An AraC mutant for sensing metal ion Cd(II) in submarines and a submarine-detecting microbial sensor constructed therefrom and applications thereof

Abstract

The present invention discloses an AraC mutant for sensing submarine metal ion Cd(II), a submarine-detecting microbial sensor constructed therefrom, and applications thereof. The AraC mutant is AraCmt-Cd, whose amino acid sequence is as shown in SEQ ID NO.10, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO.9. The mutant AraCmt-Cd can specifically bind to the metal ion Cd(II) and be induced by it to activate the P BAD promoter, thereby driving the expression of the downstream electron transfer pigment protein (CymA), and realizing the output of electrochemical signals after sensing the metal Cd(II) ions. The submarine-detecting microbial sensor constructed by the present invention, which contains a metal ion sensing element and an electrical signal reporting element, can sense the metal ions released by submarines in water and cause changes in electrochemical signals, so that submarines can be detected in real time.

Description

Technical Field

[0001] The present invention belongs to the technical fields of genetic engineering and molecular biology, and particularly relates to an AraC mutant that senses metal ion Cd(II) in submarines, a submarine-detecting microbial sensor constructed thereby, and applications thereof. Background Art

[0002] As an important part of modern navies, submarines are characterized by high concealment and strong striking power. The main structure of a submarine is composed of metal, and in a seawater salt solution system, metal ions will be released around the ship's hull through oxidation-reduction, etc. These metal ions can be sensed by microbial-related sensing elements and then detected.

[0003] In cell-based biosensors, biological reactions are converted into physicochemical signals by a sensing module. In these systems, reports such as color development, bioluminescence, and fluorescence are widely used. The selection of a reporter gene should take into account sensitivity and measurement convenience. Since the transcription of a reporter gene is controlled by a promoter / operator, the expression level of the reporter gene is designed to be proportional to the sensing signal. Currently, there are two different reporting methods. (1) The reporter gene encodes an enzyme that can catalyze the formation of a measurable product. For example, β-galactosidase and luciferase catalyze the production of colored products and light, respectively. (2) The protein itself is the product to be measured (e.g., green fluorescent protein [GFP]). In the present invention, after successfully establishing an electron metabolic transfer pathway for microorganisms to sense metal ions through the construction and optimization of a microbial electrical signal reporting element, and coupling extracellular electron transfer with an in vitro electrochemical device, the detection of electrical signals induced by metal sensing is achieved through electrochemical means such as cyclic voltammetry (CV) and chronoamperometry, and further research is conducted to explore the effects of different sensing elements, different metal ions, and different metal ion concentrations on the intensity of electrochemical signals.

[0004] Wild-type AraC-P BAD system: The transcription factor AraC can naturally respond to L-arabinose. In the absence of L-arabinose, the AraC dimer binds to the I 1 and O 2 sites, forming a DNA loop upstream of the P BAD promoter to inhibit transcription; after binding L-arabinose, the AraC dimer changes its conformation, enabling binding to adjacent I 1 and I 2 half-sites, activating transcription. An AraC mutant obtained by mutation can bind to a compound other than L-arabinose, thereby activating the transcriptional activity of the P BAD promoter, making the AraC mutant induced by other compounds. Compared with a promoter that directly senses metal ions, this AraC-P BADThe system has an amplification effect and its signal is stronger. SUMMARY OF THE INVENTION

[0005] The object of the present invention is to provide an AraC mutant for sensing metal ion Cd(II) in submarines, a microbial sensor for submarine detection constructed therefrom, and applications thereof. The AraC mutant AraCmt-Cd can specifically bind to the metal ion Cd(II), and the microbial sensor for submarine detection prepared by using the same realizes the output of an electrochemical signal after sensing the metal Cd(II) ion.

[0006] To achieve the above object of the invention, the present invention is implemented by the following technical solutions:

[0007] The present invention provides an AraC mutant, which is AraCmt-Cd, and its amino acid sequence is as shown in SEQ ID NO.10.

[0008] Furthermore, the AraC mutant AraCmt-Cd has a significant function of sensing metal ion Cd(II) in submarines.

[0009] The present invention also provides a coding gene of the above AraC mutant, and the coding gene of the AraC mutant has one of the following nucleotide sequences:

[0010] (1) The nucleotide sequence as shown in SEQ ID NO.9;

[0011] (2) A nucleotide sequence having a homology of more than 95% with the nucleotide sequence shown in SEQ ID NO.9 and capable of encoding the amino acid sequence shown in SEQ ID NO.10.

