CN114480388B - Screening and application of novel promoter element responding to explosive molecules - Google Patents

Abstract

The invention discloses a novel promoter element responsive to explosive molecules, screening and application thereof. The promoter element is derived from rhodopseudomonas palustris and is obtained by screening by transcriptome, has a nucleotide sequence shown as SEQ ID NO.4 or SEQ ID NO.7, is optimized by directed evolution, has the capability of obviously improving the detection sensitivity of 2,4-DNT, and provides a new thought and method for screening other efficient inducible promoters. In addition, compared with the traditional screening method, the screening method provided by the invention has the advantages that the purpose is stronger, the screening efficiency is higher, and the accuracy, the safety and the efficiency of explosive detection are improved by the screened novel promoter.

Description

Screening and application of novel promoter element responding to explosive molecules

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a promoter element responding to explosive molecules, a screening method and application thereof.

Background

Explosives are widely used for military and civilian purposes, which not only pose a threat to personnel life safety in conflicting areas, but also cause serious soil and water pollution. 2, 4-Dinitrotoluene (DNT) is the major component of the gas phase generated by explosive TNT and is often used as a characteristic chemical to detect the presence of explosives because of its relatively high volatility. The conventional technology for detecting explosives does not allow remote detection, and there is a great danger in the detection process, so burland et al propose a possible biological solution, namely, dispersing genetically engineered microbial bioreactors in the target area, allowing remote location of the explosives. Several attempts have been made to develop biosensors that sense 2,4-DNT, including in vivo systems with whole cell biosensors and in vitro systems using purified proteins/peptides. Currently, the main challenge in developing biosensors for detecting DNT is to achieve high sensitivity and high selectivity specificity.

Rhodopseudomonas palustris (Rhodopseudomonas palustris) is a purple non-sulfur bacterium (PNSB) which is widely distributed in nature and mainly distributed in an anaerobic water environment with sufficient illumination, and can carry out light energy heterotrophic growth under anaerobic illumination by taking low molecular organic matters such as low-level fatty acid, various dicarboxylic acid, alcohols, saccharides, aromatic compounds and the like as an electron donor for photosynthesis and can carry out aerobic heterotrophic growth under dark aerobic condition by taking the organic matters as a breathing matrix. Pseudomonas is the most common microorganism for degrading aromatic compounds, and by virtue of the unique physiological characteristics, the Pseudomonas can degrade various aromatic compounds such as aromatic carboxylic acid, nitrophenol, azobenzene and the like under aerobic and anaerobic conditions, so that the genome of the strain contains related promoter elements capable of efficiently sensing aromatic compound molecules. However, there is no report on the use of 2,4-DNT to stimulate rhodopseudomonas palustris to analyze genomic changes to screen promoter elements for highly potent induction TNT or DNT molecules.

Disclosure of Invention

The invention aims at providing a promoter element responding to explosive molecules, and a screening method and application thereof. According to the invention, the promoter element for efficiently sensing the explosive molecules is screened by utilizing the change of transcriptome of the photosynthetic bacteria stimulated by the explosive molecules, and optimized by directed evolution, and the finally obtained promoter element has the capability of remarkably improving the 2,4-DNT detection sensitivity.

In order to achieve the aim of the invention, the invention is realized by adopting the following technical scheme:

the present invention provides a promoter element responsive to an explosive molecule, said promoter element having one of the following nucleotide sequences:

(1) A nucleotide sequence as shown in SEQ ID NO. 4;

(2) The nucleotide sequence shown as SEQ ID NO. 7.

Further, the promoter element is derived from rhodopseudomonas palustris and is obtained by screening by transcriptome.

Furthermore, the promoter element with the nucleotide sequence shown in SEQ ID NO.4, which is screened according to transcriptome, can respond to explosive molecules with high sensitivity.

Furthermore, the sensitivity of the promoter element after directed evolution optimization with the nucleotide sequence shown as SEQ ID NO.7 to respond to explosive molecules is obviously improved compared with that of the promoter element without optimization.

The invention also provides a screening method of the promoter element responding to the explosive molecules, which comprises the following steps:

(1) Stimulating rhodopseudomonas palustris by using a solution containing explosive molecules, extracting RNA of the rhodopseudomonas palustris after stimulation, and carrying out transcriptomic analysis to find out genes with up-regulation or down-regulation;

(2) Predicting the promoter sequence of the gene in step (1); and amplifying the genome of rhodopseudomonas palustris as a template to obtain a promoter sequence of the gene;

(3) Synthesizing the self-luminous operon onto a pUC-57 vector to obtain a pUC-luxPleio vector;

(4) The pUC-luxpelio vector is used as a template, and positive clones are screened to obtain recombinant plasmid p-luxPleio through amplification, enzyme digestion and cloning;

(5) After enzyme cutting p-luxpleio plasmid, connecting with the promoter sequence of the step (2), and transforming competent cells by the connection product to obtain recombinant strain;

(6) Adding an explosive molecule solution into a culture bacterial liquid of the recombinant bacterial strain, and carrying out self-luminescence screening to obtain a bacterial strain with obvious self-luminescence signals, wherein a promoter carried by the bacterial strain is a promoter element for responding to the explosive molecule.

