Genetic manipulation method of sacB-mediated Pseudomonas chlororaphis

A technique for genetic manipulation of Pseudomonas chloropinus, applied in the field of genetic manipulation of biocontrol bacteria, can solve problems such as different genetic transformation conditions

Active Publication Date: 2019-06-21
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, in the genetic manipulation of Xanthomonas, Streptomyces, and Pseudomonas fluorescens, a good amphipathic mating system has been established for DNA transfer (Zou Lifang, doctoral thesis, Nanjing Agricultural University, 2007

Method used

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  • Genetic manipulation method of sacB-mediated Pseudomonas chlororaphis
  • Genetic manipulation method of sacB-mediated Pseudomonas chlororaphis
  • Genetic manipulation method of sacB-mediated Pseudomonas chlororaphis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Sensitivity determination of Pseudomonas aeruginosa YL-1, Escherichia coli DH5α and S17-1 to ampicillin, gentamicin and tetracycline

[0050] The Pseudomonas chloropinus YL-1 used in this embodiment is a strain isolated by the applicant to various important pathogenic bacteria in agriculture (Burkholderia glumae; Erwinia amylovora; Pectobacterium carotovora, etc.) and pathogenic fungi (Rhizoctonia solani, etc.) have strong inhibitory effects on biocontrol bacteria (Liu Youzhou et al., "Acta Phytopathology", 2015).

[0051] After culturing Pseudomonas aeruginosa YL-1 in LB liquid medium overnight at 28°C and 180rpm on a shaker, the concentration was about 10 8 cfu / mL bacterial solution for later use. After serially diluting the bacterial solution, spread it on LB plates with ampicillin concentrations of 10, 50, and 100 μg / mL, and LB plates with gentamicin and tetracycline concentrations of 10, 25, and 50 μg / mL, and culture at 28°C. 2d, observe the growth, the...

Embodiment 2

[0056] Example 2 Construction and connection of target gene deletion fragment

[0057] In order to excavate the active substances of Pseudomonas chloropinus YL-1 antagonizing pathogenic bacteria and fungi, and locate its synthetic gene cluster, this example uses the synthetic gene of fluorescent siderophilic iron (Pyoverdine) in Pseudomonas chloropinus YL-1 The pvdS gene on the cluster is the target gene, which is a σ factor and regulates the transcription of siderophore in the strain YL-1. Once the pvdS gene is destroyed, the strain YL-1 cannot synthesize siderophore, and it can also be cultured in LB liquid medium for 2 days. The green color characteristic of the wild-type strain cannot be displayed.

[0058] The primer table used in the embodiment 2 of table 2

[0059]

[0060] (1) Select the flanking sequences at both ends of the target gene pvdS, use Bioxm software to design upstream and downstream primers F1, R1, F2 and R2, respectively, and add HindIII, KpnI, XbaI r...

Embodiment 3

[0088] Example 3 Genetic Transformation of Escherichia coli DH5α with the Ligation Product Carrying the Fusion Gene

[0089] (1) Preparation of Escherichia coli DH5α chemically competent cells

[0090] ①Pick a fresh single colony of Escherichia coli in 25mL LB liquid medium, culture overnight at 37°C and 220rpm;

[0091] ②Transfer the cultured bacterial solution to 50mL fresh LB liquid medium at a ratio of 1:100, 37°C, 220rpm, 3-3.5h to OD 600nm ≈1.0;

[0092] ③After 10 minutes of ice-bathing the above bacterial solution, centrifuge at 4°C, 6000rpm for 10 minutes, discard the supernatant, and keep the precipitated bacteria;

[0093] ④ Add pre-cooled CaCl 2 (0.1M) 10mL, pipette gently to suspend the bacteria, and ice-bath for 30min;

[0094] ⑤ Centrifuge at 4°C, 6000rpm for 10min, discard the supernatant, and keep the precipitated bacteria;

[0095] ⑥Add pre-cooled CaCl 2 (0.1M containing 15% glycerol) 1mL, gently pipet to suspend the bacteria. Aliquot into 1.5mL centrif...

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Abstract

The invention discloses a genetic manipulation method of sacB-mediated Pseudomonas chlororaphis and belongs to the technical field of biology. Pseudomonas chlororaphis is used as a start material; flanking sequences of upstream and downstream ends of a target gene are selected and subjected to gradient PCR (polymerase chain reaction) amplification respectively; two amplified fragments and conjugative suicide plasmid pEX18 are subjected to restriction digestion and connection respectively; a recombinant plasmid is constructed; the recombinant plasmid is converted to Escherichia coli and subjected to amphiphilic mating with wild Pseudomonas chlororaphis; deletion of the target gene is successfully achieved. The genetic manipulation method of sacB-mediated Pseudomonas chlororaphis is constructed successfully herein, which is suitable for acting mechanism and function researches for related genes and gene clusters of Pseudomonas chlororaphis antibacterial active materials.

Description

technical field [0001] The invention relates to a genetic manipulation method of biocontrol bacteria, in particular to a sacB-mediated genetic manipulation method of Pseudomonas aeruginosa. Background technique [0002] Pseudomonas spp. is a class of microorganisms active in the rhizosphere of plants. Many strains in the genus can prevent and control plant diseases and promote plant growth. It belongs to Plant Growth-PromotingRhizobacteria (PGPR) kind. Pseudomonas chlororaphis belongs to the Pseudomonas family, the genus Pseudomonas, the bacteria are rod-shaped, the size is 0.3μm-0.8μm×1.0μm-1.1μm, unipolar flagella, in common Orange pigment can be produced on the bacterial culture medium, which has the biochemical characteristics common to Pseudomonas. The fungus has disease control and growth-promoting effects on a variety of plants, can enhance plant stress resistance, is environmentally friendly, and has no pathogenicity to organisms (Cho et al., 2008; Dilfuza, 2012; R...

Claims

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Application Information

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IPC IPC(8): C12N15/78C12Q1/6869
Inventor 刘邮洲周亚秋乔俊卿刘永锋
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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