[0012] The present invention also provides a microbial sensor for submarine detection containing the above AraC mutant, and the microbial sensor for submarine detection simultaneously includes a coding gene of the AraC mutant, an electron transfer pigment protein gene, and a promoter element.

[0013] Furthermore, the electron transfer pigment protein gene is the CymA gene with a nucleotide sequence as shown in SEQ ID NO.18.

[0014] Furthermore, the amino acid sequence of the electron transfer pigment protein CymA is as shown in SEQ ID NO.19.

[0015] Furthermore, the preparation method of the microbial sensor for submarine detection includes the following steps:

[0016] (1) Amplify the electron transfer pigment protein gene CymA and the promoter element pBAD;

[0017] (2) Connect the amplified fragment in step (1) with the AraC mutant coding gene fragment at a concentration ratio of 1:1:1, and clone it into the expression vector pACYCDuet-1 to obtain the recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA;

[0018] (3) Transform the recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA into the host Escherichia coli to obtain the recombinant strain E. coli / pACYC-AraCmt-Cd-PBAD-CymA, which is the submarine-detecting microbial sensor.

[0019] The present invention also provides the application of the above-mentioned AraC mutant or the above-mentioned submarine-detecting microbial sensor in the preparation of an electrochemical device for real-time detection of submarine metal ions.

[0020] Furthermore, the electrochemical device is composed of a working electrode, a reference electrode, a counter electrode and the above-mentioned submarine-detecting microbial sensor, which can sense the metal ions released by the submarine and output the electrochemical signal of the above-mentioned submarine-detecting microbial sensor through difference after sensing the metal ions, so as to achieve the purpose of real-time detection of the submarine.

[0021] Furthermore, the concentration of the metal ions of the submarine detected by the electrochemical device is not less than 5 μmol / L.

[0022] Furthermore, the submarine metal ions are Cd(II) ions.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] The present invention has screened a brand-new mutant AraCmt-Cd by means of directed evolution, which has a significant effect of sensing the submarine metal ion Cd(II), can be used for the construction of a submarine-detecting microbial sensor, and thus can be applied to the real-time monitoring of submarines and their metal ions. The screening method of the AraC mutant of the present invention can also be applied to the screening of other AraC mutants that can sense submarine metal ions, and the method is simple and fast. The AraC mutant obtained by the present invention can sense trace metal ions and has good specificity. The microbial sensor constructed by the present invention has a good effect of sensing Cd(II) and can detect trace Cd(II) through electrochemical signals, so it has great application prospects. Description of the Drawings

[0025] Figure 1 Fluorescence detection results of the mutant AraCmt-Cd screened for sensing the metal ion Cd(II).

[0026] Figure 2Plasmid map of the constructed vector pACYC-AraCmt-Cd-PBAD-CymA.

[0027] Figure 3 Electrochemical signal output diagram of the constructed potential-detecting microbial sensor containing an electrical signal element in the presence and absence of metal ion Cd(II). Specific implementation manners

[0028] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited by the embodiments.

[0029] For those not specifying specific techniques or conditions in the embodiments, the techniques described in the literature in this field or the product specifications shall be followed. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained by purchase.

[0030] Example 1: Screening of AraC mutants

[0031] 1. Construction of the basic vector pACYCDuet-1-T7-AraC-EGFP-PBAD

[0032] Using plasmid pBAD24 (GenBank: X81837.1) as a template, primer P-AraC-F and primer P-AraC-R were used for polymerase chain reaction (PCR) to amplify the Arac fragment, and primer araBAD-F and primer araBAD-R were used to amplify the araBAD promoter fragment; using plasmid pEGFP-1 (GenBank: U55761.1) as a template, primer p-EGFP-F and primer p-EGFP-R were used to amplify the EGFP fragment.

[0033] The primer sequences are shown as follows:

[0034] P-AraC-F:

[0035] P-AraC-R:

[0036] araBAD-F:

[0037] araBAD-R:

[0038] p-EGFP-F:

[0039] p-EGFP-R:

[0040] The PCR products were purified by gel extraction using a Gel Extraction and Purification Kit (Vazyme, catalog number DC301-01).

[0041] The pACYCDuet-1 plasmid (Novagen, catalog number 71147-3) was double-digested with restriction enzyme 1 Bgl II (TaKaRa, catalog number 1606) and restriction enzyme 2 Nde I (TaKaRa, catalog number 1621), and then purified by liquid recovery.

