CN105779477A - Broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application - Google Patents
Broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application Download PDFInfo
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- CN105779477A CN105779477A CN201610231173.3A CN201610231173A CN105779477A CN 105779477 A CN105779477 A CN 105779477A CN 201610231173 A CN201610231173 A CN 201610231173A CN 105779477 A CN105779477 A CN 105779477A
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Abstract
The invention discloses a broad-spectrum substituted urea herbicide degradation bacterium and amidohydrolase gene and application and provides an amidohydrolase gene of a substituted urea herbicide and hydrolase protein induced by the gene.The total length of the gene is 1377 bp, the G+C content is 63.5%, 458 amino acids are encoded, and the gene is a new amidohydrolase gene resource having a broad-spectrum hydrolysis function on the substituted urea herbicide, 30 mg/L N-methoxyl-N-methyl substituted urea herbicide (linuron) and N,N-dimethyl substituted urea herbicide (diuron, chlortoluron and fluometuron) can be completely degraded within 24 h, and produced enzymic preparations can be used for removing substituted urea herbicide residues on soil, water and crops.The gene can be used for cultivation of transgenic crops resisting substituted urea herbicide.
Description
Technical field
The invention belongs to applied environment microorganism field, relate to wide spectrum phenyl ureagroup herbicides degradation bacteria and hydroamidase gene and answer
With.
Background technology
Although the use of herbicide improves the production efficiency of agricultural, but brings serious environmental problem and chemical injury of crops problem simultaneously.
Phenyl ureagroup herbicides were commercially produced more than 50 years, became one of most widely used important herbicide in the whole world.
The use extensively repeated due to it and of a relatively high water solublity, be often detected in the habitat and agricultural byproducts of various pollutions
Arriving, herbicide chemical injury of crops also happens occasionally.Therefore, phenyl ureagroup herbicides residual has become as ecological environmental protection and crop medicine
Evil releases urgent problem.The method of microorganism remediation pollution of herbicide is a kind of simple, efficient, cheap and non-secondary pollution
Method, than chemical method, physical method repair pollution by pesticides there is more advantage.The basis that microorganism remediation herbicide residue pollutes
Matter is the enzyme of the microorganism generation Degradation to herbicide, and the degradation enzyme system extracted from microorganism eliminates herbicide residue in state
Outer existing successful precedent, the source of digestive enzyme can be by extracting from degradation bacteria, it would however also be possible to employ genetic engineering means builds height
Efficient expression bacterial strain obtains.The reparation that the acquisition of wide spectrum phenyl ureagroup herbicides hydroamidase gene is polluted at phenyl ureagroup herbicides
There is huge application potential in field.Meanwhile, Transgenic Resistant Herbicide Crops is cultivated is a major transformation in weed control history.2012
The transgenic crop of year grown worldwide 1.7 hundred million hectares has herbicide resistance trait more than 80%.Therefore, herbicide is dropped
Solve gene and proceed in crop, cultivate Transgenic Resistant Herbicide Crops new varieties, there is highly important using value.
Summary of the invention
It is an object of the invention to the above-mentioned deficiency for prior art, it is provided that wide spectrum phenyl ureagroup herbicides degradation bacteria.
It is a further object of the present invention to provide the hydroamidase gene being cloned into from this degradation bacteria.
It is yet another object of the invention to provide the application of this bacterial strain, gene or gene coded protein.
The purpose of the present invention can be achieved through the following technical solutions:
Wide spectrum phenyl ureagroup herbicides hydroamidase gene, its nucleotides sequence is classified as SEQ ID NO.1.This full length gene is (from initial
Codon is to termination codon) be 1377bp, G+C content be 63.5%, encode 458 aminoacid, its aminoacid sequence is:
SEQ ID NO.2。
Recombiant plasmid containing described wide spectrum phenyl ureagroup herbicides hydroamidase gene.
As a kind of optimal way of the present invention, pET-29a that hydroamidase genetic fragment is good with enzyme action (+) carry out enzyme and successively win containing wide
Spectrum phenyl ureagroup herbicides hydroamidase gene PET-29a (+) recombiant plasmid.
Recombinant microorganism BL21 (DE3) containing recombiant plasmid of the present invention.
