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Application of enzyme FadD1 in pseudomonas aeruginosa for degrading DSF family signal molecules

A signal molecule, family technology, applied in the application, medical preparations containing active ingredients, hydrolase, etc., can solve the problems of no effect and unsatisfactory effect.

Active Publication Date: 2018-05-04
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme RpfB in Xanthomonas campestris can only degrade DSF or BDSF in vivo, and the effect of preventing and controlling related pathogens is not satisfactory, or even has no effect at all.

Method used

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  • Application of enzyme FadD1 in pseudomonas aeruginosa for degrading DSF family signal molecules
  • Application of enzyme FadD1 in pseudomonas aeruginosa for degrading DSF family signal molecules
  • Application of enzyme FadD1 in pseudomonas aeruginosa for degrading DSF family signal molecules

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 Obtaining of fadD1 gene sequence

[0033] BLAST alignment was carried out in the genome database of Pseudomonas aeruginosa PAO1 from the known amino acid sequence of Xanthomonas rpfB, and the sequence of fadD1 having 55% homology with rpfB was obtained, figure 1 It is the amino acid sequence alignment result of fadD1 and rpfB.

[0034] Using the Pseudomonas aeruginosa PAO1 genome as a template, design primers for PCR amplification, the amplification primers are:

[0035] fadD1-F: 5'-ATGATCGAAAACTTCTGGAAGGAC;

[0036] fadD1-R: 5'-TTACTTCTGGCCCGCTTTCTT,

[0037] After cloning analysis, the fadD1 gene and its sequence of Pseudomonas aeruginosa were obtained, as shown in SEQ ID NO: 1, the full length is 1689bp, the GC% content is 63.41%, the size of the protein encoded by it is 61.65kDa, and the amino acid sequence is shown in SEQ ID NO: 2 shown.

Embodiment 2

[0038] Example 2 FadD1 degrades signals BDSF and DSF in vitro

[0039] 1. Expression and purification of FadD1

[0040] Construct the recombinant plasmid fadD1-pET-21a(+), transform the recombinant plasmid fadD1-pET-21a(+) into Ecoli.BL21(DE3), induce expression with IPTG, and analyze the expression product by SDS-PAGE, attached figure 2 Lane 1 and Lane 2 are supernatants of broken cells, and the size of the target protein FadD1 is 61.65kDa. The C-terminus of the target protein is connected with (His) 6 The tag was purified by NiSepharose 6 Fast Flow Beads by affinity chromatography.

[0041] 2. FadD1 degrades signal molecules BDSF and DSF in vitro

[0042] FadD1 degradation BDSF reaction system is: 5μM FadD1, 200μM BDSF, 30μL TME Buffer, 0.01MPBS Buffer to make up to 200μL, placed at 37°C for 6h, 12h, 18h, 24h respectively, then took out and boiled for 5min to terminate the reaction, and the reaction product was analyzed by LC-MS For detection, each treatment was repeate...

Embodiment 3

[0045] Example 3 Determination of the phenotypic effect of FadD1 on the regulation of the Burkholderia cepacia BDSF system

[0046] 1. Construction of Burkholder onion strain overexpressing gene fadD1

[0047] The overexpression plasmid fadD1-pBBRI-5 was constructed, and the plasmid was transformed into wild-type Burkholderia cepacia H111 (hereinafter referred to as H111), and a Burkholderia cepacia strain overexpressing gene fadD1 (abbreviated as H111 (fadD1 )).

[0048]2. Activation of Burkholderia cepacia

[0049] The Burkholderia cepacia wild-type strain H111, the Burkholderia cepacia mutant strain, the gene Bcam0581 related to BDSF biosynthesis in the H111 strain was knocked out (hereinafter referred to as mutant 0581), and the overexpressed gene fadD1 The strain H111 (fadD1) was activated on LB plates and cultured overnight in a 37°C incubator.

[0050] 3. Effect of FadD1 on strain biofilm

[0051] Get activated Burkholderia cepacia bacterial strain H111, mutant 0581...

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Abstract

The invention discloses application of an enzyme FadD1 in pseudomonas aeruginosa for degrading DSF family signal molecules. The enzyme FadD1 capable of degrading DSF family signal molecules produced by thalli in vitro is obtained from the pseudomonas aeruginosa, and has an amino acid sequence shown as SEQ ID NO: 2. The enzyme FadD1 disclosed by the invention is capable of degrading the DSF familysignal molecules outside the thalli, including signal molecules DSF of xanthomonas campestris and quorum sensing signaling BDSF in burkholderia cepacia. Therefore, the enzyme FadD1 can be used for preparing a preparation capable of degrading the DSF family signal molecules in vitro, so as to be applied to controlling plant or animal diseases caused by pathogenic bacteria taking the DSF or BDSF asthe quorum sensing signaling, particularly plant black rot caused by xanthomonas campestris and diseases caused by burkholderia cepacia.

Description

technical field [0001] The invention relates to the technical field of enzyme application, more specifically, relates to an application of enzyme FadD1 in Pseudomonas aeruginosa to degrade DSF family signal molecules in vitro. Background technique [0002] Quorum sensing (QS) is an important communication method between microorganisms, which assists bacteria to sense population density and regulate gene expression. Xanthomonas campestris (X. campestrispv. campestris, Xcc) is a Gram-negative plant pathogen that can infect all Brassicaceae plants and cause black rot. In Xanthomonas campestris, the QS system mediated by DSF family (Diffusible signal factor family) signaling molecules plays a key role in resisting plant immune defense system and systemic infection of plants. Burkholderia cepacia is an opportunistic pathogen that can cause respiratory tract infection, pneumonia, and urinary tract infection in humans and animals, endangering human health and life. Burkholderia c...

Claims

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Application Information

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IPC IPC(8): C12N9/52C12N15/57A01N63/02A01P1/00A61K38/48A61P11/00
CPCA01N63/10A61K38/00C12N9/52
Inventor 邓音乐司阳刘景云付书娜
Owner SOUTH CHINA AGRI UNIV
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