CN103255204B - Method for detecting whether quorum sensing system exists in type II bacteriocin producing strain - Google Patents
Method for detecting whether quorum sensing system exists in type II bacteriocin producing strain Download PDFInfo
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Abstract
The invention discloses a PCR primer set used in identifying or auxiliary identifying of whether a quorum sensing system related gene exists in a type II bacteriocin producing strain requiring detection. The PCR primer set is composed of 18 primers respectively composed of DNA single chains represented by Sequence 1, Sequence 2, Sequence 3, Sequence 4, Sequence 5, Sequence 6, Sequence 7, Sequence 8, Sequence 9, Sequence 10, Sequence 11, Sequence 12, Sequence 13, Sequence 14, Sequence 15, Sequence 16, Sequence 17 and Sequence 18 in the sequence list. According to the invention, quorum sensing system three-component gene is amplified through PCR, such that the existence of quorum sensing system is determined. Therefore, large-scale high-flux screening of strains regulated by quorum sensing from type II bacteriocin is realized.
Description
Technical field
The present invention relates to a kind of for the identification of or assistant identification II Bacteriocin-like Substance Producing Strain to be measured whether there is the PCR primer group of intervention school-based genes involved, and utilize this PCR primer group to identify or whether assistant identification II Bacteriocin-like Substance Producing Strain to be measured exists the method for intervention school-based.
Background technology
Quorum sensing (Quorum Sensing, QS) refers to that the expression of some gene of microbial population is subject to the phenomenon of the signaling molecule regulation and control relevant to population density.QS system participates in a series of biological function of regulation and control, as: thalline luminescence, antibiotic biosynthesizing, the generation of virulence factor, the synthesis of exocellular polysaccharide, bacterium gather together, biofilm formation, plasmid conjugal transfer, the entering of stable growth phase.
QS system is considered to the synthesis participating in regulation and control II bacterioid element, and this system comprises self-induction peptide (autoinducing peptide, AIP), Protein histidine kinase and induction Function protein, is also called three-component system.AIP, as signaling molecule, is used to indicate cell density.When AIP reaches a certain critical concentration threshold, the expression of a series of specific gene just can be activated.
Lactobacillus plantarum WCFS1, J51 and L.plantarum C11 have identical Quorum sensing, and this system is made up of the gene plnB of the gene plnA of self-induction peptide of encoding, encoding histidine protein kinase, gene plnC and plnD of coding induction Function protein.L.plantarum NC8 has similar operon to L.plantarum C11, wherein plNC8IF coding self-induction peptide, plNC8HK encoding histidine protein kinase, plnD coding induction Function protein.
To the study general of G-quorum sensing, by detecting, its signaling molecule---homoserine lactone class confirms, and the existing a lot of report of its detection method and patent.Quorum sensing is the important mechanisms of regulation and control II bacterioid element synthesis, and produce II bacterioid element milk-acid bacteria using little peptide as its signaling molecule, but its detection method is not yet established at present, this also becomes the huge obstacle limiting the research of its quorum sensing.Although found many product II bacterioids element milk-acid bacteria, whether the generation of its bacteriocin is subject to quorum sensing regulation and control has not yet formed a set of effective detection method.
Summary of the invention
An object of the present invention is to provide a kind of for the identification of or assistant identification II Bacteriocin-like Substance Producing Strain to be measured whether there is the PCR primer group of intervention school-based genes involved.
PCR primer group provided by the present invention is made up of 9 primer pairs, the primer pair 1 being specific to self-induction peptide gene that first primer pair is made up of two single stranded DNAs shown in the sequence 1 in sequence table and sequence 2; The primer pair 2 being specific to self-induction peptide gene that second primer pair is made up of two single stranded DNAs shown in the sequence 3 in sequence table and sequence 4; The primer pair 3 being specific to Protein histidine kinase gene that 3rd primer pair is made up of two articles of single stranded DNAs shown in the sequence 5 in sequence table and sequence 6; 4th primer pair consists of by two articles of single stranded DNAs shown in the sequence 7 in sequence table and sequence 8 primer pair 4 being specific to Protein histidine kinase gene; The primer pair 5 being specific to Protein histidine kinase gene that 5th primer pair is made up of two articles of single stranded DNAs shown in the sequence 9 in sequence table and sequence 10; The primer pair 6 being specific to induction Function protein gene that 6th primer pair is made up of two articles of single stranded DNAs shown in the sequence 11 in sequence table and sequence 12; The primer pair 7 being specific to induction Function protein gene that 7th primer pair is made up of two articles of single stranded DNAs shown in the sequence 13 in sequence table and sequence 14; The primer pair 8 being specific to induction Function protein gene that 8th primer pair is made up of two articles of single stranded DNAs shown in the sequence 15 in sequence table and sequence 16; The primer pair 9 being specific to Protein histidine kinase gene and induction Function protein gene that 9th primer pair is made up of two articles of single stranded DNAs shown in the sequence 17 in sequence table and sequence 18.