[0042] Three PCR products were ligated to the digested pACYCDuet-1 plasmid by seamless cloning using 2×Clon Express Mix (Vazyme, catalog number C115). The reaction system is as follows:

[0043]

[0044] The reaction system was incubated at 50 °C for 30 min. The products were transformed into competent E. coli DH5α cells and spread on LB solid plates containing 34 mg / L chloramphenicol. Positive clones were screened by PCR, and the recombinant plasmid pACYCDuet-1-T7-AraC-EGFP-PBAD was extracted from the positive clones.

[0045] 2. Construction of the AraC mutant library

[0046] (1) Error-prone polymerase chain reaction (error-prone PCR) was performed using the vector pACYCDuet-1-T7-AraC-EGFP-PBAD as a template to amplify the M-AraC fragment with random mutation sites by primers AraC-F and AraC-R. The PCR amplification system is as follows:

[0047]

[0048] The PCR program was: 95 °C for 3 min; 30×(95 °C for 15 s, 58 °C for 30 s, 72 °C for 20 s); 72 °C for 5 min; 16 °C ∞.

[0049] The primer sequences were:

[0050] AraC-F:

[0051] AraC-R:

[0052] The PCR product M-AraC fragment was purified by gel extraction using a Gel Extraction and Purification Kit (Vazyme, catalog number DC301-01).

[0053] (2) The pACYCDuet-1-T7-EGFP-PBAD plasmid was double-digested with restriction enzyme 1 Bgl II (TaKaRa, catalog number 1606) and restriction enzyme 2 Nde I (TaKaRa, catalog number 1621). The digestion system was as follows:

[0054]

[0055] The digestion system was incubated at 37 °C for 1 h, and then subjected to gel extraction and purification.

[0056] (3) The M-AraC fragment was cloned onto the vector fragment using seamless cloning. The system is shown below:

[0057]

[0058] The ligation system was incubated at 50 °C for 30 min. The ligation product was transformed into E. coli BL21-ΔAraC competent cells, and then spread onto an LB solid plate containing 34 mg / L chloramphenicol and incubated overnight to obtain a random AraC mutant library.

[0059] 3. Large-scale screening of the AraC mutant library responsive to Cd(II)

[0060] (1) Primary screening of the mutant library

[0061] The above-mentioned plate was transferred through a nitrocellulose microporous membrane to an M9Y plate containing 34 mg / L chloramphenicol and supplemented with 10 μmol / L Cd(II), and then incubated at 37 °C for 12 h. When obvious colonies grew on the transferred plate, the plate was observed under a UV lamp for fluorescence, and single colonies with obvious fluorescence were screened out, which were the AraC mutants responsive to Cd(II).

[0062] (2) Secondary screening by microplate reader

[0063] Single colonies obtained from the primary screening were picked and activated by overnight culture in a shaker at 37 °C. The next day, they were transferred to 10 mL of M9Y medium and cultured until OD 600 = 0.2, and then Cd(II) with concentrations of 0 μmol / L, 5 μmol / L, and 10 μmol / L were added respectively. 200 μL of the induced bacterial solution was taken and placed in a 96-well microplate, and then placed in a microplate reader (Biotek) for shaking culture at 30 °C. The fluorescence intensity (RFU) was monitored in real time at a constant temperature, and the detection was carried out once every 30 min for a total of 12 h.

[0064] The results are as Figure 1As shown, among the 20 initially screened strains, one strain M-16 was selected. Its fluorescence intensity (RFU) showed different changes under the action of different concentrations of Cd(II). The higher the concentration of Cd(II), the higher the RFU value.

[0065] 4. Identification of AraC mutants responsive to Cd(II)

[0066] The strain M-16 with significant fluorescence effect was transferred to 10 mL of LB liquid medium containing 34 mg / L chloramphenicol for cultivation, and cultured overnight at 37°C in a shaker. The plasmid was extracted using a plasmid extraction kit (Vazyme, product number DC201-01). The extracted pACYCDuet-1-T7-AraCmt-Cd-EGFP-PBAD plasmid was sent to a sequencing company for sequencing to finally determine the mutant sequence of the AraC mutant. The sequencing results showed that the nucleotide sequence of the obtained AraC mutant AraCmt-Cd was as shown in SEQ ID NO.9, and its encoded amino acid sequence was as shown in SEQ ID NO.10.

