CN107338197B - The acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade - Google Patents
The acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade Download PDFInfo
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- CN107338197B CN107338197B CN201710267837.6A CN201710267837A CN107338197B CN 107338197 B CN107338197 B CN 107338197B CN 201710267837 A CN201710267837 A CN 201710267837A CN 107338197 B CN107338197 B CN 107338197B
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- quinolinic acid
- dichloro quinolinic
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- degrade
- acinetobacter calcoaceticus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
Abstract
The invention discloses one plant of can degrade dichloro quinolinic acid degradation bacterias and its application.One plant of dichloro quinolinic acid degradation bacteria provided by the present invention is acinetobacter calcoaceticus J4(AcinetobacterSp.J4), it is preserved in China typical culture collection center (CCTCC), the deposit date is on March 16th, 2017, deposit number was CCTCC NO:M 2017129, was accredited as acinetobacter calcoaceticus using 16S rDNA.The bacterium is Gram-negative bacteria, and bacterium colony is white, round, smooth, neat in edge.The bacterium has the function of dichloro quinolinic acid of degrading.
Description
Technical field
The invention belongs to environmental pollution microbiological treatment technical fields, and in particular to one plant of pedo relict dichloro that can degrade
The acinetobacter calcoaceticus of quinolinic acid.
Background technique
Tobacco (Nicotiana tabacum) belong to that Solanaceae is annual or limited herbaceos perennial, originate from South America,
Plantation throughout world various regions at present.Tobacco is a kind of special industrial crops, China from nineteen eighty-two carry out tobacco monopoly system with
Come, tobacco business obtains tremendous development and becomes a mainstay industry of national economy.Tobacco is in China, each provinces and regions, north and south
There is plantation, main application is processing leaf tobacco production cigarette.Therefore, good tobacco leaf is the important goal of tobacco leaf production, such as
What guarantees that good tobacco leaf is also the major issue that tobacco grower is concerned about.In Fujian, tobacco planting mainly based on rice cigarette crop rotation method,
However the use of the common pesticide of first crop crop rice such as herbicide, seriously affect the growth of rear stubble tobacco.Tobacco is to weeding
Agent is more sensitive, and with herbicide being widely used in paddy field, tobacco leaf production is happened occasionally by the phenomenon that herbicide damage,
Great harm is brought to tobacco leaf production.
After existing research shows dichloro quinolinic acid application, there is researcher to detect water near paddy field in the U.S. Arkansas State
The residual quantity of domain dichloro quinolinic acid is up to g/l grades of μ;The environmental behaviour of dichloro quinolinic acid is applied using lisimeter simulated rice field,
The result shows that 95% remains in 30 cm topsoils of paddy field.Remaining dichloro quinolinic acid will affect tobacco plant in soil
The teratogenesis of strain, causes young leaves to be crispaturaed to blade back, it is leaf to be then developing progressively linear rat-tail shape.Due to dichloro quinolinic acid
Phytotoxicity, the yield and the output value of flue-cured tobacco are substantially reduced, and when serious harm can reduce by 80% or more, or even total crop failure.
Microbial method deteriorating pesticide residue have it is environmental-friendly, it is pollution-free, it is efficient the features such as, therefore using biology progress ring
Border reparation is research hotspot in recent years.Separation screening in the soil of dichloro quinolinic acid is used for a long time by field and is obtained by the present invention
Degradation bacteria strains, bacterial strain of the present invention are general Pseudomonas bacterial strain, have not yet to see general Pseudomonas for pedo relict dichloroquinoline
The report of acid degradation；Bacterium source of the present invention has the characteristics that environmental-friendly in soil.Tobacco field residual is released using bioanalysis to remove
Careless agent dichloro quinolinic acid can not only reduce the economic loss that phytotoxicity brings tobacco, and to protection environment, purification soil has not
The meaning that can be despised.
Summary of the invention
Tobacco production and the output value is caused to reduce to solve the problems, such as that above-mentioned dichloro quinolinic acid remains in the soil, the present invention mentions
For the acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade.The bacterial strain can effectively degrade remaining two chloroquine in soil
Quinoline acid, to improve tobacco production.
