CN1900283A - Gene of chlorine tolerance strain V430 - Google Patents

Gene of chlorine tolerance strain V430 Download PDF

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CN1900283A
CN1900283A CN 200610018134 CN200610018134A CN1900283A CN 1900283 A CN1900283 A CN 1900283A CN 200610018134 CN200610018134 CN 200610018134 CN 200610018134 A CN200610018134 A CN 200610018134A CN 1900283 A CN1900283 A CN 1900283A
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strain
gene
dna
rdna
chlorine
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CN100419081C (en
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信欣
王焰新
鲍建国
刘慧�
李平
杨雪芬
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China University of Geosciences
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China University of Geosciences
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Abstract

The chlorine tolerant strain gene V430 is the 16S rDNA gene of Staphylococcus saprophyticus V430. The gene of the present invention is obtained through extracting the nucleoplasm DNA, PCR amplification of 16S rDNA gene, recovery of PCR product, and whole 16S rDNA sequence determination and analysis. The present invention has the advantages of high speed and being simple.

Description

The gene of anti-chlorine strain V 430
Technical field
The present invention relates to the gene of anti-chlorine strain V 430, it is the 16S rDNA gene of Staphylococcus saprophyticus (Staphylococcussaprophyticus) V430.Wherein, by China typical culture collection center (being called for short CCTCC) preservation, preservation date is on December 9th, 2005 to Staphylococcus saprophyticus V430, and preserving number is CCTCC NO.M205145.By the method that classical phenotypic evaluation and 16S rDNA nucleotide sequence analysis combine, anti-chlorine strain V 430 has been carried out the evaluation of Physiology and biochemistry and 16S rDNA molecular level, determine that thus it is a Staphylococcus saprophyticus.
Background technology
Occurring in nature, exist some can be in the environment that contains high density or even extreme high-concentration chlorine ion the microorganism of normal growth.Through discovering that these microorganisms are not the processing that can be applied to contain high-concentration chlorine ion wastewater (chlorine ion concentration is 5000-80000mg/L), have only and just can accomplish its anti-chlorine microorganism that obtains after through strict screening and domestication.Anti-chlorine microorganism both can tolerate the high-concentration chlorine ion environment, again the organism of degradable waste water middle and high concentration.The first-selected sampling spot that screens anti-chlorine microorganism is exactly that because exist the microorganism of some amount in waste water, these microorganisms have adaptive faculty preferably to the waste water environment in institute's waste water to be processed.The anti-chlorine microorganism that just screens promptly can be used as the microorganism strains of anti-chlorine and is applied in the wastewater treatment practice through after taming.
The sorting technique of microorganism is commonly used classical sorting technique and molecular biology classification method.Classical sorting technique mainly refers to the method according to classification such as morphological specificity (Morphological characteristics), cultural characteristic (Cultural characteristics) and physiological and biochemical properties (Physiological characteristics).Molecular biology classification method claims the molecular classification method again, is meant that nucleic acid and the protein to biont is studied on molecular level, and the method for in view of the above biont being classified.Because 16S rDNA sequential analysis has outstanding effect, therefore often adopted by the molecular classification method.
At present, 16S rDNA sequencing technique mainly contains two kinds: a kind of 16S of being rDNA checks order with ThermoScript II and conservative primer after purifying, and another kind is an amplification 16S rDNA gene corresponding DNA sequences, then the PCR product is reclaimed directly order-checking of back.
Anti-chlorine bacterium is owing to have anti-chlorine characteristic, macromole such as its form, cell wall, intracellular protein, lipid acid are formed the change that can take place in various degree under the situation that has salt to exist, in the identification of strains process, only be difficult to judge their ownership from form, and very waste time and energy from physiological characteristic and chemical feature evaluation, all the more so for a large amount of isolated strains.
Summary of the invention
Technical problem to be solved by this invention is: by 16S rDNA sequence analysis method, fast anti-chlorine strain V 430 is carried out the evaluation of molecular level, determine that it is Staphylococcus saprophyticus.This bacterial strain is the newfound chlorine resisting strain that can survive under the high-concentration chlorine ion environment, and it helps the organism in the degrading waste water.