[0067] Example 2: Construction of recombinant strain E. coli / pACYC-AraCmt-Cd-PBAD-CymA

[0068] 1. Using the S. oneidensis genome as a template, with primer CymA-F and primer CymA-R, perform PCR to amplify the CymA fragment; using the plasmid pACYCDuet-1-T7-AraCmt-Cd-EGFP-PBAD as a template, with primer AraCmt-Cd-F / R and primer pBAD-F / R, perform PCR to amplify the AraCmt-Cd fragment and the pBAD promoter fragment respectively. The PCR amplification system is as follows:

[0069]

[0070] The PCR program is: 95°C for 3 min; 30×(95°C for 15 s, 55°C for 15 s, 72°C for 1 min); 72°C for 5 min; 16°C ∞.

[0071] The primer sequences are as follows:

[0072] CymA-F:

[0073] CvmA-R:

[0074] AraCmt-Cd-F:

[0075] AraCmt-Cd-R:

[0076] pBAD-F:

[0077] pBAD-R:

[0078] The PCR products were purified by gel extraction using a Gel Extraction and Purification Kit (Vazyme, catalog number DC301-01).

[0079] 2. The pACYCDuet-1 plasmid was double-digested with restriction enzyme 1 Bgl II (TaKaRa, catalog number 1606) and restriction enzyme 2 Xho I (TaKaRa, catalog number 1635). The digestion system was as follows:

[0080]

[0081] The digestion system was incubated at 37 °C for 1 h, and then purified by gel extraction.

[0082] 3. Three PCR products were ligated to the digested pACYCDuet-1 vector by seamless cloning using 2×Clon Express Mix (Vazyme, catalog number C115). The system was as follows:

[0083]

[0084] The system was incubated at 50 °C for 30 min. The products were transformed into E. coli BL21-ΔAraC competent cells and spread on LB solid plates containing 34 mg / L chloramphenicol. Positive clones were screened by PCR, and the recombinant plasmid pACYC-AraCmt-Cd-PBAD-CymA ( Figure 2 ) was extracted from the positive clones and identified by sequencing. The nucleotide sequence of the plasmid pACYC-AraCmt-Cd-PBAD-CymA is shown in SEQ ID NO.17.

[0085] Example 3: Application of recombinant strain E. coli / pACYC-AraCmt-Cd-PBAD-CymA in detecting metal ion Cd(II)

[0086] (1) Construction of the detection device

[0087] A three - electrode electrochemical system was adopted, where the working electrode, reference electrode and counter electrode were a glassy carbon electrode, an Ag / AgCl electrode and a platinum wire respectively, and a PBS solution containing various metal ions was used as the electrolyte. First, the system was left in the open - circuit state for 1 h, the scanning rate was set at 1 mV / s, the scanning range for anodic testing was - 0.7~+0.2 V (vs.SCE), and the scanning range for cathodic testing was - 0.4~+0.4 V (vs.SCE). Each scan was repeated for 3 cycles. Usually, the curves of the second and third scans basically coincided, and the data of the third scan were taken for analysis. After the constructed potential - detecting microbial sensor E.coli / pACYC - AraCmt - Cd - PBAD - CymA was cultured and the cells were collected, they were added to the electrochemical system for CV testing and signal acquisition.

[0088] (2) Detection with electrical signals as the output

[0089] A buffer solution with a metal ion Cd(II) concentration of 5 μmol / L was introduced into the constructed device, and an electrochemical workstation was used to detect the change in the electrical signal output of the microbial fuel cell, which was detected once every 1 hour for a total of 12 hours.

