CN113355267A - Organophosphorus pesticide live bacteria degrading agent and preparation method thereof - Google Patents
Organophosphorus pesticide live bacteria degrading agent and preparation method thereof Download PDFInfo
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Abstract
The invention provides a degrading agent of organophosphorus pesticide viable bacteria and a preparation method thereof, wherein the degrading agent of organophosphorus pesticide viable bacteria adopts pLEV1-ToRA-mpdThe plasmid is transformed into Escherichia coli and obtained by amplification culture, and the pLEV1-TorA-mpdThe sequence of the plasmid is shown as SEQ ID NO. 1. By adopting the technical scheme of the invention, a new escherichia coli strain containing the light-operated expression organophosphorus hydrolase gene is created, a transfer signal peptide TorA capable of transferring the expressed hydrolase to the periplasm of a cell membrane is added, the upstream of the constructed plasmid is a light-operated gene expression system, and the downstream is a transfer signal peptide sequence and a gene capable of coding the expression organophosphorus hydrolase in the periplasm of the cell. The constructed plasmid is transferred into Escherichia coliIn the dark environment, the organophosphorus hydrolase is induced to express and is transferred to the periplasm of the cells, and the experimental result proves that the organophosphorus hydrolase in the periplasm of the cells can effectively degrade the organophosphorus pesticide.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to an organophosphorus pesticide viable bacteria degrading agent and a preparation method thereof.
Background
In China, as a big agricultural country, the annual usage amount of pesticides reaches more than 120 x 104 t. Agricultural chemicals have made a great contribution to agricultural development and human food supply as important inputs for agricultural production, so that about 1/3 agricultural products are protected from disease and pest disasters every year. However, the widespread use of agricultural chemicals inevitably results in residues of soil and agricultural products. The residue of these pesticides not only affects the food safety, but also pollutes the surrounding environment, and brings serious adverse consequences to human health and ecosystem. With the enhancement of food safety consciousness and the improvement of pesticide residue detection technology, the pesticide residue degradation agent becomes a necessity for agricultural production. In addition, as people's awareness of green food is improved and various policies of governments are brought out one after another, development and production of green food are inevitable trends in agricultural production and food processing, and products serving green food production and capable of degrading pesticide residues are concerned and favored by more and more food producers. In most of soil in China, the organophosphorus pesticides in soil account for 80% of pesticide residues, and are main pesticide components. Due to the large and unreasonable use of organophosphorus pesticides, organophosphorus residues in soil seriously exceed the standard. Therefore, from the aspects of ensuring the use safety of agricultural products and developing outward agricultural economy, the degradation of pesticide residues in soil must be enhanced so as to protect food safety and the surrounding water and soil environment.
Most of pesticide residue degradation methods used in the market at present are physical and chemical methods, and products mainly comprise Subijing, Woltean, Nainalao A, Biya fruit and vegetable washing machines and the like. Physical methods such as ultrasonic technology, ionizing radiation, adsorption, peeling, washing and the like, and the action objects of the physical methods are only limited to pesticide residues on the surfaces of crops, and the requirements on technical equipment are high, so that the physical methods are difficult to popularize in a large range. The more used chemical degradation methods comprise photochemical degradation, oxidative decomposition, hydrolysis and the like, and common chemical preparations have certain degradation effect on pesticide residues in plants, but have the possibility of causing secondary pollution by themselves and products. In recent years, biodegradation methods, including methods in which enzymatic degradation is accomplished by secretory enzymes, oxidation, reduction, hydrolysis, ring cleavage, condensation, dehalogenation, decarboxylation, methylation, and the like using microorganisms, degrading enzymes, engineering bacteria, and the like, have been attracting much attention because of their safety, low cost, and convenience in use. However, the degradation speed of the pesticide degrading bacteria obtained by directly screening from the environment is slow, and the degradation capability is lost due to the fact that the pesticide degrading bacteria cannot meet the actual requirement or the pesticide degrading bacteria frequently generate variation. In addition, the degrading bacteria put into the environment may be affected by the existing bacterial flora in the environment and may not survive in the environment due to antagonism. In addition, the composition of the compounds in the external environment polluted by the pesticide is unstable, and the fluctuation of factors such as temperature, pH and humidity is large, so that the growth of the pesticide degrading bacteria is not favorable, and the growth of the degrading bacteria can be possibly inhibited.
Disclosure of Invention
Aiming at the technical problems, the invention discloses a live bacteria degrading agent of organophosphorus pesticide and a preparation method thereof, and the bacterial strain can be directly used for degrading pesticide residue in soil as the live bacteria degrading agent because a transfer signal peptide capable of transferring expressed hydrolase to periplasm of cell membrane is added.
In contrast, the technical scheme adopted by the invention is as follows:
an organophosphorus pesticide live bacteria degradation agent which adopts pLEV1-Tora-mpdThe plasmid is transformed into Escherichia coli and obtained by amplification culture, and the pLEV1-TorA-mpdThe sequence of the plasmid is shown as SEQ ID NO. 1.
By adopting the technical scheme, the escherichia coli strain containing the light-operated expression organophosphorus hydrolase gene is newly built, and the strain can be directly used for degrading pesticide residues in soil as a viable bacteria degrading agent because a transfer signal peptide TorA capable of transferring the expressed hydrolase to the periplasm of a cell membrane is added.
