Lactobacillus plantarum LuxS protein, application thereof and lactobacillus plantarum like recombinant strain
阅读说明：本技术 类植物乳杆菌LuxS蛋白、其应用及类植物乳杆菌重组菌 (Lactobacillus plantarum LuxS protein, application thereof and lactobacillus plantarum like recombinant strain ) 是由 李平兰 刘蕾 谭春明 武瑞赟 于 2021-02-01 设计创作，主要内容包括：本发明涉及微生物技术领域,具体涉及类植物乳杆菌LuxS蛋白、其应用及类植物乳杆菌重组菌。本发明成功克隆得到类植物乳杆菌L-ZS9菌株中的luxS基因,并将该基因与过表达质粒pMG76e连接,导入类植物乳杆菌L-ZS9菌株中,成功构建了过表达工程菌株luxS-pMG76e-L-ZS9。通过对比过表达luxS基因的luxS-pMG76e-L-ZS9菌株和L-ZS9菌株的AI合成和生物膜形成能力等相关生理指标发现,luxS基因的过表达会促进类植物乳杆菌L-ZS9信号分子AI-2的合成,且可增强L-ZS9菌株的被膜形成能力。(The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum LuxS protein, application thereof and a lactobacillus plantarum like recombinant bacterium. The luxS gene in the L-ZS 9-like strain of lactobacillus plantarum is successfully cloned, and is connected with an overexpression plasmid pMG76e and introduced into the L-ZS 9-like strain of lactobacillus plantarum, so that an overexpression engineering strain luxS-pMG76e-L-ZS9 is successfully constructed. Through comparing the LuxS-pMG76e-L-ZS9 strain and L-ZS9 strain which over-express the LuxS gene with relevant physiological indexes such as AI synthesis and biofilm formation capability, the over-expression of the LuxS gene can promote the synthesis of a Lactobacillus plantarum L-ZS9 signal molecule AI-2 and can enhance the envelope formation capability of the L-ZS9 strain.)
1. The lactobacillus plantarum LuxS protein is characterized by having an amino acid sequence shown as SEQ ID No. 1.
2. The gene encoding the lactobacillus plantarum LuxS protein according to claim 1, having the nucleotide sequence shown in SEQ ID No. 2.
3. Biomaterial containing the gene according to claim 2, characterized in that it is an expression cassette, a vector or a recombinant microorganism.
4. Use of the lactobacillus plantarum LuxS protein of claim 1 or the gene of claim 2 or the biomaterial of claim 3 for enhancing the biofilm-forming ability of lactobacillus plantarum.
5. Use of the lactobacillus plantarum LuxS protein according to claim 1 or the gene according to claim 2 or the biomaterial according to claim 3 for enhancing the signal molecule AI-2 synthesizing capacity of lactobacillus plantarum.
6. Use of a lactobacillus plantarum LuxS protein according to claim 1 or a gene according to claim 2 or a biomaterial according to claim 3 for regulating the expression of the glutamate-protein co-transporter glutamate protein symporter, RNA pseudouridine synthase, N-acetylglucosaminyltransferase, acetyltransferase or methionine ABC transporter permease in lactobacillus plantarum.
7. The use according to any one of claims 4 to 6, wherein the Lactobacillus plantarum is Lactobacillus plantarum L-ZS 9.
8. The lactobacillus plantarum-like recombinant strain is characterized in that lactobacillus plantarum is used as a starting strain, the recombinant strain has the advantage that the expression level of LuxS protein is improved compared with the starting strain, and the LuxS protein has an amino acid sequence shown as SEQ ID No. 1.
9. The lactobacillus plantarum recombinant bacterium according to claim 8, wherein the outbreak is lactobacillus plantarum L-ZS9, and the recombinant bacterium contains a pMG76e expression vector of a LuxS protein encoding gene.
10. The method of constructing a recombinant bacterium according to claim 8 or 9, comprising the steps of:
(1) using the genome DNA of the lactobacillus plantarum L-ZS9 as a template, and adopting a primer shown in SEQ ID NO.3-4 to amplify the luxS gene;
(2) connecting the luxS gene to a pMG76e expression vector to construct an over-expression recombinant plasmid luxS-pMG76 e;
(3) the over-expression recombinant plasmid luxS-pMG76e is transformed into Lactobacillus plantarum L-ZS9, and the recombinant strain of the over-expression luxS gene Lactobacillus plantarum L-ZS9 is obtained.
Technical Field
The invention relates to the technical field of microorganisms, in particular to a lactobacillus plantarum LuxS protein, application thereof and a lactobacillus plantarum like recombinant bacterium.
Background
Biofilm is a growth mode of most bacteria in a natural state, is beneficial to resisting external environmental stress of thalli, and is regulated and controlled by a quorum sensing QS system in formation and development. However, the research on the biofilm is mostly focused on pathogenic bacteria, and the related research on the probiotic biofilm is very deficient.
The lactobacillus plantarum L-ZS9 is separated from the traditional fermented meat product, produces IIb bacteriocin, has an inhibiting effect on colorectal cancer cells, and has the potential of being developed as probiotics and leavening agents. The coated lactobacillus plantarum L-ZS9 has stronger heat resistance, acid resistance and cholate resistance than the floating state; the signal molecule AI-2 has the function of promoting the biofilm, and can relieve the inhibition of pepsin and trypsin on the initial biofilm, and also relieve the damage of the two on the formed biofilm. The discovery of the protein related to biofilm formation regulation of the lactobacillus plantarum L-ZS9 and the development of a high-activity enveloped probiotic preparation are of great significance for the production and utilization of probiotics.
Disclosure of Invention
The invention aims to provide a lactobacillus plantarum LuxS protein, application thereof and a lactobacillus plantarum like recombinant strain.
The invention carries out whole genome sequencing analysis on the lactobacillus plantarum L-ZS9, finds that L-ZS9 has a key gene luxS for synthesizing AI-2, has complete AI-2 synthesis pathway and does not have SAH hydrolase encoding gene sahH, indicates that the SAH metabolic pathway of L-ZS9 is unique, has a molecular basis for synthesizing AI-2, and verifies that L-ZS9 has the capacity of synthesizing and secreting AI-2, and the envelope formation of L-ZS9 has correlation with signal molecules AI-2 and luxS genes. The luxS gene of the Lactobacillus plantarum-like L-ZS9 strain is cloned, the Lactobacillus plantarum-like L-ZS9 engineering strain with the gene over-expression is constructed, and the biofilm formation capacity and the AI-2 synthesis capacity of the engineering strain are both obviously enhanced. Further, by analyzing the transcriptome and protein expression conditions of the engineering strain, genes which are differentially expressed in response to luxS gene overexpression in the lactobacillus plantarum L-ZS9 are found.