The technical solution adopted in the present invention is as follows:
The gene of anti-chlorine strain V 430, its feature are levied the 16S rDNA gene that is to Staphylococcus saprophyticus (Staphylococcussaprophyticus) V430 CCTCC NO.M205145, and the 16S rDNA complete sequence of its bacterial strain is as follows:
GTACGATGCGGACAGCATATGGTGCGTCTCGTTCCTTTGCCGTCAGGGGCGGACGGCGCG 60
GTTCCATGTGGGTAACCTACCTATAAGACTGTTCTAACTCCGGGATACCGGGGCTAATGC 120
CGGATGGCATTAGAACCGGTTGCCCGGAGTGTGAAAGATGGTTTTGCTATCTCTTATACG 180
TGGACCCGCGCCGTATGAGGTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATACGTA 240
GCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAACTCTCTACGGG 300
AGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAG 360
TGATGAAGGGTTTCGGCTCGTAAAACTCTGTTATTAGGGAAGAACAAACGTGTAAGTAAC 420
TGTGCACGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGC 480
GGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGG 540
TTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGA 600
GACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATA 660
TGGAGGAACACCAGTGGCGAAAGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAA 720
GCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTA 780
AGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG 840
GAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAG 900
CATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACCA 960
CTCTAGAGATAGAGTTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGT 1020
CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAACTTAGT 1080
TGCCAGCATTTAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGG 1140
GATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAA 1200
TACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAAAGTTGTTCTCAGTTCGGA 1260
TTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGC 1320
TACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAA 1380
CACCCGAAGCCGGTGGAGTAACCATTAATGGAGCTAGCCGTCG 1423
1423 Nucleotide of above-mentioned 16S rDNA sequence total length.
Adopt the BLAST analytical method, with the 16S rDNA complete sequence and the comparison of GenBank database of the bacterial strain of Staphylococcus saprophyticus V430 CCTCC NO.M205145, the homology of this bacterial strain and Staphylococcus saprophyticus is 98.0-99.0%.
A kind of method of obtaining the gene of anti-chlorine strain V 430, it has adopted and has extracted bacterium nuclear DNA, the pcr amplification of 16S rDNA gene, the recovery of PCR product, and the complete sequence determination of 16S rDNA and analytical procedure, it is characterized in that:
(1) in the process of extracting bacterium nuclear DNA, adopted following damping fluid,
TE damping fluid: 10mmol/L Tris-Cl, pH8.0; 1mmol/L EDTA-Na2, pH8.0,
STE damping fluid: 0.1mol/L NaCl; 10mmol/L Tris-Cl, pH8.0; 1mmol/L EDTA, pH8.0,
(2) in the pcr amplification process of 16S rDNA gene, adopted the following amplification condition and the primer of amplification:
The pcr amplification condition is: 94 ℃ of pre-sex change 5min; At 94 ℃ of sex change 30s, 61-65 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min eventually,
The primer of pcr amplification is: the P1 forward primer is 5 ' AGAGTTTGATCCTGGCTCAG3 ', and the P2 reverse primer is 5 ' GGTTACCTTGTTACGACTT3 ',
(3) recovery of PCR product:
After the PCR product carried out electrophoresis with 1% sepharose, under ultraviolet lamp, cut and contain desire and reclaim segmental gel, put into the 1.5ml centrifuge tube and add 2 times of volume TE, 65 ℃ of water-baths add the extracting of equal-volume water-saturated phenol and get the upper strata water after once centrifugal and use that phenol-chloroform-the primary isoamyl alcohol extracting once again after 10 minutes, reset and add 10mol/L ammonium acetate and 2 times of volume dehydrated alcohol precipitations of 0.1 times of volume in the collection, centrifugal, wash precipitation once with 70% ethanol, be dissolved in an amount of sterilization distilled water after air-dry
(4) complete sequence determination of 16S rDNA and analysis:
Add 1 μ L template DNA,＜0.1 μ g; 30 circulations of pcr amplification, its condition are 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 2min; Adopt dyestuff terminator termination reaction in the ABI PRISM sequencing kit; Then, on AppliedBiosystem 373ADNA sequenator, check order; Adopt BLAST software then,, finally determine that it is the gene complete sequence of Staphylococcus saprophyticus strain 16S rDNA complete sequence and the comparative analysis of GenBank database that records,
When PCR checks order, use following forward and reverse primer:
P-f：CACGACGTTGTAAAACGACAGTTTGATCCTGGCTC，
P-r：GGATAACAATTTCACACAGGAAGGAGGTGATCCAGCC。
The 16S rDNA gene of Staphylococcus saprophyticus V430 CCTCC NO.M205145 is provided.Gene order of the present invention, by extracting bacterium nuclear DNA, the pcr amplification of 16S rDNA gene, the recovery of PCR product, and the complete sequence determination of 16S rDNA and analysis and obtain.The present invention has quick, easy advantage.