The invention also discloses a preparation method of the organophosphorus pesticide viable bacteria degrading agent, which comprises the following steps:
s11, constructing a light-operated gene expression vector for producing organophosphorus hydrolase;
step S12, inserting a promoter bba _ J23116, a light-sensitive fusion protein LEV1 and two Escherichia coli terminators rrnB into RI and II enzyme cutting sites of the light-operated gene expression vector to obtain a pLEV1 plasmid;
step S13, mixing pCole promotor and methyl parathion hydrolase genempdAnd a transfer signal peptide inserted into the cleavage sites of KpnI and Bglll in the pLEV1 plasmid to obtain pLEV1-Tora-mpdA plasmid;
step S14, transforming pLEV1-TorA-mpd into colibacillus strains for amplification culture to obtain the organophosphorus pesticide live bacteria degradation agent.
As a further improvement of the invention, in step S11, the backbone of the expression vector for producing the organophosphorus hydrolase is pCDFDuet1, and the expression vector does not contain a promoter and an MCS sequence.
As a further modification of the present invention, in step S13, the Escherichia coli terminator rrnB is obtained from a pBAD/His vector; the methyl parathion hydrolase genempdObtained from Pseudomonas WBC-3; the transfer signal peptide is TorA.
As a further improvement of the present invention, in step S14, the Escherichia coli strain is BL21 strain.
An organophosphorus pesticide live bacteria degradation agent which adopts pLEV1-Tora-mpd-supernovaThe plasmid is transformed into Escherichia coli and obtained by amplification culture, and the pLEV1-TorA-mpd-supernovaIs at pLEV1-Tora-mpdIntroduction of suicide Gene on plasmidsupernovaObtained, the plasmid pLEV1-Tora-mpd-supernovaThe sequence of (A) is shown in SEQ ID NO. 2.
By adopting the technical scheme, the phototoxic protein SUPERNOVA is harmless to bacteria in dark environment, and does not influence division and propagation of the bacteria. Once leaving the dark environment and entering the natural environment (such as soil environment), under the action of natural light, SUPERNOVA protein releases a large amount of active oxygen to initiate the breaking of the bacterial self genome DNA chain, so that the bacterial self genome DNA chain can not be copied and die. But the organophosphorus pesticide live bacteria degradation agent still has the function of degrading pesticide residues under the condition that bacteria die. The establishment of the mechanism skillfully utilizes two natural alternate phenomena of darkness and illumination, not only transforms live bacteria into a pesticide residue degradation agent, but also effectively avoids the biological leakage of transgenosis and the pollution to the natural environment by introducing the photosensitive suicide gene.
The invention also discloses a preparation method of the organophosphorus pesticide viable bacteria degrading agent, which comprises the following steps:
step S21, constructing a light-operated gene expression vector for producing the organophosphorus hydrolase, wherein the light-operated gene expression vector does not contain a promoter and an MCS sequence;
step S22, inserting a constitutive promoter, a photosensitive fusion protein LEV1 and two Escherichia coli terminators rrnB into RI and II enzyme cutting sites of the light-operated gene expression vector to obtain a pLEV1 plasmid;
step S23, mixing pCole promotor and methyl parathion hydrolase genempdAnd a transfer signal peptide inserted into the pLEV1 plasmidKpnI AndBglllin the cleavage site of (a), pLEV1-TorA-mpdA plasmid; in pLEV1-Tora-mpdIntroduction of suicide Gene on plasmidsupernovaTo obtain the plasmid pLEV1-TorA-mpd-supernova；
Step S24, plasmid pLEV1-TorA-mpd-supernovaAnd (3) transforming the bacillus subtilis into an escherichia coli strain, and carrying out dark amplification culture at 37 ℃ to obtain the organophosphorus pesticide viable bacteria degrading agent.
By adopting the technical scheme, under the induction of a dark environment, the organophosphorus hydrolase can be expressed and transferred to the periplasm of the cell membrane, so that the escherichia coli cell becomes a viable bacteria pesticide residue degradation agent and can be directly used for pesticide residue degradation in soil.
As a further improvement of the invention, in step S21, the backbone of the expression vector for producing the organophosphorus hydrolase is pCDFDuet1, and the expression vector does not contain a promoter and an MCS sequence.
As a further modification of the present invention, in step S23, the Escherichia coli terminator rrnB is obtained from a pBAD/His vector; the methyl parathion hydrolase genempdObtained from Pseudomonas WBC-3; the transfer signal peptide is TorA.
As a further improvement of the present invention, in step S24, the Escherichia coli strain is BL21 strain.
Compared with the prior art, the invention has the beneficial effects that:
by adopting the technical scheme of the invention, a new escherichia coli strain containing the light-operated expressed organophosphorus hydrolase gene is created, and the strain can be directly used for degrading pesticide residues in soil as an active bacteria degradation agent due to the addition of a transfer signal peptide TorA capable of transferring the expressed hydrolase to the periplasm of a cell membrane. Furthermore, the upstream of the constructed plasmid is a light-operated gene expression system, and the downstream is a transfer signal peptide sequence and a gene which can code the expression of the organophosphorus hydrolase in the periplasm of the cell. The constructed plasmid is transferred into escherichia coli, is induced to express organophosphorus hydrolase in a dark environment and is transferred to periplasm of cells, and experimental results prove that the organophosphorus hydrolase in the periplasm of cells can effectively degrade organophosphorus pesticide. The genetically modified escherichia coli can be used as a viable bacteria degradation device, and can be directly sprayed in soil containing organophosphorus pesticide residues after being cultured and expressed so as to achieve the purpose of degradation.
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FIG. 1 is a schematic diagram of a light-controlled inducible expression system of Escherichia coli according to an embodiment of the present invention.
