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Quorum sensing signaling molecule degradation gene aidf, application thereof, catabolic enzyme AidF coded by quorum sensing signaling molecule degradation gene aidf and application of catabolic enzyme AidF

A technology for quorum sensing signal and enzyme degradation, applied in the field of enzyme genetic engineering, which can solve the problems of complex microbial interactions, many uncontrollable factors, and unsatisfactory effects.

Inactive Publication Date: 2020-10-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, a large number of quorum-sensing quenching bacteria have been isolated from nature (such as CN201810843540.4, CN201910837948.5), but because the interaction between microorganisms is very complicated, if the quorum-sensing quenching bacteria are directly used to prevent and control diseases, then There are too many uncontrollable factors that arise, and many experiments and investigations are required to ensure the safe application of a certain quenching bacteria
In addition, the genetically engineered quenched bacteria constructed by biotechnology also have QQ activity, but because they have not undergone evolution and adaptation in nature, the effect is not ideal (Liu P F et al.AhlX, an N-acylhomoserine lactonase with unique properties[J].Marine Drugs,2019)

Method used

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  • Quorum sensing signaling molecule degradation gene aidf, application thereof, catabolic enzyme AidF coded by quorum sensing signaling molecule degradation gene aidf and application of catabolic enzyme AidF
  • Quorum sensing signaling molecule degradation gene aidf, application thereof, catabolic enzyme AidF coded by quorum sensing signaling molecule degradation gene aidf and application of catabolic enzyme AidF
  • Quorum sensing signaling molecule degradation gene aidf, application thereof, catabolic enzyme AidF coded by quorum sensing signaling molecule degradation gene aidf and application of catabolic enzyme AidF

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Expression and purification of degrading enzyme AidF

[0040] 1. Experimental method

[0041] In this example, a quorum sensing signal molecule (AHLs) degradation gene aidf was cloned from Ochrobactrum intermedium D-2 (which has been disclosed and preserved in the invention patent with the application number CN201810843540.4), and its nucleoside The acid sequence is shown as SEQ ID No: 1, the sequence length is 816bp, and the G+C content is 62%.

[0042] The degradation gene aidf can encode the degradation enzyme AidF, which consists of 271 amino acids (aa), the predicted molecular weight is 29.3 kDa, and the isoelectric point is 4.95. The method for expressing and purifying the degrading enzyme AidF specifically comprises the following steps:

[0043] (1) According to the nucleotide sequence (SEQ ID No: 1) of the degradation gene aidf, the primer aidF-F / R whose sequence is shown in Table 1 is designed, and the primer is used as a template to carry out A...

Embodiment 2

[0050] Embodiment 2 Degradative enzyme AidF quenching activity assay

[0051] 1. Experimental method

[0052] In order to verify whether the degrading enzyme AidF has AHLs (3OC6-HSL) quenching activity, this embodiment uses the reporter strain Agrobacterium tumefaciens (Agrobacterium tumefaciens) CF11 to carry out verification experiments, specifically including the following steps:

[0053] Set up 4 experimental groups: (1) Experimental group A: (3OC6-HSL only); (2) Experimental group B: E.coli BL21(DE3)pET28b crude enzyme solution + 3OC6-HSL (+IPTG); (3 ) Experimental group C: E.coli BL21(DE3) / pET28b-aidF crude enzyme solution + 3OC6-HSL(-IPTG); (4) Experimental group D: E.coli BL21(DE3) / pET28b-aidF crude enzyme solution + 3OC6-HSL(+IPTG).

[0054] The reaction system is: 10 μL crude enzyme solution + 189.9 μL PBS + 0.1 μL 3OC6-HSL mother solution. After standing at 28°C for 30 minutes, take 5 μL of the reaction mixture and apply it to the top of a MM agar strip with a wi...

Embodiment 3

[0058] Example 3 Biocontrol Effect Determination of Degrading Enzyme AidF on Radish Soft Rot

[0059] 1. Experimental method

[0060] In this example, taking the pathogen Pectinbacterium carotovorum subsp. carotovora (Pcc) Z3-3, which causes soft rot in cruciferous crops, as an example, the effect of the degrading enzyme AidF on AHLs-dependent pathogenicity was studied. The biocontrol effect of germs specifically includes the following steps:

[0061] (1) Inoculate a single colony of Pcc Z3-3 into LB medium, culture at 30°C and 200 rpm for 8 hours, and then centrifuge to obtain the bacteria. After washing the bacteria twice with PBS buffer, resuspend to make the OD of Pcc Z3-3 in the resuspension 600 = 0.3. Take 50 μL of Pcc Z3-3 resuspension, and evenly spread it on each white radish slice (the white radish slices in the first experimental group were coated with 50 μL PBS).

[0062] (2) Take 100 μL each of PBS buffer solution, E.coli BL21(DE3)pET-28b crude enzyme solution...

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Abstract

The invention discloses a quorum sensing signaling molecule degradation gene aidf, an application thereof, a catabolic enzyme AidF coded by the quorum sensing signaling molecule degradation gene aidfand an application of the catabolic enzyme AidF. The nucleotide sequence of the quorum sensing signaling molecule degradation gene aidf is shown as SEQID No:1. The invention provides the catabolic enzyme AidF coded by the quorum sensing signaling molecule degradation gene aidf, which can quench a quorum sensing signaling molecule, and has notable control effects on soft rot caused by pectobacterium sp., through further construction, a recombinant plasmid and a recombinant microorganism are obtained, and have quenching activity of the quorum sensing signaling molecule. Therefore, the degradation gene aidf, the catabolic enzyme AidF, the recombinant plasmid and the recombinant microorganism have the potential being used as a target for controlling plant diseases, can be widely applied to quenching the quorum sensing signaling molecule, can be used for controlling plant diseases mediated by the quorum sensing signaling molecule, can be used to a preparation for controlling the pathogenicbacteria caused by the quorum sensing signaling molecule, and provides an effective technique means for degradation of the quorum sensing signaling molecule.

Description

technical field [0001] The invention belongs to the technical field of enzyme gene engineering. More specifically, it relates to a quorum sensing signal molecule degrading gene aidf and its coded degrading enzyme AidF and applications. Background technique [0002] Bacterial soft rot caused by Pectobacterium carotovorum subsp. carotovora (Pcc) has become a worldwide disease, which occurs in many countries and leads to the reduction of related crops and significant economic losses. Pcc infects host plants mainly by synthesizing and secreting extracellular enzymes (pectinase, polygalacturonase, protease), which can degrade the cell walls of host plants and destroy their tissue structure, so that Pcc can obtain water and nutrients to support its own growth and reproduction. After successful infection, the host plant will show symptoms related to soft rot. At present, for the soft rot caused by Pcc, in actual production, the main control measures are the use of chemical pestic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N9/18C12N15/70C12N1/21A01N63/50A01N63/20A01P1/00C12R1/19
CPCA01N63/20A01N63/50C12N9/18C12N15/70
Inventor 陈少华范兴辉张文平叶田李海鹏许旭丹张炼辉
Owner SOUTH CHINA AGRI UNIV
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