CN105713857B

CN105713857B - A kind of Atrazine degradation bacterium and its application

Info

Publication number: CN105713857B
Application number: CN201610115396.3A
Authority: CN
Inventors: 马利民; 陈松松; 杨盼盼
Original assignee: Tongji University
Current assignee: Tongji University
Priority date: 2016-03-01
Filing date: 2016-03-01
Publication date: 2019-07-05
Anticipated expiration: 2036-03-01
Also published as: CN105713857A

Abstract

The present invention relates to a kind of Atrazine degradation bacterium and its application, which belongs to Rhizobiaceae sword Pseudomonas, and in China typical culture collection center preservation, deposit number is: CCTCC No:M2015741, the deposit date is on December 14th, 2015.The degradation bacteria has efficient degradation characteristic, the biological prosthetic purposes of water body and soil for being polluted by atrazine to atrazine.The bacterium can lead to Cell Division Mode progress fast breeding in biological prosthetic engineering, stable community is established rapidly, when being grown by atrazine-contaminated soil and water body, can use atrazine is that only nitrogen source achievees the purpose that Atrazine biodegrade to administer pollution by pesticides, this bacterial strain can survive in soil and water body, and tolerance atrazine concentration is high, has broad application prospects and good environmental benefit.

Description

A kind of Atrazine degradation bacterium and its application

Technical field

The present invention relates to a kind of bacterial strains, more particularly, to a kind of Atrazine degradation bacterium and its application.

Background technique

The whole world has 12,500,000 tons of pesticide activity component to be used for agricultural production every year according to estimates, the wherein use of herbicide Amount is maximum, accounts for 40% of Pesticide use amount or so.Atrazine (2- chlorine 4-2 isopropylamino -6- ethylamino-s- triazine) and triazine Herbicide Simanex, Garagard and propazine active structure having the same, as the maximum triazine herbicide quilt of usage amount It is widely used for controlling emergence front and back annual gramineae and broadleaf weeds in the agricultural production of the crops such as corn, sorghum.Ah Te Lajin enters environment mainly due to improper use, controls weeds and subsequent rainwash enters surface water and penetrates into or pass through Soil enters underground water.Behavior of the Atrazine in soil environment is related to several different processes, including chemistry, biology and Photochemical degradating, transport, accumulation and absorption.The transmission of Atrazine occurs through diffusion, mass flow, or even is adsorbed to load Organic substance or soil colloid of the body substance such as dissolution.Being adsorbed onto stationary phase such as clay mineral or the soil organism makes herbicide exist Pedosphere causes to accumulate and pollute in the soil.Although forbidding in the nineties, atrazine still can be in European Environment sample Detect, the migration diafiltration of Atrazine and its metabolin after the use of many years, cause in global range underground water pollution and Soil pollution.It was once mass produced and used in Chinese Atrazine, Atrazine and its metabolite is caused to be widely present In soil environment and natural water.Atrazine is widely paid close attention to as a kind of environment incretion interferent, toxicity, Research shows that Atrazine and its metabolite have development genotoxicity, genetoxic, immunotoxicity, neurotoxicity, carcinogenic Property etc..Atrazine leads to food and drinking water safety problem in the pollution of soil and water body.For the pollution of area source of pesticide, by Wide in its pollution range, contaminated area is big, and the difficulty that other methods are administered is big, at high cost to be not easy to implement.Therefore Atrazine ring The biological prosthetic of border pollution is a kind of effective governing measure, and wherein repairing method of microorganism is easy to operate because its is at low cost Can in the environment large-area applications and attract attention, wherein the breeding of Atrazine-degrading Bacteria from Soil become key.

102492637 A of Chinese patent CN discloses one plant of Atrazine degradation bacterium, it is related to one plant of degradation bacteria.It is Acinetobacter calcoaceticus (Acinetobacter sp.) DNS32, in China Committee for Culture Collection of Microorganisms's common micro-organisms Heart preservation, deposit number are CGMCC NO.5365, and the deposit date is on October 21st, 2011.Bacterial strain DNS32 has efficiently drop The ability of Atrazine is solved, degradation rate is up to 95.88% within 36 hours；The optimum temperature range of bacterial strain DNS32 is 25-30 DEG C, than Report that bacterial strain is 5-10 DEG C low；Bacterial strain DNS32 is stronger to the tolerance of salinity, degrades within 36 hours under the conditions of salinity is 1-4% Rate is above 60%, is better than reported other congenerous bacterial strains, the reparation atrazine-contaminated suitable for time-sharing environment with high salt.

Summary of the invention

It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of Atrazine degradations Bacterium and its application.

The purpose of the present invention can be achieved through the following technical solutions:

A kind of Atrazine degradation bacterium, the entitled CX-T of degradation bacteria belong to sword Pseudomonas (Ensifer sp.), in China Type Tissue Collection preservation, deposit number are: CCTCC No:M2015741, and the deposit date is December 14 in 2015 Day.

The Atrazine degradation bacterium is Gram-negative, contact enzyme positive, oxidase positive bacterium.The Aunar is drawn Saliva degradation bacteria is aerobic bacteria, should be used under aerobic conditions.

The degradation bacteria includes degrading genes atzA, atz B, atz C and the atz D of Atrazine, is separately encoded Aunar drawing Saliva degrading enzyme AtzA, Atz B, Atz C, Atz D.Degradation pathway is atz A-atz B-atz C-atz D, mesostate Respectively hydroxyatrazine (Hydroxyatrazine) takes off ethyl Atrazine (N-Isopropylammelide), cyanogen urine Sour (Cyanurica Acid), biuret (Biuret).It is complete to Atrazine degradation, it can be effectively de- to Atrazine Poison.

The base sequence of degrading genes atzA is as shown in SEQ ID NO.1, the base sequence such as SEQ of degrading genes atz B Shown in ID NO.2, the base sequence of degrading genes atz C is as shown in SEQ ID NO.3, the base sequence of degrading genes atz D As shown in SEQ ID NO.4.

The Atrazine degradation bacterium can quickly be bred by Cell Division Mode, can be formed in 10 hours steady Fixed bacterium colony, for the Atrazine of concentration 100mg/L can in 45h it is degradable, have very high degradation efficiency, have very Good engineer application potentiality and application prospect.