FIG. 2 is the plasmid pLEV1-TorA-mpdSchematic structural diagram of (1).
FIG. 3 is the plasmid pLEV1-TorA-mpd-supernovaSchematic structural diagram of (1).
FIG. 4 is a graph showing the results of detecting the MPH protein content by the enzyme-linked immunosorbent assay of parathion-methyl hydrolase in example 2 of the present invention, wherein (a) is a graph showing the results of detecting the MPH protein content by the enzyme-linked immunosorbent assay of parathion-methyl hydrolase, and (b) is a standard curve.
FIG. 5 is a graph showing the results of enzyme activity measurements of the methyl parathion hydrolase MPH in different crude enzyme solutions of example 2 of the present invention.
FIG. 6 is a graph showing the results of stability of MPH activity in living cells of example 2 of the present invention.
FIG. 7 shows the growth status of colonies re-cultured after light (light) or dark (dark) treatment of the resuspended cells in example 2 of the present invention, wherein (a) the cells are re-cultured after light and (b) the cells are re-cultured after dark.
Detailed Description
Preferred embodiments of the present invention are described in further detail below.
Example 1
An organophosphorus pesticide live bacteria degradation agent which adopts pLEV1-Tora-mpdThe plasmid is transformed into Escherichia coli and obtained by amplification culture, and the pLEV1-TorA-mpdThe sequence of the plasmid is shown as SEQ ID NO.1, and the structural schematic diagram of the plasmid is shown as figure 2.
The organophosphorus pesticide live bacteria degrading agent is prepared by the following steps:
step S11, constructing an expression vector for producing the organophosphorus hydrolase, wherein the skeleton of the expression vector for producing the organophosphorus hydrolase is pCDFDuet1, and the expression vector does not contain a promoter and an MCS sequence.
Step S12, inserting a promoter bba _ J23116, a light-sensitive fusion protein LEV1 and two Escherichia coli terminators rrnB into RI and II enzyme cutting sites of the light-operated gene expression vector to obtain a pLEV1 plasmid;
step S13, mixing pCole promotor and methyl parathion hydrolase genempdAnd a transfer signal peptide inserted into the cleavage sites of KpnI and Bglll in the pLEV1 plasmid to obtain pLEV1-Tora-mpdA plasmid; in step S13, the Escherichia coli terminator rrnB is obtained from the pBAD/His vector; the methyl parathion hydrolase genempdObtained from Pseudomonas WBC-3; the transfer signal peptide is TorA.
Step S14, pLEV1-TorA-mpdAnd transforming the bacillus subtilis into escherichia coli BL21 strain for amplification culture to obtain the organophosphorus pesticide live bacteria degrading agent.
Example 2
An organophosphorus pesticide live bacteria degradation agent which adopts pLEV1-Tora-mpd-supernova plasmid is transformed into Escherichia coli for amplification culture, and the pLEV1-Tora-mpd-supernovaIs at pLEV1-Tora-mpdIntroduction of suicide Gene on plasmidsupernovaObtained, the plasmid pLEV1-Tora-mpd-supernovaThe sequence of (A) is shown in SEQ ID NO. 2.
The organophosphorus pesticide live bacteria degrading agent is prepared by the following steps:
construction of light-operated Gene expression vector for production of organophosphorus hydrolase the vector backbone used for the light-operated gene expression vector was pCDFDuet1, in which the promoter and MCS sequences were removed. The RI and II cleavage sites into which the constitutive promoter bba _ J23116, light-sensitive fusion protein LEV1 and two Escherichia coli terminators rrnB (obtained from pBAD/His vector) were inserted, yielded pLEV1 plasmid. On the basis, pCole promoter (containing an operator sequence for binding light sensitive protein) and methyl parathion hydrolase gene are addedmpd(from Pseudomonas sp.)WBC-3Obtained in (1), and a transfer signal peptide (TorA) are inserted into the cleavage sites of KpnI and Bglll in the pLEV1 plasmid after the gene synthesis, obtaining pLEV1-TorA-mpdA plasmid. As shown in the schematic diagram of the light-operated inducible expression system of the Escherichia coli in FIG. 1, under the illumination condition, the light-sensitive fusion protein LEV1(LexA-LOV) forms a homodimer and is combined on an operator sequence, and the function of a promoter is inhibited by blocking the combination of RNA polymerase; after the light source is deprived, LEV1 dimer is gradually separated, and downstream genesmpdIs initiated. Plasmid pLEV1-Tora-mpdThe structure of (a) is shown in FIG. 2 and verified by colony PCR. The primer sequence of colony PCR is:mpdA-F(5'-GCGGCGTAGCTTTTATGCTG-3', SEQ shown in ID NO. 3),mpdA-R(5' -CCATGTTATCCTCCTCGCCC-3, shown in SEQ ID NO. 4). The PCR product was detected by agarose gel electrophoresis.
Further, in this example, suicide gene was introducedsupernovaTo obtain the plasmid pLEV1-TorA-mpd- supernova，supernovaGenes were synthesized commercially according to published gene sequences. Plasmid pLEV1-Tora-mpd- supernovaIs shown in fig. 3. The plasmid has the function of promoting apoptosis in order to avoid ecological pollution when the engineering bacteria are used as a viable bacteria degradation device in the field.