As a kind of optimal way of the present invention, the pET-29a of the amide containing hydrolase gene that enzyme has connected (+) recombinant plasmid transformed is to table
Reach Host Strains BL21 (DE3) and obtain recombinant microorganism BL21 (DE3).
Described wide spectrum phenyl ureagroup herbicides hydroamidase gene is removing crops, soil, the phenyl ureagroup herbicides of water body
Application in terms of residual.
Described phenyl ureagroup herbicides preferably are selected from N-methoxy-. N-methyl phenyl ureagroup herbicides, N, N-dimethyl substituted urea class removes
Grass agent；The described N-further preferred Du Pont Herbicide 326 of methoxy-. N-methyl phenyl ureagroup herbicides；Described N, N-dimethyl substituted urea
The further preferred diuron of class herbicide, chlortoluron, fluometuron.
Amide hydrolysis zymoprotein of the present invention is in terms of removing the phenyl ureagroup herbicides residual of crops, soil, water body
Application.
Described phenyl ureagroup herbicides preferably are selected from N-methoxy-. N-methyl phenyl ureagroup herbicides, N, N-dimethyl substituted urea class removes
Grass agent；The described N-further preferred Du Pont Herbicide 326 of methoxy-. N-methyl phenyl ureagroup herbicides；Described N, N-dimethyl substituted urea
The further preferred diuron of class herbicide, chlortoluron, fluometuron.
Beneficial effect
1. the present invention utilizes antibacterial genome sequencing technology successfully to clone from bacterial strain LR2014-1 (CCTCC NO:M 2015236)
Go out Du Pont Herbicide 326 hydroamidase gene.
2. this full length gene (from start codon to termination codon) is 1377bp, and G+C content is 63.5%, encodes 458 amino
Acid.
3. by round pcr amplification end containing BamH I and the complete Du Pont Herbicide 326 hydroamidase genetic fragment of Xho I restriction enzyme site,
Connect it to escherichia coli high-level expression carrier pET-29a (+) BamH I and the Xho I restriction enzyme site of (purchased from Novegen company)
On, convert and express Host Strains BL21 (DE3) (purchased from invitrogen company), carry out IPTG abduction delivering.
4. the present invention product to Du Pont Herbicide 326 amide hydrolysis enzyme gene expression, has done enzyme assay, Du Pont Herbicide 326 of degrading efficiently, right
The Du Pont Herbicide 326 of 30mg/L can be degradable in 24h.
5. the present invention has carried out substrate spectrum research to the expression product of Du Pont Herbicide 326 hydroamidase gene, finds that this hydroamidase can drop
Solve most phenyl ureagroup herbicides, including Du Pont Herbicide 326, diuron, chlortoluron, fluometuron etc., for the substituted urea weeding of wide spectrum
Agent hydroamidase.
6. utilizing engineered strain high efficient expression wide spectrum substituted urea herbicide hydroamidase of energy that this is gene constructed, the enzyme preparation of production can be used
The degraded of Du Pont Herbicide 326 or removal in soil, water body and crops residual.
Accompanying drawing explanation
Fig. 1 is that the bacterial strain LR2014-1 in wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention and encoding gene source thereof is solid
Bacterium colony figure in body culture medium and Electronic Speculum figure and added with the hydrolysis circle produced on Du Pont Herbicide 326 pesticide flat board.
Fig. 2 is the high-efficient liquid phase chromatogram detection figure of wide spectrum phenyl ureagroup herbicides hydrolase Du Pont Herbicide 326 of the present invention, and figure A is profit
The liquid chromatographic detection figure of the grand standard substance of paddy, figure B is the liquid chromatographic detection figure of 3,4-DCA standard substance；The C present invention is wide for figure
The high-efficient liquid phase chromatogram detection figure of spectrum phenyl ureagroup herbicides hydrolytic enzyme crude enzyme liquid degraded Du Pont Herbicide 326.
Fig. 3 is the policy map of wide spectrum phenyl ureagroup herbicides hydrolase gene functional verification of the present invention.