The Atrazine degradation bacterium is used for the biology by atrazine-contaminated upper soll layer and natural water surface layer It repairs.The Atrazine degradation bacterium can breed rapidly in by atrazine-contaminated soil or water body, and with soil Or the Atrazine in water body is only nitrogen source, utilizes contained degrading genes atz A, atz B, atz C, atz D encoding transcription Degrading enzyme atz A, Atz B, Atz C, Atz D Atrazine degradation is metabolized, the biology for reaching soil and water pollution is repaired Multiple purpose, degradation Atrazine carry out the ecological restoration function of contaminated soil by degrading genes express corresponding biological enzyme into A series of biological respinses of row are completed.The bacterial strain has high tolerable concentration (being greater than 100mg/L) and high drop to atrazine-contaminated Efficiency is solved, there is effective Governance Ability for atrazine-contaminated soil and water body.The use of bacterial strain can effectively change Kind and recovery soil and water ecological setting, have a good application prospect.

It is proliferated using Atrazine degradation strain is added in soil or water body environment by Cell Division Mode.

The Atrazine degradation strain is dry powder-shaped or bacteria suspension state.

The Atrazine degradation bacterium is in use, best degradation temperature range is 25-35 DEG C, the pH of best environment of degrading Range is 5-9, can be good at reform of nature environment.

The present invention with Atrazine orientation domestication by Long-term selection by, by the bacterium in atrazine-contaminated soil, being trained It supports, filters out a kind of efficient degrading bacteria (Ensifer sp.) of Atrazine, its base of degrading is identified by molecular biology method Because of atz A, atz B, atz C, atzD, studying its degradation pathway is atz A-atz B-atz C-atz D, can be by Aunar Draw saliva degradation-detoxification.Preliminary soil degrading experiment show the bacterial strain can within the scope of 25-35 DEG C of temperature in 45h by concentration It is degradable for the Atrazine of 100mg/L.By application of the bacterial strain in contaminated soil can effectively reach administer Ah The mesh of Te Lajin pollution, has broad application prospects and good environmental benefit.

Compared with prior art, the present invention has the following advantages and beneficial effects:

1, bacterial strain of the invention (Ensifer sp.) has good to Atrazine degradation characteristic, and degradation characteristic is used In the biological prosthetic purposes by atrazine-contaminated soil and water body.It can be under the conditions of room temperature normal ph by Atrazine Efficient degradation achievees the purpose that Atrazine detoxification repairing polluted soil.

2, bacterial strain of the invention (Ensifer sp.) is the substance of domestication breeding in soil, is added to soil as engineering bacteria It not will receive the repulsion of native bacterium in earth, can quickly breed the mesh for increasing and realizing that degradation Atrazine administers soil pollution 's.

3, the best degradation temperature of bacterial strain of the invention (Ensifer sp.) is 25-35 DEG C, Optimal pH 5-9, can be fine Engineer application under the conditions of ground reform of nature.

4, bacterial strain of the invention (Ensifer sp.) include atrazine-degrading gene atz A, atz B, atz C, AtzD can encode four kinds of enzymes of degradation process, more thorough to the detoxification degradation of Atrazine.

5, bacterial strain of the invention can carry out being formed in quick breeding 10 hours by Cell Division Mode stable Bacterium colony can have well the Atrazine of concentration 100mg/L with degradable in 45h again, with very high degradation efficiency Engineer application potentiality and application prospect.

Detailed description of the invention

Fig. 1 is the PCR amplification map of Atrazine degradation bacterium 16S rRNA gene；

Fig. 2 is the systematic evolution tree of the bacterial strain CX-T based on 16S rRNA gene order；

Fig. 3 is the systematic evolution tree of the bacterial strain H based on 16S rRNA gene order；

Fig. 4 is chadogram for the bacterial strain K's based on 16S rRNA gene order；

Fig. 5 is the systematic evolution tree of the bacterial strain W based on 16S rRNA gene order；

Fig. 6 is hydroxyatrazine mass spectrogram；

Fig. 7 is cyanuric acid mass spectrogram；

Fig. 8 is growth curve chart；

Fig. 9 is degradation curve figure；

Figure 10 is influence result of the temperature to degradation efficiency；

Figure 11 is influence result of the pH to degradation efficiency；

Figure 12 is influence result of the revolving speed to degradation efficiency；

Figure 13 is that atz A and trz N expand electrophoretogram；

Figure 14 is that atz B, atz C expand electrophoretogram；

Figure 15 is that atz D and trz D expand electrophoretogram；

Figure 16 is that atz E and atz F expand electrophoretogram.

Specific embodiment

The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.

Embodiment 1

CX-T bacteria selection: using enrichment culture, the isolated method of scribing line, isolated from pedotheque can degrade Ah The bacterial strain of Te Lajin, and preliminary assessment is carried out to its degradation efficiency, filter out efficient degrading bacterial strain.

1, required reagent: Atrazine standard items (ATR): Dr.Ehrenstorfer, 99.8%；Atrazine raw medicine: TCI, 99.6%；The chromatographies pure reagent such as acetonitrile purchases Town in Shanghai spectrum company；Yeast extract, peptone, agar powder etc. are domestic point Analyse pure reagent.

2, culture medium

Basal medium (MSM, g/L): K₂HPO₄·3H₂O 1.60g、KH₂PO₄0.40g, sucrose 3.00g, MgSO₄· 7H₂O 0.20g、NaCl 0.10g、CaCl₂0.02g, Atrazine (1g/L methanol solution) 10mL, microelement 5mL, dH₂O 1000mL

LB culture medium: yeast extract 5g, peptone 10g, NaCl 5g, dH₂O 1000mL

For the above culture medium at 121 DEG C, 0.1MPa steam sterilizing 20min, solid medium adds 20g agar wherein Powder.

Trace element solution: Fe₂SO₄·7H₂O 2.75g、MnSO₄·7H₂O 0.33g、CoCl₂·6H₂O 0.24g、 Na₂MoO₄·2H₂O 0.17g、ZnSO₄·7H₂O 0.08g、CuSO₄·5H₂O 0.05g、dH₂O 1000mL。

3, soil collection method: acquisition surface layer (0~20cm) soil sample is stored at room temperature, part soil is taken after sending to laboratory Start to test, remaining soil air-dries, and saves after removing the wherein impurity such as branch, grit.