Test kit containing above-mentioned PCR primer group also belongs to protection scope of the present invention.
The preparation method of described PCR primer group also belongs to protection scope of the present invention.This preparation method comprises the step of individually being packed by described two single stranded DNAs of each primer pair in described PCR primer group.
The preparation method of above-mentioned PCR kit also belongs to protection scope of the present invention.After this preparation method comprises the steps: described two single stranded DNAs of each primer pair in described PCR primer group individually to pack, be packaged in same reagent box with following substances: PCR reaction buffer, archaeal dna polymerase, 4 kinds of dNTP and ddH
2o.
Whether above-mentioned PCR primer group is also belonging to protection scope of the present invention containing the application in the test kit of intervention school-based genes involved for the preparation of qualification or assistant identification II Bacteriocin-like Substance Producing Strain to be measured.Described intervention school-based genes involved is self-induction peptide gene, Protein histidine kinase gene and induction Function protein gene.
The method whether qualification or assistant identification II Bacteriocin-like Substance Producing Strain to be measured contain intervention school-based genes involved also belongs to protection scope of the present invention.Described intervention school-based genes involved is self-induction peptide gene, Protein histidine kinase gene and induction Function protein gene.
Described method comprises the step of following (1) and (2):
Described (1) is following A)-I):
A) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 1;
B) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 2;
C) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 3;
D) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 4;
E) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 5;
F) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 6;
G) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 7;
H) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 8;
I) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with described primer pair 9;
(2) size of PCR primer that obtains of detecting step (1), determine that whether described II Bacteriocin-like Substance Producing Strain to be measured is containing described intervention school-based genes involved, i.e. described self-induction peptide gene, described Protein histidine kinase gene and described induction Function protein gene according to PCR primer as follows:
The DNA fragmentation of if the PCR primer of primer pair 1 contains (or for) 450bp, and/or the PCR primer of primer pair 2 contains the DNA fragmentation of (or for) 114bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains self-induction peptide gene or candidate contains self-induction peptide gene; If the DNA fragmentation of the PCR primer of primer pair 1 not containing 450bp, the DNA fragmentation of the PCR primer of primer pair 2 also not containing 114bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain self-induction peptide gene containing self-induction peptide gene or candidate; The DNA fragmentation of if the PCR primer of primer pair 3 contains (or for) 165bp, and/or the PCR primer of primer pair 4 contains the DNA fragmentation of (or being) 758bp, and/or the PCR primer of primer pair 5 contains the DNA fragmentation of (or being) 927bp, and/or the PCR primer of primer pair 9 contains the DNA fragmentation of (or for) 1727bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains Protein histidine kinase gene or candidate contains Protein histidine kinase gene; If the DNA fragmentation of the PCR primer of primer pair 3 not containing 165bp, the DNA fragmentation of the PCR primer of primer pair 4 not containing 758bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 5 not containing 927bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 9 not containing 1727bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain Protein histidine kinase gene containing Protein histidine kinase gene or candidate; The DNA fragmentation of if the PCR primer of primer pair 6 contains (or for) 108bp, and/or the PCR primer of primer pair 7 contains the DNA fragmentation of (or being) 414bp, and/or the PCR primer of primer pair 8 contains the DNA fragmentation of (or being) 1249bp, and/or the PCR primer of primer pair 9 contains the DNA fragmentation of (or for) 1727bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains induction Function protein gene or candidate contains induction Function protein gene; If the DNA fragmentation of the PCR primer of primer pair 6 not containing 108bp, the DNA fragmentation of the PCR primer of primer pair 7 not containing 414bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 8 not containing 1249bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 9 not containing 1727bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain induction Function protein gene containing induction Function protein gene or candidate
The method whether above-mentioned qualification or assistant identification II Bacteriocin-like Substance Producing Strain to be measured contain intervention school-based genes involved is utilized also to belong to protection scope of the present invention in the application that acquisition exists or candidate exists in the II Bacteriocin-like Substance Producing Strain of intervention school-based.
Described being applied as meets following a)-c using what utilize aforesaid method to identify to obtain) the II Bacteriocin-like Substance Producing Strain of condition is as to exist or candidate exists the II Bacteriocin-like Substance Producing Strain of intervention school-based:
A) containing or candidate contain self-induction peptide gene;
B) containing or candidate contain Protein histidine kinase gene;
C) containing or candidate contain induction Function protein gene.
