LU500402B1

LU500402B1 - QUORUM SENSING SIGNAL-REGULATING ENZYME GENE ZD03-aiiA, PROTEIN ENCODED THEREBY, AND CLONING METHOD THEREOF

Info

Publication number: LU500402B1
Application number: LU500402A
Authority: LU
Inventors: Xian Ding
Original assignee: South China Sea Fisheries Res Inst Chinese Acad Fishery Sciences
Priority date: 2021-07-07
Filing date: 2021-07-07
Publication date: 2022-01-07

Abstract

The present disclosure relates to a quorum sensing (QS) signal-regulating enzyme gene ZD03-aiiA, a protein encoded thereby, and a cloning method thereof. With molecular biotechnology, the ZD03-aiiA gene is studied and a function of a protein encoded thereby is analyzed, which lays the foundation for further research and use of the ZD03-aiiA gene. Moreover, a ZD03 strain is a microorganism with the activity of regulating bacterial QS signals in a breeding ecological environment, which is conducive to the development of microbial resources and the construction of new disease prevention and control technologies that target QS signals of pathogenic bacteria.

Description

DESCRIPTION 7500402 QUORUM SENSING SIGNAL-REGULATING ENZYME GENE ZD03-aiiA, PROTEIN ENCODED THEREBY, AND CLONING METHOD THEREOF

TECHNICAL FIELD The present disclosure relates to the technical field of bacterial quorum sensing (QS), and in particular to a QS signal-regulating enzyme gene ZD03-aiiA, a protein encoded thereby, and a cloning method thereof.

BACKGROUND Studies have proved that QS systems are widely present in many animals, plants, and microorganisms in the nature. Bacteria monitor their own or other bacterial quantity changes by sensing the concentration changes of specific signal molecules in the extracellular environment and initiate the expression of related genes to make bacterial behaviors adapt to environmental changes. Most Gram-negative (G°) bacteria use acyl-homoserine lactone (AHL) as an inter-bacterial signal molecule, and Gram-positive (G”) bacteria use autoinducing peptides (AIPs) as inter-bacterial mediation signals. AHL is a small-molecule substance that is synthesized under the catalysis of acyl-carrier protease (ACP), enoyl-ACP reductase Fabl, and AHL synthase in bacteria. AHL released in the extracellular environment is recognized by a bacterial QS system and binds to a protein to form a dimer or multimer with transcription-regulatory activity, which activates the expression of a target gene and regulates the transfer of the Ti plasmid, the expression of a virulence gene, the formation of a biomembrane, and so on. Researches of proteomics and DNA microarray technology show that the expression of virulence genes is activated by signal-mediated regulation of transcript activity, thus realizing different functional regulations. A QS system of pathogenic bacteria can be interfered with such that a QS signal is present in the cell environment at a concentration that can hardly be sensed by the QS system of pathogenic bacteria, thus inhibiting the expression of pathogenic factors and achieving the purpose of preventing and treating pathogenic bacteria.

In the present disclosure, a strain ZDO3 is isolated and purified from a breeding ecological environment. With Agrobacterium tumefaciens (A. tumefaciens) W CF47 500402 as a reporter strain, it is found that the ZD03 has bacterial QS signal quenching activity, and after a spore is formed, the ZD03 has heat-resistant stability. According to the colony morphology, biochemical characteristics, and 16S rDNA sequence alignment analysis, it can be known that the strain is Bacillus subtilis (B. subtilis). It indicates that there are microorganisms with the activity of quenching bacterial QS signals in breeding ecological environments, which is conducive to the development of microbial resources and the construction of new disease prevention and control technologies that target QS signals of pathogenic bacteria.

A QS signal-regulating enzyme gene ZDO03-aiiA is subjected to cloning and sequence analysis, and results show that a positive clone ZD03-aiiA has a full length of 753 bp and an open reading frame (ORF), which encodes a polypeptide that is composed of 250 amino acid residues and is expected to have a molecular weight of

28.1 KDa and a protein isoelectric point of 4.78. An amino acid residue sequence deduced from the sequence above includes a conserved domain (Lactamase-B) (34AA-235AA). The research result lays the foundation for recombinant expression of the sequence and its related activity research.

SUMMARY A technical problem solved by the present disclosure is to provide a QS signal-regulating enzyme gene ZD03-aiiA; another objective of the present disclosure is to provide a protein encoded by the QS signal-regulating enzyme gene ZD03-aii A; and the present disclosure also provides a cloning method of the QS signal-regulating enzyme gene ZD03-aiiA.