4, the enrichment culture of degradation bacteria strains: each soil sample takes 5g, and sterile water, shaken overnight is added, takes soil dilution respectively Liquid 10mL is added in Atrazine basal salt media, and using 250ml triangular flask, liquid amount 100ml takes 10mL every 5~7d Culture solution is inoculated into fresh basal medium, because solubility is smaller in aqueous solution for Atrazine, is drawn by observation Aunar The culture situation that bacterium is judged whether disappearance of saliva powder, continuous culture 4 weeks.Using dilution-plate method, scribing line partition method Separation and purifying bacterial strain.

5, enrichment culture liquid the isolation and purification of degradation bacteria strains: is diluted to 10 respectively^-1、10^-2、10^-3、10^-4、10^-5、10^-6、10^-7It respectively takes 100 μ l to be respectively coated on basic solid medium, is placed in 30 DEG C of constant incubators and cultivates；Culture terminates Afterwards, the best single colonie of picking growing way on basic culture dish, continuously scribing line is separated 3~5 times, is purified on LB culture medium Bacterial strain.

6, prepared by bacteria suspension: by the strain inoculated of purifying in LB culture medium, being incubated overnight.It is transferred to sterile centrifuge tube In, it is centrifuged 20min under the conditions of 6000r/min, throws aside supernatant, adds suitable phosphate buffer, vortex oscillation, again Centrifugation, this process are repeated 3 times.Finally bacteria suspension OD is configured with phosphate buffer₆₀₀It is spare 2.0 or so.

7, above-mentioned purifying bacterial strain the preliminary assessment of microbial degradation performance: is utilized into LB liquid medium rapid amplifying Afterwards, it is configured to OD₆₀₀In 2.0 or so bacteria suspension, dosage 1% is added in the basal medium of 100mg/L, and 30 DEG C 180rpm shaking table shaken cultivation, each processing set 3 repetitions, the basal medium of thallus is not added as control.After cultivating 96h The content for measuring Atrazine in culture medium, filters out the stronger bacterial strain of degradation capability.

8, the measuring method of Atrazine: culture solution directly passes through 0.45 μm of miillpore filter and is packed into sample injection bottle, to be measured.

HPLC testing conditions: CNWSIL C18 chromatographic column (250mm × 4.6mm i.d., 5 μm)；Mobile phase: V_Acetonitrile∶V_Water= 60:40, flow velocity 0.8mL/min, sample volume 20 μ L, 25 DEG C of column temperature；UV detector, wavelength 220nm.It is legal using peak area external standard Amount.

9, result and analysis: from 4 pedotheques for picking up from Zhejiang, Jiangsu Province, by the enrichment of 4 weeks basal mediums It after culture, separated, purify the bacterial strain that 4 different shapes are obtained, after the amplification of LB culture medium, be prepared into OD₆₀₀2.0 The bacteria suspension of left and right, is seeded in basal medium, assesses its degradation property, to filter out efficient degrading bacterial strain.

The separation of degradation bacteria strains and degradation property are evaluated

It is total to obtain 4 plants of degradation bacteria strains, the degradation to 4 plants of bacterial strains by 4 weeks enrichment secondary cultures, scribing line 3~5 times Performance (96h) measurement result is shown in Table 1.

Degradation property (96h) of 1 bacterial strain of table to basal medium

As can be seen from Table 1, the bacterial strain isolated from Changxing, Jiaxing Pinghu, Jiande, Kunshan and other places can incite somebody to action in 96h The Atrazine of 100mg/L is all degraded；From bacterium source, the pedotheque of Atrazine-degrading Bacteria from Soil strain is isolated Greatly mostly from soil near the plant area of production Atrazine, point of soil physico-chemical property and Atrazine degradation bacterium is thought in research Without directly contacting between, the generation of degradation bacteria may be related with domestication is directed under specific environment, for a long time by high concentration Aunar It draws in saliva contaminated soil, certain micro-organisms is reduced, or even is withered away, but certain micro-organisms quantity gradually increases, and is enriched with Phenomenon.These researchs all provide many suggestion and method to the separation of bacterial strain, purifying.

2 efficient degrading bacterial strain colonial morphology of table and culture medium feature

From the pedotheque for picking up from Zhejiang Province, Jiangsu Province, 9 plants of Atrazine drops are isolated by enrichment culture, scribing line Bacterial strain is solved, and is come in every shape；By the Preliminary Determination of the degradation property to this 9 plants of bacterial strains, 4 high-efficiency degradation bacterial strains are filtered out (96h), wherein the soil sampled near production or the once factory site of production Atrazine, because by long-term Atrazine M8003 line As a result, can be easily separated out the efficient degrading bacteria of Atrazine, optimal selection is CX-T bacterial strain.

The storage conditions of each bacterial strain are as follows: cryogenic freezing saves (- 20 DEG C) protective agent: glycerine (glycerol).

Embodiment 2

The identification of degradation bacteria strains

Main agents:SPIN Kit for Soil (MP), Taq archaeal dna polymerase, dNTPs, agarose,18-T Vector connection kit, plasmid extraction kit, H₂O₂, crystal violet, sarranine, oxidizing ferment etc..

The present embodiment does simple analysis only for features such as bacterial strain Gram's staining, catalase and oxidizing ferment, specific former Reason is referring to " common bacteria system identification handbook ".

Genome extracts: purifying bacterial strain is incubated overnight using LB culture medium inoculated, according toSPIN Kit Strain cultured solution is added in for Soil operation manual, and 3~4h can get template DNA.

Analysis of physio biochemical characteristics is carried out to bacterial strain, as shown in table 3, Physiology and biochemistry qualification result shows CX-T and K for leather Lan Shi negative bacterium, catalase, oxidase positive bacterium；H and W is Gram-positive, catalase negative bacterium.