Described II Bacteriocin-like Substance Producing Strain is milk-acid bacteria, can be Bacterium lacticum (as plant lactobacillus (L.plantarum)), is specially class plant lactobacillus (L.paraplantarum) L-XM1 in an embodiment of the present invention.
The present invention by the gene of pcr amplification intervention school-based three components, thus confirms the existence of intervention school-based, and this makes the bacterial strain that extensive high flux screening regulates and controls by quorum sensing from II bacterioid element become possibility.
Accompanying drawing explanation
Fig. 1 is the schema whether qualification or assistant identification II Bacteriocin-like Substance Producing Strain to be measured exist intervention school-based genes involved.Wherein, QS represents intervention school-based.
Fig. 2 is the electrophoresis result whether qualification or assistant identification class plant lactobacillus (L.paraplantarum) L-XM1 exist intervention school-based.Wherein, A is the electrophorogram of the self-induction peptide gene specific fragment that primer pair 1 amplifies; B is the electrophorogram of the Protein histidine kinase gene-specific fragment amplified with primer pair 3; C is the electrophorogram of the Protein histidine kinase gene-specific fragment amplified with primer pair 5; D is with primer pair 9 the Protein histidine kinase gene amplified and the electrophorogram responding to Function protein gene-specific fragment; E is the electrophorogram of the induction Function protein gene-specific fragment amplified with primer pair 8.In A-E, swimming lane M is BM2000DNA ladder; Swimming lane 1 is corresponding gene PCR primer; Swimming lane 2 is negative control.
Fig. 3 utilizes liquid skimmed milk substratum to detect or the plain schema that whether there is quorum sensing regulation and control phenomenon of auxiliary detection bacterium to be checked generation II bacterioid.
Fig. 4 is the bacteriostatic activity test result that Autoinducer induction II bacterioid element produces.Wherein, A experimental group (fungistatic effect through the liquid skimmed milk culture medium culturing thing of Autoinducer induction); B is control group 1 (replacing the fungistatic effect of the liquid skimmed milk culture medium culturing thing of Autoinducer induction with equivalent MRS liquid nutrient medium); C is control group 2 (fungistatic effect of Autoinducer, concentration is 5 μ L/10mL, dilutes with fresh MRS medium)); D is control group 3 (fungistatic effect of the mixture of L.paraplantarum L-XM160h culture and Autoinducer (final concentration 5 μ L/10mL)).
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
MRS substratum: take peptone 10g, extractum carnis 10g, yeast leaching powder 5g, K
2hPO
43H
2o 2g, Triammonium citrate 2g, NaAC 5g, glucose 20g, MgSO
47H
2o 0.58g, MnSO
4h
2o 0.25g, tween 80 1mL, be dissolved in 1000mL distilled water, 121 DEG C of sterilizing 20min.
Whether embodiment 1, qualification or assistant identification class plant lactobacillus (L.paraplantarum) L-XM1 exist intervention school-based
One, method of the present invention is utilized to identify or assistant identification class plant lactobacillus (L.paraplantarum) L-XM1 (is formerly numbered Y1, now through identifying that definite designation is L.paraplantarum L-XM1, see: Gui Meng, Zhang Xiaoqiong, Yu Muyang, military auspicious Yun, Li Pinglan. the Screening and Identification that thermotolerant lactic acid bacteria meat is natural fermented dose. meat is studied, 2011,25 (8): 1-5) whether there is intervention school-based (experiment flow as shown in Figure 1).
Concrete operations are as follows:
1, extracting genome DNA
Class plant lactobacillus (L.paraplantarum) L-XM1 is inoculated in MRS substratum with the inoculum size of 5% (volume ratio), cultivates 24h, get 2mL, the centrifugal 1min of 13800 × g for 37 DEG C, collect thalline.
Adopt bacterial genomes DNA extraction kit (TIANamp Bacteria DNA kit) (TIANGEN Biotech's catalog number (Cat.No.): DP302-02) to extract genomic dna, concrete operation method is see test kit specification sheets.
2, pcr amplification
The goal gene of pcr amplification relates to following gene: the gene of coding intervention school-based component self-induction peptide (or self-induction peptide precursor); The gene of coding intervention school-based component Protein histidine kinase; The gene of coding intervention school-based component induction Function protein.