Description of drawings
Fig. 1 is the 16S rDNA complete sequence figure of the bacterial strain of Staphylococcus saprophyticus V430 CCTCC NO.M205145 of the present invention
Fig. 2-1a is a strain V 430 bacterial strain Electronic Speculum picture
Fig. 2-1b is a strain V 430 bacterial strain Electronic Speculum picture
Fig. 2-1c is a strain V 430 bacterial strain Electronic Speculum picture
Fig. 2-the 2nd, strain V 430 is growing state figure on the SC substratum of the different chlorine ion concentrations of liquid
Fig. 2-the 3rd, and strain V 430 pcr amplification agarose electrophoresis figure (1: nuclear DNA, 2: amplified production, M:Marker)
Fig. 2-4a is the following degraded efficiency diagram of strain V 430 under high chloride ion, high COD concentration
Fig. 2-4b is the following degraded efficiency diagram of strain V 430 under high chloride ion, high COD concentration
Fig. 2-4c is the following degraded efficiency diagram of strain V 430 under high chloride ion, high COD concentration
Fig. 2-4d is the following degraded efficiency diagram of strain V 430 under high chloride ion, high COD concentration
Embodiment
The invention will be further described below in conjunction with embodiment and accompanying drawing.
The gene of Staphylococcus saprophyticus V430 CCTCC NO.M205145 provided by the invention, its 16S rDNA complete sequence as shown in Figure 1.
Adopt the BLAST analytical method, with the 16S rDNA complete sequence of the bacterial strain of Staphylococcus saprophyticus V430 CCTCC NO.M205145 and GenBank database relatively, the homology of this bacterial strain and Staphylococcus saprophyticus is 98.0-99.0%, can determine that this bacterial strain is a Staphylococcus saprophyticus from molecular level thus.Simultaneously, in conjunction with form and the physiological and biochemical property that classical sorting technique obtained, determine that finally this bacterial strain is a Staphylococcus saprophyticus.
The present invention adopts and extracts bacterium nuclear DNA, the pcr amplification of 16S rDNA gene, the recovery of PCR product, and the complete sequence determination of 16S rDNA and analytical procedure, obtains the 16S rDNA gene complete sequence of CCTCC NO.M205145 bacterial strain.
Above-mentioned steps is specific as follows:
(1) extraction of bacterium nuclear DNA
1) choose single colony inoculation overnight incubation in LB liquid pipe with autoclaved toothpick, get 1.5ml bacterium liquid in the Eppendorf pipe, 8, the centrifugal 5min of 000rpm room temperature thoroughly removes supernatant.
2) add STE solution 1.5ml washing once, the centrifugal supernatant of abandoning adds 0.6mlTE solution again, resuspended bacterium.
3) add the N,O-Diacetylmuramidase of 30ul10mg/ml, 37 ℃ of water-bath 45min
4) add the SDS of 65 μ l10%, the Proteinase K 3 μ l of 20mg/ml, 50 ℃ of water-baths 2 hours, clear to solution becomes.