To achieve the above objective, the present disclosure adopts the following technical solutions: In a first aspect of the present disclosure, a QS signal-regulating enzyme gene ZD03-aiiA is provided, with a nucleotide sequence shown in SEQ ID NO.: 1.

In a second aspect of the present disclosure, a protein encoded by the QS signal-regulating enzyme gene ZD03-aiiA according to the first aspect of the present disclosure, with an amino acid sequence shown in SEQ ID NO.: 2.

In a third aspect of the present disclosure, a cloning method of the QS 500402 signal-regulating enzyme gene ZD03-aiiA according to the first aspect of the present disclosure, including the following steps: (1) degenerate primer design and primer pair for the ZD03-aiiA gene: aiiF: SEQ ID NO: 3 5'-ATG GGA TCC ATG ACA GTA AAG AAG CTT TAT-3’; aiiR: SEQ ID NO.: 4 5'-GTC GAA TTC CTC AAC AAG ATA CTC CTA ATG-3’; (2) total DNA extraction for a ZDO03 strain: collecting bacteria at logarithmic growth phase by centrifuging at 12,000 r/min for 1 min, adding a lysis buffer and then mol/L NaCl, thoroughly mixing, and centrifuging a resulting mixture at 12,000 r/min for 10 min; collecting a resulting supernatant, and conducting extraction twice with an equal volume of phenol/chloroform/isoamyl alcohol (at a volume ratio of 25:24:1); centrifuging a resulting extract solution at 12,000 r/min for 10 min, and collecting a resulting supernatant; adding 2.5-fold volume of pre-cooled absolute ethanol to the supernatant, thoroughly mixing, and placing a resulting mixture in a refrigerator at -20°C for 5 min; centrifuging the mixture at 12,000 r/min for 15 min, discarding a resulting supernatant, and washing a resulting precipitate with 70% ethanol; centrifuging to make residual liquid gather at a bottom of a tube, and removing the liquid with a pipette; and air-drying a resulting precipitate, dissolving the precipitate with 225 uL of TE Buffer, and storing a resulting solution at 4°C for later use; and (3) with aiiF and aiiR as primers and total DNA of the ZDO03 strain as a template, conducting PCR amplification, subjecting an amplification product to agarose gel electrophoresis, and sequencing to obtain the ZD03-aiiA gene.

Compared with the prior art, the present disclosure has the following beneficial effects: The present disclosure relates to a QS signal-regulating enzyme gene ZD03-aiiA, a protein encoded thereby, and a cloning method thereof. With the RT-PCR technique, the ZD03-aiiA gene and a protein encoded thereby are studied, which lays the foundation for further research of the ZD03-aiiA gene. Moreover, a ZDO03 strain is a microorganism with the activity of regulating bacterial QS signals in a breeding 500402 ecological environment, which is conducive to the development of microbial resources and the construction of new disease prevention and control technologies that target QS signals of pathogenic bacteria.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 shows an agarose gel electrophoresis result of an amplification product of the ZD03-ail A gene in Example 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present disclosure is described in detail below in conjunction with specific examples, but the present disclosure can be implemented in a variety of different ways defined and covered by the claims.

Example 1 Step 1. Strain cultivation: an experimental sample with the strain ZD03 was collected from a breeding ecological environment, diluted appropriately, coated on a 2216E plate, and cultivated at 30°C for 2 d to 3 d; and single colonies were picked and inoculated on a slope for purification, and then stored at 4°C for later use.

Step 2. Total DNA extraction for a ZDO03 strain: bacteria at logarithmic growth phase were collected by centrifuging at 12,000 r/min for 1 min, and added with a lysis buffer and then with 5 mol/L NaCl; a resulting mixture was thoroughly mixed and centrifuged at 12,000 r/min for 10 min; a resulting supernatant was collected, and extraction was conducted twice with an equal volume of phenol/chloroform/isoamyl alcohol (at a volume ratio of 25:24:1); a resulting extract solution was centrifuged at 12,000 r/min for 10 min, and a resulting supernatant was collected; the supernatant was added with 2.5-fold volume of pre-cooled absolute ethanol, and a resulting mixture was thoroughly mixed, placed in a refrigerator at -20°C for 5 min, and centrifuged at 12,000 r/min for 15 min; a resulting supernatant was discarded, and a resulting precipitate was washed with 70% ethanol; a resulting mixture was centrifuged to make residual liquid gather at a bottom of a tube, and the liquid was removed with a pipette; and a resulting precipitate was air-dried and dissolved with 225 uL of TE Buffer, and a resulting solution was stored at 4°C for later use.