(7) And (3) performing directed evolution on the promoter element in the step (6) by using error-prone PCR, and performing primary screening and enzyme-labeled secondary screening on a plurality of obtained single colonies respectively to obtain the promoter element with stronger detection effect.

Further, the solution containing the explosive molecules in the step (1) is a 2,4-DNT solution with the final concentration of 0.1mM-0.5 mM.

Further, the self-luminous operon in the step (3) is luxABCDE operon from Photobacterium spinosus, and the nucleotide sequence of the luxABCDE operon is shown as SEQ ID NO. 1.

Further, the nucleotide sequence of the promoter element in the step (6) is shown as SEQ ID NO. 4; the nucleotide sequence of the promoter element screened after directed evolution in the step (7) is shown as SEQ ID NO. 7.

The invention also provides application of the promoter element in preparing an expression cassette, a kit or a preparation for detecting explosive molecules.

Further, the detection concentration of the explosive molecules is not lower than 1mg/L.

The invention also provides application of the promoter element in preparation of a preparation for improving the detection sensitivity of explosive molecules.

Further, the method for using the promoter element comprises the following steps: the promoter element is amplified, digested, cloned and transformed into competent cells, the obtained recombinant strain is cultured to obtain a culture solution, and the culture solution is directly or mixed with other culture mediums to be added into a solution containing explosive molecules, so that the purposes of improving the detection sensitivity and detecting the explosive molecules in real time can be achieved.

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

according to the invention, the rhodopseudomonas palustris is stimulated by using 2,4-DNT, the transcriptome change is analyzed, the promoter element for inducing explosive molecules is screened, the detection sensitivity is further improved by directed evolution, and the obtained promoter element has the capability of remarkably improving the 2,4-DNT detection sensitivity, and the screening method is simple, convenient to use and high in safety; the method overturns the traditional detection technology, can purposefully and selectively screen the sensing element which can be induced by explosives from the genome of the rhodopseudomonas palustris through the change of transcriptome of the rhodopseudomonas palustris after 2,4-DNT stimulation, further combines error-prone PCR to perform directed evolution, not only provides a new thought and method for obtaining other efficient inducible promoters, but also can be used for detecting explosives such as mine in military, can be used for detecting environmental pollutants in an ecological system, and has important significance for military, anti-terrorism, environmental protection and the like.

Drawings

FIG. 1 shows the growth curves of rhodopseudomonas palustris after stimulation with different concentrations of 2,4-DNT.

FIG. 2 is a plasmid map of the constructed vector p-luxpleio.

FIG. 3 is a constructed vector P-P ₀₁₄₂₅ Plasmid map of luxpleio.

FIG. 4 is a filtered P ₀₁₄₂₅ And (5) detecting a result by a novel promoter enzyme label instrument.

FIG. 5 is an optimized P _01425-1 And (5) detecting a result by a novel promoter enzyme label instrument.

Detailed Description

The technical scheme of the invention is further described in detail below with reference to specific embodiments.

Example 1: determination of 2,4-DNT stimulation concentration and extraction of RNA

1. Determination of 2,4-DNT stimulation concentration

Anaerobic culture of rhodopseudomonas palustris by illumination, and activation to OD ₆₆₀ 0.8, transferred to fresh anaerobic medium and cultured to OD ₆₆₀ Rhodopseudomonas palustris was stimulated at 0.2 with 2,4-DNT at final concentrations of 0mM, 0.1mM, 0.25mM, 0.5mM, 5 replicates each, and OD was measured every 2 hours ₆₆₀ Growth curves were made (FIG. 1) and 0.25mM stimulation concentration was selected that did not significantly differ from growth by the addition of 0mM 2,4-DNT.

2. Extraction of RNA

1) Rhodopseudomonas palustris (L.) VahlCulturing the bacteria to OD ₆₆₀ Rhodopseudomonas palustris is stimulated with 0.25mM 2,4-DNT at a final concentration, 5 parallel groups of ethanol are added as a control group, after 6h of stimulation with 0.25mM 2,4-DNT, centrifugation is carried out at 10000rpm for 2min, 3mL of bacterial liquid is collected from each tube, and the supernatant is removed.

2) 1mL of Trizol was added to resuspend the cells, and the cells were transferred to a 2mL shaking tube pre-cooled with 1g of 0.1mm glass beads, shaking at 4200rpm for 90s, water-bathing for 90s, repeating 5 times, and water-bathing at 22℃for 5min after completion.

3) 200. Mu.L of chloroform was added, and the mixture was manually shaken for 15s and water-bath at 22℃for 3min.

4) 12000 Xg, centrifuge at 4℃for 15min, transfer 560. Mu.L of supernatant to a fresh 1.5mL EP tube, add 500. Mu.L of isopropanol, mix upside down, and water bath at 22℃for 10min.