5) add equivalance body phenol chloroform extracting three times, till can't see egg white layer.
6) NaAc (pH5.2) of the 3M of adding 1/10 volume in supernatant liquor.
7) add isopyknic Virahol, deposit D NA.Twine with glass stick and to choose the DNA filament, once with 70% washing with alcohol, natural airing, and DNA is dissolved in the 100 μ l TE solution.
8) adding final concentration is the RNase of 50 μ g/ml, and 37 ℃, 30min removes RNA, and-20 ℃ of preservations are standby.
(2) pcr amplification of 16S rDNA gene:
In 50 μ L reaction volumes, add: 1 μ L template DNA, 0.1 μ g; 0.5 μ LP1 and P2, final concentration are 0.5 μ M; 1 μ LdNTP, every kind of NTP0.2mM; 0.5 μ LTaq polysaccharase, 2U; 5 μ L, 10 * PCR damping fluid.
The pcr amplification condition is: 94 ℃ of pre-sex change 5min; At 94 ℃ of sex change 30s, 61-65 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min eventually.
The primer of pcr amplification is: the P1 forward primer is 5 ' AGAGTTTGATCCTGGCTCAG3 ', and the P2 reverse primer is 5 ' GGTTACCTTGTTACGACTT3 '.
(3) recovery of PCR product:
After the PCR product carried out electrophoresis with 1% sepharose, under ultraviolet lamp, cut and contain desire and reclaim segmental gel, put into the 1.5ml centrifuge tube and add 2 times of volume TE, 65 ℃ of water-baths add the extracting of equal-volume water-saturated phenol and get the upper strata water after once centrifugal and use that phenol-chloroform-the primary isoamyl alcohol extracting once again after 10 minutes, reset and add 10mol/L ammonium acetate and 2 times of volume dehydrated alcohol precipitations of 0.1 times of volume in the collection, centrifugal, wash precipitation once with 70% ethanol, be dissolved in an amount of sterilization distilled water after air-dry.
(4) complete sequence determination of 16S rDNA and analysis:
Add 1 μ L template DNA,＜0.1 μ g; 30 circulations of pcr amplification, its condition are 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 2min; Adopt dyestuff terminator termination reaction in the ABI PRISM sequencing kit; Then, on AppliedBiosystem 373ADNA sequenator, check order; Adopt BLAST software then,, finally determine that it is the gene complete sequence of Staphylococcus saprophyticus strain 16S rDNA complete sequence and the comparative analysis of GenBank database that records.
When PCR checks order, use following forward and reverse primer:
P-f：CACGACGTTGTAAAACGACAGTTTGATCCTGGCTC，
P-r：GGATAACAATTTCACACAGGAAGGAGGTGATCCAGCC。
The present invention adopts the method for 16S rDNA sequencing and analysis is carried out the evaluation of molecular level to bacterium.Nuclear DNA with bacterium is a template, is primer with the universal primer of the pcr amplification of 16S rDNA gene, carries out pcr amplification, and (detecting with 1% agarose gel electrophoresis) is shown in Fig. 2-3.
The screening method of anti-chlorine strain V 430 and use as follows:
One, the screening method of a chlorine-resisting strain V430:
(1), material is prepared:
1, bacterium source and experiment waste water
(1) bacterium source: the active sludge in the biochemical system of processing saponin waste water;
(2) experiment waste water (promptly mixing saponin waste water): this experiment waste water is taken from the female Home Co., Ltd in Shiyan City side, Hubei Province diosgenin wastewater; After interior electrolysis and CaO allotment, specific targets are: pH=7.5-8.2, COD=4000-17000mg/L, Cl-=8000-30000mg/L, NH 4-N 140-300mg/L.
2, substratum:
Separate (fully) substratum: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml.