Specifically, the invention provides the following technical scheme:
in a first aspect, the invention provides a lactobacillus plantarum LuxS protein having an amino acid sequence shown as SEQ ID No. 1.
In a second aspect, the invention provides a gene encoding the lactobacillus plantarum LuxS protein, which has a nucleotide sequence shown as SEQ ID No. 2.
In a third aspect, the invention provides a biological material containing a gene encoding the lactobacillus plantarum LuxS-like protein, which is an expression cassette, a vector or a recombinant microorganism.
In a fourth aspect, the invention provides the application of the lactobacillus plantarum LuxS-like protein or the gene or the biological material in enhancing the biofilm formation capacity of lactobacillus plantarum.
In a fifth aspect, the invention provides the application of the lactobacillus plantarum LuxS protein or the gene or the biological material in enhancing the synthesis capacity of the lactobacillus plantarum-like signal molecule AI-2.
In a sixth aspect, the invention provides the use of the Lactobacillus plantarum LuxS-like protein or the gene or the biological material for regulating the expression of the glutamic-protein co-transporter glutamate, protein symporter, RNA pseudouridine synthase, N-acetylglucosaminyltransferase, acetyltransferase or methionine ABC transporter permease in Lactobacillus plantarum.
In the application, the lactobacillus plantarum is preferably lactobacillus plantarum L-ZS 9.
L-ZS9 of Lactobacillus plantarum is isolated from the fermented meat SAUCISSON SEC PUR of Belgium, and is now deposited in the China Committee for culture Collection of microorganisms (CGMCC, address: No.3 Hopkin West Lu 1 of the sunward area of Beijing, institute of microbiology, China academy of sciences, postal code 100101) with the deposit number of CGMCC No.11669, which is disclosed in the patent application CN 111374278A.
In a seventh aspect, the invention provides a lactobacillus plantarum-like recombinant bacterium, the strain takes lactobacillus plantarum-like bacteria as a starting bacterium, the recombinant bacterium has an improved expression level of a LuxS protein compared with the starting bacterium, and the LuxS protein has an amino acid sequence shown in SEQ ID No. 1.
Preferably, the starting bacterium is lactobacillus plantarum L-ZS9, and the recombinant bacterium contains a pMG76e expression vector of a LuxS protein encoding gene.
In an eighth aspect, the invention provides a method for constructing the lactobacillus plantarum recombinant bacterium, which comprises the following steps:
(1) using the genome DNA of the lactobacillus plantarum L-ZS9 as a template, and adopting a primer shown in SEQ ID NO.3-4 to amplify the luxS gene;
(2) connecting the luxS gene to a pMG76e expression vector to construct an over-expression recombinant plasmid luxS-pMG76 e;
(3) the over-expression recombinant plasmid luxS-pMG76e is transformed into Lactobacillus plantarum L-ZS9, and the recombinant strain of the over-expression luxS gene Lactobacillus plantarum L-ZS9 is obtained.
Specifically, the above construction method includes the steps of:
(1) designing a primer: designing luxS gene PCR amplification primers according to the luxS gene sequence of the lactobacillus plantarum L-ZS9, wherein the sequences are as follows:
luxS-F：5’-TGC TCTAGA ATGGCTAAAGTAGAAAGTTT-3’；
luxS-R：5’-CCG CTCGAG CTATTCAACGACTTTGCGAA-3’；
(2) carrying out PCR amplification on the luxS gene segment in the L-ZS9 genome of the lactobacillus plantarum by using the primer in the step (1);
(3) construction of the overexpression recombinant plasmid: connecting the linearized plasmid pMG76e fragment with the amplified luxS target gene by double digestion with pMG76e as an expression vector to construct an over-expressed luxS-pMG76e recombinant plasmid, and then verifying the recombinant plasmid;
(4) transformation of recombinant plasmid: preparing a Lactobacillus plantarum L-ZS9 competent cell, and introducing a recombinant plasmid luxS-pMG76e into a Lactobacillus plantarum L-ZS9 protoplast by an electric shock transformation method to obtain a luxS overexpression strain;
(5) and verifying the construction result of the luxS gene overexpression strain.
The construction of the overexpression recombinant plasmid specifically comprises the following steps:
a. firstly, linking the recovered product of the luxS gene gel with a pMD-18T vector, wherein the linking system comprises: 8 mu L of luxS gene glue recovery product, pMD-18T 2 mu L, Solution I10 mu L; the connection temperature is 18 ℃, and the connection time is 2 h;
b. chemically transforming the ligation product into escherichia coli DH5 alpha competent cells after ligation, carrying out shake culture at 37 ℃ for 60min, centrifuging 2mL of thallus at 6000rpm for 2min, removing 1.8mL of supernatant, suspending the thallus in the residual supernatant, uniformly coating the thallus in an LB solid plate containing 100 mu g/mL of ampicillin, carrying out culture at 37 ℃ until the thallus is visible to the naked eye, and screening out a positive monoclonal colony;
c. inoculating the screened positive strains into an LB liquid culture medium containing 100 mu g/mL ampicillin, carrying out shake culture at 37 ℃ for 10h, extracting recombinant plasmid luxS-pMD-18T by adopting a high-purity small-extraction medium-amount plasmid extraction kit, and carrying out agarose gel electrophoresis verification;
d. carrying out double enzyme digestion on the recombinant plasmid luxS-pMD-18T, wherein the enzyme digestion system comprises: plasmid luxS-pMD-18T 3. mu.L, XbaI Fastdigest 1. mu.L, XhoI Fastdigest 1. mu.L, 10 XBuffer 5. mu.L, ddH2O40 mu L; the enzyme digestion temperature is 22 ℃, and the enzyme digestion time is 10 min; after double enzyme digestion, agarose gel electrophoresis is carried out, and the luxS gene fragment is subjected to gel cutting and recovery;
e. plasmid pMG76e was subjected to a double digestion system comprising: plasmid pMG76e 5 μ L, XbaI Fastdigest 1 μ L, XhoI Fastdigest 1 μ L, 10 XBuffer 5 μ L, ddH2O38 mu L; the enzyme digestion temperature is 22 ℃, and the enzyme digestion time is 10 min; after the double enzyme digestion is finished, carrying out agarose gel electrophoresis, and carrying out gel cutting recovery on the linearized plasmid pMG76e fragment;
f. connecting the luxS gene segment recovered by cutting the gel with the linearized plasmid pMG76e segment, wherein the connecting system is as follows: 8 muL of luxS gene fragment, 2 muL of plasmid pMG76e 2 and 10 muL of Solution I; the connection temperature is 18 ℃, and the connection time is 2 h;
g. chemically transforming the ligation product into escherichia coli DH5 alpha competent cells after the ligation is finished; the transformation step is the same as the step b; after the transformation is finished, uniformly coating the strain in an LB solid plate containing 200 mu g/mL erythromycin, culturing at 37 ℃ for 48h, and screening positive monoclonal colonies;
h. and (3) inoculating the positive strain into an LB liquid culture medium containing 200 mu g/mL of erythromycin for amplification culture, extracting the plasmid luxS-pMG76e according to the method in the step c, and then performing double-enzyme digestion identification on the recombinant plasmid.