3 bacterial strain physiological and biochemical property of table

The amplification of bacterial strain: respectively using the total DNA of 4 plants of bacterial strains as template, through PCR amplification.Experimental result is shown in Fig. 1.Succeed To the amplified production of 4 plants of degradation bacteria 16S rRNA genes, about between 1300bp~1500bp.Remaining primer fails amplification Target gene length out, details are not described herein.

Phylogenetic tree analysis:

Since the 16S rRNA gene of same kind, category bacterium has the conservative of height, so 16S rRNA gene sequence The homology analysis of column is commonly used for the network analysis of bacterial strain.

After purification by the cutting of gained band, it send and gives the raw work sequencing in Shanghai, gained sequence is as shown in SEQ ID NO.5, bacterial strain After sequencing, the sequence of 4 plants of bacterial strains, which is submitted GeneBank database and carries out Blast with the sequence in database, to be compared, Search out with the highest correlated series of its homology, determine the classification of bacterial strain, it may be determined that belong to.With the Neighbor- of MEGA5.0 Joining formation function phylogenetic tree knows that CX-T bacterial strain qualification result is by the development tree information of CX-T Ensifer.sp.Fig. 2~Fig. 5 is the systematic evolution tree of above 4 plants of bacterial strains.According to the homology of 16S rRNA gene: by 4 plants of bacterium Strain be divided into two classes, wherein CX-T, K in 16S rRNA gene order with sword Pseudomonas (Ensifer.sp) and Sinorhizobium (Sinorhizobium sp.) has 98% or more similarity, in GenBank matching, CX-T and Ensifer sp.SB2 Matching degree reaches 99.2%, reaches 99.6% with Sinorhizobium americanum DSS1 matching degree, same K with Sinorhizobium sp.SB2 matching degree reaches 98.5%, is 98.8% with Ensifer adhaerens 4CCS7 matching degree. We make phylogenetic tree with Mega, and using nearest bacterial strain as reference, CX-T belongs to Rhizobiaceae (Family Rhizobiaceae) sword Pseudomonas (Ensifer sp.), K belong to Rhizobiaceae (Family Rhizobiaceae) Sinorhizobium Pseudomonas (Sinorhizobium sp.)；H, W in 16S rRNA gene order with streptomyces (Streptomyces sp.) 97% matching degree is kept, therefore H, W are belonged into streptomyces.

Embodiment 3

1/10LB culture medium: yeast extract 0.5g, peptone 1.0g, NaCl 0.5g, dH₂O 1000mL.Culture medium exists 121 DEG C, 0.1MPa steam sterilizing 20min.Other culture mediums are same as above.

The analysis of catabolite: optimizing cyanuric acid, hydroxyatrazine standard substance first with ion source, Culture solution sample 0.22 μm of miillpore filter of direct mistake, using the Mass Spectrometry Conditions optimized, direct injected detects Targeting groups.Ion Source is the source ESI.

1, the measurement of growth curve:

Bacteria suspension is inoculated into 1/10LB culture medium by inoculum concentration for 1%, is cultivated under the conditions of 30 DEG C, 180r/min, preceding The every 4h sampling of 28h is primary, measures OD₆₀₀Value, every plant of bacterial strain repeat three.A blank control is done simultaneously.

2, the measurement of degradation curve:

Degradation bacteria strains bacteria suspension is inoculated with into the basal medium (pH=7.0) of the Atrazine containing 100mg/L, inoculum concentration is 1%, shake culture under the conditions of 30 DEG C, 180rpm starts sample interval 12h, subsequent dense according to the Atrazine of measurement Degree adjusts sample time, with the concentration of HPLC detection Atrazine.

3, the influence of temperature, pH value and revolving speed to degradation:

Degradation bacteria strains bacteria suspension is inoculated with into the basal medium (pH=7.0) of the Atrazine containing 100mg/L, inoculum concentration is 1%.25 DEG C, 30 DEG C, 35 DEG C, shake culture under the conditions of 180rpm select time sampling to measure Atrazine according to degradation curve.

Degradation bacteria is inoculated with into the basal medium (pH=5.0, pH=7.0, pH=9.0) of the Atrazine containing 100mg/L Strain bacteria suspension, inoculum concentration 1%, shake culture under the conditions of 30 DEG C, 180rpm select time sampling measurement according to degradation curve Atrazine.

Degradation bacteria strains bacteria suspension is inoculated with into the basal medium (pH=7.0) of the Atrazine containing 100mg/L, inoculum concentration is 1%, shake culture under the conditions of 30 DEG C, 100r/min, 180rpm, 260r/min selects time sampling to survey according to degradation curve Determine Atrazine.

All of above sample does three repetitions, and experiment has a blank cultures as control every time.

4, using cyanuric acid as the strain growth situation of only nitrogen source:

Atrazine in basal medium is replaced as cyanuric acid, other operate same atrazine degradation experiments.

5, result and analysis

5.1, the analysis of catabolite

Reported Atrazine degradation product hydroxy Atrazine, cyanuric acid are carried out using LC-MS Atrazine Analysis.To the biological sample of culture, after culture solution crosses 0.22 μm of filtering with microporous membrane, qualitative point is done to its product using LC-MS Analysis.Testing result is shown in Fig. 6 and Fig. 7.

After testing, the group in bacterial strain product all containing molecular weight 128 and 198 or so, it can therefore be concluded that going out Contain cyanuric acid and hydroxyatrazine in the catabolite of bacterial strain.

According to the detection to product, hydroxyatrazine is detected in 4 samples, and showing 4 plants of bacterial strains can generate Atrazine dechlorination hydrolase, dechlorination hydrolysis enzymatic Atrazine are hydrolyzed to hydroxyatrazine, illustrate to contain in 4 plants of bacterial strains There are gene trz N or atz A.

In addition, also showing 4 plants of bacterial strains to the testing result of cyanuric acid can convert Atrazine to cyanuric acid, Thus the deduction before, degradation bacteria strains should also contain gene atz B and atz C other than containing gene trz N or atz A.

5.2, the growth of bacterial strain, degradation characteristic research

5.2.1 growth curve

Fig. 8 show 4 plants of bacterial strains in 1/10LB culture medium growing state, in 4 initial~8h, bacterial strain be in adjust Whole phase, Adaptable growth environment, 8~20h are in increased logarithmic phase, and bacterial strain is quickly bred.OD₆₀₀It dramatically increases；Then into steady Periodically, in this stage, each strain bacterial strain is different, and decline phase is just entered after about 184h.