Pcr amplification primer used relates to 9 primer pairs altogether: the primer pair 1 being specific to self-induction peptide gene that first primer pair is made up of two single stranded DNAs shown in the sequence 1 in sequence table and sequence 2; The primer pair 2 being specific to self-induction peptide gene that second primer pair is made up of two single stranded DNAs shown in the sequence 3 in sequence table and sequence 4; The primer pair 3 being specific to Protein histidine kinase gene that 3rd primer pair is made up of two articles of single stranded DNAs shown in the sequence 5 in sequence table and sequence 6; 4th primer pair consists of by two articles of single stranded DNAs shown in the sequence 7 in sequence table and sequence 8 primer pair 4 being specific to Protein histidine kinase gene; The primer pair 5 being specific to Protein histidine kinase that 5th primer pair is made up of two articles of single stranded DNAs shown in the sequence 9 in sequence table and sequence 10; The primer pair 6 being specific to induction Function protein gene that 6th primer pair is made up of two articles of single stranded DNAs shown in the sequence 11 in sequence table and sequence 12; The primer pair 7 being specific to induction Function protein gene that 7th primer pair is made up of two articles of single stranded DNAs shown in the sequence 13 in sequence table and sequence 14; The primer pair 8 being specific to induction Function protein gene that 8th primer pair is made up of two articles of single stranded DNAs shown in the sequence 15 in sequence table and sequence 16; The primer pair 9 being specific to Protein histidine kinase gene and induction Function protein gene that 9th primer pair is made up of two articles of single stranded DNAs shown in the sequence 17 in sequence table and sequence 18.
For the difference of above-mentioned 9 primer pairs, 9 different PCR reaction systems are set.Each reaction system comprises 2 × Taq PCR Master Mix, and template DNA (genomic dna that step (1) is extracted) 10ng, (concentration is 10pmol μ L to upstream and downstream primer
-1) each 1 μ L, ddH
2o complements to 25 μ L.When upstream primer is for primer shown in sequence 1, downstream primer is primer shown in sequence 2; When upstream primer is for primer shown in sequence 3, downstream primer is primer shown in sequence 4; When upstream primer is for primer shown in sequence 5, downstream primer is primer shown in sequence 6; When upstream primer is for primer shown in sequence 7, downstream primer is primer shown in sequence 8; When upstream primer is for primer shown in sequence 9, downstream primer is primer shown in sequence 10; When upstream primer is for primer shown in sequence 11, downstream primer is primer shown in sequence 12; When upstream primer is for primer shown in sequence 13, downstream primer is primer shown in sequence 14; When upstream primer is for primer shown in sequence 15, downstream primer is primer shown in sequence 16; When upstream primer is for primer shown in sequence 17, downstream primer is primer shown in sequence 18.Above-mentioned 9 different PCR reaction systems, are arranged with ddH all simultaneously
2o replaces the negative control of template.
Amplification program is 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, (annealing temperature of different primers is different for annealing 30s, specifically in table 1), 72 DEG C of extensions, extend basis of time expanding fragment length and determine, about 1000bp/min, totally 30 circulations, last 72 DEG C extend 10min, obtain PCR reaction product.
Table 1PCR amplification the primer
3, electrophoresis detection
(1) judge that the method that whether II Bacteriocin-like Substance Producing Strain to be measured (L.paraplantarum L-XM1) exists or candidate exists intervention school-based is according to electrophoresis result |:
First, utilize above-mentioned 9 pairs of primer sequences to carry out PCR reaction, judge whether II Bacteriocin-like Substance Producing Strain to be measured (L.paraplantarum L-XM1) contains or candidate contains self-induction peptide gene, Protein histidine kinase gene or induction Function protein gene.Specific as follows: if the PCR primer of primer pair 1 contains the DNA fragmentation of 450bp, and/or the PCR primer of primer pair 2 contains the DNA fragmentation of 114bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains self-induction peptide gene or candidate contains self-induction peptide gene; If the DNA fragmentation of the PCR primer of primer pair 1 not containing 450bp, the DNA fragmentation of the PCR primer of primer pair 2 also not containing 114bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain self-induction peptide gene containing self-induction peptide gene or candidate; If the PCR primer of primer pair 3 contains the DNA fragmentation of 165bp, and/or the PCR primer of primer pair 4 contains the DNA fragmentation of 758bp, and/or the PCR primer of primer pair 5 contains the DNA fragmentation of 927bp, and/or the PCR primer of primer pair 9 contains the DNA fragmentation of 1727bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains Protein histidine kinase gene or candidate contains Protein histidine kinase gene; If the DNA fragmentation of the PCR primer of primer pair 3 not containing 165bp, the DNA fragmentation of the PCR primer of primer pair 4 not containing 758bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 5 not containing 927bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 9 not containing 1727bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain Protein histidine kinase gene containing Protein histidine kinase gene or candidate; If the PCR primer of primer pair 6 contains the DNA fragmentation of 108bp, and/or the PCR primer of primer pair 7 contains the DNA fragmentation of 414bp, and/or the PCR primer of primer pair 8 contains the DNA fragmentation of 1249bp, and/or the PCR primer of primer pair 9 contains the DNA fragmentation of 1727bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains induction Function protein gene or candidate contains induction Function protein gene; If the DNA fragmentation of the PCR primer of primer pair 6 not containing 108bp, the DNA fragmentation of the PCR primer of primer pair 7 not containing 414bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 8 not containing 1249bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 9 not containing 1727bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain induction Function protein gene containing induction Function protein gene or candidate.