The specific steps for transformation of the recombinant plasmid described above are as follows:
a. mixing 1 mu L of recombinant plasmid luxS-pMG76e DNA and 20 mu L of Lactobacillus plantarum L-ZS9 competent cells, placing the mixture on ice, transferring the mixture into a 2mm electric shock cup, and carrying out ice bath for 5-10 min to ensure that no bubbles exist;
b. setting a click program: 1.5KV, 25 μ L, 400 Ω, clicking, and standing on ice for 5 min;
c. adding 0.5mL of MRSSM recovery culture medium, transferring the mixture into a 1.5mL centrifuge tube, and culturing for 2h at 37 ℃;
d. and centrifuging the recovered bacterial liquid at the normal temperature of 5000rpm for 5min, removing 400 mu L of supernatant, coating the supernatant on an MRS plate containing 3 mu g/mL erythromycin, and culturing at 37 ℃ for 36-48 h.
The specific steps for verifying the construction result of the luxS gene overexpression strain are as follows:
a. selecting a monoclonal colony, placing the colony in a test tube containing a MRS liquid culture medium containing 3 mu g/mL erythromycin, performing static culture at 37 ℃ for 36-48 h, and extracting a recombinant plasmid luxS-pMG76e according to the same method;
b. taking 4 microliter of recombinant plasmid to carry out electrophoresis detection on 1% agarose gel;
c. and (3) double enzyme digestion verification: and (3) carrying out enzyme digestion on the recombinant plasmid by utilizing XbaI Fastdigest and XhoI Fastdigest, and if the enzyme digestion product has two bands, and the size of the bands is consistent with the size of the plasmid and the size of a target fragment, successfully constructing the recombinant plasmid and the luxS gene overexpression strain.
The invention has the beneficial effects that:
the luxS gene in the L-ZS 9-like strain of lactobacillus plantarum is successfully cloned, and is connected with an overexpression plasmid pMG76e and introduced into the L-ZS 9-like strain of lactobacillus plantarum, so that an overexpression engineering strain luxS-pMG76e-L-ZS9 is successfully constructed.
Through comparing the LuxS-pMG76e-L-ZS9 strain and L-ZS9 strain which over-express the LuxS gene with relevant physiological indexes such as AI synthesis and biofilm formation capability, the over-expression of the LuxS gene can promote the synthesis of a Lactobacillus plantarum L-ZS9 signal molecule AI-2 and can enhance the envelope formation capability of the L-ZS9 strain.
qRT-PCR and differential transcriptome analysis show that more than 2 times of differential expression occurs to 35 genes in the recombinant strain luxS-pMG76e-L-ZS9, wherein 9 genes are up-regulated, and 26 genes are down-regulated; and the iTRAQ test shows that 35 proteins in the recombinant strain luxS-pMG76e-L-ZS9 are differentially expressed with the expression value of more than or equal to 1.2 or less than or equal to 0.83, wherein 11 proteins are down-regulated, and 24 proteins are up-regulated. The luxS gene overexpression influences the transport and membrane-associated proteins, the proportion of the luxS gene in the known function difference proteins is more than 50%, and the adaptation and response of thalli to the extracellular environment are regulated and controlled by regulating cell membrane proteins and transport proteins on the membrane, so that the formation of a biofilm is influenced. In addition, luxS gene overexpression affects the expression of AraC family transcriptional regulators, LacI family transcriptional regulators, and PadR transcriptional regulators, which are associated with biofilm formation and QS regulatory mechanisms.
Drawings
FIG. 1 is an electrophoretogram of a PCR product of luxS gene in example 1 of the present invention, wherein M represents DL5000 DNA Marker; luxS stands for the PCR amplification product of the luxS gene.
FIG. 2 is a schematic diagram showing the cleavage site analysis of the luxS gene of Lactobacillus plantarum L-ZS9 in example 1 according to the present invention.
FIG. 3 shows the results of PCR identification of the empty pMG76e vector-introduced strain in example 1 of the present invention, in which M represents DNA Marker and pMG76e represents the PCR amplification product of the empty pMG76e vector-introduced strain.
FIG. 4 is a PCR identification result of the luxS gene overexpression strain luxS-pMG76e-L-ZS9 in example 1 of the present invention, wherein M represents a DNA Marker, and luxS-pMG76e-L-ZS9 represents a PCR amplification product of the luxS-pMG76e-L-ZS9 strain.
FIG. 5 shows the transcription levels of the luxS gene of recombinant strain luxS-pMG76e-L-ZS9, empty-load strain pMG76e-L-ZS9 and wild-type strain L-ZS9 in example 2 of the present invention.
FIG. 6 shows AI-2 activity assays of recombinant strain luxS-pMG76e-L-ZS9, empty-carrier strain pMG76e-L-ZS9 and wild-type strain L-ZS9 in example 3 of the present invention.