5.2.2 degradation curve

The part determines degradation of 4 plants of bacterial strains to Atrazine.The result shows that: with the extension of incubation time, Aunar Saliva content is drawn to gradually decrease, the time used in the degradable culture solution of bacterial strain is not exactly the same, wherein CX-T, W and K degradation energy Power is suitable, Atrazine degradation in culture medium, bacterial strain H degradation efficiency can be better than CX-T and W and K in 45h, culture medium of degrading It is 42h that middle Atrazine, which needs the time, is detailed in Fig. 9.But it as it can be seen in figure 9 that the degradation curve of CX-T, W essentially coincide, drops Solution is more steady；And K is between preceding 24~30h, degradation rate is more steady, then rapidly increases, and degradation rate increases.H There is degradation situation similar with K.

5.2.3 the influence of temperature, pH and revolving speed to degradation

The temperature of different strains type suitable growth is different.It utilizes the activity with degradation of organic substances also different from.? After obtaining degradation curve, the influence of temperature, pH, revolving speed to Atrazine removal effect is investigated respectively and determines each strain through overtesting The sample time of bacterial strain is as follows: CX-T (42h), H (42h), W (42h), K (42h).

From Figure 10 it can be found that under the conditions of revolving speed 180r/min, pH=7, for CX-T, H, W, 30 DEG C of condition declines Solution rate is compared with 25 DEG C and 35 DEG C of height, and at 30 DEG C, atrazine degradation rate, which is differed close to 100%, 25 DEG C with 30 DEG C of degradation effects, to exist 43% or more, at 35 DEG C, degradation efficiency is differed also 10% or more.It can be seen that temperature change influences the degradation of each bacterial strain Though it is different, it is still apparent.And K, in 25 DEG C and 35 DEG C, degradation effect is better than 30 DEG C, and difference is obvious, Difference is all 14% or more.

Acid-base condition is one of the important environmental factor for influencing strain growth development.Therefore, the pH value of strain growth environment It is utilized and the ability of degradation of organic substances have significantly affect.

From in Figure 11 it can be found that: for CX-T, degradation efficiency all tends to 100% when pH=5,7, is higher than pH=9, phase Difference about 8%, shows under acid and neutrallty condition, CX-T is easy to grow；W also has same trend, but at these three Under part, because of the relationship of sample time, otherness is unobvious, and the degradation rate of pH=5 and pH=9 have differed only by 4%；But we have Reason is believed, in the case where shifting to an earlier date sample time, the degradation rate of acid condition is higher than neutrallty condition and alkaline condition.To H Speech, pH=7 degradation rate tend to 100%, pH=9 and have degraded completely, and degradation rate differs not poor mistake 0.1%, and otherness is unobvious, but It is to differ about 47% with the degradation rate of pH=5, illustrates that this bacterium is easy to Atrazine of degrading under neutral, alkaline condition；To K Speech, when pH=5 degradation rate tends to 100%, pH=9 degradation rate is suitable therewith, and degradation rate difference is no more than 1%, and pH=7 Under the conditions of degradation rate be only 80.1%, show that the bacterium has very strong adaptability to acid-base condition.

Revolving speed influences contact situation of the bacterial strain with culture solution, and influences coming into full contact with for bacterial strain and culture medium, and oxygen Contact of the mass transfer by bacterial strain with culture medium influenced, can also influence the degradation of bacterial strain indirectly.

As can see from Figure 12: for CX-T, when the degradation efficiency of 180rpm is 99.71%, in 100rpm and The degradation effect of 260rpm is 99% and 61% respectively, and degradation efficiency is significant lower when high revolving speed, and W also has similar experiment As a result, degradation efficiency (78%) is higher than CX-T still when 260rpm；For H, the degradation efficiency of 180rpm is It is 98% and 100% respectively in the degradation effect of 100rpm and 260rpm when 100%；The difference of degradation efficiency under the conditions of these three It is anisotropic unobvious；For K, under three conditions, atrazine degradation rate is followed successively by 54%, 80%, 100%, occurs very Apparent gradient phenomenon.

5.3, strain growth situation in cyanuric acid culture medium

From 5.1 parts it can be inferred that bacterial strain should contain gene trz N or atz A and atz B, atzC, but can bacterial strain Permineralization Atrazine, namely whether contain atz DEF or trz D, we are still uncertain.Research thinks that cyanuric acid is The important intermediate of the degradable Atrazine of degradation bacteria strains energy and final product that cannot be degradable, can be by atz D or trz The enzymatic open loop of D coding, generates biuret.Can it be nitrogen source growth using cyanuric acid that this section mainly investigates degradation bacteria strains, As bacterial strain the reference index of Atrazine permineralization can be provided to reference for subsequent gene magnification, the result is shown in tables 4。

Strain growth situation in 4 cyanuric acid culture medium of table

Bacterial strain	CX-T	K	W	H
					Growing state	+	+	+	+

Note :+indicating bacterial strain well-grown in the medium, culture medium is gradually muddy, OD₆₀₀Increase；Indicate that bacterial strain is being trained It supports and is not grown in base, culture medium OD₆₀₀It is held essentially constant in cultivation cycle.

Table 4 shows upgrowth situation of the bacterial strain in cyanuric acid culture medium, thus it is speculated that degradation bacteria strains are using sucrose as carbon source In the case where, it is that nitrogen source is grown using cyanuric acid.From this phenomenon it can be inferred that degradation bacteria strains have to draw Aunar The ability of saliva permineralization.This also indicates that 4 plants of bacterial strains should contain cyanuric acid hydrolase gene trz D or atz D.

Embodiment 4

The research of strains for degrading approach

The process of microbial degradation Atrazine, the enzymatic Atrazine dechlorination water mainly generated by microorganism Solution, de- alkyl, open loop.Up to the present, 4 approach are reported completely, relevant 8 kinds of enzymes in degradation process And related gene, primer etc. are well-known.Hereafter correlative study discovery, atz A, atz B and atz C are that have height Spend the gene of conservative.Therefore, with the conserved sequence primer of reported atz A, atz B and atz C, degrading genes are carried out Amplification.