Secondly, judge whether described II Bacteriocin-like Substance Producing Strain to be measured (L.paraplantarum L-XM1) is contained or candidate is contained self-induction peptide gene, Protein histidine kinase gene or responded to the result of Function protein gene, judges whether described II Bacteriocin-like Substance Producing Strain to be measured exists or candidate exists intervention school-based further according to above-mentioned.Specific as follows: if described II Bacteriocin-like Substance Producing Strain to be measured contains or candidate contains self-induction peptide gene, Protein histidine kinase gene and induction Function protein gene simultaneously, then described II Bacteriocin-like Substance Producing Strain existence to be measured or candidate exist intervention school-based; If to contain when described II Bacteriocin-like Substance Producing Strain to be measured is different or candidate contains self-induction peptide gene, Protein histidine kinase gene and induction Function protein gene, then described II Bacteriocin-like Substance Producing Strain to be measured does not exist or candidate does not exist intervention school-based.
(2) prepare 1.0% sepharose, the PCR primer of above-mentioned steps 2 gained is carried out electrophoresis detection.Voltage is 100V, and electrophoresis time is 20min.Electrophoresis result as shown in Figure 2, has amplified with primer pair 1 the object band that size is about 450bp, has conformed to expected results; Amplify with primer pair 3 the object band that size is about 170bp, conformed to expected results; Amplify with primer pair 5 the object band that size is about 900bp, conformed to expected results; Amplify with primer pair 9 the object band that size is about 1700bp, conformed to expected results; Amplify with primer pair 8 the object band that size is about 1200bp, conformed to expected results.And the negative control corresponding to above-mentioned four primer pairs does not all amplify corresponding object band.Comprehensive analysis the above results, described 9 primer pairs are utilized to carry out pcr amplification, screening class plant lactobacillus (L.paraplantarum) L-XM1 quorum sensing genes involved, result has amplified the specific fragment (with primer pair 8,9) as the specific fragment (with primer pair 1) of intervention school-based self-induction peptide (or self-induction peptide precursor) encoding gene, the specific fragment (with primer pair 3,5,9) of histidine kinase encoding gene and induction Function protein encoding gene simultaneously.Then not increase object band with other primer pairs.Reach a conclusion thus, in class plant lactobacillus (L.paraplantarum) L-XM1 there is intervention school-based in existence or candidate.
Two, whether another kind of method detects or regulates and controls by quorum sensing regulator control system in auxiliary detection class plant lactobacillus (L.paraplantarum) L-XM1
The formula of this method various liquid nutrient medium used is as follows:
Liquid skimmed milk substratum: take skimming milk (Harbin Meihua Biologic Technology Co., Ltd.'s raw material skimmed milk powder) 11g, be dissolved in 100mL distilled water, 6.5 ~ 7.0,115 DEG C of sterilizing 10 ~ 13min.
Liquid MRS substratum: take peptone 10g, extractum carnis 10g, yeast leaching powder 5g, K
2hPO
43H
2o 2g, Triammonium citrate 2g, NaAC 5g, glucose 20g, MgSO
47H
2o 0.58g, MnSO
4h
2o 0.25g, tween 80 1mL, be dissolved in 1000mL distilled water, pH6.5 ~ 7.0,121 DEG C of sterilizing 20min.
Liquid 1/2MRS substratum: except distilled water, other various MRS medium component × 1/2, pH6.5 ~ 7.0,121 DEG C of sterilizing 20min.
Liquid 1/5MRS substratum: except distilled water, other various MRS medium component × 1/5, pH6.5 ~ 7.0,121 DEG C of sterilizing 20min.
Liquid 1/10MRS substratum: except distilled water, other various MRS medium component × 1/10, pH6.5 ~ 7.0,121 DEG C of sterilizing 20min.
Solid MRS substratum: for MRS liquid nutrient medium adds 15g/L agar powder.
Below for other method detects or auxiliary detection class plant lactobacillus (L.paraplantarum) L-XM1 produces the concrete operation step whether II bacterioid element is subject to quorum sensing regulator control system regulation and control (experiment flow is as shown in Figure 3):
1, Autoinducer (fermented supernatant fluid) is prepared
Stroke-physiological saline solution (10 will be suspended in
9cfu/mL) class plant lactobacillus (L.paraplantarum) L-XM1 in is inoculated in liquid MRS substratum with the inoculum size of 0.5% (volume ratio), cultivate 24h (class plant lactobacillus (L.paraplantarum) L-XM1 is in latter stage stationary phase) for 37 DEG C, the centrifugal 5min of 13800 × g, collect fermented supernatant fluid, as Autoinducer.