Step 3. Gene cloning: according to a known aiiA gene sequence on GenBank, 500402 highly-homologous regions of the sequence were used to design a pair of degenerate primers aiiF and aiiR (with BamHI and EcoRI enzyme digestion sites) for the amplification of the ZD03-aiiA gene.

aiiF: SEQ ID NO: 3 S'-ATG GGA TCC ATG ACA GTA AAG AAG CTT TAT-3’, (BamHI enzyme digestion site); and aiiR: SEQ ID NO.:4 5S'-GTC GAA TTC CTC AAC AAG ATA CTC CTA ATG-3", (EcoRI enzyme digestion site).

Step 4. PCR amplification: with aiiF and aiiR as primers and total DNA of the ZD03 strain as a template, the ZD03-aiiA gene was amplified by PCR, and an amplification product was subjected to agarose gel electrophoresis (as shown in FIG. 1). A result shows that a DNA fragment amplified from the ZD03 genome has a length of about 750 bp and there are obvious non-specific bands, which is in line with an expected result. Sequence analysis shows that the positive clone ZD03-aiiA gene sequence has a full length of 753 bp and a nucleotide sequence shown in SEQ ID NO ::

1.

There is one ORF in the sequence, which encodes a polypeptide that is composed of 250 amino acid residues and has an amino acid sequence shown in SEQ ID NO.: 2.

It is expected that the polypeptide has a molecular weight of 28.1 KDa and a protein isoelectric point of 4.78. An amino acid residue sequence deduced from the sequence includes a conserved domain (Lactamase-B) (34AA-235AA). This lays the foundation for recombinant expression of the sequence and its related activity research.

The above are only preferred examples of the present disclosure and thus do not limit the scope of the present disclosure. Any simple modification or change made according to the contents of the specification in the present disclosure, no matter whether it is directly or indirectly used in any other related technical fields, should be included within the protection scope of the present disclosure.

Claims

CLAIMS LU500402

1. À quorum sensing (QS) signal-regulating enzyme gene ZD03-aiiA, wherein the gene has a nucleotide sequence shown in SEQ ID NO.: 1.

2. A protein encoded by the QS signal-regulating enzyme gene ZDO03-aiiA according to claim 1, wherein the protein has an amino acid sequence shown in SEQ ID NO: 2.

3. A cloning method of the QS signal-regulating enzyme gene ZDO03-aiiA according to claim 1, comprising the following steps: (1) degenerate primer design and primer pair for the ZD03-aiiA gene: aiiF: SEQ ID NO : 3 5'-ATG GGA TCC ATG ACA GTA AAG AAG CTT TAT-3", and aiiR: SEQ ID NO.:4 5'-GTC GAA TTC CTC AAC AAG ATA CTC CTA ATG-3"; (2) total DNA extraction for a ZDO03 strain: collecting bacteria at logarithmic growth phase by centrifuging at 12,000 r/min for 1 min, adding a lysis buffer and then mol/L NaCl, thoroughly mixing, and centrifuging a resulting mixture at 12,000 r/min for 10 min; collecting a resulting supernatant, and conducting extraction twice with an equal volume of phenol/chloroform/isoamyl alcohol (at a volume ratio of 25:24:1); centrifuging a resulting extract solution at 12,000 r/min for 10 min, and collecting a resulting supernatant; adding 2.5-fold volume of pre-cooled absolute ethanol to the supernatant, thoroughly mixing, and placing a resulting mixture in a refrigerator at -20°C for 5 min; centrifuging the mixture at 12,000 r/min for 15 min, discarding a resulting supernatant, and washing a resulting precipitate with 70% ethanol; centrifuging to make residual liquid gather at a bottom of a tube, and removing the liquid with a pipette; and air-drying a resulting precipitate, dissolving the precipitate with 225 uL of TE Buffer, and storing a resulting solution at 4°C for later use; and (3) with aiiF and aiiR as primers and total DNA of the ZD03 strain as a template, conducting PCR amplification, subjecting an amplification product to agarose gel electrophoresis, and sequencing to obtain the ZD03-aiiA gene.