To achieve the above object, the present invention adopts the following technical scheme:
The acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade, identified, the bacterial strain is acinetobacter calcoaceticus J4
(Acinetobacter Sp. J4), be preserved in China typical culture collection center CCTCC, address: Wuhan, China is military
Chinese university, postcode: 430072；The deposit date is on March 16th, 2017, deposit number was CCTCC NO:M 2017129.
Bacterial strain J4 of the present invention is enriched with by the following method to be obtained:
This test acquires soil from Fujian tobacco field, is screened and is obtained using concentration method, and screening process is as follows:
(1) it the enrichment of dichloro quinolinic acid degradation bacteria: takes 0.1g soil to mix with 100 ml MM culture mediums, and dichloro is added
Quinolinic acid makes its final concentration of 100 μ g/ml, and in 30 DEG C, 150 r/min are cultivated 1 week；It takes 1 ml of culture solution that 100 ml are added to contain
In the fresh culture of same concentration herbicidal agent, while crossing in solid medium to confirm having bacterial growth, similarity condition training
It supports 1 week；It after continuous subculture 10 times, is separated through plate streaking, obtains single colonie bacterial strain.
(2) liquid chromatogram measuring bacterium degradation rate: obtained strains are respectively placed in containing 5 μ g/ml dichloro quinolinic acids and not
It is cultivated in MM culture medium containing dichloro quinolinic acid, MM culture medium of the bacterium solution containing only same concentrations dichloro quinolinic acid is not added as ginseng
According to being measured using high performance liquid chromatography；Sample time is respectively the 6th d and the 23rd d, and sample volume is 1 ml；By sample
Product are centrifuged 10 min under the conditions of 8000 r/min, and supernatant is analyzed after 0.22 μm of membrane filtration for HPLC, each place
Reason repeats three times.HPLC testing conditions are as follows: stationary phase is C18 column (4.6 × 250 nm, 5 μm, Agilent)；Mobile phase is methanol
+ 0.5% acetic acid water (volume ratio 65:35)；Flow velocity is 1.0 ml/min；Column temperature is 30 DEG C；Detection wavelength is 240 nm；Sample introduction
Volume is 10 μ l.
It is detected using dichloro quinolinic acid degradation capability of the testing conditions described in (2) to the bacterial strain, and by following
Formula acquires its dichloro quinolinic acid degradation rate:
Bacterial strain J4 of the present invention can be used for dichloro quinolinic acid of degrading.
The present invention has the advantages that
Separation screening in the soil of dichloro quinolinic acid is used for a long time by field and obtains degradation bacteria strains by the present invention, using biology
Method degradation tobacco field residual herbicide dichloro quinolinic acid can not only reduce the economic loss that phytotoxicity brings tobacco, and to protection ring
Border, purification soil have the meaning that can not be despised.
Detailed description of the invention
Fig. 1 concentration method screens the microbial strains flow chart of degradable dichloro quinolinic acid；
Fig. 2 J4 colonial morphology figure: bacterium colony is white, round, smooth, neat in edge；
Chromatogram of Fig. 3 standard items dichloro quinolinic acid (5 mg/L) in MM culture medium and methanol: A is MM culture medium, B
For the MM culture medium for adding bacterium solution, C is the MM culture medium for adding dichloro quinolinic acid, and D is the dichloro quinolinic acid of dissolution in methyl alcohol；
The standard curve of Fig. 4 high performance liquid chromatography detection dichloro quinolinic acid；
To the degradation rate of dichloro quinolinic acid after addition bacterial strain J4 culture 6d and 23d in Fig. 5 MM culture medium.
Specific embodiment
The screening of 1 bacterial strain J4 of embodiment
This test acquires soil from tobacco field, and the microbial strains of degradable dichloro quinolinic acid, process are screened using concentration method
As shown in Figure 1.This experiment sieving obtains the bacterial strain of one plant of soil dichloro quinolinic acid that can degrade, and is named as J4；It is screened
Process is as follows:
(1) it the enrichment of dichloro quinolinic acid degradation bacteria: takes 0.1g soil to mix with 100 ml MM culture mediums, and dichloro is added
Quinolinic acid makes its final concentration of 100 μ g/ml, and in 30 DEG C, 150 r/min are cultivated 1 week；It takes 1 ml of culture solution that 100 ml are added to contain
In the fresh culture of same concentration herbicidal agent, while crossing in solid medium to confirm having bacterial growth, similarity condition training
It supports 1 week；It after continuous subculture 10 times, is separated through plate streaking, obtains single colonie bacterial strain.The bacterium is Gram-negative bacteria, bacterium
Fall white, round, smooth, neat in edge；Its colonial morphology figure is shown in Fig. 2.