Plasmid transformation and cell culture: pLEV1-Tora-mpd-supernovaTransformed into Escherichia coli BL21 strain, and amplified and cultured overnight in the dark at 37 ℃. Single colonies were picked from the plates and cultured in 5mL LB liquid medium (containing 1. mu.g/mL streptomycin) in the dark at 37 ℃ to OD600 of 0.6-0.8. The bacterial liquid in the culture medium is diluted by 100 times into two LB liquid culture media (containing streptomycin) with the volume of 20 mL, and the two LB liquid culture media are respectively cultured overnight in the light (natural light, the illumination intensity is common sunshine) and the dark (wrapped by double-layer tinfoil paper), and the temperature is 37 ℃. Crude protease liquid produced by the recombinant BL21 was extracted by ultrasonication (Total protein extraction kit for Escherichia coli, Beeber). The BCA protein quantitative kit is used for determining soluble protein solution, polyacrylamide gel electrophoresis (SDS-PAGE) is used for detecting whole protein solution, and enzyme linked immunosorbent assay is used for determining the content of methyl parathion hydrolase in crude enzyme solution, and the result is shown in figure 4. Crude enzyme solutions were obtained from the periplasm or cytoplasm of dark-induced strains, and periplasmic proteins of whole cells and light-induced strains were also detected. Abbreviations in the figures: LF/lightoff: peri-illumination, periplast: dark periplasm, white cell: whole cell, cytoplasms: cytoplasm. The absorbance (OD) in the graph was measured at 450nm with a microplate reader.
Activity assay of Methyl Parathion Hydrolase (MPH): the overnight-cultured BL21 bacterial solution (OD 600= 1.0) after transformation was collected by centrifugation and suspended in PBS buffer (ph 7.4). The activity of Methyl Parathion Hydrolase (MPH) in live bacteria was measured by UV-Vis photometer (405 nm, 30), and methyl parathion was hydrolyzed into p-nitrophenol (p-nitrophenol), and the activity was expressed as the amount of p-nitrophenol produced in 1umol per minute in 0D600 broth. The mixture (1 ml) to be tested contained 100. mu.l of the bacterial suspension, 50ug/ml of methyl parathion (dissolved in 10mg/ml of methanol), and 870. mu.l of PBS buffer (pH 7.4). The results of enzyme activity measurement of methyl parathion hydrolase MPH in different crude enzyme solutions are shown in FIG. 5, and it can be seen that the crude enzyme solutions were obtained from the periplasm or cytoplasm of the dark-induced strain, and periplasmic proteins (lightOFF) of whole cells and light-induced strains were also detected. Wherein the MPH activity is determined using paraoxon as a substrate.
Viable bacteria stability experiment: containing pLEV1-Tora-mpd-supernovaColi of plasmid or control vector (pLEV 1)BL2After the cells were placed in a wrapped aluminum foil flask and induced to culture at 37 ℃ for 24 hours in the dark, the cells were washed twice with 50 mL of 150 mM NaCl solution, then resuspended in 5mL of 100mM phosphate buffer (pH 7.4), and incubated at 30 ℃ on a shaker. Samples were taken at 0.1 mL/day over a one week period. The samples were centrifuged and resuspended in 0.1mL of 100mM phosphate buffer (pH 7.4). MPH activity assays were performed as described above. The activity of MPH was measured in one week using resuspended whole cells with paraoxon as substrate. The stability analysis of MPH activity in living cells is shown in FIG. 6.
Bacterial suicide experiments: containing pLEV1-Tora-mpd-supernovaOr the control vector (pLEV 1) in BL21 cells in a wrapped aluminum foil bottle, at 37 degrees C under dark induction culture for 24 hours, collecting the induced cells and 100mm phosphate buffer (ph 7.4) washing. The cell suspension was diluted 10-fold, plated on LB agar plates again after 10 hours in direct sunlight, and grown at 37 ℃ for 12 hours. The resuspended cells after dark induction were cultured again after light (light) or dark (dark) treatment. A comparison of the growth state of colonies after LB plate display culture is shown in FIG. 7. Therefore, the cells after dark induction are cultured again after being processed in a dark place, the cells grow well, and the cells after dark induction are cultured again after being processed in a light place, and only a small amount of cells grow.
The technical proposal of the invention is that a coding gene of organophosphorus hydrolase is connected with a gene which can induce cell suicidesupernovaA gene. Under the irradiation of visible light, the suicide gene synthesizes red protein and releases a large amount of active oxygen to cause the cell to die due to oxygen deficiency. The whole mechanism is characterized in that the constructed plasmid can produce organophosphorus hydrolase which can be expressed on the cell surface under the dark condition and is used for degrading organophosphorus pesticide residues; under the condition of illumination, the SUPERNOVA protein product releases active oxygen to initiate the breakage of bacterial DNA chains, so that the bacterial DNA chains cannot be copied, and cells are further promoted to die. The viable bacteria degradation device can still degrade pesticide residues in a death state, and effectively avoids the leakage of transgenic organisms and natural environmentEnvironmental pollution.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Sequence listing