FIG. 7 is a graph showing the biofilm-forming ability of recombinant strain luxS-pMG76e-L-ZS9, empty-carrier strain pMG76e-L-ZS9 and wild-type strain L-ZS9 in example 4 of the present invention.
FIG. 8 is the volcanic plot of the differentially expressed genes of the recombinant strains pMG76e-L-ZS9 and luxS-pMG76e-L-ZS9 in example 5 of the present invention.
FIG. 9 is the volcano plots of the differential proteins of the recombinant strains pMG76e-L-ZS9 and luxS-pMG76e-L-ZS9 in example 6 of the present invention.
FIG. 10 is a GO cell fraction analysis of the differential protein of example 6 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
EXAMPLE 1 construction of L-ZS9 luxS Gene-overexpressing Strain of Lactobacillus plantarum
The embodiment provides a lactobacillus plantarum L-ZS9 luxS gene overexpression strain and a construction method thereof, and the specific steps are as follows:
1. experimental Material
1.1 Experimental strains and plasmid vectors
Lactobacillus plantarum (Lactobacillus paracasei) L-ZS9 is separated from the fermented meat SAUCISSON SEC PUR in Belgium, and is currently preserved in China General Microbiological Culture Collection Center (CGMCC), with the preservation number of CGMCC No. 11669; vibrio harveyi (Vibrio harveyi) BB170 and BB152 were provided by Han Xian researchers of Shanghai veterinary institute of Chinese academy of agricultural sciences; escherichia coli (Escherichia coli) DH5 α was purchased from Takara Bio Inc.; the construction of the recombinant strain of the synthetic trehalose lactic acid bacteria and the evaluation of its stress resistance [ J ] biotechnology, 2013(02):29-34 ] of the vector pMG76e (Chengling, Wei-Yanjie, Zuofangli, etc.) were provided by Chenshangwu professor of the Chinese academy of agricultural sciences.
1.2 Primary reagents and materials
MRS culture medium, LB culture medium, culture medium for Vibrio harveyi, AB culture medium, glycine purchasing culture mediumPurchased from Qingdao Haibo Biometrics, Inc.; PrimeSTAR HS DNA Polymerase (R010Q), TaKaRa Ex Taq (RR001A), pMD 18-T Vector Cloning Kit (6011) from Takara-Bao bioengineering (Dalian) Inc.; t4 DNA Ligase (EL0021), protein quantification stain, Bovine Serum Albumin (Bovine Serum Albumin, BSA), DNase I (RNase-free) and RNase inhibitor were purchased from Thermo Scientific; a common agarose gel DNA recovery kit (DP209) and a high-purity plasmid small-extraction medium-volume kit (DP107) are purchased from Tiangen Biotechnology technology (Beijing) Co., Ltd; reverse transcription kits TUREscript 1st Strand cDNA Synthesis Kit and TRIpure were purchased from Beijing Edela Biotech Ltd; SYBGreen dye was purchased from Kapa Biosystems; RNeasy MinElute clear Kit is available from QIAGEN, Germany; Ribo-ZeroTMMagnetic Kit (Gram-Negative or Gram-positive Bacteria) was purchased from Epicentre, USA;UltraTM directed RNA Library Prep Kit for Illumina from NEB, USA; AMPure XP Beads are available from Agencour, USA; XbaI and XhoI rapid restriction endonucleases were purchased from Fermentas, USA; urea, CHAPS, was purchased from Bio-Rad, Inc., USA; protease Inhibitor Cocktail was purchased from Roche, usa; other reagents such as thiourea were purchased from Sigma-Aldrich, USA.
1.3 Main instruments
The main instrumentation is shown in table 1.
TABLE 1 Main instrumentation
2. Experimental methods
2.1 Strain culture and genome extraction
The activated strain L-ZS9 is inoculated in MRS liquid culture medium, and after shaking culture at 37 ℃ and 200rpm for 24h, the strain is collected by centrifugation at 12000g for 2 min. Then, after lysozyme wall breaking, bacterial genome DNA extraction kit (DP302) is adopted to extract the strain L-ZS9 genome DNA, and the strain L-ZS9 genome DNA is frozen and stored at the temperature of minus 20 ℃.
2.2 amplification of luxS Gene fragment
A luxS gene identification primer is designed by taking the luxS gene sequence of Lactobacillus plantarum WCFS1 in GenBank as a reference. The primer sequence is luxS-F': 5'-ATGGCTAAAGTAGAAAGTTT-3', luxS-R ': 5'-CTATTCAACGACT TTGCGTA-3' are provided. The luxS gene segment is amplified by taking the Lactobacillus plantarum L-ZS 9-like genome DNA as a template (figure 1), and an amplification system comprises: PrimeSTAR HS DNA Polymerase 15. mu.L, luxS-F and luxS-R each 1. mu.L, template 1. mu.L, sterile ddH2O12. mu.L. PCR reaction procedure: 3min at 95 ℃; 30s at 95 ℃, 30s at 55 ℃ and 1min at 72 ℃ (30 cycles); 10min at 72 ℃; storing at 4 ℃.
2.3 Whole genome sequencing and analysis
Constructing a library of the extracted Lactobacillus plantarum L-ZS 9-like genome DNA, and performing high-throughput sequencing on an Illumina Hiseq 2000 sequencing platform, wherein the sequencing depth is 311 x. All DNA reading short fragments are collected by SOAPdenovo, and the whole genome sequence of Lactobacillus plantarum WCFS1 in GenBank is taken as a reference genome, gap closure is carried out by SOAP GapCloser, and a large fragment (without gap) spliced whole genome draft is obtained. The gap between large fragments was filled by multiple rounds of PCR and Sanger sequencing. Rapid genome annotation was then performed using Subsystem Technology. After the complete genome map is generated, the full-length genome size, GC content, protein coding sequences CDSs (coding sequences), genes, rRNA operons and the like of the lactobacillus plantarum L-ZS9 are analyzed. Sequence size and position analysis was performed on the luxS gene based on the whole genome sequence and annotation.