Hereafter, Mulbry W W etc. constructed the specificity amplification primer of gene trz N in 2002, in subsequent article It is widely used, this primer is also used in the present invention.

For atz D and trz D gene, Fruchey I etc. is using more plants of Atrazine degradation bacterium as research object, discovery drop Solution bacterial strain cannot contain atz D and trz D gene simultaneously, while also report corresponding primer, this document is also used in the present invention In sequence.

For subsequent two degrading genes atz E and atz F, subsequent bacterial strain can seldom amplify corresponding gene, therefore Study it is less, mostly with the research of Pseudomonas sp.ADP be reference.Therefore the present invention uses the overall length of atz E and atz F Primer respectively expands gene atz E and atz F.

It is to sum up told, the present invention is with the primer of 8 kinds of above-mentioned degrading genes, respectively to the efficient drop of the invention isolated and purified The degrading genes of solution bacterial strain are expanded, and are understood the degrading genes composition of each strain bacterial strain, are analyzed involved in each strains for degrading process The enzyme arrived describes the degradation process of bacterial strain.

The amplification of target gene segment

The synthesis of 1.PCR amplimer

Primer needed for PCR reacts is incorporated in the document delivered, and is shown in Table 5, by the raw limited public affairs of work bioengineering share in Shanghai Department's synthesis.

The target gene primer sequence of 5 Atrazine degradation bacterium of table

2.PCR amplification

PCR reaction system is 30uL, and each group is grouped as: Taq archaeal dna polymerase (5U/ μ L), 1 μ L；10 × buffer, 3 μ L； 2.5mmol/L dNTP, 1 μ L；25mmol/L MgCl₂, 3 μ L, each 1 μ L of forward primer, reverse primer, template DNA, 1 μ L steams again 19 μ L of water.

Amplification condition are as follows: 94 DEG C, initial denaturation 3min；94 DEG C, it is denaturalized 60s；Annealing 60s is required according to primer in table 5；72℃ Lower extension 120s, 35~50 circulations；72 DEG C of extension 10min, 4 DEG C of preservations.PCR product passes through 1.0% agarose gel electrophoresis After EB dyeing, is observed and taken pictures under uv analyzer.

As a result with analysis

It has been found that atrazine-degrading gene degradation bacteria in be it is very conservative, especially degrading genes atz A, Atz B and atz C, the present invention expand degrading genes according to the gene primer sequence delivered.Amplification is detailed in figure 13-16。

Figure 13 is shown in the amplification electrophoretogram of chlorine hydrolase gene trz N and atz A, wherein CX-T and H amplifies trz N base Cause, K and W amplify atz A gene.Institute's amplification gene length is in target length or so, thus may determine that CX-T and H contain Trz N gene, K and W contain atz A.

To degrading genes atz B, the mrna length expanded in bacterial strain H is in 300bp or so, and amplification length is more than in K 500bp, and occur the gene of target fragment length in W and CX-T, it is contemplated that the specificity of atz 1 B gene, therefore Think that 4 plants of bacterial strains all amplify gene atz B.

Equally to gene atz C, the length of each strain bacterial strain amplification is slightly different, it is contemplated that the specificity of each strain bacterial strain, Think that 4 plants of bacterial strains all amplify atz C.

Amplification to gene atz D and trz D, CX-T and H amplify atz D, and discovery bacterial strain K and W amplify trz D.

To degrading genes atz E and atz F, the amplified band of specific length is all arrived without expanding, therefore these bacterial strains are equal Do not contain atz E and atz F.

As previously mentioned, less for the Study on degradation of Rhizobiaceae and streptomycete, especially in terms of its mechanism of degradation Research, although being determined in chapter 3 to the catabolite of bacterial strain, and do not carried out the analysis of gene composition, but still not The degradation pathway that bacterial strain can be understood completely, cannot be fully described the degradation process of bacterial strain, this section is according to atrazine-degrading gene Conserved sequence, amplification obtains Atrazine degradation enzyme gene, the degrading genes of each strain bacterial strain counted, are shown in Table 6.

6 degrading genes amplification of table

Bacterial strain CX-T and H amplifies gene trz N, atz BCD band as can be seen from Table 6, and fails to amplify gene Atz A and atz EF band, this two bacterial strain are different from the Psendomonas sp.ADP, Arthrobacter to have registered Aurescens TC1 and Alcalegenes sp.SG1, and it is identical as Arthrobacter sp.MCM B-436, it may infer that Out Atrazine can mineralising be CO₂And water, this result can be using cyanuric acids as the culture medium of nitrogen source by CX-T, H bacterial strain Middle growth is supported, therefore the degradation pathway of CX-T and H is: Atrazine is catalyzed Atrazine dechlorination under the action of Trz N It is hydrolyzed to hydroxyatrazine, then under Atz B effect, catalysis hydroxyatrazine sloughs ethylamino group, generates de- Ethylamino hydroxyatrazine sloughs isopropylamino group under Atz C effect again, generates cyanuric acid, finally exist Atz D effect is lower to generate biuret.

Bacterial strain K and W amplify gene atz ABC and trz D band, and do not amplify other bands, be inferred to bacterial strain K and Atrazine mineralization can be CO by W₂And water, this result can be using cyanuric acids as life in the culture medium of nitrogen source by K and W bacterial strain Length is supported, therefore the degradation pathway of K and W is: under the action of Atz A, catalysis Atrazine dechlorination is hydrolyzed to Atrazine Hydroxyatrazine, then under Atz B effect, catalysis hydroxyatrazine sloughs ethylamino group, generates de- ethyl ammonia Base hydroxyatrazine sloughs isopropylamino group under Atz C effect, generates cyanuric acid, finally under Trz D effect Generate biuret.