Screening culture medium: MgSO 47H 2O, 0.2g/L, KH 2PO 40.5g/L, K 2HPO 41.5g/L, NH 4Cl 0.5/L, CaCl 2135g/L, the 1ml trace element solution dissolves in 1L saponin waste water (COD concentration is 1000mg/L), pH=7.5;
Trace element solution: FeCl 36H 2O 2g, CoCl 22g, MnCl 20.5g, CuCl 20.03g, ZnCl 20.05g, NiCl 2.6H 2O 0.05g, EDTA 1g, pH=7.0.
Enlarged culturing base: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml; CaCl 218.0g/L.
SC substratum: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Distilled water 1000ml; CaCl 2Add-on is 12.6g-140g; Work as CaCl 2When add-on is 12.6g, [Cl in the SC substratum -]=10000mg/L.
Slant medium: peptone 10g; Extractum carnis 5g; Sodium-chlor; 5g; Potassium primary phosphate 2g; Ammonium chloride 1g; Agar 20g; Distilled water 1000ml; CaCl 218g/L, pH=7.5.
Above substratum is standby after respectively at 121 ℃ of autoclaving 30min.
3, laboratory apparatus and equipment:
State China SHA-B constant temperature oscillator,
SHH150G illumination bio-incubator,
Horizontal pressure steam sterilizer,
WMX-1 type microwave seal is cleared up the COD tacheometer,
The 722S spectrophotometer,
Opticmicroscope,
PH meter,
Bechtop,
Air dry oven,
Ultramicrotome,
The H-7000FA of Hitachi projection microscope,
PTC200 type PCR instrument,
Electrophoresis apparatus and electrophoresis chamber,
Gel ultraviolet visualizer etc.
(2), the separation of bacterial strain and screening:
1, zoogleic fragmentation:
Get the anaerobic activated sludge in the 2g saponin waste water treatment system, add in the triangular flask of the 250ml that the 100ml sterilized water is housed that sterilization is good in advance, and adding weight is the trisodium phosphate of sterilized water 0.01%, the shaking table vibration, zoogloea is smashed, got mud mixture, standby.
2, the separation of anti-chlorine ion dominant bacteria:
Utilize the above-mentioned mud mixture of aseptic pipette, extract 0.5ml, add isolation medium 4.5ml, 35 ℃ of anaerobism constant temperature culture 5-7 days, treat that the test tube substratum becomes muddy, illustrate that bacterium grows, dull and stereotyped then dilution coating, picking has single bacterium of notable difference on colony characteristics, until obtaining single bacterial strain, and preserve in the slant medium ramp.
3, the screening of anti-chlorine ion dominant bacteria:
The above-mentioned isolated single bacterium colony of picking is inoculated in respectively in the screening culture medium, 35 ℃ of anaerobism constant temperature culture 5-7 days, and the muddy situation of observing bacteria suspension becomes the muddy high chlorine resistant ionic bacterial strain that filters out that is.Through screening, obtain the bacterial strain that a strain is numbered strain V 430 at last, i.e. anti-chlorine strain V 430.Anti-chlorine strain V 430 growing state on the liquid SC substratum that adds the different concns chlorion (is aided with [Cl simultaneously shown in Fig. 2-2 -OD during]=3000mg/L 600nmBe control value).
Two, the colonial morphology of anti-chlorine strain V 430, physiology and biochemical character see Table 1.Cellular form is seen Fig. 2-1a, Fig. 2-1b, Fig. 2-1c.
Table 1:
The colony morphology characteristic of strain V 430 and main physiological characteristic
Project Bacterium V430
Colony morphology characteristic Bacterium colony is rounded, children's oyster white in age, aged safran is opaque; Bacterium colony projection, edge-smoothing.
Physiological characteristic Cell spheroid, d=0.6～single or paired, the in heaps appearance of 0.9 μ m, atrichia.Do not move no endogenous spore, Gram-positive, anaerobism.The suitableeest growth pH value: 6.0～7.5, growth temperature: 25-45 ℃.
Glucose produces sour grapes sugar aerogenesis wood sugar arabinose fructose sucrose maltose sweet mellow wine mannose oxidizing ferment cromoci catalase arginine hydrolysed casein gelatin hydrolysate hydrolyzed starch citrate + - - - + - - - + - - - + - - - -
NO 3 -Reduction +
Nomenclature in the table: "+": the bacterial strain more than 90% is positive, "-": the bacterial strain more than 90% is negative.