2.4 Lactobacillus plantarum L-ZS9 luxS gene sequence analysis and enzyme cutting site analysis
Based on the analysis of the whole genome information of the lactobacillus plantarum L-ZS9, the luxS gene was mapped, and the fragment size and the cleavage site were analyzed (FIG. 2). Meanwhile, the restriction enzyme sites of the vector pMG76e were analyzed, and the appropriate restriction enzyme sites were selected to design the vector pMG76e identifying primer and luxS gene amplification primer (Table 2).
TABLE 2 primer sequences
2.5 construction of an overexpression vector, connecting the linearized plasmid pMG76e fragment with the amplified luxS target gene by double digestion with pMG76e as an overexpression vector to construct an overexpression luxS-pMG76e recombinant plasmid, and then verifying the recombinant plasmid, wherein the steps are as follows:
a. firstly, linking the recovered product of the luxS gene gel with a pMD-18T vector, wherein the linking system comprises: 8 mu L of luxS gene glue recovery product, pMD-18T 2 mu L, Solution I10 mu L; the connection temperature is 18 ℃, and the connection time is 2 h;
b. chemically transforming the ligation product into escherichia coli DH5 alpha competent cells after ligation, carrying out shake culture at 37 ℃ for 60min, centrifuging 2mL of thallus at 6000rpm for 2min, removing 1.8mL of supernatant, resuspending the thallus in the residual supernatant, uniformly coating the thallus in an LB solid plate containing 100 mu g/mL of ampicillin, carrying out culture at 37 ℃ until the thallus is visible to naked eyes, and screening out a positive monoclonal colony;
c. inoculating the screened positive strains into an LB liquid culture medium containing 100 mu g/mL ampicillin, carrying out shake culture at 37 ℃ for 10h, extracting recombinant plasmid luxS-pMD-18T by adopting a high-purity small-extraction medium-amount plasmid extraction kit, and carrying out agarose gel electrophoresis verification;
d. carrying out double enzyme digestion on the recombinant plasmid luxS-pMD-18T, wherein the enzyme digestion system comprises: plasmid luxS-pMD-18T 3. mu.L, XbaI Fastdigest 1. mu.L, XhoI Fastdigest 1. mu.L, 10 XBuffer 5. mu.L, ddH2O40 mu L; the enzyme digestion temperature is 22 ℃, and the enzyme digestion time is 10 min. After double enzyme digestion, agarose gel electrophoresis is carried out, and the luxS gene fragment is subjected to gel cutting and recovery;
e. plasmid pMG76e was subjected to a double digestion system comprising: plasmid pMG76e 5 μ L, XbaI Fastdigest 1 μ L, XhoI Fastdigest 1 μ L, 10 XBuffer 5 μ L, ddH2O38. mu.L. The enzyme cutting temperature is 22 ℃, and the enzyme cutting time is 10min; after the double enzyme digestion is finished, carrying out agarose gel electrophoresis, and carrying out gel cutting recovery on the linearized plasmid pMG76e fragment;
f. connecting the luxS gene segment recovered by cutting the gel with the linearized plasmid pMG76e segment, wherein the connecting system is as follows: 8 muL of luxS gene fragment, 2 muL of plasmid pMG76e 2 and 10 muL of Solution I; the connection temperature is 18 ℃, and the connection time is 2 h;
g. chemically transforming the ligation product into escherichia coli DH5 alpha competent cells after the ligation is finished; the transformation procedure was as described above; after the transformation is finished, uniformly coating the strain in an LB solid plate containing 200 mu g/mL erythromycin, culturing at 37 ℃ for 48h, and screening positive monoclonal colonies;
h. the positive strains are inoculated into LB liquid culture medium containing 200 mug/mL of erythromycin for amplification culture, the plasmid luxS-pMG76e is extracted according to the same method, and then the recombinant plasmid is subjected to double enzyme digestion identification.
2.6 competent preparation of Lactobacillus plantarum L-ZS9
a. Add 500. mu.L of 20% glycine stock to 9.5mL of MRSS (MRSS +0.3M glucose); inoculating Lactobacillus plantarum L-ZS9 to the above culture medium at an inoculation amount of 1%, and allowing the strain to grow to OD600nmAround 0.6;
b. placing the cultured bacterial liquid into a 4mL centrifuge tube, centrifuging at 5000rpm and 4 ℃ for 10min, and collecting thalli;
c. remove supernatant, add 2mL Washing Buffer (0.3M sucrose, 0.1M MgCl)2) Washing the precipitate, centrifuging at 5000rpm and 4 deg.C for 10min, and collecting thallus; repeating the steps;
d. removing supernatant, adding 2mL of 30% PEG-1500, resuspending, centrifuging at 5000rpm and 4 deg.C for 10 min;
e. removing supernatant, adding 200 μ L30% PEG-1500, resuspending, dividing into 20 μ L tubes, and placing on ice or quick freezing with liquid nitrogen and storing in-80 deg.C refrigerator.
2.7 transformation of recombinant plasmids
The overexpression plasmid is introduced into a Lactobacillus plantarum L-ZS 9-like competent cell by an electric shock transformation method to obtain a pMG76e no-load introduction strain (figure 3) and a luxS overexpression strain (figure 4), and the specific implementation steps are as follows:
a. mixing 1. mu.L of recombinant plasmid luxS-pMG76e DNA with 20. mu.L of competent cells, placing on ice, transferring into a 2mm electric shock cup, and carrying out ice bath for 6min to ensure that no bubbles exist;
b. setting a click program: 1.5KV, 25 μ L, 400 Ω; after clicking, standing for 5min on ice;
c. adding 0.5mL of MRSSM recovery culture medium, transferring the mixture into a 1.5mL centrifuge tube, and culturing for 2h at 37 ℃;
d. centrifuging the recovered bacteria liquid at the normal temperature of 5000rpm for 5min, removing 400 mu L of supernatant, coating the supernatant on an MRS plate containing 3 mu g/mL erythromycin, and culturing at 37 ℃ for 48 h.
2.8 validation of construction results of luxS Gene overexpression Strain
a. Picking a monoclonal colony, placing the colony in a test tube containing a MRS liquid culture medium containing 3 mu g/mL erythromycin, standing and culturing at 37 ℃ for 48h, and extracting the recombinant plasmid luxS-pMG76e according to the same method;
b. taking 4 microliter of recombinant plasmid to carry out electrophoresis detection on 1% agarose gel;
c. and (3) double enzyme digestion verification: the recombinant plasmid is digested by XbaI Fastdigest and XhoI Fastdigest, if the digestion product has two bands, and the size of the bands is consistent with the size of the plasmid and the size of a target fragment, the successful construction of the recombinant plasmid is proved, and the construction of the over-expressed luxS gene of the lactobacillus plantarum luxS-pMG76e-L-ZS9 is successful.