<110> Shenzhen technologist college (Shenzhen senior technologist school)
<120> organophosphorus pesticide viable bacteria degrading agent and preparation method thereof
<160> 4
<170> SIPOSequenceListing 1.0
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ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct cggtaccccc 60
ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag 120
cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat 180
gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 240
agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc 300
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agtccacagt cttgacaggg aaaatgcagc ggcgtagctt ttatgctgta tataaaacca 420
gtggttatat gtacagtatt tattttaact tattgtttta aaagtcaaag aggattttat 480
aataatcgat atgaacaata acgatctctt tcaggcatca cgtcggcgtt ttctggcaca 540
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gactgcggcg caagcggcgc atatgccgct gaagaaccgt ctgctggcgc gtctgagctg 660
cgtggcggcg gtggttgcgg cgaccgcggc ggtggcgccg ctgaccctgg ttagcaccgc 720
gcacgcggcg gcgccgcagg ttcgtaccag cgcgccgggc tactatcgta tgctgctggg 780
tgacttcgag atcaccgcgc tgagcgatgg taccgtggcg ctgccggttg acaagcgtct 840
gaaccaaccg gcgccgaaaa cccaaagcgc gctggcgaag agcttccaaa aagcgccgct 900
ggaaaccagc gtgaccggct acctggttaa caccggtagc aagctggtgc tggttgatac 960
cggtgcggcg ggtctgtttg gtccgaccct gggtcgtctg gcggcgaacc tgaaagcggc 1020
gggttaccag ccggagcaag tggatgaaat ctatattacc catatgcacc cggaccatgt 1080
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gcagaagcgg tctgataaaa cagaatttgc ctggcggcag tagcgcggtg gtcccacctg 1740
accccatgcc gaactcagaa gtgaaacgcc gtagcgccga tggtagtgtg gggtctcccc 1800
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gcctttcgtt ttatctgttg tttgcgctag cggatccgcc tatgcagcga caaatattga 1920
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acttcctagg tattattatg aaagcgttaa cggccaggca acaagaggtg tttgatctca 2040
tccgtgatca catcagccag acaggtatgc cgccgacgcg tgcggaaatc gcgcagcgtt 2100
tggggttccg ttccgcgtcc tcggctgaag aacatctgaa ggcgctggca cgcaaaggcg 2160
ttattgaaat tgtttccggc gcatcacgcg ggattcgtct gttgcaggaa gaggaagaag 2220
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gttatgacat tatgggctat ctgattcaga ttatgaagag gccaaacccc caagtagaac 2340
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ggaagtacgt cgactccaac acgatcaata cgatgaggaa agcgattgat aggaacgccg 2580
aggtgcaggt tgaggtggtc aattttaaga agaacggcca acggtttgtc aacttcttga 2640
cgatgattcc ggtgcgagat gaaacagggg aataccggta cagcatgggt ttccagtgcg 2700
aaacggaatg agaattcccc ctgttttggc ggatgagaga agattttcag cctgatacag 2760
attaaatcag aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg 2820
gtggtcccac ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt 2880
gtggggtctc cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca 2940
gtcgaaagac tgggcctttc gttttatctg ttgtttgcct cgagctgcca ccgctgagca 3000
ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaacc 3060
tcaggcattt gagaagcaca cggtcacact gcttccggta gtcaataaac cggtaaacca 3120
gcaatagaca taagcggcta tttaacgacc ctgccctgaa ccgacgaccg ggtcatcgtg 3180
gccggatctt gcggcccctc ggcttgaacg aattgttaga cattatttgc cgactacctt 3240
ggtgatctcg cctttcacgt agtggacaaa ttcttccaac tgatctgcgc gcgaggccaa 3300
gcgatcttct tcttgtccaa gataagcctg tctagcttca agtatgacgg gctgatactg 3360
ggccggcagg cgctccattg cccagtcggc agcgacatcc ttcggcgcga ttttgccggt 3420
tactgcgctg taccaaatgc gggacaacgt aagcactaca tttcgctcat cgccagccca 3480
gtcgggcggc gagttccata gcgttaaggt ttcatttagc gcctcaaata gatcctgttc 3540
aggaaccgga tcaaagagtt cctccgccgc tggacctacc aaggcaacgc tatgttctct 3600
tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg gctggctcga agatacctgc 3660
aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg cgcttagctg gataacgcca 3720
cggaatgatg tcgtcgtgca caacaatggt gacttctaca gcgcggagaa tctcgctctc 3780
tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa gctcgccgcg ttgtttcatc 3840
aagccttacg gtcaccgtaa ccagcaaatc aatatcactg tgtggcttca ggccgccatc 3900
cactgcggag ccgtacaaat gtacggccag caacgtcggt tcgagatggc gctcgatgac 3960
gccaactacc tctgatagtt gagtcgatac ttcggcgatc accgcttccc tcatactctt 4020
cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 4080
tgaatgtatt tagaaaaata aacaaatagc tagctcactc ggtcgctacg ctccgggcgt 4140
gagactgcgg cgggcgctgc ggacacatac aaagttaccc acagattccg tggataagca 4200
ggggactaac atgtgaggca aaacagcagg gccgcgccgg tggcgttttt ccataggctc 4260
cgccctcctg ccagagttca cataaacaga cgcttttccg gtgcatctgt gggagccgtg 4320
aggctcaacc atgaatctga cagtacgggc gaaacccgac aggacttaaa gatccccacc 4380
gtttccggcg ggtcgctccc tcttgcgctc tcctgttccg accctgccgt ttaccggata 4440
cctgttccgc ctttctccct tacgggaagt gtggcgcttt ctcatagctc acacactggt 4500
atctcggctc ggtgtaggtc gttcgctcca agctgggctg taagcaagaa ctccccgttc 4560
agcccgactg ctgcgcctta tccggtaact gttcacttga gtccaacccg gaaaagcacg 4620
gtaaaacgcc actggcagca gccattggta actgggagtt cgcagaggat ttgtttagct 4680