(2) liquid chromatogram measuring bacterium degradation rate: obtained strains are respectively placed in containing 5 μ g/ml dichloro quinolinic acids and not
It is cultivated in MM culture medium containing dichloro quinolinic acid, MM culture medium of the bacterium solution containing only same concentrations dichloro quinolinic acid is not added as ginseng
According to being measured using high performance liquid chromatography；Sample time is respectively the 6th d and the 23rd d, and sample volume is 1 ml；By sample
Product are centrifuged 10 min under the conditions of 8000 r/min, and supernatant is analyzed after 0.22 μm of membrane filtration for HPLC, each place
Reason repeats three times.HPLC testing conditions are as follows: stationary phase is C18 column (4.6 × 250 nm, 5 μm, Agilent)；Mobile phase is methanol
+ 0.5% acetic acid water (volume ratio 65:35)；Flow velocity is 1.0 ml/min；Column temperature is 30 DEG C；Detection wavelength is 240 nm；Sample introduction
Volume is 10 μ l.Its chromatogram is shown in Fig. 3, the results showed that under this condition, can be very good by the target peak of dichloro quinolinic acid
It is separated with impurity.
(3) it makes standard curve: being prepared to obtain 500 μ g/ml dichloro quinolinic acid Standard Stock solutions with chromatography methanol, then
0.10,0.20,0.50,1.0,2.0,5.0 and 10.0 μ g/ml dichloro quinolinic acid series standard works are obtained with chromatography methanol dilution
Make solution.Standard solution is distinguished into sample introduction, the peak area of acquisition figure related to liquor strength work seeks related coefficient.Standard curve
As shown in Figure 4, the results showed that the linearly dependent coefficient r between various concentration dichloro quinolinic acid and peak area is 0.9980；Show two
Chloro-quinolinic acid content and response are in good linear relationship.
Testing conditions described in (2) are utilized to be measured the dichloro quinolinic acid degradation capability of the bacterial strain, and by following
Formula acquires its dichloro quinolinic acid degradation rate:
For measurement result as shown in figure 5, showing after cultivating 6d, bacterial strain J4 is 12.1% to the degradation rate of dichloro quinolinic acid；Culture
After 23d, bacterial strain J4 is 38.6% to the degradation rate of dichloro quinolinic acid.It can be seen that bacterial strain J4 has the dichloro quinolinic acid in soil
There is preferable degradation capability.
2 Molecular Identification of embodiment
Using bacterial 16 S rDNA universal primer fD2/ rP1, after the 16s rDNA sequencing that bacterial strain is expanded by bacterium colony PCR
Compare and analyze identification bacterial strain:
FD2:5 '-AGAGTTTGATCATGGCTCAG-3 ',
RP1:5 '-ACGGTTACCTTGTTACGACTT-3 '；
PCR reaction system: PCR reaction system are as follows: 1 × PCR MIX reaction solution (Quan Shijin Biotechnology Co., Ltd, north
Capital), 0.4 μM of primer, and using a small amount of bacterium colony of toothpick picking as template；
PCR response procedures: carrying out 32 circulations after 95 DEG C of 4 min of initial denaturation, circulation includes 95 DEG C of denaturation 30 s every time, and 58
DEG C annealing 30 s and 72 DEG C of 1 min of extension；Finally 10 min are re-extended at 72 DEG C.
PCR product J4-16S rDNA carried out after agarose gel electrophoresis is verified DNA recycling, sequencing, sequencing result with
Genbank database is compared.The results show that the present invention screens the acinetobacter in obtained strains J4 and Genbank
(Acinetobacter oleivorans) for homology up to 100%, homologous sequence accession number is NR_102814.1.It is identified,
Bacterial strain J4 is acinetobacter calcoaceticus J4(Acinetobacter Sp. J4), number J4 has been preserved in China typical culture collection
Heart CCTCC, address: Wuhan, China Wuhan University, postcode: 430072；The deposit date is on March 16th, 2017, preservations
Number is CCTCC NO:M 2017129.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
SEQUENCE LISTING
<110>Fujian Prov. Co., China Tobacco Corp.