Example 2 expression analysis of the luxS Gene in the luxS-pMG76e-L-ZS9 Strain
qRT-PCR is utilized to verify the expression condition of the luxS gene in the successfully constructed luxS-pMG76e-L-ZS9 strain for over-expressing the luxS gene, and the details are as follows:
1. experimental methods
a. Inoculating a lactobacillus plantarum L-ZS9 wild strain, an empty vector pMG76e introduced strain and a luxS overexpression recombinant strain into an MRS culture medium, and standing and culturing for 8 hours at 37 ℃; after the culture is finished, centrifuging 12,000g to collect thalli, grinding by liquid nitrogen, adding TRIpure, and freezing and storing at-80 ℃;
b. extracting total RNA by referring to a TRIpure extraction step of Beijing Erdela Biotechnology GmbH;
c, synthesizing cDNA according to the specification of TUREscript 1st Strand cDNA Synthesis Kit of the company, wherein the specific method comprises the following steps: 50 ng-5 μ g of Total RNA, 1 μ L of Random Primer, 4 μ L of 5 × RT Reaction Mix, 1 μ L of TURescript H-RTase, and then diluting to 20 μ L with RNase free H2O; incubating at 25 ℃ for 10min, then incubating at 42 ℃ for 30-50 min, and heating at 65 ℃ for 15min to inactivate TURescript H-Rtase; treating the synthesized cDNA with DNase I, and then placing at-80 ℃ for later use;
d. the luxS gene sequence was analyzed with reference to the whole genome sequence of strain L-ZS9, 16s RNA was used as an internal reference gene, primers were designed using Primer 3Input (version0.4.0) for qRT-PCR, and the Primer sequences are shown in Table 3:
TABLE 3 primer sequences
qRT-PCR reaction system: mu.L of template cDNA, 10. mu.L of 2 XSSYB green, 1. mu.L each of the forward and reverse primers (10. mu. mol/L), and 7. mu.L of RNase-free water. The amplification procedure was: 10min at 95 ℃; at 95 ℃ for 15s and at 60 ℃ for 30s for 40 cycles; analyzing data by adopting a Livak method;
2. results of the experiment
The fold expression of the luxS gene of the luxS overexpressing recombinant strain was determined by qRT-PCR (FIG. 5), the empty vector pMG76e had no effect on the luxS gene expression of strain L-ZS9, while the transcription level of the luxS gene of the recombinant overexpressing strain luxS-pMG76e-L-ZS9 was significantly up-regulated. Meanwhile, the success of the construction of the overexpression strain is further illustrated.
Example 3 Effect of luxS Gene overexpression on the ability of the Strain L-ZS9 to synthesize the Signal molecule AI-2
1. Experimental methods
a. Preparing a supernatant: inoculating a lactobacillus plantarum L-ZS9 wild strain, an empty vector pMG76e introduced strain and a luxS overexpression recombinant strain into a skim milk culture medium, collecting bacterial liquids for 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 hours respectively, preparing a supernatant, and freezing and storing at-80 ℃; preparing Vibrio harveyi BB152 supernatant as a positive control, preparing E.coli DH5 alpha supernatant as a negative control, and freezing and storing at-80 ℃;
b. detection of signal molecules: inoculating Vibrio harveyi BB170 into an MB culture medium, and carrying out shaking culture at the temperature of 28 ℃ and the rpm of 200 until yellow-green fluorescence can be seen in a dark room; inoculating the enzyme-linked immunosorbent assay solution into a freshly prepared AB culture medium according to the ratio of 1:5000, adding the prepared supernatant according to the proportion of 10% (v/v), carrying out shaking culture at 28 ℃ and 200rpm for 5h, taking 100 mu L of the supernatant to a white enzyme-linked immunosorbent assay plate, detecting the luminous intensity by using a multifunctional enzyme-linked immunosorbent assay bioluminescence mode, and repeating for 5 holes; 3 biological replicates were performed;
2. results of the experiment
Since the luxS gene is responsible for the synthesis of the signal molecule AI-2, AI-2 activity in the supernatant of the over-expressed recombinant strain luxS-pMG76e-L-ZS9 was tested, and the test results (FIG. 6) show that the introduction of the empty vector pMG76e has no effect on AI-2 activity in the supernatant of the strain L-ZS9, but that the over-expression of the luxS gene can significantly up-regulate AI-2 production, indicating that the over-expression of the luxS gene can significantly enhance AI-2 synthesis ability of the strain L-ZS 9.
Example 4 Effect of luxS Gene overexpression on the biofilm Forming ability of the Strain L-ZS9
1. Experimental methods
The biofilm formation capacity is detected by adopting a crystal violet staining method, which comprises the following steps: inoculating an activated lactobacillus plantarum L-ZS9 wild strain, an empty vector pMG76e introduced strain and a luxS overexpression recombinant strain into an MRS culture medium, adding a culture solution into a 96-well cell culture plate, keeping stand at 37 ℃ for 36 hours, wherein each well contains 200 mu L of the culture solution; after the culture is finished, the cells are washed by PBS for 3 times, suspended bacteria are removed, and the cells are dried at room temperature; adding 0.1% crystal violet solution into each well, dyeing at room temperature for 30min, washing with sterile water until the lotion is colorless, and air drying at room temperature; adding 100 mu L of absolute ethyl alcohol to fully dissolve the crystal violet; the absorbance value of OD595nm was measured.
2. Results of the experiment
The biofilm-forming ability of each of the above strains was examined by crystal violet staining, and the results (FIG. 7) showed that the introduction of the empty vector pMG76e had no effect on the biofilm-forming ability of strain L-ZS 9. However, luxS overexpression enhanced biofilm formation by strain L-ZS 9.