[0090] The results are as Figure 3 shown. In the presence of 5 μmol / L metal ion Cd(II), there was a significant difference in the electrical signal output of the microbial sensor containing the electrical signal reporting element compared with the case without metal ions. The detected voltage reached above 0.25 V after 6 hours, and the current density decreased to a steady value after all metal ions were excluded later.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, for those of ordinary skill in the art, it is still possible to modify the technical solutions recorded in the foregoing embodiments, or perform equivalent substitution on some of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions required to be protected by the present invention. Sequence Listing <110> Qingdao Agricultural University <120> An AraC mutant for sensing submarine metal ion Cd(II) and its constructed potential - detecting microbial sensor and application <141> 2022 - 06 - 08 <160> 19 <170> SIPOSequenceListing 1.0 <210> 1 <211> 41 <212> DNA <213> Artificial Sequence <400> 1 gtataagaag gagatataca tatggctgaa gcgcaaaatg a 41 <210> 2 <211> 41 <212> DNA <213> Artificial Sequence <400> 2 ccgatatcca attgagatct ttatgacaac ttgacggcta c 41 <210> 3 <211> 42 <212> DNA <213> Artificial Sequence <400> 3 gtttctttac cagactcgag aagaaaccaa ttgtccatat tg 42 <210> 4 <211> 42 <212> DNA <213> Artificial Sequence <400> 4 ctagcccaaa aaaacgggta tggagaaaca gtagagagtt gc 42 <210> 5 <211> 52 <212> DNA <213> Artificial Sequence <400> 5 tcaattggat atcggccggc cacgcgatcg ttacttgtac agctcgtcca tg 52 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 gggctagaaa taattttgtt taactttaag 30 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <400> 7 catatggctg aagcgcaaaa tgatc 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <400> 8 gagatcttta tgacaacttg acggc 25 <210> 9 <211> 879 <212> DNA <213> Artificial Sequence <400> 9 atggctgaag cgcaaaatga tcccctgctg ccgggatact cgtttaacgc ccatctggtg 60 gcgggtttaa cgccgattga ggccaacggt tatctcgatt tttttatcga ccgaccgctg 120 ggaatgaaag gttatattct caatctcacc attcgcggtc agggggtggt gaaaaatcag 180 ggacgagaat ttgcctgccg accgggtgat attttgctgt tcccgccagg taagattcat 240 cactacggtc gtcatccgga ggctcgcgaa tggtatatcc agtgggttta ctttcgtccg 300 cgcgcctact ggcatgaatg gcttaactgg ccgtcaatac ctgccaatac gggtttcttt 360 cgcccggatg aagcgcacca gccgcatttc agcgacctgt ttgggcaaat cattaacgcc 420 gggcaagggg aagggcgcta ttcggagctg ctggcgataa atctgcttga gcaattgtta 480 ctgcggcgca tggaagcgat taacgagtcg ctccatccac cgatggataa tcgggtacgc 540 gaggcttgtc agtacatcag cgatcacctg gcagacagca attttgatat cgccagcgtc 600 gcacagcatg tttgcttgtc gccgtcgcgt ctgtcacatc ttttccgcca gcagttaggg 660 attagcgtct taagctggcg cgaggaccaa cgcattagtc aggcgaagct gcttttgagc 720 actacccgga tgcctatcgc caccgtcggt cgcaatgttg gttttgacga tcaactctat 780 ttctcgcgag tatttaaaaa atgcaccggg gccagcccga gcgagtttcg tgccggttgt 840 gaagaaaaag tgaatgatgt agccgtcaag ttgtcataa 879 <210> 10 <211> 292 <212> PRT <213> Artificial Sequence <400> 10 Met Ala Glu Ala Gln Asn Asp Pro Leu Leu Pro Gly Tyr Ser Phe Asn 1 5 10 15 Ala His Leu Val Ala Gly Leu Thr Pro Ile Glu Ala Asn Gly Tyr Leu 20 25 30 Asp Phe Phe Ile Asp Arg Pro Leu Gly Met Lys Gly Tyr Ile Leu Asn 35 40 45 Leu Thr Ile Arg Gly Gln Gly Val Val Lys Asn Gln Gly Arg Glu Phe 50 55 60 Ala Cys Arg Pro Gly Asp Ile Leu Leu Phe Pro Pro Gly Lys Ile His 65 70 75 80 His Tyr Gly Arg His Pro Glu Ala Arg Glu Trp Tyr Ile Gln Trp Val 85 90 95 Tyr Phe Arg Pro Arg Ala Tyr Trp His Glu Trp Leu Asn Trp Pro Ser 100 105 110 Ile Pro Ala Asn Thr Gly Phe Phe Arg Pro Asp Glu Ala His Gln Pro 115 120 125 His Phe Ser Asp Leu Phe Gly Gln Ile Ile Asn Ala Gly Gln Gly Glu 130 135 140 Gly Arg Tyr Ser Glu Leu Leu Ala Ile Asn Leu Leu Glu Gln Leu Leu 145 150 155 160 Leu Arg Arg Met Glu Ala Ile Asn Glu Ser Leu His Pro Pro Met Asp 165 170 175 Asn Arg Val Arg Glu Ala Cys Gln Tyr Ile Ser Asp His Leu Ala Asp 180 185 190 Ser Asn Phe Asp Ile Ala Ser Val Ala Gln His Val Cys Leu Ser Pro 195 200 205 Ser Arg Leu Ser His Leu Phe Arg Gln Gln Leu Gly Ile Ser Val Leu 210 215 220 Ser Trp Arg Glu Asp Gln Arg Ile Ser Gln Ala Lys Leu Leu Leu Ser 225 230 235 240 Thr Thr Arg Met Pro Ile Ala Thr Val Gly Arg Asn Val Gly Phe Asp 245 250 255 Asp Gln Leu Tyr Phe Ser Arg Val Phe Lys Lys Cys Thr Gly Ala Ser 260 265 270 Pro Ser Glu Phe Arg Ala Gly Cys Glu Glu Lys Val Asn Asp Val Ala 275 280 285 Val Lys Leu Ser 290 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <400> 