2, the L.paraplantarum L-XM1 having and do not produce II bacterioid element cell phenotype is obtained
Stroke-physiological saline solution (10 will be suspended in
9cfu/mL) the L.paraplantarum L-XM1 in is inoculated in liquid skimmed milk substratum (simultaneously arranging the contrast of following four kinds of liquid nutrient mediums as liquid skimmed milk substratum: liquid MRS substratum, liquid 1/2MRS substratum, liquid 1/5MRS substratum and liquid 1/10MRS substratum) using the inoculum size of 0.5% (volume ratio) respectively, and cultivate 12h, 24h, 36h, 48h, 60h, 72h under 37 DEG C of conditions after, detect respectively in the culture of now five kinds of liquid nutrient mediums and whether there is II bacterioid element.Concrete grammar is: the culture collecting five kinds of liquid nutrient mediums of different incubation time respectively, and the centrifugal 5min of 13800 × g, gets supernatant liquid filtering degerming, regulates pH to 6.5 ~ 7.0, gets 100 μ L for subsequent use.With can produce by described bacterium to be checked II bacterioid element Developing restraint plant lactobacillus (L.plantarum) PL2 be the bacteriostatic activity of five kinds of liquid nutrient medium culture supernatants that indicator cylinder plate method measures above-mentioned different incubation time, thus whether to judge in described culture containing II bacterioid element.Concrete operations are: fresh indicator plant lactobacillus (L.plantarum) PL2 getting incubated overnight, is diluted to 10 by stroke-physiological saline solution
6cFU/mL, gets 1mL and mixes with the MRS solid medium of 20mL being cooled to 50 DEG C and be down flat plate.After to be solidified, put into Oxford cup, according to the consumption of 100 μ L/ cups, add the supernatant liquor through above-mentioned process, cultivate 24h for 37 DEG C, observe fungistatic effect.
Result shows, cultivate through 72h, the flat board adding liquid skimmed milk culture medium culturing thing supernatant liquor produces inhibition zone not yet, and four kinds of liquid nutrient mediums (MRS substratum, 1/2MRS substratum, 1/5MRS substratum and the 1/10MRS substratum) flat board added in contrast all produces inhibition zone (table 2).This result shows, comprise in totally five kinds of substratum of control medium, only have in the culture supernatants of described liquid skimmed milk substratum and there is not II bacterioid element, and then the L.paraplantarum L-XM 1 utilizing described liquid skimmed milk substratum to obtain not produce II bacterioid element cell phenotype is described.
Table 2 different culture media synthesizes the impact of II bacterioid element to L.paraplantarum L-XM1
| Incubation time | 12h | 24h | 36h | 48h | 60h | 72h |
| Degreasing milk medium | - | - | - | - | - | - |
| MRS | + | ++ | ++ | ++ | ++ | ++ |
| 1/2MRS | - | + | ++ | ++ | ++ | ++ |
| 1/5MRS | - | + | ++ | ++ | ++ | ++ |
| 1/10MRS | - | + | ++ | ++ | ++ | ++ |
Note: "-" indicates without inhibition zone; "+" represents that antibacterial circle diameter is between 6mm and 10mm; " ++ " represents that antibacterial circle diameter is greater than 10mm.
3, induce
According to the consumption adding 5 μ L in every 10mL culture, cultivate to step 2 Autoinducer (fermented supernatant fluid) (experimental group) that have in the culture of the liquid skimmed milk substratum of L.paraplantarum L-XM1 (L.paraplantarum L-XM1 be in logarithmic phase latter stage) and add step 1 and prepare, carry out inducing and (contrast of carrying out inducing to wait quantity of fluid MRS substratum to substitute Autoinducer is set simultaneously, control group 1), continue to be cultured to 60h.
4, detect
Collect the culture of liquid nutrient medium in step 3 respectively, the centrifugal 5min of 13800 × g, gets supernatant liquid filtering degerming, regulates pH to 6.5 ~ 7.0, gets 100 μ L for subsequent use.With can produce by described bacterium to be checked II bacterioid element Developing restraint plant lactobacillus (L.plantarum) PL2 (Wu Pengpeng, Liu Guorong, smooth Xiao Yuan, Zhang Xiangmei, Li Pinglan. the purifying of anti-streptococcus suis Lactobacillus pentosus element and characteristic research. China Agricultural University's journal, 2011, 16 (5): 121-126) be indicator, the bacteriostatic activity of above-mentioned culture supernatants is measured with cylinder plate method, thus whether judge in described culture containing II bacterioid element, and then judge that described L.paraplantarum L-XM1 is after Autoinducer (fermented supernatant fluid) process, whether recovery produces II bacterioid element.Concrete operations are: fresh indicator plant lactobacillus (L.plantarum) PL2 getting incubated overnight, is diluted to 10 by stroke-physiological saline solution
6cFU/mL, gets 1mL and mixes with the MRS solid medium of 20mL being cooled to 50 DEG C and be down flat plate.After to be solidified, put into Oxford cup, according to the consumption of 100 μ L/ cups, add the supernatant liquor through above-mentioned process, simultaneously with isocyatic Autoinducer (5 μ L/10mL, diluting with fresh MRS medium) (control group 2) and L.paraplantarum L-XM160h culture add Autoinducer (5 μ L/10mL) (control group 3) in contrast, and cultivate 24h for 37 DEG C, observe fungistatic effect.