Example 5 Effect of luxS Gene overexpression on the transcriptional information of the L-ZS9 Gene of Strain
1. Experimental methods
1.1 transcriptome sequencing
a. Total RNA extraction, quality inspection and purification: inoculating the lactobacillus plantarum L-ZS9 empty vector pMG76e introduced strain and the luxS overexpression recombinant strain into an MRS culture medium, and standing and culturing for 8 hours at 37 ℃;
b. after completion of the culture, 12,000g of the cells were collected by centrifugation, ground with liquid nitrogen, and TRIpure was added. Extracting total RNA according to TRIpure extraction step of Beijing Erdela biotechnology, Inc.;
c. the extracted RNA was subjected to quality inspection by agarose gel electrophoresis and 2100 bioanalyzer. 10 μ L of qualified total RNA was digested with 5U of DNase I for 30min at 37 ℃ and then purified with the RNeasy MinElute clear Kit, eluting with 15 μ L of RNase-free water;
DNase digested RNA Using Ribo-ZeroTMThe Magnetic Kit (Gram-Negative or Gram-positive) removes rRNA: adding Ribo-Zero Reaction Buffer and Ribo-Zero rRNA Removal Solution (Gram-Negative Bacteria or Gram-positive Bacteria), complementing the volume to 40 mu L, reacting at 68 ℃ for 10min, then placing at room temperature for 5min, adding the treated RNA into pre-washed magnetic beads, immediately and fully mixing, placing at room temperature for 5min, then reacting at 50 ℃ for 5min, immediately placing on a magnetic frame for more than 1min, sucking the supernatant, adding water to complement to 180 mu L, adding 3M Sodium Acetate and Glycogen (10mg/mL), adding 600 mu L of absolute ethyl alcohol, placing at-20 ℃ for more than 1h, centrifuging to obtain a precipitate, and adding water to dissolve to obtain rRNA-deleted RNA.
1.2RNA-Seq library construction
100ng of rRNA-deppleted RNA was taken and NEB was usedUltraTM directed RNA Library Prep Kit for Illumina construction of libraries: adding Random Primers and First Strand Synthesis Reaction Buffer (5X) to break mRNA, reacting at 94 deg.C for 15min, and rapidly placing on ice; murine RNase Inhibitor, Actinomycin D (0.1. mu.g/. mu.L) and ProtoScript II Reverse Transcri were addedperforming cDNA first strand synthesis by ptase, wherein the reaction program is 25 ℃ for 10min, 42 ℃ for 15min and 70 ℃ for 15 min; second Strand cDNA Synthesis was performed by adding Second Strand Synthesis Reaction Buffer with dUTP Mix (10 ×) and Second Strand Synthesis Enzyme Mix, and reacting at 16 ℃ for 1 h; purifying by using AMPure XP Beads (Agencourt, USA), adding End Repair Buffer (10 x) and End Prep Enzyme Mix for End Repair, wherein the Reaction program is 20 ℃ for 30min and 65 ℃ for 30 min; adding NEBNext adapter and Blunt/TA Ligase Master Mix junction, and reacting at 20 ℃ for 15 min; adding AMPure XP Beads, uniformly mixing, standing for 5min, placing on a magnetic frame for 5min, carefully absorbing supernate, adding the AMPure XP Beads, and purifying to obtain ligated cDNA with the range of 300-500 bp; then carrying out PCR amplification, wherein the primers are Universal PCR Primer and index (X) Primer, and simultaneously adding USER Enzyme, and the PCR reaction conditions are 37 ℃ for 15min, 98 ℃ for 10Sec, 65 ℃ for 30Sec and 72 ℃ for 30 Sec; and finally, purifying by adopting AMPure XP Beads to obtain a library for sequencing.
1.3 library quality inspection and sequencing
After the library construction is finished, triple inspection, namely Qubit quantification, 2% agarose gel electrophoresis detection and High-sensitivity DNA chip detection is carried out to ensure the quality of the library. Then performing bidirectional sequencing by adopting an Illumina Hiseq TM 4000(PE150) platform, wherein the data volume is 2-3G.
1.4 data analysis
a. Data volume statistics and quality assessment
The original image Data file obtained by high-throughput sequencing is converted into an original sequencing sequence, namely Raw Data or Raw Reads, through CASAVA Base recognition (Base Calling) analysis; performing quality filtration on the Raw Reads to generate Filtered Reads;
b. analysis of Gene expression
Mapping each sample Filtered reads with a reference genome by using Tophat, and then calculating the count and RPKM value of each gene by using a Cufflinks program and relying on a GTF annotation file of the reference genome according to a Tophat comparison result; differential expression analysis of the treatment group and the control group is carried out by Cuffdiff, and differential expression genes more than 2 times are screened out. Performing cluster analysis on the differentially expressed genes according to the RPKM value;
GO classification, enrichment analysis and KEGG analysis
Performing classification statistics on differential genes according to an annotation list on a GO database, and performing GO enrichment analysis on differentially expressed genes between two samples by using super-geometric distribution; performing pathway classification statistics on differentially expressed genes, and performing KEGG enrichment analysis on the differentially expressed genes by using hyper-geometric distribution according to the classification result of the KEGG.
2. Results of the experiment
Differential expression gene analysis showed (FIG. 8) that 35 genes of recombinant strain luxS-pMG76e-L-ZS9 were differentially expressed by more than 2 fold compared to control strain pMG76e-L-ZS9, 9 of which were up-regulated and 26 of which were down-regulated. Of these genes, 6 genes were expressed only in strain luxS-pMG76e-L-ZS9, and 4 genes were expressed only in strain pMG76e-L-ZS 9. The specific gene numbers, differential expression fold and encoded proteins are shown in Table 4.
TABLE 4 differentially expressed Gene information from transcriptome analysis
Example 6 Effect of luxS Gene overexpression on Strain L-ZS9 protein expression
1. Experimental methods
1.1 sample protein extraction and quantitation
Grinding the centrifuged thallus by liquid nitrogen, adding the powder into lysate lysine Buffer (7M urea, 2M thiourea, 4% CHAPS, tablet/50 mL Protease Inhibitor Cocktail) according to a ratio of 1:10(W/V), and uniformly mixing by vortex; ultrasonic 60s, 0.2s on, 0.2s off, amplitude 22%; extracting at room temperature for 30 min; centrifuging at 10 deg.C for 1h at 15,000g, taking out supernatant, packaging, and freezing at-80 deg.C.