5) The RNA was precipitated by centrifugation at 15000 Xg for 10min at 4 ℃.

6) The whole supernatant was aspirated, and 1mL of 75% ethanol was added to wash RNA,15000 Xg, and centrifuged at 4℃for 5min.

7) The supernatant was removed, and 1mL of 75% ethanol was added to wash RNA once, 15000 Xg was centrifuged at 4℃for 5min.

8) Sucking all the supernatant as much as possible, and airing at room temperature for 5min.

9) RNA pellet was resuspended with 20. Mu.L RNase-free water.

Example 2: gene acquisition and vector construction

1. Gene acquisition

A luxABCDE operon derived from the A.mori (Photobacterium leiognathi) is chemically synthesized on pUC-57 vector by Huada genes company to obtain pUC-luxPleio vector, wherein the nucleotide sequence of the luxABCDE operon is shown as SEQ ID NO. 1.

The promoter fragment was derived from the rhodopseudomonas palustris (Rhodopseudomonas palustris CGA 009) genome.

2. Construction of vectors

2.1 primers luxpelio-F and primer luxpelio-R, polymerase Chain Reaction (PCR) was performed to amplify the luxpelio fragment, the PCR amplification system was as follows:

the PCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ C. 15s,58 ℃ C. 15s,72 ℃ C. 6 min); 72 ℃ for 5min;16 ℃ infinity. The primer sequences are shown below:

luxPleio-F：

5’-TCGACAAGCTTGCGGCCGCATGATTAAGAAGATCCCAATG’(SEQ ID NO.2)；

luxPleio-R：

5’-TACCAGACTCGAGGGTACCTTACGTATAGCTAAATGCATCA-3’(SEQ ID NO.3)。

the PCR product was subjected to gel recovery and purification using gel recovery and purification kit (Vazyme, cat. DC 301-01).

The pACYCDuet-1 plasmid was digested simultaneously with restriction enzyme 1Not I (TaKaRa, cat# 1623) and restriction enzyme 2Kpn I (TaKaRa, cat# 1618), and the digestion system is as follows:

the enzyme digestion system is incubated for 1h at 37 ℃ for gel recovery and purification.

The luxpleio fragment was cloned into the pacycdat-1 plasmid using seamless cloning, the system of which is as follows:

the ligation system was incubated at 50℃for 30min. The ligation product was transformed into E.coli DH 5. Alpha. Competent, spread on LB solid plates containing 34mg/L chloramphenicol, PCR screened positive clones, recombinant plasmid p-luxpleio was extracted from positive clones (FIG. 2), and identified by restriction enzyme digestion and sequencing.

2.2 extraction of bacterial genomic DNA

Obtaining rhodopseudomonas palustris Rhodopseudomonas palustris CGA009 and culturing, and extracting genome DNA from the obtained bacterial liquid by using a bacterial genome extraction kit:

1) 2.5mL of fresh bacterial liquid is taken and added into a 1.5mL centrifuge tube, and the bacterial liquid is collected by centrifugation to remove the supernatant. Adding 200 mu L Buffer Digestion and 2 mu L of beta-mercaptoethanol, shaking and mixing uniformly, and carrying out water bath at 80 ℃ for 5min until cells are completely lysed;

2) To obtain RNA-free DNA, 2. Mu.L of RNase A (10 mg/mL) was added after the water bath, and the mixture was left at 37℃for 1 hour, and the mixture was inverted and mixed once every 10 minutes during the water bath;

3) Then adding 20 mu L of protease K solution, shaking and mixing uniformly, and carrying out water bath at 55 ℃ for 2 hours, wherein in the water bath process, the mixing is reversed and uniform every 10 minutes, so that the sample cracking can be promoted;

4) Cooling to room temperature, adding 100 μL Buffer PF, mixing, and standing at-20deg.C for 5min;

5) Centrifuging at 10,000rpm for 5min at room temperature, transferring the supernatant to a new 1.5mL centrifuge tube;

6) 200 mu L Buffer BD is added, and the mixture is fully inverted and uniformly mixed; after adding Buffer BD, if precipitate exists, water bath is performed for 10min at 70 ℃;

7) Adding 200 mu L of absolute ethyl alcohol, fully reversing and uniformly mixing; semitransparent fibrous suspended matters can be generated after absolute ethyl alcohol is added, and extraction and application of DNA are not affected;

8) Placing the adsorption column into a collecting pipe, adding the solution and semitransparent fibrous suspension into the adsorption column by using a liquid transfer device, standing for 2min, centrifuging at room temperature for 1min at 10,000rpm, and pouring out waste liquid in the collecting pipe;

9) Placing the adsorption column back into a collection tube, adding 500 mu L PW Solution, centrifuging at 10,000rpm for 30s, and pouring out the waste liquid in the collection tube; before use, paying attention to whether isopropanol is added into the solution in proportion;