University Of Agriculture and Forestry In Fujian
The acinetobacter calcoaceticus of<120>one plants of pedo relict dichloro quinolinic acids that can degrade
<130> 3
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> fD2
<400> 1
agagtttgat catggctcag 20
<210> 2
<211> 21
<212> DNA
<213> rP1
<400> 2
acggttacct tgttacgact t 21
<210> 3
<211> 940
<212> DNA
<213> J4-16S rDNA
<400> 3
aggtagcttg ctactgatct tagcggcgga cgggtgagta atgcttagga atctgcctat 60
tagtggggga caacatttcg aaaggaatgc taataccgca tacgtcctac gggagaaagc 120
aggggatctt cggaccttgc gctaatagat gagcctaagt cggattagct agttggtggg 180
gtaaaggcct accaaggcga cgatctgtag cgggtctgag aggatgatcc gccacactgg 240
gactgagaca cggcccagac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300
ggaagcctga tccagccatg ccgcgtgtgt gaagaaggcc ttatggttgt aaagcacttt 360
aagcgaggag gaggctactt tagttaatac ctagagatag tggacgttac tcgcagaata 420
agcaccggct aactctgtgc cagcagccgc ggtaatacag agggtgcaag cgttaatcgg 480
atttactggg cgtaaagcgc gcgtaggcgg ctaattaagt caaatgtgaa atccccgagc 540
ttaacttggg aattgcattc gatactggtt agctagagtg tgggagagga tggtagaatt 600
ccaggtgtag cggtgaaatg cgtagagatc tggaggaata ccgatggcga aggcagccat 660
ctggcctaac actgacgctg aggtgcgaaa gcatggggag caaacaggat tagataccct 720
ggtagtccat gccgtaaacg atgtctacta gccgttgggg cctttgaggc tttagtggcg 780
cagctaacgc gataagtaga ccgcctgggg agtacggtcg caagactaaa actcaaatga 840
attgacgggg gcccgcacaa gcggtggagc atgtggttta atttcgatgc aacgcgagaa 900
tcttacctgg tcttgacata gtagactttc cagagatgga 940
Claims (2)
1. the acinetobacter calcoaceticus of one plant of pedo relict dichloro quinolinic acid that can degrade, it is characterised in that: the bacterial strain is acinetobacter calcoaceticus
J4(Acinetobacter Sp. J4), it is preserved in China typical culture collection center CCTCC, the deposit date is 2017
March 16, deposit number are CCTCC NO:M 2017129.
2. using the acinetobacter calcoaceticus of the described in claim 1 one plant pedo relict dichloro quinolinic acid that can degrade in two chloroquines of degrading
Application in quinoline acid.
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| CN108220211B (en) * | 2018-04-02 | 2019-12-31 | 中国科学院微生物研究所 | Acinetobacter oleophilic NMB17 and application thereof in plant disease control |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154141A (en) * | 2010-10-19 | 2011-08-17 | 中国科学院武汉病毒研究所 | Pseudomonas sp. NyZ42 strain capable of degrading tribenuron-methly and preparation method thereof |
| CN103243060A (en) * | 2013-05-27 | 2013-08-14 | 湖南农业大学 | Quinclorac degrading bacteria and application thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154141A (en) * | 2010-10-19 | 2011-08-17 | 中国科学院武汉病毒研究所 | Pseudomonas sp. NyZ42 strain capable of degrading tribenuron-methly and preparation method thereof |
| CN103243060A (en) * | 2013-05-27 | 2013-08-14 | 湖南农业大学 | Quinclorac degrading bacteria and application thereof |
Non-Patent Citations (3)
| Title |
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| 1株二氯喹啉酸降解菌QC06的筛选鉴定及其降解特性;范俊 等;《中国生物防治学报》;20130831;第29卷(第3期);第431-436页 * |
| Bacterial community dynamics and enhanced degradation of di-n-octyl phthalate (DOP) by corncob-sodium alginate immobilized bacteria;Ke Zhang 等;《Geoderma》;20170622;第305卷;第264-274页 * |
| Stress response of Burkholand the biodegradation of quincloracderia cepacia WZ1 exposed to quinclorac;Zimu Li 等;《Soil Biology & Biochemistry》;20091231;第41卷;第984-990页 * |
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