Fig. 4 is wide spectrum phenyl ureagroup herbicides hydrolase gene of the present invention at BL21 (pET-29a (+)) in high efficient expression experimental program figure；
Fig. 5 is wide spectrum phenyl ureagroup herbicides hydrolase gene of the present invention express in expressive host bacterium BL21 (DE3) and after purification
Protein SDS-PAGE electrophoretogram.
Fig. 6 is optimum temperature and the result of study figure of optimum pH of wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention.
The result of study figure that wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention is affected by Fig. 7 metal ion.
Fig. 8 is wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention to degrade the efficient liquid phase of Du Pont Herbicide 326 in standard enzyme live body system
Chromatogram and Mass Spectrometer Method figure.
Fig. 9 is wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention to degrade the efficient liquid phase of diuron in standard enzyme live body system
Chromatogram and Mass Spectrometer Method figure.
Figure 10 is wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention to degrade the high-efficient liquid of chlortoluron in standard enzyme live body system
Phase chromatogram and Mass Spectrometer Method figure.
Figure 11 is wide spectrum phenyl ureagroup herbicides hydrolase protein matter of the present invention to degrade the high-efficient liquid of fluometuron in standard enzyme live body system
Phase chromatogram and Mass Spectrometer Method figure.
Biomaterial preservation information
LR2014-1, Classification And Nomenclature is Diaphorobacter sp.LR2014-1, is preserved in China typical culture collection
The heart, culture presevation number is CCTCC NO:M 2015236, and preservation date is on 04 15th, 2015, and preservation address is that China is military
Chinese Wuhan University.
Detailed description of the invention
The recruitment evaluation of embodiment 1 bacterial strain LR2014-1 degraded Du Pont Herbicide 326 and Methanogenesis
Prepared by 1.1 seed liquor
Bacterial strain LR2014-1 (CCTCC NO:M 2015236) is accessed in the 100mL LB culture medium containing 30mg/L Du Pont Herbicide 326,30 DEG C,
150rpm shaking table is cultivated, and after 48h, 6000rpm is centrifugal collects thalline, uses MM washing thalline twice, finally uses 10mL MM
Resuspended, standby as seed liquor.
The 1.2 bacterial strain LR2014-1 degraded to herbicide Du Pont Herbicide 326
Bacterial strain LR2014-1 is connected in the 100mL MM containing 30mg/L Du Pont Herbicide 326 by the inoculum concentration of 5%, 30 DEG C, 150rpm
Shaking table is cultivated, and after cultivating 12h, takes 3ml culture fluid and extracts with isopyknic dichloromethane, and anhydrous sodium sulfate is except taking 2ml after water
Dichloromethane dries up mutually, and then 1ml methanol redissolves, and filters with filter membrane (aperture 0.22 μm), uses high performance liquid chromatography to examine
Survey.Liquid chromatographic detection condition: Shimadzu RID-10A；C18 reversed-phase column；Flowing phase: acetonitrile: water (volume ratio 65/35), flow velocity:
1ml·min-1, UV-detector, wavelength 210nm and 250nm；Sample size: 20 μ l.
Test result indicate that described bacterial strain LR2014-1 can degrade Du Pont Herbicide 326, after cultivating 12h, the described bacterial strain fall to Du Pont Herbicide 326
Solution rate is up to more than 80%.HPLC testing result shows that Du Pont Herbicide 326 is degraded to 3,4-dichloroaniline (Fig. 2).
The clone of embodiment 2 Du Pont Herbicide 326 hydroamidase gene
The extraction of 2.1 bacterial genomes STb gene
Bacterial strain Diaphorobacter sp.LR2014-1 (Diaphorobacter sp.LR2014-1, bolter from Du Pont Herbicide 326 contaminated soil
The Du Pont Herbicide 326 efficient degrading bacterial strain that choosing obtains).After mass propgation, CTAB method is used to extract high-purity, the Diaphorobacter of large fragment
The genome DNA of sp.LR2014-1, is dissolved in TE (pH8.0), is placed in-20 DEG C of preservations, and concrete grammar is with reference to F Ao Sibai etc.
" the fine works molecular biology experiment guide " compiled.
