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Diffusible signal factor (DSF) quorum sensing signal degradation genes and application thereof

A quorum-sensing signal and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve problems such as economic loss and little research on prevention and control methods, and achieve the effect of improving degradation ability

Active Publication Date: 2019-12-24
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pathogenic bacteria mediated by DSF family signals involve multiple species, including Xanthomonas campestris pv. campestris, Xcc, Xanthomonas oryzaepv. Klebsiella (Burkholderia cepacia complex, Bcc), Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, mutans (Streptococcus mutans), Xylella fastidiosa (Xylella fastidiosa) and Lysobacter enzymogenes, etc. Hazards to important economic crops, resulting in huge economic losses
At present, there is little research on the prevention and control methods of DSF-mediated pathogens except pesticide control.

Method used

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  • Diffusible signal factor (DSF) quorum sensing signal degradation genes and application thereof
  • Diffusible signal factor (DSF) quorum sensing signal degradation genes and application thereof
  • Diffusible signal factor (DSF) quorum sensing signal degradation genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Acquisition and identification of DSF degradation genes in Pseudomonas nitroreducing strain HS-18

[0048] 1. Experimental materials

[0049] The inventors obtained a high-efficiency DSF-degrading strain Pseudomonas nitroreducing by using DSF as the sole carbon source to separate, purify and identify 16s rDNA from soil samples that have been contaminated with oil for a long time near South China Agricultural University. (Pseudomonas nitroreducens) strain HS-18, this strain HS-18 has been deposited in the China Type Culture Collection on May 12, 2017, and the deposit number is CCTCC NO: M2017257 (see patent 201710764814.6).

[0050] 2. In the present invention, the DSF degradation function of the above-mentioned strain HS-18 is explored and analyzed at the gene level, and DSF degradation related genes dig are obtained through gene analysis and amplification methods, including dig1, dig2, dig3, and dig4. And by designing primers to amplify dig1, dig2, dig3, dig4 seque...

Embodiment 2

[0060] Example 2 Construction of four gene deletion mutants of dig1, dig2, dig3, and dig4 in HS-18

[0061] The pK18 vector was used to construct knockout vectors for the four genes of dig1, dig2, dig3, and dig4, and transformed into DH5α. After PCR verification, the positive transformants were confirmed, namely knockout donor bacteria DH5α (pK18-dig1), DH5α (pK18- dig2), DH5α (pK18-dig3) and DH5α (pK18-dig4). Cultivate the helper strain E.coli HB101 (pRK2013), donor bacteria, and recipient bacteria overnight. Using three-parent binding technology and the principle of homologous recombination, the correct knockout mutations of dig1, dig2, dig3, and dig4 were obtained by PCR body.

Embodiment 3

[0062] Example 3 HS-18 test for the growth of each successfully constructed knockout mutant in the MM* medium with DSF as the sole carbon source and the DSF degradation ability

[0063] After each mutant successfully constructed in Example 2 was activated on the LB solid plate, a single colony was picked and cultured in the LB liquid medium overnight. Use MM* liquid medium to OD 600 Resuspend to uniformity, about 0.5. Inoculate at a ratio of 1:100 into 3ml of MM* medium with 0.5mM DSF as the sole carbon source, and culture at 30℃ and 150rpm in a constant temperature shaker. After culturing for 0h, 3h, 6h, 9h, 12h, 24h After that, use an ultraviolet spectrophotometer (NANODROP) to measure the absorbance value at a wavelength of 600nm to indicate the growth of microorganisms; then add ethyl acetate to the sample for extraction, take the upper organic phase, repeat twice, and combine the extracts. After rotary evaporation to dryness, the volume was adjusted to 200 μl with methanol ...

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Abstract

The invention discloses diffusible signal factor (DSF) quorum sensing signal degradation genes and application thereof. The degradation genes are degradation genes dig1, dig2, dig3 and dig4 separatedfrom pseudomonas nitroreducens bacterial strains HS-18 and having a remarkable degradation effect on quorum sensing signal DSF family signals. The degradation genes are subjected to heterologous expression in nonpathogenic bacteria or biocontrol bacteria, and the degradation ability of the nonpathogenic bacteria or the biocontrol bacteria to DSFs can be remarkably improved; the degradation genes are subjected to heterologous expression in DSF family signal mediated pathogenic bacteria, expression of virulent factors regulated and controlled by the DSF family signals can be remarkably influenced, and the pathogenicity of the pathogenic bacteria to plant hosts is lowered; and thus the DSF quorum sensing signal degradation genes have important practical application value and application prospects in the aspect of biological prevention and treatment of the DSF family signal mediated pathogenic bacteria.

Description

Technical field [0001] The invention belongs to the technical field of molecular biological control. More specifically, it relates to DSF quorum sensing signal degradation genes and their applications. Background technique [0002] The quorum sensing signal DSF (Diffusible signal factor) was first discovered in Xanthomonas campestris pv. campestris (Xcc), and it participates in the regulation of multiple virulence factors in Xcc, such as: biofilm, antioxidant activity, and Drug properties, extracellular polysaccharides, extracellular enzymes, motility, etc. Bacteria have the ability to adjust population density by sensing and responding to quorum sensing signals. DSF family signal-mediated pathogenic bacteria involve multiple species, including X. campestris pv. campestris (Xcc), Xanthomonas oryzaepv. oryzae (Xoo), onion Burkholderia cepacia complex (Bcc), Pseudomonas aeruginosa (Pseudomonas aeruginosa) PAO1, Streptococcus mutans, Xylella fastidiosa and Lysobacter enzymogenes,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/21C12N15/70C12N1/21A01N47/44A01P1/00C12R1/38
CPCA01N47/44C07K14/21C12N15/70
Inventor 张炼辉王惠杉廖立胜陈少华
Owner SOUTH CHINA AGRI UNIV
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