Three, strain V 430 is new bacterial strain:
The 16S rDNA gene order of strain V 430 and the sequence in the international GenBank database are carried out online homology find that relatively the homology of a plurality of bacterial strains of bacterium V430 and Staphylococcus saprophyticus (Staphylococcus saprophyticus) is up to more than the 98-99%.The Physiology and biochemistry and the Molecular Identification result of comprehensive strain V 430, can determine that bacterium V430 is Staphylococcus saprophyticus (Staphylococcus saprophyticus), strain V 430 called after Staphylococcus saprophyticus (Staphylococcus saprophyticus) V430 (being anti-chlorine strain V 430), consult pertinent data, do not have still that Staphylococcus saprophyticus belongs to organic wastewater with difficult degradation thereby under the relevant high-concentration chlorine ion condition and the report of its anti-chlorine capability study.Staphylococcus saprophyticus (Staphylococcus saprophyticus) V430 was new bacterial strain, had been preserved in Chinese typical culture collection center (being called for short CCTCC), preserving number: CCTCC NO.M205145 on December 9th, 2005.
The strain V 430 that the present invention separates, filters out has the function of better degrading high concentration chlorion difficult degradation saponin waste water.This has just widened people to the applied research thinking of Staphylococcus saprophyticus (Staphylococcus saprophyticus) in its function aspects, and provide useful bacterium source and technology for degraded contains the high-concentration chlorine ion saponin waste water, have stronger actual application value.
Four, the application of anti-chlorine strain V 430:
The single bacterium colony of picking Staphylococcus saprophyticus (Staphylococcus saprophyticus) V430, in the enlarged culturing base, cultivate 24h, get saponin composite waste ([Cl-]=8000-20000/L, COD=8000-10000mg/L) 50ml adds respectively in the Erlenmeyer flask of 250ml, adds the bacteria suspension 1ml of bacterial strain respectively, 35 ℃, pH value=7.5, anaerobic reaction 5 days, calculate the removal situation of bacterial strain to the COD of waste water, degradation factors is tested except the factor of being investigated, and all the other are all with above condition.Strain V 430 is removed the excellent in efficiency of COD, is 50-60%; The results are shown in Figure 2-4a, Fig. 2-4b, Fig. 2-4c, Fig. 2-4d.
Described enlarged culturing base is: peptone 10g; Extractum carnis 5g; Distilled water 1000ml; CaCl 218.0g/L, pH=7.5.
1),, in waste water, adds CaCl to the removal effect of COD in order to investigate bacterial strain under different chlorine ion concentrations 29864.2,14980,21061,24780,30080mg/L the adjusting chlorine ion concentration is respectively:.Anti-chlorine strain V 430 removal effect result such as Fig. 1-4a.
From Fig. 2-4a as can be seen, as [Cl -During]=9864.2mg/L, reaction times is 3d, strain V 430 only is 39.31% to the clearance of COD, but rising along with chlorine ion concentration in the waste water, strain V 430 increases the clearance of COD, when chlorine ion concentration in the waste water reaches 2.5 ten thousand mg/L, strain V 430 can reach 64.38% to the clearance of COD, and strain V 430 increases and changes little to the clearance of COD along with the continuation of chlorine ion concentration in the waste water, when chlorine ion concentration in the waste water during up to 30,000 mg/L, strain V 430 still remains on 60% to the clearance of COD.
2) in order to investigate bacterial strain (6610.4,7906.4,11209.6,13420.0,16196.2mg/L) under different COD concentration,, in waste water, add CaCl to the removal effect of COD 2Regulate chlorine ion concentration and be respectively 20000mg/L.Anti-chlorine strain V 430 removal effect result such as Fig. 2-4b.By experiment as can be known: [Cl in waste water -]=20000mg/L, when COD concentration reaches 10,000 mg/L, strain V 430 is better to the clearance of COD, can reach 62.37%.