11 ttctccatgg taccctcgag atgaactggc gtgcactatt 40 <210> 12 <211> 41 <212> DNA <213> Artificial Sequence <400> 12 gcagcggttt ctttaccaga ttatcctttt ggatatgggt g 41 <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <400> 13 gatatacata tggcagatct ttatgacaac ttgacggcta ca 42 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <400> 14 tggtttctta tatccaattg atggctgaag cgcaaaatga 40 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <400> 15 ttcagccatc aattggatat aagaaaccaa ttgtccatat tg 42 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <400> 16 gttcatctcg agggtaccat ggagaaacag tagagagt 38 <210> 17 <211> 5000 <212> DNA <213> Artificial Sequence <400> 17 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag 60 gagatatacc atgggcagca gccatcacca tcatcaccac agccaggatc cgaattcgag 120 ctcggcgcgc ctgcaggtcg acaagcttgc ggccgcataa tgcttaagtc gaacagaaag 180 taatcgtatt gtacacggcc gcataatcga aattaatacg actcactata ggggaattgt 240 gagcggataa caattcccca tcttagtata ttagttaagt ataagaagga gatatacata 300 tggcagatct ttatgacaac ttgacggcta catcattcac tttttcttca caaccggcac 360 gaaactcgct cgggctggcc ccggtgcatt ttttaaatac tcgcgagaaa tagagttgat 420 cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtagtg ctcaaaagca 480 gcttcgcctg actaatgcgt tggtcctcgc gccagcttaa gacgctaatc cctaactgct 540 ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg acgctggcga 600 tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg cgtacccgat 660 tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt aacaattgct 720 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 780 caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg gcgttaatga 780 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaacccg 840 tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga aagaaacccg 840 tattggcagg tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 900 tattggcagg tattgacggc cagttaagcc attcatgcca gtaggcgcgc ggacgaaagt 900 aaacccactg gatataccat tcgcgagcct ccggatgacg accgtagtga tgaatcttac 960 aaacccactg gatataccat tcgcgagcct ccggatgacg accgtagtga tgaatcttac 960 ctggcgggaa cagcaaaata tcacccggtc ggcaggcaaa ttctcgtccc tgatttttca 1020 ctggcgggaa cagcaaaata tcacccggtc ggcaggcaaa ttctcgtccc tgatttttca 1020 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 1080 ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc agcggtcggt 1080 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 1140 cgataaaaaa atcgagataa ccgttggcct caatcggcgt taaacccgcc accagatggg 1140 cgttaaacga gtatcccggc agcaggggat cattttgcgc ttcagccatc aattggatat 1200 cgttaaacga gtatcccggc agcaggggat cattttgcgc ttcagccatc aattggatat 1200 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 1260 aagaaaccaa ttgtccatat tgcatcagac attgccgtca ctgcgtcttt tactggctct 1260 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 1320 tctcgctaac caaaccggta accccgctta ttaaaagcat tctgtaacaa agcgggacca 1320 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 1380 aagccatgac aaaaacgcgt aacaaaagtg tctataatca cggcagaaaa gtccacattg 1380 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 1440 attatttgca cggcgtcaca ctttgctatg ccatagcatt tttatccata agattagcgg 1440 atcctacctg acgcttttta tcgcaactct ctactgtttc tccatggtac cctcgagatg 1500 aactggcgtg cactatttaa acctagcgca aaatattcaa ttctggcgct gatggtcgtc 1560 ggtattgtta tcggcgtagt gggttatttc gcaacccaac aaaccttaca tgcgacaagt 1620 acggatcagt tctgtatgtc ctgtcacagc aaccattcac ttaaggatga agttcttgca 1680 tctgcccatg gtggtggtcg tgcgggtgtg accgttcaat gtcaagactg tcacttacca 1740 catggccctg tagattattt gatcaaaaaa atcattgtat ctaaagactt atatggtttc 1800 ttaacgattg atggctttaa cactcaagct tggttagatg aaaaccgtaa agagcaagcc 1860 gatctagcac ttaagtattt ccgcagtaat gactccgcta actgtcaaca ctgccatact 1920 cgcatttatg aaaaccagcc agaaactatg aagcctatgg ctgtcagaat gcacactaat 1980 aactttaaga aagatccaga agccagaaaa acctgtgttg actgccataa aggtgtagct 2040 cacccatatc caaaaggata atctggtaaa gaaaccgctg ctgcgaaatt tgaacgccag 2100 cacatggact cgtctactag cgcagcttaa ttaacctagg ctgctgccac cgctgagcaa 2160 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaacct 2220 caggcatttg agaagcacac ggtcacactg cttccggtag tcaataaacc ggtaaaccag 2280 caatagacat aagcggctat ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg 2340 ctttcgaatt tctgccattc atccgcttat tatcacttat tcaggcgtag caccaggcgt 2400 ttaagggcac caataactgc cttaaaaaaa ttacgccccg ccctgccact catcgcagta 2460 ctgttgtaat tcattaagca ttctgccgac atggaagcca tcacagacgg catgatgaac 2520 ctgaatcgcc agcggcatca gcaccttgtc gccttgcgta taatatttgc