2.2 bacterial genomes sketch order-checkings
The qualified Diaphorobacter sp.LR2014-1 genomic DNA of detection is delivered to Shanghai Mei Ji biological medicine science and technology limited
Company carries out the scanning of antibacterial full-length genome sketch.Gene order-checking result shows, described bacterial strain LR2014-1 draft genome is big
Little for 3825957bp, 87 scaffold, there are 87 more than the scaffold of 1000bp, by de novo prediction totally 4238
Individual open reading frame (ORF).
2.3 sequencing result analyses
According to antibacterial full-length genome sketch scanning result, in conjunction with it has been reported that substituted urea class hydrolase gene, use biological software
OMIGA3.0 Yu LR2014-1 whole genome sequence carries out local comparison, it is thus achieved that the doubtful sequence of Du Pont Herbicide 326 hydroamidase gene.
2.4 doubtful sequence verification
By on the Du Pont Herbicide 326 hydroamidase doubtful sequence directed cloning of acquisition to wide host cell pBBR1MCS-2, with Du Pont Herbicide 326 it is
Substrate, verifies whether doubtful sequence has function.
The PCR amplification of 2.5 doubtful sequences
With forward primer F1:5 '-GAACCTCGAGGCTGACCCTGACACGACCTA-3 ' (SEQ ID NO.3) and reverse primer R1:
5 '-TCAGGATCCCGGCGGTCTTTTTCGTTATTG-3 ' (SEQ ID NO.4) are primer, with PCR from Diaphorobacter sp.
LR2014-1 genome amplifies Du Pont Herbicide 326 hydroamidase genetic fragment.
Amplification system:
PCR amplification program:
A.98 DEG C degeneration 2min
B.98 DEG C degeneration 15s, 64 DEG C of annealing 15s, 68 DEG C extend 1min45s, carry out 30 circulations
C.68 DEG C extension 10min
D.10 DEG C 5min, is cooled to room temperature.
2.6 PCR primer and carrier BamH I and XhoI substep enzyme action.
Enzyme action system:
Digestion products reclaims kits with purification and reclaims.
Enzyme is even
Set up following reaction system:
16 DEG C of incubations 12 hours.
2.7 prepare bacillus coli DH 5 alpha Efficiency Competent Cells
Concrete grammar is with reference to " fine works molecular biology experiment guide " P 22-23 of the volumes such as F. Ao Sibai.
2.8 convert
Taking 10 μ l enzymes to connect product and convert 200 μ l competent cells, concrete grammar is with reference to " the fine works molecular biosciences of the volumes such as F. Ao Sibai
Learn experiment guide " P 23.The coating LB flat board containing 50mg/L kanamycin, cultivates 24h and waits picking positive colony.
The enzyme that 2.9 checking positive transformants the are expressed degradation function to Du Pont Herbicide 326
Positive transformant is cultivated to 0D in LB culture medium600Between 0.6-0.8, centrifugal collection thalline.With Tris-HCl (pH 8.0)
Washing thalline, ultrasonic disruption, centrifuging and taking supernatant.Take 1ml supernatant and join the 2ml containing final concentration of 30mg/L Du Pont Herbicide 326
In Tris-HCl (pH 8.0) buffer, arranging the zero load being not added with in supernatant and Host Strains without genes of interest is comparison, 30 DEG C of water-baths
12h。
After reaction terminates, with equal-volume dichloromethane whole volume extraction buffer, remove supernatant aqueous phase, with anhydrous sodium sulfate except water, directly
Connecing and survey effect with ultraviolet spectrophotometer or dichloromethane volatilization eliminated, then methanol redissolves, and crosses 0.22 μm organic facies filter membrane,
Finally use high performance liquid chromatography detection.Liquid chromatographic detection condition: Shimadzu RID-10A；C18 reversed-phase column；Flowing phase: acetonitrile:
Water (volume ratio 65/35), flow velocity: 1ml min-1, UV-detector, wavelength 210nm and 250nm；Sample size: 20 μ l.
High performance liquid chromatography testing result (Fig. 2) shows that the crude enzyme liquid that this positive transformant is expressed can be by 80% Li Gu in 12h
Basket strainer urea bridge is converted into 3,4-dichloroaniline.Therefore, test result indicate that this nucleotide sequence coded albumen is Du Pont Herbicide 326 amide
Hydrolytic enzyme.