10 Placing the adsorption column back into the collection tube, adding 500 mu L of Wash Solution, centrifuging at 10,000rpm for 30s, pouring out the waste liquid in the collection tube, and paying attention to whether absolute ethyl alcohol is added into the Solution according to a certain proportion before use; if it is desired to increase the yield of DNA, the procedure may be repeated;

11 Placing the adsorption column back into the collection tube again, centrifuging at 12,000rpm at room temperature for 2min, and removing residual Wash Solution; the adsorption column is opened and the cover is placed at room temperature for a plurality of minutes, so that the Wash Solution remained in the adsorption material is thoroughly dried, and the Wash Solution residue can influence the yield of the genome DNA and the subsequent experiment;

12 Taking out the adsorption column, placing into a new 1.5mL centrifuge tube, adding 50 μl ddH ₂ O was allowed to stand for 3min, centrifuged at 12,000rpm at room temperature for 2min, and the DNA solution was collected.

2.3 obtaining promoter fragments

And (3) analyzing transcriptomics results, finding up-and down-regulated genes, predicting promoter sequences of the genes, amplifying the promoter sequences of the up-and down-regulated genes by taking the extracted rhodopseudomonas palustris CGA009 genome as a template, and performing gel recovery and purification on PCR products by using a gel recovery and purification kit (Vazyme, product number DC 301-01). Meanwhile, the p-luxpleio plasmid is digested by using restriction enzyme PstI (TaKaRa, product number 1624), and the digestion system is as follows:

the enzyme digestion system is incubated for 1h at 37 ℃ for gel recovery and purification.

The up-down-regulated gene promoter fragment was cloned onto a p-luxpleio plasmid using seamless cloning. The ligation system was incubated at 50℃for 30min. The ligation product was transformed into E.coli DH 5. Alpha. Competent, spread on LB solid plates containing 34mg/L chloramphenicol, PCR screened positive clones, recombinant plasmid was extracted from positive clones, and identified by restriction enzyme digestion and sequencing.

Example 3: enzyme-labeled instrument screening promoter element for inducing 2,4-DNT

1. Strain activation and culture

The strain containing the predicted promoter fragment plasmid vector is transferred into LB liquid medium containing 34mg/L chloramphenicol, and cultured overnight at 37 ℃ to obtain bacterial liquid.

10mL of M9 liquid medium containing 34mg/L chloramphenicol is added with 330 mu L of 60% glucose, 10 mu L of 1M magnesium sulfate stock solution, 200 mu L of bacterial liquid, and shaking culture is carried out at 37 ℃ to give a raw materialAs long as OD ₆₀₀ =0.2 or so.

2. Preparation of 2,4-DNT solution

Preparing 20mg/mL mother liquor 2,4-DNT (100 mg 2,4-DNT dissolved in 5mL absolute ethanol);

the diluted 2,4-DNT solution was prepared in the following proportions:

100mg/L: 5. Mu.L of mother liquor+980. Mu. L M9 medium+15. Mu.L of absolute ethanol;

50mg/L: 2.5. Mu.L of mother liquor +980. Mu. L M9 Medium +17.5. Mu.L of absolute ethanol;

10mg/L: 0.5. Mu.L of mother liquor +980. Mu. L M9 Medium +19.5. Mu.L of absolute ethanol;

0mg/L: 980. Mu. L M9 Medium+20. Mu.L absolute ethanol.

The concentration of ethanol in each DNT solution was 2%, and the final concentration of ethanol after 10-fold dilution was 0.2%.

3. Sample addition

The 2,4-DNT mother liquor with concentration of 90 mu L of bacterial liquid plus 10 mu L of 0mg/L, 1mg/L, 10mg/L, 50mg/L and 100mg/L is added into a 96 Kong Chunbai ELISA plate, so that the final concentration of 2,4-DNT is respectively 0mg/L, 1mg/L, 5mg/L and 10mg/L, 4 parallel concentrations are respectively carried out, and the sealing plate film (Biored, product number MSB 1001) is used for sealing.

4. Luminescence detection

The fluorescence intensity (RLU) of the strain was monitored in real time at a constant temperature of 30℃using a microplate reader (Biotek), and the detection was performed every 10min for a total of 12 hours.

Construction of 125 promoters Using transcriptomic data in total, P ₀₁₄₂₅ The promoter has stronger response under the stimulation of 2,4-DNT at different concentrations, and the nucleotide sequence is shown as SEQ ID NO. 4. As shown in fig. 4, P ₀₁₄₂₅ The promoter produces a higher RLU value and also has a very significant difference in RLU value at a minimum DNT concentration of 1mg/L compared to 0 DNT concentration. The invention is shown that a promoter element P for inducing 2,4-DNT is screened out by a method for carrying out transcriptomics research on rhodopseudomonas palustris stimulated by 2,4-DNT with a certain concentration ₀₁₄₂₅ The promoter element obtained by the invention can be prepared into an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic cell line and the like so as to achieve wider application scenes.