Result shows, inhibition zone is not produced with waiting control group 1 of quantity of fluid MRS substratum induction, Autoinducer (control group 2) and L.paraplantarum L-XM1 60h culture add Autoinducer (5 μ L/10mL) (control group 3) do not produce inhibition zone yet, and inhibition zone (Fig. 4) appears in experimental group.This result shows, there is II bacterioid element in the culture supernatants of described liquid skimmed milk substratum, and this II bacterioid element induces generation by Autoinducer.
The experimental result of the present embodiment shows, utilize degreasing milk medium to obtain cell phenotype that L.paraplantarum L-XM1 does not produce II bacterioid element, again by the induction of Autoinducer, L.paraplantarum L-XM1 can be made to recover to produce II bacterioid element (namely there is II bacterioid element in corresponding culture supernatant), and then demonstrate L.paraplantarum L-XM1 bacterial strain and produce II bacterioid element and there is quorum sensing and regulate and control phenomenon.
Claims (16)
1. for the identification of or assistant identification II Bacteriocin-like Substance Producing Strain to be measured whether there is the PCR primer group of intervention school-based genes involved, be made up of 9 primer pairs, the primer pair 1 being specific to self-induction peptide gene that first primer pair is made up of two single stranded DNAs shown in the sequence 1 in sequence table and sequence 2; The primer pair 2 being specific to self-induction peptide gene that second primer pair is made up of two single stranded DNAs shown in the sequence 3 in sequence table and sequence 4; The primer pair 3 being specific to Protein histidine kinase gene that 3rd primer pair is made up of two articles of single stranded DNAs shown in the sequence 5 in sequence table and sequence 6; 4th primer pair consists of by two articles of single stranded DNAs shown in the sequence 7 in sequence table and sequence 8 primer pair 4 being specific to Protein histidine kinase gene; The primer pair 5 being specific to Protein histidine kinase that 5th primer pair is made up of two articles of single stranded DNAs shown in the sequence 9 in sequence table and sequence 10; The primer pair 6 being specific to induction Function protein gene that 6th primer pair is made up of two articles of single stranded DNAs shown in the sequence 11 in sequence table and sequence 12; The primer pair 7 being specific to induction Function protein gene that 7th primer pair is made up of two articles of single stranded DNAs shown in the sequence 13 in sequence table and sequence 14; The primer pair 8 being specific to induction Function protein gene that 8th primer pair is made up of two articles of single stranded DNAs shown in the sequence 15 in sequence table and sequence 16; The primer pair 9 being specific to Protein histidine kinase gene and induction Function protein gene that 9th primer pair is made up of two articles of single stranded DNAs shown in the sequence 17 in sequence table and sequence 18.
2. PCR primer group according to claim 1, is characterized in that: described II Bacteriocin-like Substance Producing Strain is milk-acid bacteria.
3. PCR primer group according to claim 2, is characterized in that: described II Bacteriocin-like Substance Producing Strain is Bacterium lacticum.
4. PCR primer group according to claim 3, is characterized in that: described II Bacteriocin-like Substance Producing Strain is class plant lactobacillus (L.paraplantarum) L-XM1 or plant lactobacillus (L.plantarum).
5. the test kit containing PCR primer group according to claim 1.
6. the preparation method of PCR primer group described in claim 1, is characterized in that: described preparation method comprises the step of individually being packed by described two single stranded DNAs of primer pair each in PCR primer group described in claim 1.
7. the preparation method of PCR kit described in claim 5, after comprising the steps: described two single stranded DNAs of primer pair each in PCR primer group described in claim 1 individually to pack, be packaged in same reagent box with following substances: PCR reaction buffer, archaeal dna polymerase, 4 kinds of dNTP and ddH
2o.
8. whether PCR primer group according to claim 1 is containing the application in the test kit of intervention school-based genes involved for the preparation of qualification or assistant identification II Bacteriocin-like Substance Producing Strain to be measured, and described intervention school-based genes involved is self-induction peptide gene, Protein histidine kinase gene and induction Function protein gene.