Embodiment 3 Du Pont Herbicide 326 hydroamidase gene is at BL21 (pET-29a (+)) in high efficient expression
The PCR amplification of 3.1 Du Pont Herbicide 326 hydroamidase enzyme genes
With forward primer F2:5 '-TTAGGATCCCCAACGGACTGGAGAGTTGAA-3 ' (SEQ ID NO.5) and reverse primer R2:
5 '-AACCTCGAGTTGCGGTTCCGAACGCGGATA-3 ' (SEQ ID NO.6) are primer, with PCR from Diaphorobacter sp.
LR2014-1 amplifies Du Pont Herbicide 326 hydroamidase genetic fragment.
Amplification system:
PCR amplification program:
A.98 DEG C degeneration 2min
B.98 DEG C degeneration 15s, 63 DEG C of annealing 15s, 68 DEG C extend 1min30s, carry out 30 circulations
C.68 DEG C extension 10min,
D.10 DEG C 5min, is cooled to room temperature.
3.2 PCR primer and carrier BamH I and XhoI substep enzyme action.
Enzyme action and enzyme disjunctor system (with reference to 1.6)
3.3 convert and express
The pET-29a containing Du Pont Herbicide 326 hydroamidase gene that enzyme has been connected (+) recombinant plasmid transformed is to expressive host bacterium BL21 (DE3)
Obtain recombinant microorganism BL21 (DE3).Coat the flat board containing 50mg/L kanamycin, picking positive transformant.
The enzyme that 3.4 checking positive transformants the are expressed degradation function to Du Pont Herbicide 326
Positive transformant is cultivated to OD in LB culture medium600About 0.5, in culture medium, add the IPTG of final concentration of 0.5mM,
After 16 DEG C of low temperature inductions 15h, centrifugal collection thalline.Thalline, ultrasonic disruption, centrifuging and taking supernatant is washed with Tris-HCl (pH 8.0).
Taking in Tris-HCl (pH 8.0) buffer that 1ml supernatant joins containing final concentration of 30mg/L Du Pont Herbicide 326, enzyme live body system is 3ml,
Arranging the zero load being not added with in supernatant and Host Strains without genes of interest is comparison, 30 DEG C of water-bath 12h.
With equal-volume dichloromethane whole volume extraction buffer, remove supernatant aqueous phase, with anhydrous sodium sulfate except water, dichloromethane is volatilized
Eliminating, then methanol redissolves, and crosses 0.22 μm organic facies filter membrane, finally uses high performance liquid chromatography tandem mass spectrum detect and identify generation
Thank to product.High performance liquid chromatography testing conditions: flowing phase: acetonitrile: water (volume ratio 65/35), Zorbax XDB-C18,5cm × 0.46
Cm, 1.8mm reversed-phase column (5 μm, 4.6mm × 250mm, Agilent, USA), flow velocity is 0.25mL/min.MS analyzes
Using ESI pattern, detector is Agilent G6410B Triple Quad Mass Spectrometer.
HPLC-MS (high performance liquid chromatography and mass spectrometry) testing result shows, the crude enzyme liquid of described positive transformant can be by profit
Gu Long is converted into 3,4-dichloroaniline (Fig. 8).
The substrate spectrum research of 3.5 Du Pont Herbicide 326 hydroamidases
Positive transformant is cultivated to 0D in LB culture medium600About 0.5, in culture medium, add the IPTG of final concentration of 0.5mM,
After 16 DEG C of low temperature inductions 15h, centrifugal collection thalline.Thalline, ultrasonic disruption, centrifuging and taking supernatant is washed with Tris-HCl (pH 8.0).
Take 1ml supernatant and be added separately to Tris-HCl (pH 8.0) buffering containing final concentration of 30mg/L diuron, chlortoluron and fluometuron
In liquid, enzyme live body system is 3ml, and arranging the zero load being not added with in supernatant and Host Strains without genes of interest is comparison, 30 DEG C of water-baths
12h。
The detection method of experimental result is with 3.4.
Test result indicate that, substrate diuron, chlortoluron and fluometuron can be all converted into its correspondence by Du Pont Herbicide 326 hydroamidase
Aniline compound (see respectively Fig. 9,10,11).