Example 4: directed evolution of promoters

Primer P ₀₁₄₂₅ -F and primer P ₀₁₄₂₅ R, error-prone PCR, amplification of P ₀₁₄₂₅ The error-prone PCR amplification system of the promoter fragment is as follows:

the PCR procedure was: 3min at 95 ℃;30 cycles X (95 ℃ 15s,58 ℃ 15s,72 ℃ 20 s); 72 ℃ for 5min;16 ℃ infinity. The primer sequences are shown below:

P ₀₁₄₂₅ -F：

5’-AGCTCGGCGCGCCTGCAGCCGACTGGCACGGCATTT-3’(SEQ ID NO.5)；

P ₀₁₄₂₅ -R：

5’-CAAGCTTGTCGACCTGCAGTTGTCGTCTCCTGAGTGAAG-3’(SEQ ID NO.6)。

the PCR product was subjected to gel recovery and purification using gel recovery and purification kit (Vazyme, cat. DC 301-01).

P Using seamless cloning ₀₁₄₂₅ The promoter fragment was cloned into the p-luxpleio plasmid. The ligation system was incubated at 50℃for 30min. The ligation product was transformed into E.coli DH 5. Alpha. Competent, and the E.coli DH 5. Alpha. Competent was applied to LB solid plates containing 34mg/L chloramphenicol, and 50 LB solid plates were obtained by each transformation and application, and about 150 clones were obtained for 1 plate, and 7500 transformants were grown well in total.

Example 5: initial screening and rescreening of promoter directed evolution library

1. Initial screening of promoter directed evolution library by using plant living body fluorescence detector

The above obtained 50 plates with good growth vigor were subjected to membrane transfer of nitric acid microporous membrane, and the nitric acid microporous membrane was transferred to LB solid plate containing 10 mg/L2, 4-DNT (100. Mu.L mother liquor concentration of 20mg/mL 2,4-DNT was added to 200mL LB) and LB solid plate containing 100. Mu.L absolute ethyl alcohol, respectively, and the mixture was kept in a constant temperature incubator at 37℃for 12 hours. When obvious colony growth exists on the LB solid plate with the transfer film, 2 plates are correspondingly placed into a plant living body fluorescence detector, the exposure is carried out for 30 seconds for observation, bacterial colony with stronger fluorescence on the LB solid plate with the transfer film containing 10 mg/L2, 4-DNT is picked by using a sterile toothpick, and is cultivated into 96 deep hole plates containing 1mL LB liquid culture medium in each hole, and the initial screening of the promoter directed evolution library is completed.

2. Rescreening the promoter directed evolution library by using an enzyme-labeled instrument

The primary screening to obtain 15 single colonies with stronger fluorescence on a transfer membrane LB solid plate containing 10 mg/L2, 4-DNT, transferring the strains which are cultured to a 96 deep well plate containing 1mL LB liquid culture medium to a 96 deep well plate containing 1mL M9 liquid culture medium (50 mL M9 liquid culture medium containing 34mg/L chloramphenicol is added with 1.65mL 60% glucose and 50 mu L1M magnesium sulfate storage solution, 1mL of culture medium is respectively absorbed into 98 deep well plates after uniform mixing), and shaking the mixture at 37 ℃ to OD ₆₀₀ =0.2 or so. The preparation, sample application and luminescence detection of the 2,4-DNT solution are the same as those of the method described above. The detection result is shown in FIG. 5, and P is subjected to directed evolution screening _01425-1 The promoter has the nucleotide sequence shown in SEQ ID NO.7, the fluorescence intensity of response 2,4-DNT is obviously enhanced, wherein the fluorescence intensity of response 5 mg/L2, 4-DNT is enhanced by nearly 30 times, and when the lowest concentration is detected, compared with the non-oriented evolution promoter, the fluorescence value is obviously enhanced, the difference between the fluorescence value and the fluorescence value is larger when the fluorescence value is 0mg/L, and the detection limit is obviously enhanced.

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.

Sequence listing

<110> Qingdao university of agriculture

<120> screening and use of novel promoter elements responsive to explosive molecules

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 6533

<212> DNA

<213> Acidovorax facilis (Photobacterium leiognathi)