ccatagtgaa 2580 aacgggggcg aagaagttgt ccatattggc cacgtttaaa tcaaaactgg tgaaactcac 2640 ccagggattg gctgagacga aaaacatatt ctcaataaac cctttaggga aataggccag 2700 gttttcaccg taacacgcca catcttgcga atatatgtgt agaaactgcc ggaaatcgtc 2760 gtggtattca ctccagagcg atgaaaacgt ttcagtttgc tcatggaaaa cggtgtaaca 2820 agggtgaaca ctatcccata tcaccagctc accgtctttc attgccatac ggaactccgg 2880 atgagcattc atcaggcggg caagaatgtg aataaaggcc ggataaaact tgtgcttatt 2940 tttctttacg gtctttaaaa aggccgtaat atccagctga acggtctggt tataggtaca 3000 ttgagcaact gactgaaatg cctcaaaatg ttctttacga tgccattggg atatatcaac 3060 ggtggtatat ccagtgattt ttttctccat tttagcttcc ttagctcctg aaaatctcga 3120 taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt tggaacctct 3180 tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc cggtatcaac 3240 agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta tttattcggc 3300 gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg tgtttttgag 3360 gtgctccagt ggcttctgtt tctatcagct gtccctcctg ttcagctact gacggggtgg 3420 tgcgtaacgg caaaagcacc gccggacatc agcgctagcg gagtgtatac tggcttacta 3480 tgttggcact gatgagggtg tcagtgaagt gcttcatgtg gcaggagaaa aaaggctgca 3540 ccggtgcgtc agcagaatat gtgatacagg atatattccg cttcctcgct cactgactcg 3600 ctacgctcgg tcgttcgact gcggcgagcg gaaatggctt acgaacgggg cggagatttc 3660 ctggaagatg ccaggaagat acttaacagg gaagtgagag ggccgcggca aagccgtttt 3720 tccataggct ccgcccccct gacaagcatc acgaaatctg acgctcaaat cagtggtggc 3780 gaaacccgac aggactataa agataccagg cgtttcccct ggcggctccc tcgtgcgctc 3840 tcctgttcct gcctttcggt ttaccggtgt cattccgctg ttatggccgc gtttgtctca 3900 ttccacgcct gacactcagt tccgggtagg cagttcgctc caagctggac tgtatgcacg 3960 aaccccccgt tcagtccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc 4020 cggaaagaca tgcaaaagca ccactggcag cagccactgg taattgattt agaggagtta 4080 gtcttgaagt catgcgccgg ttaaggctaa actgaaagga caagttttgg tgactgcgct 4140 cctccaagcc agttacctcg gttcaaagag ttggtagctc agagaacctt cgaaaaaccg 4200 ccctgcaagg cggttttttc gttttcagag caagagatta cgcgcagacc aaaacgatct 4260 caagaagatc atcttattaa tcagataaaa tatttctaga tttcagtgca atttatctct 4320 tcaaatgtag cacctgaagt cagccccata cgatataagt tgtaattctc atgttagtca 4380 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 4440 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 4500 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 4560 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 4620 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 4680 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 4740 tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 4800 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4860 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4920 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4980 cgcagacgcg ccgagacaga 5000 <210> 18 <211> 564 <212> DNA <213> Artificial Sequence <400> 18 atgaactggc gtgcactatt taaacctagc gcaaaatatt caattctggc gctgatggtc 60 gtcggtattg ttatcggcgt agtgggttat ttcgcaaccc aacaaacctt acatgcgaca 120 agtacggatc agttctgtat gtcctgtcac agcaaccatt cacttaagga tgaagttctt 180 gcatctgccc atggtggtgg tcgtgcgggt gtgaccgttc aatgtcaaga ctgtcactta 240 ccacatggcc ctgtagatta tttgatcaaa aaaatcattg tatctaaaga cttatatggt 300 ttcttaacga ttgatggctt taacactcaa gcttggttag atgaaaaccg taaagagcaa 360 gccgatctag cacttaagta tttccgcagt aatgactccg ctaactgtca acactgccat 420 actcgcattt atgaaaacca gccagaaact atgaagccta tggctgtcag aatgcacact 480 aataacttta agaaagatcc agaagccaga aaaacctgtg ttgactgcca taaaggtgta 540 gctcacccat atccaaaagg ataa 564 <210> 19 <211> 187 <212> PRT <213> Artificial Sequence <400> 19 Met Asn Trp Arg Ala Leu Phe Lys Pro Ser Ala Lys Tyr Ser Ile Leu 1 5 10 15 Ala Leu Met Val Val Gly Ile Val Ile Gly Val Val Gly Tyr Phe Ala 20 25 30 Thr Gln Gln Thr Leu His Ala Thr Ser Thr Asp Gln Phe Cys Met Ser 35 40 45 Cys His Ser Asn His Ser Leu Lys Asp Glu Val Leu Ala Ser Ala His 50 55 60 Gly Gly Gly Arg Ala Gly Val Thr Val Gln Cys Gln Asp Cys His Leu 65 70 75 80 Pro His Gly Pro Val Asp Tyr Leu Ile Lys Lys Ile Ile Val Ser Lys 85 90 95 Asp Leu Tyr Gly Phe Leu Thr Ile Asp Gly Phe Asn Thr Gln Ala Trp 100 105 110 Leu Asp Glu Asn Arg Lys Glu Gln Ala Asp Leu Ala Leu Lys Tyr Phe 115 120 125 Arg Ser Asn Asp Ser Ala Asn Cys Gln His Cys His Thr Arg Ile Tyr 130 135 140 Glu Asn Gln Pro Glu Thr Met Lys Pro Met Ala Val Arg Met His Thr 145 150 155 160 Asn Asn Phe Lys Lys Asp Pro Glu Ala Arg Lys Thr Cys Val Asp Cys 165 170 175 His Lys Gly Val Ala His Pro Tyr Pro Lys Gly 180 185