The enzymatic property research of 3.6 Du Pont Herbicide 326 hydroamidases
Positive transformant is cultivated to 0D in LB culture medium600About 0.5, in culture medium, add the IPTG of final concentration of 0.5mM,
After 16 DEG C of low temperature inductions 15h, centrifugal collection thalline.Thalline, ultrasonic disruption, centrifuging and taking supernatant is washed with Tris-HCl (pH 8.0).
Taking in Tris-HCl (pH 8.0) buffer that 0.5ml supernatant joins containing final concentration of 30mg/L Du Pont Herbicide 326, enzyme live body system is
3ml.In the water-bath of different temperatures, carry out enzymatic reaction, add isopyknic dichloromethane after 15 minutes and terminate enzymatic reaction,
Finally use high performance liquid chromatography detection enzymatic reaction result, with the degradation efficiency under optimum temperature for 100%, calculate relative enzyme and live
Power.Separately take 0.5ml supernatant and be added separately to there are four kinds of different pH value range delay containing final concentration of 30mg/L Du Pont Herbicide 326
Rush liquid acetic acid-sodium acetate buffer solution (pH 3.6,4.0,4.4,5.0,5.8), Na2HPO3-citrate buffer solution (pH 5.0,6.0,6.4,7.0,
8.0), in Tris-HCl buffer (pH 7.5,7.8,8.6) and glycine-NaOH buffer (pH 8.6,9.0,9.4,10.0), enzyme live body system is
3ml.Under the optimum temperature of enzyme, carry out enzymatic reaction, add isopyknic dichloromethane after 15 minutes and terminate enzymatic reaction, finally
Use high performance liquid chromatography detection enzymatic reaction result, with the highest degradation efficiency for 100%, calculate enzyme activity.Finally take
0.5ml supernatant is added separately to the Tris-HCl (pH 8.0) containing final concentration of 30mg/L Du Pont Herbicide 326 and 0.1mM different metal ion
In buffer, enzyme live body system is 3ml.Under the optimum temperature of enzyme, carry out enzymatic reaction, after 15 minutes, add isopyknic dichloro
Methane terminates enzymatic reaction, finally uses high performance liquid chromatography detection enzymatic reaction result, with the highest degradation efficiency for 100%,
Calculate enzyme activity.
Test result indicate that, the optimum temperature of Du Pont Herbicide 326 hydroamidase is 35 ± 2 DEG C, and optimum pH is 7.5 ± 0.5 (Fig. 6).Different
Metal ion is on test result indicate that Du Pont Herbicide 326 hydroamidase affects, the Zn of 0.1mM2+、Cd2+And Ni2+Shadow that can be serious
Ring this hydrolytic enzyme enzyme to live, and the Al of 0.1mM3+The enzyme that can be obviously promoted this hydrolytic enzyme lives (Fig. 7).
Claims (8)
1. wide spectrum phenyl ureagroup herbicides hydroamidase gene, it is characterised in that nucleotides sequence is classified as SEQ ID NO.1.
2. the amide hydrolysis zymoprotein of the hydroamidase gene code described in claim 1, it is characterised in that ammonia
Base acid sequence is: SEQ ID NO.2.
3. containing the recombiant plasmid of the wide spectrum phenyl ureagroup herbicides hydroamidase gene described in claim 1.
4. containing the recombinant microorganism BL21 (DE3) of the recombiant plasmid described in claim 3.
5. the bacterial strain LR2014-1 described in claim 1 is in degraded soil, water body or the substituted urea class weeding of crops
Application in terms of agent residual.
6. the wide spectrum phenyl ureagroup herbicides hydroamidase gene described in claim 1 remove soil, water body or
Genetic engineering application in terms of the phenyl ureagroup herbicides residual of crops.
7. the wide spectrum phenyl ureagroup herbicides hydroamidase gene described in claim 1 is at anti-phenyl ureagroup herbicides
Application in genetically modified crops cultivation.
8. amide hydrolysis zymoprotein described in claim 2 removes at removal crops, soil, the substituted urea class of water body
Application in terms of grass agent residual.
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