aaacacgcgg ttgctcttga agtgtgcgcc aaagtccggc tacactggaa ggacagattt 4740
ggttgctgtg ctctgcgaaa gccagttacc acggttaagc agttccccaa ctgacttaac 4800
cttcgatcaa accacctccc caggtggttt tttcgtttac agggcaaaag attacgcgca 4860
gaaaaaaagg atctcaagaa gatcctttga tcttttctac tgaaccgctc tagatttcag 4920
tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat aagttgtaat 4980
tctcatgtta gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa 5040
gggcatcggt cgagatcccg gtgcctaatg agtgagctaa cttacattaa ttgcgttgcg 5100
ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca 5160
acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg 5220
agacgggcaa cagctgattg cccttcaccg cctggccctg agagagttgc agcaagcggt 5280
ccacgctggt ttgccccagc aggcgaaaat cctgtttgat ggtggttaac ggcgggatat 5340
aacatgagct gtcttcggta tcgtcgtatc ccactaccga gatgtccgca ccaacgcgca 5400
gcccggactc ggtaatggcg cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca 5460
tcgcagtggg aacgatgccc tcattcagca tttgcatggt ttgttgaaaa ccggacatgg 5520
cactccagtc gccttcccgt tccgctatcg gctgaatttg attgcgagtg agatatttat 5580
gccagccagc cagacgcaga cgcgccgaga cagaacttaa tgggcccgct aacagcgcga 5640
tttgctggtg acccaatgcg accagatgct ccacgcccag tcgcgtaccg tcttcatggg 5700
agaaaataat actgttgatg ggtgtctggt cagagacatc aagaaataac gccggaacat 5760
tagtgcaggc agcttccaca gcaatggcat cctggtcatc cagcggatag ttaatgatca 5820
gcccactgac gcgttgcgcg agaagattgt gcaccgccgc tttacaggct tcgacgccgc 5880
ttcgttctac catcgacacc accacgctgg cacccagttg atcggcgcga gatttaatcg 5940
ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca 6000
acgactgttt gcccgccagt tgttgtgcca cgcggttggg aatgtaattc agctccgcca 6060
tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc 6120
gggaaacggt ctgataagag acaccggcat actctgcgac atcgtataac gttactggtt 6180
tcacattcac caccctgaat tgactctctt ccgggcgcta tcatgcc 6227
<210> 2
<211> 6950
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ataccgcgaa aggttttgcg ccattcgatg gtgtccggga tctcgacgct cggtaccccc 60
ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag aacgcagaag 120
cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat 180
gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc cccatgcgag 240
agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc 300
gttttatctg ttgtttgcgc tagcggatcc tgtttttttg atcgttttca caaaaatgga 360
agtccacagt cttgacaggg aaaatgcagc ggcgtagctt ttatgctgta tataaaacca 420
gtggttatat gtacagtatt tattttaact tattgtttta aaagtcaaag aggattttat 480
aataatcgat atgaacaata acgatctctt tcaggcatca cgtcggcgtt ttctggcaca 540
actcggcggc ttaaccgtcg ccgggatgct ggggccgtca ttgttaacgc cgcgacgtgc 600
gactgcggcg caagcggcgc atatgccgct gaagaaccgt ctgctggcgc gtctgagctg 660
cgtggcggcg gtggttgcgg cgaccgcggc ggtggcgccg ctgaccctgg ttagcaccgc 720
gcacgcggcg gcgccgcagg ttcgtaccag cgcgccgggc tactatcgta tgctgctggg 780
tgacttcgag atcaccgcgc tgagcgatgg taccgtggcg ctgccggttg acaagcgtct 840
gaaccaaccg gcgccgaaaa cccaaagcgc gctggcgaag agcttccaaa aagcgccgct 900
ggaaaccagc gtgaccggct acctggttaa caccggtagc aagctggtgc tggttgatac 960
cggtgcggcg ggtctgtttg gtccgaccct gggtcgtctg gcggcgaacc tgaaagcggc 1020
gggttaccag ccggagcaag tggatgaaat ctatattacc catatgcacc cggaccatgt 1080
gggtggcctg atggttggcg agcagctggc gttcccgaac gcggtggttc gtgcggacca 1140
gaaggaagcg gatttttggc tgagccaaac caacctggac aaagcgccgg acgatgagag 1200
caagggcttc tttaaaggtg cgatggcgag cctgaacccg tacgtgaagg cgggcaagtt 1260
caaaccgttt agcggtaaca ccgatctggt tccgggtatc aaggcgctgg cgagccatgg 1320
tcacaccccg ggtcacacca cctatgtggt tgaaagccag ggtcaaaaac tggcgctgct 1380
gggcgacctg attctggtgg cggcggttca gttcgacgat ccgagcgtta ccacccaact 1440
ggacagcgat agcaagagcg tggcggttga gcgtaagaaa gcgtttgcgg atgcggcgaa 1500
aggtggctat ctgattgcgg cgagccacct gagcttcccg ggtatcggcc acattcgtgc 1560
ggaaggcaag ggctaccgtt ttgtgccggt taactatagc gtggttaacc cgaaataaat 1620
gggtagcgaa gttggcccgg ccctgtttca gagtgatatg acctttaaaa ttttcatcga 1680
cggtgaagtt aacggtcaga aattcactat tgttgccgat ggtagcagta aatttccgca 1740
tggcgatttt aatgtgcatg cagtttgtga aaccggcaaa ctgccgatga gctggaaacc 1800
gatttgtcat ctgattcagt atggtgaacc gtttttcgcc cgctatccgg atggtattag 1860
tcattttgcc caggaatgtt ttccggaagg cctgagcatt gatcgcaccg ttcgttttga 1920
aaatgatggt accatgacca gtcatcatac ctatgaactg gatgatacct gtgttgttag 1980
tcgtattacc gttaattgtg atggctttca gccggatggc ccgattatgc gtgatcagct 2040
ggttgatatt ctgccgagtg aaacccatat gtttccgcat ggtccgaatg cagtgcgcca 2100
gaccgcaacc attggtttta ccaccgcaga tggcggcaaa atgatgggtc attttgatag 2160
taaaatgacc tttaacggta gtcgcgcaat tgaaattccg ggtccgcatt ttgtgaccat 2220
tattaccaaa cagacccgtg ataccagcga taaacgtgat catgtgtgcc agcgtgaagt 2280
ggcatatgcc catagcgtgc cgcgtattac cagtgccatt ggtagtgatg aagattaata 2340
aagatctccc ctgttttggc ggatgagaga agattttcag cctgatacag attaaatcag 2400
aacgcagaag cggtctgata aaacagaatt tgcctggcgg cagtagcgcg gtggtcccac 2460
ctgaccccat gccgaactca gaagtgaaac gccgtagcgc cgatggtagt gtggggtctc 2520
cccatgcgag agtagggaac tgccaggcat caaataaaac gaaaggctca gtcgaaagac 2580
tgggcctttc gttttatctg ttgtttgcgc tagcggatcc gcctatgcag cgacaaatat 2640
tgatagcctg aatcagtatt gacagctagc tcagtcctag ggactatgct agcaaacact 2700
ataacttcct aggtattatt atgaaagcgt taacggccag gcaacaagag gtgtttgatc 2760