<400> 1

atgattaaga agatcccaat gattattggg ggtgtagttc aaaacacgtc tggatatggc 60

atgcgtgaac taacgctcaa caataataaa gtgaatatcc ctatcatcac ccaaagtgat 120

gttgaagcta ttcaatcact aaatatagaa aacaaattga ctataaatca gatagttaat 180

ttcttatata cagtgggaca aaaatggaag agcgaaactt acagccgacg actcacttat 240

attcgagatc ttattaagtt cctcggttac tcacaagaga tggcaaaact tgaagctaac 300

tggatctcaa tgattctgtg tagcaaaagt gcgttgtacg atattgttga gaatgatctt 360

agctcacggc atattattga tgagtggatc ccccaaggtg aatgttatgt caaagcgctc 420

ccaaaaggaa aatctgtaca cctattagct ggtaacgtac cactatctgg tgtgacttct 480

attcttcgtg cgattttgac caaaaacgag tgcatcataa aaacgtcatc agctgatcct 540

tttacagcta ctgcgctagt taatagtttt atcgatgtag atgcagaaca cccgatcaca 600

cgttcaatct cagttatgta ttggtcacat agcgaggatc ttgctattcc aaaacaaata 660

atgagctgtg ctgatgtggt tattgcatgg ggtggtgatg atgcaattaa atgggctaca 720

gaacatgcac catcacacgc agatattcta aaatttggtc ccaaaaagag tatatccatt 780

gttgacaacc caacagatat taaggctgct gctatcggtg tagcacatga tatctgtttt 840

tacgatcagc aagcatgttt ctccacccaa gatatttatt atattggcga tagcatagac 900

atattttttg atgaattagc tcagcaatta aataaatata aagacatatt gcctaaaggt 960

gagcggaatt ttgatgaaaa agcagctttt tctttaacgg aaagagaatg tttgtttgcc 1020

aaatataaag ttcaaaaagg tgaaagccaa tcttggttat taacgcaatc acctgcggga 1080

tcatttggta atcagccgtt atcacgctcg gcttatattc atcaagtaaa tgacatttca 1140

gaagtcattc cattcgtgca taaggcggta acgcaaaccg tcgcaatagc gccgtgggag 1200

tcgtctttca aatatagaga tatattagca gaacatggtg cagaacgaat tatagaagcc 1260

ggaatgaata atatatttcg agtaggtggc gcccatgatg ggatgcgtcc ccttcaacgg 1320

cttgttaact atatatcaca tgaaaggccg tcaacatata ccactaaaga tgtctcggtg 1380

aaaatcgaac agactcgtta tcttgaggaa gataagttcc tcgtatttgt accgtagaaa 1440

gagatatatc atggaaaata cacaacattc attacctatt gatcacgtaa ttgatattgg 1500

tgataaccgt tatattcgag tatgggaaac caagccgaaa aataaagaaa ccaagcgtaa 1560

taataccatc gttatcgcct caggctttgc tcgacgcatg gatcattttg ctggtcttgc 1620

cgaatattta gcaaataatg gttttcgtgt tattcgttat gattcgttaa atcatgtcgg 1680

tcttagtagc ggagagatca aacagttctc gatgtcagta ggtaaacaca gtttgctaac 1740

tgttattgat tggctaaaag aacgaaatat taacaatata ggtcttattg cttcgagtct 1800

ttctgctcga attgcttatg aagtggcagc agaaattgat ttgtcatttt taattaccgc 1860

cgtcggtgtt gtcaatttaa gaagtacgct agaaaaagca ctgaaatatg attatctaca 1920

aatggaagta aatactattc ctgaagattt aatttttgaa ggacacaatc taggttcaaa 1980

agtctttgtg acagattgtt ttgaaaataa ttgggactca ttagattcga caataaataa 2040

aatttgtgaa ctagatattc catttattgc tttcacttca gatggcgatg attgggtttg 2100

ccaacatgaa gtaaaacatt tagtcagtaa cgttaaatct gacaaaaaga aaatttactc 2160

actcgttggc tcatctcatg atttgggcga aaacctagtg gtgcttcgta acttctatca 2220

atcaatgacg aaagctgctg tgagcttaga tcgtcaatta gtagagcttg ttgatgaaat 2280

tattgaacca aattttgaag acctaacagt tattacggta aatgaacggc gcctcaaaaa 2340

taaaatcgaa aatgaaatta ttaatagatt agctgatcgc gtattggcta gtgtctaaat 2400

agtacttacc taagtacagc caaaaggaag aaataatgaa aattagtaat atctgtttct 2460

cataccaacc accaggtgaa tcacatcaag aggtaatgga gcgctttatt cgtttaggcg 2520

ttgcatcaga agagctcaac tttgatggtt tctatacact tgaacaccat ttcactgagt 2580

ttggtattac aggtaacctt tatattgcct gtgccaatat tcttggtcga accaaaagga 2640

tccaagtcgg taccatgggg atagtgttac cgacagagca cccagcacga catgtagaaa 2700

gtcttctcgt tttagatcaa ctgtctaaag ggcgctttaa ctacggtact gttcgcggac 2760

tctaccataa agattttcgt gtttttggta catcacagga agattctcgt aagaccgcag 2820

aaaatttcta ctctatgatc ttggatgcat caaaaacagg tgtgctacat actgacggtg 2880

aagtagtaga gttcccagat gtcaatgttt atccagaagc ttacagcaaa aaacaaccca 2940

cctgcatgac agccgaatca tccgagacca tcacttattt agctgaacgt ggtttaccaa 3000

tggtgttaag ttggattatt ccggtcagtg agaaagtctc acaaatggaa ttgtacaatg 3060