Research thinks that gene atz A is present in Gram-negative bacteria more, Arthrobacter AD1 exception.Rhizobium are that leather is blue Family name's negative bacterium, gene trz N is by successful clone mistake, but atz A has not been reported, and present invention success is expanded in Rhizobiaceae bacterial strain K Increase gene atz A band out；Gene trz N is at first in Nocardioides sp.C190 discovery, reported Gram-positive Efficient degrading bacterial strain had not amplified gene atz A based on actinomyces Nocardia so far, and present invention success is blue in leather Gene atz A band has been amplified in family name's positive bacteria streptomycete bacterial strain W.In Gram-negative bacteria and gram-positive bacteria, though The distribution of right gene atz A and trz N is presented different difference, but Recent study gradually increases, in two class bacterial strains All successfully different hydrolase genes is arrived in amplification, this research is no exception, has broken hydrolase gene in gram-positive bacteria With the distribution hedge in negative bacterium, there is directive function to subsequent research.

Research thinks, microorganism removes triazine in environment and in carbon that they are participated in, Nitrogen Cycling, The cracking of triazine ring is a critically important step.But up to the present, related gene atz D and trz D are not yet in root nodule It is found in Rhizobiaceae bacterium, the present invention is that amplification is to gene atz D and trz D in Rhizobiaceae for the first time, and the conclusion is not It is same as the degradation pathway of reported Rhizobiaceae bacterial strain Sinorhizobium sp.NEA-B, the degradation pathway of the bacterium is trz N-atz BC。

Though the present invention is not the efficient degrading bacterial strain for isolating streptomyces from pedotheque for the first time, to being at present Only, the degrading genes of streptomyces degradation bacteria and degradation pathway research still belong to blank.The streptomyces isolated drops in the present invention Solution gene and degradation pathway have carried out more in-depth study, compensate for the blind spot of Pseudomonas research, have to subsequent research Higher reference value.

In short, according to the viewpoint and research achievement of the invention of Ralebitso TK, to Rhizobiaceae and streptomycete For belonging to actinomyces, research of the invention is all two novel degradation pathways for them.

With reported atrazine-degrading gene primer amplification degrading genes, gene trz N and atz BCD are in bacterial strain It amplifies and in CX-T and H, and gene atz AEF is cloned not successfully, deduction bacterial strain CX-T and H degradation pathway is trz N-atz BCD；To bacterial strain K and W, while gene atz ABC and trz D is amplified, other genes also do not clone success, infer bacterial strain K and W Degradation pathway be atz ABC-trz D.To hydrolyzable group because research think: gene atz A and trz N are in Gram-negative bacteria Though the distribution different with presentation in gram-positive bacteria, the present invention successfully expands in Gram-negative bacteria and positive bacteria Increase and atz A and trz N gene；Furthermore for the first time to streptomyces bacterial strain carry out degrading genes and degradation pathway research, be To the supplement and extension of the mechanism of degradation of Atrazine-degrading Strains.

The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention. Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein general Principle is applied in other embodiments without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be of the invention Within protection scope.