tcatccgtga tcacatcagc cagacaggta tgccgccgac gcgtgcggaa atcgcgcagc 2820
gtttggggtt ccgttccgcg tcctcggctg aagaacatct gaaggcgctg gcacgcaaag 2880
gcgttattga aattgtttcc ggcgcatcac gcgggattcg tctgttgcag gaagaggaag 2940
aagggttgcc gctggtaggt cgtgtggctg ccggtgaacc gcatacgctc tacgctcccg 3000
gcggttatga cattatgggc tatctgattc agattatgaa gaggccaaac ccccaagtag 3060
aactgggacc tgttgacacg tcagttgctc tgattctgtg cgacctgaag caaaaagaca 3120
cgccaattgt gtacgcctcg gaagcttttc tctatatgac aggatacagc aatgcggagg 3180
tcttggggag aaactgccgt tttcttcagt cacccgacgg aatggtcaag ccgaaatcga 3240
caaggaagta cgtcgactcc aacacgatca atacgatgag gaaagcgatt gataggaacg 3300
ccgaggtgca ggttgaggtg gtcaatttta agaagaacgg ccaacggttt gtcaacttct 3360
tgacgatgat tccggtgcga gatgaaacag gggaataccg gtacagcatg ggtttccagt 3420
gcgaaacgga atgagaattc cccctgtttt ggcggatgag agaagatttt cagcctgata 3480
cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 3540
gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 3600
agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 3660
tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg cctcgagctg ccaccgctga 3720
gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa 3780
acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa 3840
ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcatc 3900
gtggccggat cttgcggccc ctcggcttga acgaattgtt agacattatt tgccgactac 3960
cttggtgatc tcgcctttca cgtagtggac aaattcttcc aactgatctg cgcgcgaggc 4020
caagcgatct tcttcttgtc caagataagc ctgtctagct tcaagtatga cgggctgata 4080
ctgggccggc aggcgctcca ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc 4140
ggttactgcg ctgtaccaaa tgcgggacaa cgtaagcact acatttcgct catcgccagc 4200
ccagtcgggc ggcgagttcc atagcgttaa ggtttcattt agcgcctcaa atagatcctg 4260
ttcaggaacc ggatcaaaga gttcctccgc cgctggacct accaaggcaa cgctatgttc 4320
tcttgctttt gtcagcaaga tagccagatc aatgtcgatc gtggctggct cgaagatacc 4380
tgcaagaatg tcattgcgct gccattctcc aaattgcagt tcgcgcttag ctggataacg 4440
ccacggaatg atgtcgtcgt gcacaacaat ggtgacttct acagcgcgga gaatctcgct 4500
ctctccaggg gaagccgaag tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc 4560
atcaagcctt acggtcaccg taaccagcaa atcaatatca ctgtgtggct tcaggccgcc 4620
atccactgcg gagccgtaca aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat 4680
gacgccaact acctctgata gttgagtcga tacttcggcg atcaccgctt ccctcatact 4740
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 4800
atttgaatgt atttagaaaa ataaacaaat agctagctca ctcggtcgct acgctccggg 4860
cgtgagactg cggcgggcgc tgcggacaca tacaaagtta cccacagatt ccgtggataa 4920
gcaggggact aacatgtgag gcaaaacagc agggccgcgc cggtggcgtt tttccatagg 4980
ctccgccctc ctgccagagt tcacataaac agacgctttt ccggtgcatc tgtgggagcc 5040
gtgaggctca accatgaatc tgacagtacg ggcgaaaccc gacaggactt aaagatcccc 5100
accgtttccg gcgggtcgct ccctcttgcg ctctcctgtt ccgaccctgc cgtttaccgg 5160
atacctgttc cgcctttctc ccttacggga agtgtggcgc tttctcatag ctcacacact 5220
ggtatctcgg ctcggtgtag gtcgttcgct ccaagctggg ctgtaagcaa gaactccccg 5280
ttcagcccga ctgctgcgcc ttatccggta actgttcact tgagtccaac ccggaaaagc 5340
acggtaaaac gccactggca gcagccattg gtaactggga gttcgcagag gatttgttta 5400
gctaaacacg cggttgctct tgaagtgtgc gccaaagtcc ggctacactg gaaggacaga 5460
tttggttgct gtgctctgcg aaagccagtt accacggtta agcagttccc caactgactt 5520
aaccttcgat caaaccacct ccccaggtgg ttttttcgtt tacagggcaa aagattacgc 5580
gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tactgaaccg ctctagattt 5640
cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt 5700
aattctcatg ttagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct 5760
caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt 5820
gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 5880
ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca 5940
gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc 6000
ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga 6060
tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc 6120
gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca 6180
gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca 6240
tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt 6300
tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg 6360
cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat 6420
gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa 6480
cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga 6540
tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc 6600
cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa 6660
tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca 6720
gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg 6780
ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca 6840
cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg 6900
gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc 6950
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccatgttatc ctcctcgccc 20
Claims (10)
1. An organophosphorus pesticide live bacteria degrading agent is characterized in that: it adopts pLEV1-Tora-mpdThe plasmid is transformed into Escherichia coli and obtained by amplification culture, and the pLEV1-TorA-mpdThe sequence of the plasmid is shown as SEQ ID NO.1Shown in the figure.