3) from Fig. 2-4c and 2-4d as can be seen, strain V 430 increases afterwards earlier with the increase of pH the clearance of COD and descends, and its highest clearance reaches 62% when pH is 7.5-8.Waste water after the centrifugal treating, when finding that pH is 7-8, the wet thallus amount is 9,10,11 o'clock apparently higher than pH, as seen during in neutrality, helps the growth of bacterial strain at pH; When inoculum size changed between 1-4ml, strain V 430 did not have considerable change to the clearance of COD, was 60% substantially, and when inoculum size was 1ml, its clearance was best.This is the certain nutritive substance of growth needs because of bacterial strain, and when bacteria suspension was 1ml, its material that needs was met easily.
＜110〉China Geological Univ. Wuhan
＜120〉gene of anti-chlorine strain V 430
<160>1423
<210>1
<211>1423bp
<212>DNA
＜213〉Staphylococcus saprophyticus (Staphylococcus saprophyticus)
<220>
<221>misc_feature
<222>
<400>1
GTACGATGCGGACAGCATATGGTGCGTCTCGTTCCTTTGCCGTCAGGGGCGGACGGCGCG 60
GTTCCATGTGGGTAACCTACCTATAAGACTGTTCTAACTCCGGGATACCGGGGCTAATGC 120
CGGATGGCATTAGAACCGGTTGCCCGGAGTGTGAAAGATGGTTTTGCTATCTCTTATACG 180
TGGACCCGCGCCGTATGAGGTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATACGTA 240
GCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAACTCTCTACGGG 300
AGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAG 360
TGATGAAGGGTTTCGGCTCGTAAAACTCTGTTATTAGGGAAGAACAAACGTGTAAGTAAC 420
TGTGCACGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGC 480
GGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGG 540
TTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGA 600
GACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATA 660
TGGAGGAACACCAGTGGCGAAAGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAA 720
GCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTA 780
AGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG 840
GAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAG 900
CATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACCA 960
CTCTAGAGATAGAGTTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGT 1020
CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAACTTAGT 1080
TGCCAGCATTTAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGG 1140
GATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAA 1200
TACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAAAGTTGTTCTCAGTTCGGA 1260
TTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGC 1320
TACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAA 1380
CACCCGAAGCCGGTGGAGTAACCATTAATGGAGCTAGCCGTCG 1423

Claims (3)

1. the gene of anti-chlorine strain V 430, its feature is levied the 16S r DNA gene that is to Staphylococcus saprophyticus (Staphylococcussaprophyticus) V430 CCTCC NO.M205145, and the 16S r DNA complete sequence of its bacterial strain is as follows:
GTACGATGCGGACAGCATATGGTGCGTCTCGTTCCTTTGCCGTCAGGGGCGGACGGCGCG 60
GTTCCATGTGGGTAACCTACCTATAAGACTGTTCTAACTCCGGGATACCGGGGCTAATGC 120
CGGATGGCATTAGAACCGGTTGCCCGGAGTGTGAAAGATGGTTTTGCTATCTCTTATACG 180
TGGACCCGCGCCGTATGAGGTAGTTGGTAAGGTAATGGCTTACCAAGGCAACGATACGTA 240
GCCGACCTGAGAGGGTGATCGGCCACACTGGAACTGAGACACGGTCCAACTCTCTACGGG 300
AGGCAGCAGTAGGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAG 360
TGATGAAGGGTTTCGGCTCGTAAAACTCTGTTATTAGGGAAGAACAAACGTGTAAGTAAC 420
TGTGCACGTCTTGACGGTACCTAATCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGC 480
GGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGTAGGCGG 540
TTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGA 600
GACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGCAGAGATA 660
TGGAGGAACACCAGTGGCGAAAGCGACTTTCTGGTCTGTAACTGACGCTGATGTGCGAAA 720
GCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTA 780
AGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGG 840
GAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAG 900
CATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAATCTTGACATCCTTTGACCA 960
CTCTAGAGATAGAGTTTTCCCCTTCGGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGT 1020
CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAAACTTAGT 1080
TGCCAGCATTTAGTTGGGCACTCTAGGTTGACTGCCGGTGACAAACCGGAGGAAGGTGGG 1140
GATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAA 1200
TACAAAGGGCAGCTAAACCGCGAGGTCATGCAAATCCCATAAAGTTGTTCTCAGTTCGGA 1260
TTGTAGTCTGCAACTCGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGC 1320
TACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAA 1380
CACCCGAAGCCGGTGGAGTAACCATTAATGGAGCTAGCCGTCG 1423
1423 Nucleotide of above-mentioned 16S r dna sequence dna total length.
2. the gene of anti-chlorine strain V 430 according to claim 1, it is characterized in that: adopt the BLAST analytical method, with the 16S r DNA complete sequence of the bacterial strain of Staphylococcus saprophyticus V430 CCTCC NO.M205145 and GenBank database relatively, the homology of this bacterial strain and Staphylococcus saprophyticus is 98.0-99.0%.
3. method of obtaining the gene of claim 1 or 2 described anti-chlorine strain V 430s, it has adopted and has extracted bacterium nuclear DNA, the pcr amplification of 16S rDNA gene, the recovery of PCR product, and the complete sequence determination of 16S rDNA and analytical procedure, it is characterized in that:
(1) in the process of extracting bacterium nuclear DNA, adopted following damping fluid,
TE damping fluid: 10mmol/L Tris-Cl, pH8.0; 1mmol/L EDTA-Na2, pH8.0,
STE damping fluid: 0.1mol/L NaCl; 10mmol/L Tris-Cl, pH8.0; 1mmol/L EDTA, pH8.0,
(2) in the pcr amplification process of 16S rDNA gene, adopted the following amplification condition and the primer of amplification:
The pcr amplification condition is: 94 ℃ of pre-sex change 5min; At 94 ℃ of sex change 30s, 61-65 ℃ of annealing 30s, 72 ℃ are extended 1min, circulate 30 times; Last 72 ℃ are extended 10min eventually,
The primer of pcr amplification is: the P1 forward primer is 5 ' AGAGTTTGATCCTGGCTCAG3 ', and the P2 reverse primer is 5 ' GGTTACCTTGTTACGACTT3 ',
(3) recovery of PCR product:
After the PCR product carried out electrophoresis with 1% sepharose, under ultraviolet lamp, cut and contain desire and reclaim segmental gel, put into the 1.5ml centrifuge tube and add 2 times of volume TE, 65 ℃ of water-baths add the extracting of equal-volume water-saturated phenol and get the upper strata water after once centrifugal and use that phenol-chloroform-the primary isoamyl alcohol extracting once again after 10 minutes, reset and add 10mol/L ammonium acetate and 2 times of volume dehydrated alcohol precipitations of 0.1 times of volume in the collection, centrifugal, wash precipitation once with 70% ethanol, be dissolved in an amount of sterilization distilled water after air-dry
(4) complete sequence determination of 16S rDNA and analysis:
Add 1 μ L template DNA,＜0.1 μ g; 30 circulations of pcr amplification, its condition are 94 ℃ of 1min, 55 ℃ of 30s, 72 ℃ of 2min; Adopt dyestuff terminator termination reaction in the ABI PRISM sequencing kit; Then, on AppliedBiosystem 373 A dna sequencing instrument, check order; Adopt BLAST software then,, finally determine that it is the gene complete sequence of Staphylococcus saprophyticus strain 16S rDNA complete sequence and the comparative analysis of GenBank database that records,
When PCR checks order, use following forward and reverse primer:
P-f：CACGACGTTGTAAAACGACAGTTTGATCCTGGCTC，
P-r：GGATAACA ATTTCACACAGGAAGGAGGTGATCCAGCC。
CNB2006100181341A 2006-01-10 2006-01-10 Gene of chlorine tolerance strain V430 Expired - Fee Related CN100419081C (en)

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