9. whether qualification or assistant identification II Bacteriocin-like Substance Producing Strain to be measured contain the method for intervention school-based genes involved, described intervention school-based genes involved is self-induction peptide gene, Protein histidine kinase gene and induction Function protein gene, it is characterized in that: described method comprises the step of following (1) and (2):
Described (1) is following A)-I):
A) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 1 described in claim 1;
B) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 2 described in claim 1;
C) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 3 described in claim 1;
D) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 4 described in claim 1;
E) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 5 described in claim 1;
F) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 6 described in claim 1;
G) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 7 described in claim 1;
H) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 8 described in claim 1;
I) with the genomic dna of II Bacteriocin-like Substance Producing Strain to be measured for template, carry out PCR reaction with primer pair 9 described in claim 1;
(2) size of PCR primer that obtains of detecting step (1), determine that whether described II Bacteriocin-like Substance Producing Strain to be measured is containing described intervention school-based genes involved according to PCR primer as follows:
If the PCR primer of primer pair 1 contains the DNA fragmentation of 450bp, and/or the PCR primer of primer pair 2 contains the DNA fragmentation of 114bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains self-induction peptide gene or candidate contains self-induction peptide gene; If the DNA fragmentation of the PCR primer of primer pair 1 not containing 450bp, the DNA fragmentation of the PCR primer of primer pair 2 also not containing 114bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain self-induction peptide gene containing self-induction peptide gene or candidate; If the PCR primer of primer pair 3 contains the DNA fragmentation of 165bp, and/or the PCR primer of primer pair 4 contains the DNA fragmentation of 758bp, and/or the PCR primer of primer pair 5 contains the DNA fragmentation of 927bp, and/or the PCR primer of primer pair 9 contains the DNA fragmentation of 1727bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains Protein histidine kinase gene or candidate contains Protein histidine kinase gene; If the DNA fragmentation of the PCR primer of primer pair 3 not containing 165bp, the DNA fragmentation of the PCR primer of primer pair 4 not containing 758bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 5 not containing 927bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 9 not containing 1727bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain Protein histidine kinase gene containing Protein histidine kinase gene or candidate; If the PCR primer of primer pair 6 contains the DNA fragmentation of 108bp, and/or the PCR primer of primer pair 7 contains the DNA fragmentation of 414bp, and/or the PCR primer of primer pair 8 contains the DNA fragmentation of 1249bp, and/or the PCR primer of primer pair 9 contains the DNA fragmentation of 1727bp, then described II Bacteriocin-like Substance Producing Strain to be measured contains induction Function protein gene or candidate contains induction Function protein gene; If the DNA fragmentation of the PCR primer of primer pair 6 not containing 108bp, the DNA fragmentation of the PCR primer of primer pair 7 not containing 414bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 8 not containing 1249bp simultaneously, the DNA fragmentation of the PCR primer of primer pair 9 not containing 1727bp simultaneously, then described II Bacteriocin-like Substance Producing Strain to be measured does not contain induction Function protein gene containing induction Function protein gene or candidate.
10. method according to claim 9, is characterized in that: described II Bacteriocin-like Substance Producing Strain is milk-acid bacteria.
11. methods according to claim 10, is characterized in that: described II Bacteriocin-like Substance Producing Strain is Bacterium lacticum.
12. methods according to claim 11, is characterized in that: described II Bacteriocin-like Substance Producing Strain is class plant lactobacillus (L.paraplantarum) L-XM1 or plant lactobacillus (L.plantarum).
The method of 13. claims 9 to exist or candidate exists application in II Bacteriocin-like Substance Producing Strain of intervention school-based obtaining, described in be applied as meet following a)-c using what utilize the method for claim 9 to identify to obtain) II Bacteriocin-like Substance Producing Strain of condition is as to exist or candidate exists II Bacteriocin-like Substance Producing Strain of intervention school-based:
A) containing or candidate contain self-induction peptide gene;
B) containing or candidate contain Protein histidine kinase gene;
C) containing or candidate contain induction Function protein gene.
14. application according to claim 13, is characterized in that: described II Bacteriocin-like Substance Producing Strain is milk-acid bacteria.
15. application according to claim 14, is characterized in that: described II Bacteriocin-like Substance Producing Strain is Bacterium lacticum.
16. application according to claim 15, is characterized in that: described II Bacteriocin-like Substance Producing Strain is class plant lactobacillus (L.paraplantarum) L-XM1 or plant lactobacillus (L.plantarum).
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| Genetic diversity of the pln locus among oenological Lactobacillus plantarum strains;Yolanda Sáenz et al;《International Journal of Food Microbiology》;20091231;第134卷;第176－183页 * |
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