aagttgcggc agagcatggt catgacatta acaacattga acatatccta actttcattt 3120

gctctgtaaa tgaagacggt gaaaaagcag acagcgtatg ccgtaatttc ctagaaaatt 3180

ggtacgactc ttacaaaaat gcaaccaaca tcttcaacga cagtaaccaa actcgtggct 3240

acgattacct caaagctcag tggcgtgagt gggtaatgaa ggggttggct gatcctcgtc 3300

gccgacttga ttacagtaac gaattaaacc ctgtcggcac gccagaacga tgcattgaga 3360

tcattcaaag taatattgat gccactggaa ttaagcatat tactgttgga tttgaagcga 3420

atggttctga acaagaaatt cgtgaatcca tggagctatt tatggaaaaa gtagcgccac 3480

acttaaaaga tcctcagtaa gctgttcttt ttaaactatt caatatcaag gcataaggaa 3540

taaaatatga atttcgggtt atttttccta aatttccagc ctgaaggtat gacttcagaa 3600

atggttttag acaacatggt agatactgtc gcattagtgg ataaagatga ttaccacttt 3660

aaaagagtgc tcgtcagcga gcatcatttt tctaaaaacg gcattatcgg agaacctttg 3720

acagcgatta gcttcttact tggtttgact aaacgtatag aaattggttc tttaaatcaa 3780

gtgattacca cccatcatcc tgtacgtatc ggagaacaaa cgggcttact tgatcaaatg 3840

tcttacggtc gtttcgtttt aggcttaagt gactgtgtca atgacttcga aatggatttc 3900

tttaagagaa aacgtagctc tcaacagcaa caattcgaag catgttacga aattttaaat 3960

gaagcgctga cgacaaacta ttgtcaggca gatgatgact tctttaactt cccacgtatt 4020

tctgttaacc cgcattgtat tagcgaagta aaacaatata ttttagcttc aagcatgggc 4080

gtggttgaat gggcagcaag aaaaggattg ccactcactt accgctggag tgacagccta 4140

gcagaaaaag aaaaatacta tcagcgttat ctcgctgttg ctaaagagaa taatattgat 4200

gtatcaaata ttgaccacca attcccactg ctcgttaata tcaatgaaaa tcgtcgtatt 4260

gctcgagatg aagtaaggga gtatatacaa agttatgtga gtgaagccta ccctactgac 4320

cccaacattg agctaagagt agaagagctt attgagcagc atgctgtcgg caaagtggat 4380

gagtactacg actcaacaat gcacgcagta aaagttacag gttcaaaaaa tttattactc 4440

tcttttgaat caatgaaaaa taaagacgat gttaccaagc ttataaatat gtttaatcaa 4500

aaaatcaaag ataaccttat taaataattt aattacggat agatattttc gatatatcta 4560

agtcttacta ccatttatat aaactattta tacagataac gtttcatttg attaagtcag 4620

taaataattg ccattaatta atggcagtgc agatccttac actgccattt ataaattaaa 4680

taagggttaa catgtcaaca ttattaaata tagatgcaac tgaaattaag gtgagtacag 4740

aaatagatga tattattttt acatcatcac cgctaacgtt actatttgaa gatcaagaaa 4800

aaatacagaa agaacttatt ttggagtctt tccattatca ttacaatcat aataaagatt 4860

ataagtacta ttgtaatata caaggcgtag atgagaatat acagtccatt gacgatattc 4920

ctgtttttcc tacttcaatg ttcaagtact caagattaca tactgctgat gaatcaaata 4980

ttgaaaattg gtttactagt agtggtacaa agggagtcaa aagtcatata gctcgagatc 5040

ggcagagtat tgaacgcttg ctaggttctg ttaattacgg catgaaatac ttgggtgaat 5100

ttcacgagca tcaattagaa ctagtgaata tggggccaga tcgtttcagt gcgtcaaatg 5160

tttggtttaa atatgtaatg agcttagttc aattacttta cccaacaaca tttaccgttg 5220

aaaacgatga aatcgatttt gaacaaacca tcttagcgtt aaaagcaatt cagcgtaaag 5280

gaaaaggaat ttgtttaatt ggccctccgt attttattta tttgttatgc cactacatga 5340

aagagcataa tatcgaattt aatgctggtg cacatatgtt tatcattaca ggtgggggat 5400

ggaaaaccaa acaaaaagaa gcgctaaacc gacaagattt caatcaacta ttgatggaga 5460

cttttagcct tttccatgaa agtcaaattc gagatatctt taaccaagta gagctaaaca 5520

cttgtttctt tgaagacagc ctacagcgta aacatgtacc accgtgggta tatgctcgtg 5580

cgcttgatcc tgtcacttta acgcccgtag aagatggcca agagggcttg atgagttata 5640

tggatgcctc atctaccagc tacccgacat ttattgttac cgacgatatt ggtattgttc 5700

gccatctaaa agaaccagat ccattccaag gaacaacggt tgaaattgtt cgtcgtttaa 5760

atacgcgaga acaaaaagga tgttcactct caatggccac gagcctgaaa taaaagcagg 5820

gcttaatcat gatttttaat tgcaaggtta aaaaagtcga agcatctgac agccatattt 5880

acaaagtgtt tattaagcct gacaaatgct ttgattttaa agcgggtcaa tatgtaattg 5940

tgtatctcaa tggaaaaaat ttgccgtttt ctattgctaa ctgcccaact tgtaatgagc 6000

tccttgaatt acatgtagga ggttcggtaa aagaatccgc cattgaagct atttcgcact 6060

ttattaatgc atttatttat caaaaagaat ttacaatcga tgcaccacac ggtgatgcat 6120