2. The method for preparing the organophosphorus pesticide live bacteria degrading agent of claim 1, wherein the method comprises the following steps: which comprises the following steps:
step S11, constructing an expression vector for producing organophosphorus hydrolase;
step S12, promoter bba _ J23116, light-sensitive fusion protein LEV1 and two Escherichia coli terminatorsrrnBInserting into RI and II enzyme cutting sites of the light-operated gene expression vector to obtain pLEV1 plasmid;
step S13, mixing pCole promotor and methyl parathion hydrolase genempdAnd a transfer signal peptide inserted into the cleavage sites of KpnI and Bglll in the pLEV1 plasmid to obtain pLEV1-Tora-mpdA plasmid;
step S14, pLEV1-TorA-mpdAnd transforming the bacillus subtilis into an escherichia coli strain for amplification culture to obtain the organophosphorus pesticide live bacteria degrading agent.
3. The preparation method of the organophosphorus pesticide live bacteria degrading agent according to claim 2, wherein the preparation method comprises the following steps: in step S11, the backbone of the expression vector for producing organophosphorus hydrolase is pCDFDuet1, and the expression vector does not contain promoter and MCS sequence.
4. The preparation method of the organophosphorus pesticide live bacteria degrading agent according to claim 2, wherein the preparation method comprises the following steps: in step S13, the Escherichia coli terminator rrnB is obtained from the pBAD/His vector; the methyl parathion hydrolase genempdObtained from Pseudomonas WBC-3; the transfer signal peptide is TorA.
5. The preparation method of the organophosphorus pesticide live bacteria degrading agent according to claim 2, wherein the preparation method comprises the following steps: in step S14, the Escherichia coli strain is BL21 strain.
6. An organophosphorus pesticide live bacteria degrading agent is characterized in that: it adopts pLEV1-Tora-mpd-supernovaTransformation of the plasmid into E.coliObtained by medium amplification culture, the pLEV1-TorA-mpd-supernovaIs at pLEV1-Tora-mpdIntroduction of suicide Gene on plasmidsupernovaObtained, the plasmid pLEV1-Tora-mpd-supernovaThe sequence of (A) is shown in SEQ ID NO. 2.
7. The method for preparing the organophosphorus pesticide live bacteria degrading agent of claim 6, wherein the method comprises the following steps: which comprises the following steps:
s21, constructing a light-operated gene expression vector for producing organophosphorus hydrolase;
step S22, inserting a constitutive promoter, a photosensitive fusion protein LEV1 and two Escherichia coli terminators rrnB into RI and II enzyme cutting sites of the light-operated gene expression vector to obtain a pLEV1 plasmid;
in the step of S23,will be provided withpCole promoter, methyl parathion hydrolase genempdAnd a transfer signal peptide is inserted into the restriction enzyme cutting sites of KpnI and Bglll in the pLEV1 plasmid to obtain a pLEV1-TorA-mpd plasmid; in pLEV1-Tora-mpdIntroduction of suicide Gene on plasmidsupernovaTo obtain the plasmid pLEV1-TorA-mpd-supernova；
Step S24, plasmid pLEV1-TorA-mpd-supernovaAnd (3) transforming the bacillus subtilis into an escherichia coli strain, and carrying out dark amplification culture at 37 ℃ to obtain the organophosphorus pesticide viable bacteria degrading agent.
8. The preparation method of the organophosphorus pesticide live bacteria degrading agent according to claim 7, wherein the preparation method comprises the following steps: in step S21, the backbone of the expression vector for producing organophosphorus hydrolase is pCDFDuet1, and the expression vector does not contain promoter and MCS sequence.
9. The preparation method of the organophosphorus pesticide live bacteria degrading agent according to claim 7, wherein the preparation method comprises the following steps: in step S23, the Escherichia coli terminator rrnB is obtained from the pBAD/His vector; the parathion-methyl hydrolase gene mpd is obtained from pseudomonas WBC-3; the transfer signal peptide is TorA.
10. The preparation method of the organophosphorus pesticide live bacteria degrading agent according to claim 7, wherein the preparation method comprises the following steps: in step S24, the Escherichia coli strain is BL21 strain.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114774339A (en) * | 2022-04-12 | 2022-07-22 | 深圳大学 | Whole-cell biosensor for detecting p-nitrophenol and detection method |
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| CN114774339B (en) * | 2022-04-12 | 2023-10-20 | 深圳大学 | Whole-cell biosensor for detecting p-nitrophenol and detection method |
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