability, easy artificial cultivation, stable preparation, convenient application, no pollution to the environment, and prevention and treatment of various plant diseases, the Ministry of Agriculture of my country has listed it as a microbial pesticide and can be registered. One of the types of fertilizers, it is widely used nationwide. Because it can produce one or more antibiotics, such as 2,4-diacetylphloroglu-cinol (2,4-DAP...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A01N63/00A01P1/00A01P21/00C12R1/39A01N63/27
CPCC07K14/21A01N63/27C12N15/52C12R2001/38C12N1/205C12N15/90
Inventor 张友明涂强荆晓姝于芳楠
Owner SHANDONG UNIV
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