Sequence table

110 > Tongji University of <

A kind of Atrazine degradation bacterium of 120 > of < and its application

〈160〉5

〈170〉Patent Inversion

〈210〉1

〈211〉459

〈212〉DNA

213 > Rhizobiaceae sword Pseudomonas (Rhizobiaceae Ensifer sp.) of <

〈220〉

The base sequence of 223 > degrading genes atz A of <

〈400〉1

tccttcactg tcaccgccgt ggtagtggca ggagcgggcc aaactgatat acgacctcct 60

gccgtgccat gatactgatc tgacagggct gtgatccgat ctttggccac agccgtttcc 120

tccatgatcg agcacagttc gacttgggga gagcgagcct tcaaggcgtc cacataccct 180

tgaatgcgcc cgtccatccg atcaaagaac atgcgggcgt agacgaccct cacacccacc 240

tcaccataga ccgccatcgc ggcctcgatg ttgcctgggt agatggccga atcggcgttc 300

tcgttgatcg tcgtaatccc gctgcgcaca gcttccgcac aatacaacct caccgccacc 360

gctacgtcct ccggtctcat cgccttttgt cccggaaacg taacgttgaa cagccagtca 420

tagaattgac gcccgtgcga gggccctccg cgcaggagg 459

〈210〉2

〈211〉435

〈212〉DNA

213 > Rhizobiaceae sword Pseudomonas (Rhizobiaceae Ensifer sp.) of <

〈220〉

The base sequence of 223 > degrading genes atz B of <

〈400〉2

gcagccccag caggttagcg gcatcaaact ggcggtccac cagaaagggt ccaccccggc 60

ggctgtagtt gtactggtga tcaaacgcgg tggtgcagcc gtgcttgatc agctccgcca 120

tggacaccac tgtgctgtgg tagatgcagt cttcatccac cagggcaaac accgggtaga 180

tcttgcgcag ccaggccagc acatcaagct gggtccagtc gaggggggca agattgcgga 240

caaaggcctg aaaaaaatgg tgatgggtgt tgatcagccc cggataggcg gtcagccccc 300

ggcagttcac cacctcaaca ccctgttcct ggccaacgcc aattggggcc agagttcggg 360

gcagatccgg gccaaggcca ataatctccc cgtctctcac cagaatatcg acgccattaa 420

gcaccgtgcc cgcga 435

〈210〉3

〈211〉553

〈212〉DNA

213 > Rhizobiaceae sword Pseudomonas (Rhizobiaceae Ensifer sp.) of <

〈220〉

The base sequence of 223 > degrading genes atz C of <

〈400〉3

gccaagattg atgccagctt caagcagctt tatcaccggc atagtaggcg gtgtactact 60

aaaacaggta acaaatttca tacccgaatc cttgtacaat gggattgcct catcgagcca 120

ttcggacgga gcatctgcaa aacaccaggc atgactcgta gttactctac ccttataccc 180

attttcaatt gtcttttggg caagacgatt tatcgaatat actccaacag ttccaatatc 240

atgtatgtga tagtcgatat caacatcgta ttcctttgct aatttaaagc ataggtctaa 300

agaaccctca acattatttt cccgcgtagc aggatcaact cccccaacta aatcacagcc 360

catatccaag gattttctaa tcaatgattc agattccaaa tcaacgaaaa atccactctg 420

tgcaaaggct acgacttgta tatcgataag atcctttaac tcttccttgg cttctaaaac 480

tgcttccact gcttttgttt tagcaactga atctacatct acatgggtcc gggtgtataa 540

agtcccatgg aga 553

〈210〉4

〈211〉491

〈212〉DNA

213 > Rhizobiaceae sword Pseudomonas (Rhizobiaceae Ensifer sp.) of <

〈220〉

The base sequence of 223 > degrading genes atz D of <

〈400〉4

ctcgcctccg ctttggcgaa tacgttgacg atgcgatcca tctcgtcatc gctacgtatg 60

cccagcgatt caatcgttcg ccgcaccgag gccgcgtcga tcgcgtcgct catcacgccg 120

tgggcaatga ccagttcact ggtggcctgc tcgctcatgc caatagcgat caccacgttg 180

tgctccagtt cgatgcctgc agacgccgac gccagtgacg atgagagact ccagtcattc 240

agcactgatt cgtctacgag catcgaggag ggcacctctt ctgtagcgag agcgatgccc 300

agggccgaag cgccgcgcga atagcccatc gattcatacg tatccgtcgt gactggagcg 360

catccgcgtg atcgcgccga ggcgatcttt gctggtgtca gcagcggaca cttcacctgc 420

acaaaatgca gatcgtcaat cgaagcgatc ccggcatctc gcattgcgcg tttgacggcg 480

ccggctgtct c 491

〈210〉5

〈211〉1398

〈212〉DNA

213 > Rhizobiaceae sword Pseudomonas (Rhizobiaceae Ensifer sp.) of <

〈220〉

The 16S rRNA gene order of 223 > bacterial strain CX-T of <

〈400〉5

ttaaacatgc aagtcgagcg ccccgcaagg ggagcggcag acgggtgagt aacgcgtggg 60

aatctaccct tttctacgga ataacgcagg gaaacttgtg ctaataccgt ataagccctt 120

cgggggaaag atttatcggg aaaggatgag cccgcgttgg attagctagt tggtggggta 180

aaggcctacc aaggcgacga tccatagctg gtctgagagg atgatcagcc acattgggac 240

tgagacacgg cccaaactcc tacgggaggc agcagtgggg aatattggac aatgggcgca 300

agcctgatcc agccatgccg cgtgagtgat gaaggcccta gggttgtaaa gctctttcac 360

cggtgaagat aatgacggta accggagaag aagccccggc taacttcgtg ccagcagccg 420

cggtaatacg aagggggcta gcgttgttcg gaattactgg gcgtaaagcg cacgtaggcg 480

gacatttaag tcaggggtga aatcccagag ctcaactctg gaactgcctt tgatactggg 540

tgtctagagt atggaagagg tgagtggaat tccgagtgta gaggtgaaat tcgtagatat 600

tcggaggaac accagtggcg aaggcggctc actggtccat tactgacgct gaggtgcgaa 660

agcgtgggga gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgaatgtt 720

agccgtcggg cagtttactg ttcggtggcg cagctaacgc attaaacatt ccgcctgggg 780

agtacggtcg caagattaaa actcaaagga attgacgggg gcccgcacaa gcggtggagc 840

atgtggttta attcgaagca acgcgcagaa ccttaccagc ccttgacatc ccgatcgcgg 900

attacagaga tgttttcctt cagttcggct ggatcggaga caggtgctgc atggctgtcg 960

tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc tcgcccttag 1020

ttgccagcat ttagttgggc actctaaggg gactgccggt gataagccga gaggaaggtg 1080

gggatgacgt caagtcctca tggcccttac gggctgggct acacacgtgc tacaatggtg 1140

gtgacagtgg gcagcgagac cgcgaggtcg agctaatctc caaaagccat ctcagttcgg 1200

attgcactct gcaactcgag tgcatgaagt tggaatcgct agtaatcgca gatcagcatg 1260

ctgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccatg ggagttggtt 1320

ctacccgaag gtagtgcgct aaccgcaagg aggcagctaa ccacggtagg gtcagcgact 1380

ggggtgaagt cgaacaag 1398

Claims

1. a kind of Atrazine degradation bacterium, which is characterized in that the degradation bacteria belongs to sword Pseudomonas (Ensifer sp.), in China Type Tissue Collection preservation, deposit number are: CCTCC No:M2015741, and the deposit date is December 14 in 2015 Day.

2. a kind of Atrazine degradation bacterium according to claim 1, which is characterized in that it is Gram-negative, catalase Positive, oxidase positive bacterium.

3. a kind of Atrazine degradation bacterium according to claim 1, which is characterized in that the degradation bacteria includes Atrazine Degrading genes atz B, atz C and atz D are separately encoded Atrazine degradation enzyme Atz B, Atz C, Atz D.

4. a kind of Atrazine degradation bacterium according to claim 3, which is characterized in that the base sequence of degrading genes atz B Column as shown in SEQ ID NO.2, the base sequence of degrading genes atz C as shown in SEQ ID NO.3, degrading genes atz D's Base sequence is as shown in SEQ ID NO.4.

5. a kind of Atrazine degradation bacterium according to claim 1, which is characterized in that the Atrazine degradation bacterium passes through Cell Division Mode is quickly bred, and stable bacterium colony can be formed in 10 hours, for the Atrazine of concentration 100mg/L It can be degradable in 45h.

6. a kind of application of such as Atrazine degradation bacterium according to any one of claims 1 to 5, which is characterized in that described Atrazine degradation bacterium is used for by the biological prosthetic of atrazine-contaminated upper soll layer and natural water surface layer.

7. the application of Atrazine degradation bacterium according to claim 6, which is characterized in that the Atrazine degradation bacterium Using the Atrazine in soil or water body as only nitrogen source, Atrazine degradation is metabolized, reaches the life of soil and water pollution Object repairs purpose.

8. the application of Atrazine degradation bacterium according to claim 6, which is characterized in that add Atrazine degradation strain In soil or water body environment, Atrazine degradation bacterium is proliferated by Cell Division Mode.

9. the application of Atrazine degradation bacterium according to claim 8, which is characterized in that the Atrazine degradation bacterium Kind is dry powder-shaped or bacteria suspension state.

10. the application of Atrazine degradation bacterium according to claim 6, which is characterized in that the Atrazine degradation The degradation temperature range of bacterium is 25-35 DEG C, and the pH range for environment of degrading is 5-9.