ggctgagaga tgaaagccaa tcacctttac tacttatagc aggagggaca ggtttatcat 6180

atatcaatag cattttaagt tgttgtatta gtaaacagtt atctcagcct atctatcttt 6240

attggggagt aaataactgt aatttactct atgctgatca acaactaaaa acactcgccg 6300

cacaatacag aaatataaat tatattcctg tggtagagaa tttaaatact gactggcagg 6360

gaaaaattgg taatgttatt gacgcggtta ttgaagattt ttcagattta tctgactttg 6420

atatctatgt ctgcgggcca tttggtatga gccggactgc gaaagatatt ctgatctcac 6480

agaaaaaggc gaatatagga aaaatgtatt ctgatgcatt tagctatacg taa 6533

<210> 2

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

tcgacaagct tgcggccgca tgattaagaa gatcccaatg 40

<210> 3

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

taccagactc gagggtacct tacgtatagc taaatgcatc a 41

<210> 4

<211> 500

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ccgactggca cggcatttga ttctaagggg ccggctgtgc ccgcgtagtg atgctccgcg 60

tggggaattt cagtcgagat catttcccgg ccagttcggt cgggacctag cggggatagg 120

acccatgaaa attgttatgg cgatcattaa gccattcaaa ctcgaagaag tccgtgacgc 180

cctgaccgcc atcggcgttc acggtttgac ggtgacggaa gtgaagggat acggccgcca 240

gaaaggccac acggaaatct accgcggcgc tgaatacgcg gtgagcttcc tgcccaagat 300

caagatcgag gtcgcgatcg ccaacgagca ggtcgagaag accatcgagg cgatcaccag 360

cgccgccaag accggccaga tcggcgacgg caagatcttc gtcatcggcc tcgaccacgc 420

ggtgcgcatc cgcaccggcg aggccgacgc ggccgccctc tgatcccctc accttcattg 480

cttcactcag gagacgacaa 500

<210> 5

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

agctcggcgc gcctgcagcc gactggcacg gcattt 36

<210> 6

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

caagcttgtc gacctgcagt tgtcgtctcc tgagtgaag 39

<210> 7

<211> 500

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ccgactggca cggcatttga ttctaaggcg ccggctgtgc ccgcgtagtg atgctccgcg 60

tggggaattt cagtcgagat catttcccgg ccagttcggt cgggacctag cggggatagg 120

acccatgaaa attgttatgg cgatcattaa gccattcaaa ctcgaagaag tccgcgacgc 180

cctgaccgcc atcggcgttc acggtttgac ggtgacggaa gtgaagggat acggccgcca 240

gaaaggccac acggaaatct accgcggcgc tgaatacgct gtgagcttcc tgcccaagat 300

caagatcgag gtcgcgagcg ccaacgagca ggtcgagaag accatcgagg cgatcaccag 360

cgacgccaag accggccaga tcggcgacgg caagttcttc gtcatcggcc tcgaccacgc 420

agtgcgcatc cgcaccggcg aggccgacgc ggccgccctc tgatcccctc accttcattg 480

cttcactcag gagacgacaa 500

Claims (6)

1. A promoter element responsive to an explosive molecule, wherein the nucleotide sequence of said promoter element is shown in SEQ ID No.4 or SEQ ID No. 7.

2. The explosive molecule-responsive promoter element of claim 1, wherein the promoter element is derived from rhodopseudomonas palustris and is selected by transcriptomics.

3. Use of the promoter element according to claim 1 for the preparation of an expression cassette, kit or formulation for detecting an explosive molecule, characterized in that the explosive molecule is 2,4-DNT.

4. The use according to claim 3, wherein the detected concentration of the explosive molecules is not less than 1mg/L.

5. Use of the promoter element of claim 1 for the preparation of a formulation for increasing the detection sensitivity of an explosive molecule, wherein the explosive molecule is 2,4-DNT.

6. The use according to claim 5, wherein the promoter element is used in the following way: the promoter element is amplified, digested, cloned and transformed into competent cells, the obtained recombinant strain is cultured to obtain a culture solution, and the culture solution is directly or mixed with other culture mediums to be added into a solution containing explosive molecules, so that the purposes of improving the detection sensitivity and detecting the explosive molecules in real time can be achieved.

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