Claims

1. An AraC mutant, characterized in that, the AraC mutant is AraCmt-Cd, and its amino acid sequence is as shown in SEQ ID NO.

10.

2. The AraC mutant according to claim 1, characterized in that, the AraC mutant AraCmt-Cd has a significant function of sensing the submarine metal ion Cd(II).

3. The coding gene of the AraC mutant according to claim 1, characterized in that, the coding gene of the AraC mutant has one of the following nucleotide sequences: (1) The nucleotide sequence as shown in SEQ ID NO.9; (2) A nucleotide sequence having more than 95% homology with the nucleotide sequence shown in SEQ ID NO.9 and capable of encoding the amino acid sequence shown in SEQ ID NO.

10.

4. A submarine-detecting microbial sensor containing the AraC mutant according to claim 1, characterized in that, The submersible microbial sensor simultaneously includes the coding gene of the AraC mutant, the electron transfer pigment protein gene, and the promoter element, and the electron transfer pigment protein gene is the one with the nucleotide sequence shown in SEQ ID NO.18 CymA gene, and the promoter element is the promoter element pBAD.

5. The submarine-detecting microbial sensor according to claim 4, characterized in that, the preparation method of the submarine-detecting microbial sensor comprises the following steps: (1) Amplify the electron transfer pigment protein gene CymA and the promoter element pBAD; (2) Connect the amplified fragment in step (1) with the AraC mutant coding gene fragment at a concentration ratio of 1:1:1, and clone it into an expression vector to obtain a recombinant plasmid; (3) Transform the recombinant plasmid into competent cells, and the obtained recombinant strain is the submarine-detecting microbial sensor.

6. The application of the AraC mutant according to claim 1 or the submarine-detecting microbial sensor according to claim 4 in the preparation of an electrochemical device for real-time detecting Cd(II) ions released by a submarine, characterized in that, the concentration of Cd(II) ions detected by the electrochemical device is not less than 5 μmol / L.

7. The application according to claim 6, characterized in that, the electrochemical device is composed of a working electrode, a reference electrode, a counter electrode and the submarine-detecting microbial sensor, can sense the Cd(II) ions released by the submarine, and can differentially output the electrochemical signal of the submarine-detecting microbial sensor after sensing the Cd(II) ions.

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