The Bradford method was used to determine the protein concentration extracted from the samples: diluting a sample by a certain multiple with lysine Buffer (7M urea, 2M thiourea and 0.1% CHAPS) to enable the final concentration to fall within a standard curve range, taking 10 mu L of each diluted sample and a standard product (BSA is dissolved into standard protein with a series of concentrations by the lysine Buffer) to react with 300 mu L of protein quantitative dye in a dark place for 20min, simultaneously measuring the light absorption values of the standard product and the sample at 595nm by an enzyme labeling instrument, and drawing a standard curve according to the relation between the light absorption value and the concentration of each tube of the standard product. And calculating the protein concentration of each sample according to a curve formula.
1.2 proteolytic and iTRAQ labelling
Adding 25mM DTT into 200 μ g of protein solution, and reacting at 60 deg.C for 1 h; then, 50mM iodoacetamide was added thereto and the reaction was carried out at room temperature for 10 min. Putting the protein solution after reductive alkylation into a 10K ultrafiltration tube, centrifuging for 20min at 12,000g, and discarding the solution at the bottom of the collection tube; adding 100 μ L of precipitation Buffer, centrifuging at 12,000g for 20min, discarding the solution at the bottom of the collection tube, and repeating for 3 times; changing the collection tube, adding trypsin with the total amount of 4 mug (1: 50 of the weight ratio of the trypsin to the protein) into the ultrafiltration tube, and reacting overnight at 37 ℃; centrifuging at 12,000g for 20min to collect peptide fragments after enzymolysis and digestion; adding 50 μ L Dissolution Buffer, centrifuging at 12,000g for 20min, mixing with the previous step, and collecting the sample after enzymolysis at the bottom of the tube.
Taking out iTRAQ reagent from refrigerator, balancing at room temperature, and centrifugingReagent is added to the bottom of the tube; to each tubeAdding 150 mu L of isopropanol into the reagent, and centrifuging to the bottom of the tube after vortex oscillation; taking 50 mu L of sample, namely 100 mu g of enzymolysis product, and transferring the sample into a new centrifuge tube; adding an iTRAQ reagent into a sample, carrying out vortex oscillation, centrifuging to the bottom of the tube, and reacting at room temperature for 2 h; 100 μ L of water was added to terminate the reaction; mu.L of each sample was mixed, desalted with Ziptip, and subjected to MALDI-TOF(AB SCIEX 4800Plus) to confirm that the labeling reaction is successful; the mixed labeled sample is vortexed and centrifuged to the bottom of the tube. Then vacuum freezing, centrifugal drying.
1.3 offline Pre-separation of enzymatic peptide fragments and LC-MS/MS mass spectrometry
a. Reverse phase chromatographic separation at high pH
The mixed labeled sample was dissolved with 100. mu.L of mobile phase A1, centrifuged at 14,000g for 20min, and the supernatant was collected for use. The conditions of the system were measured by separating 400. mu.g of BSA after enzymatic hydrolysis (column temperature 45 ℃ C., detection wavelength 214 nm). A100. mu.L aliquot was taken and loaded. The flow rate was 0.7mL/min and the separation gradient was as shown in Table 5.
TABLE 5 reversed phase chromatographic separation gradient
b. Protein analysis by nano-upgrading reversed phase chromatography-Q active
The high pH reverse phase separated fractions were reconstituted with 20. mu.L of methanol (2%) and formic acid (0.1%). Centrifuge at 12,000g for 10min and aspirate the supernatant. The sample was loaded by the sandwich method in a volume of 10. mu.L. The Loading Pump flow rate was 350nL/min, 15 min. The separation flow rate was 300nL/min, and the separation gradient was as shown in Table 6.
TABLE 6 reversed phase chromatographic separation gradient
1.4 Mass Spectrometry data analysis and statistics of quantitative information of differential proteins
The database in this study is the NCBI database (http:// www.ncbi.nlm.nih.gov /), the local version of the database is Lactobacillus Paraplantrum _ pro database. RAW is processed by searching a library by Mascot software, and quality control is carried out on the result of searching the library by adopting scaffold software. And (3) performing group t test on the protein quantitative data of the experimental control group and the experimental group samples, and screening the differential protein with the P value less than or equal to 0.5 and the protein ratio greater than or equal to 1.2 or the ratio less than or equal to 0.83 in the 2 groups of samples.
1.5 hierarchical clustering, functional annotation and functional Classification of Difference proteins
Hierarchical clustering analysis (Hierarchical clustering) was performed on the protein quantification information using perseus software. Centralizing each protein quantitative value and eliminating data deviation; and calculating Euclidean distance between the proteins to obtain a correlation matrix thereof, and realizing hierarchical clustering.
GO annotation analysis was performed on the differential proteins using Blast2GO software, including annotation information such as GO classification and pathway. Due to the limitations of the background annotation libraries, not all proteins will have the corresponding annotation information available.
2. Results of the experiment
A total of 1630 proteins were identified in the iTRAQ assay. 35 differentially expressed proteins with the expression rate of more than or equal to 1.2 or less than or equal to 0.83, 11 proteins with down-regulation and 24 proteins with up-regulation compared with pMG76e-L-ZS9 were expressed in luxS-pMG76e-L-ZS9 (FIG. 9). In the Volcano Volcano Plot, the green dots represent the differential protein with the expression of luxS-76e-L-ZS9 down-regulated, and the red dots represent the differential protein with the expression of luxS-76e-L-ZS9 up-regulated. Specific information of the differential proteins, including protein product name, accession number, molecular weight and fold differential expression, are shown in table 7.
TABLE 7 differentially expressed protein message
GO classification analysis was performed on the differential proteins (fig. 10), 11 of the 35 differentially expressed proteins were membrane proteins, accounting for 35.48%, and 10 were membrane fraction proteins, accounting for 32.26%. The proportion of the membrane protein or the membrane-associated protein in the differentially expressed protein is the largest, which shows that the LuxS gene overexpression has the most obvious influence on the thallus membrane protein and the membrane-associated protein. Meanwhile, luxS gene overexpression affects the expression of AraC family transcriptional regulators, LacI family transcriptional regulators, and PadR transcriptional regulators, which are associated with biofilm formation and QS regulatory mechanisms.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
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