Cadmium and lead heavy metal adsorption recombinant bacterium as well as construction method and application thereof

Abstract

The invention provides cadmium and lead heavy metal adsorption recombinant bacteria as well as a construction method and application thereof. The method comprises the following steps: fusing an inner membrane positioning peptide fragment LPP-OmpA of Escherichia coli, and specific heavy metal binding protein PbBD and MTT5 of Cupriavidus metallidurans, and introducing a Deinococcus radiodurans R1 genome, so as to construct a new genetic engineering strain. The obtained strain has high radiation resistance, strong oxidation resistance and high heavy metal absorption capacity, and has application value in the aspect of reducing toxicity of heavy metals such as cadmium, lead and the like of crops in special areas with strong oxidation, high radiation and the like.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to a cadmium and lead heavy metal adsorption recombinant bacterium and a construction method and application thereof. Background Art

[0002] Both natural processes and human activities can lead to the accumulation of toxic heavy metals in the environment and food chain. Even low concentrations of heavy metals can be toxic and pose a serious threat to life. For example, 1 million people die from lead poisoning each year worldwide, and millions of people (especially children) are exposed to low doses of lead, causing lifelong health problems. Therefore, heavy metal pollution control has always been a focus of global attention. Traditional physical and chemical methods for heavy metal removal are relatively costly, have long cycles, and are non-selective. Bioremediation degrades or reduces heavy metal pollutants through biological processes, has substrate diversity, higher efficiency and lower cost, and has become an emerging hotspot.

[0003] Some microorganisms can also grow in the presence of toxic heavy metals, so they can be used to remove heavy metals from polluted areas and protect the human living environment. Microorganisms can interact with metals, change the state of heavy metals through different biological processes such as biosorption, bioaccumulation, redox, and resistance gene expression, and repair soluble and insoluble toxic and harmful heavy metals. It has been found that bacteria, fungi, and algae have different detoxification abilities for various heavy metal soils or wastewaters. However, there are few studies on the modification of selected natural strains to obtain the ability to mitigate or remove heavy metals under special conditions. For example, extreme microorganisms can be modified so that they still have strong heavy metal tolerance under strong irradiation or high oxidation environments. Therefore, the use of natural characteristic microorganisms to construct heavy metal adsorption recombinant strains has great potential in the removal of harmful heavy metals.

[0004] Mars is known as the "twin sister" of the Earth and has become the first choice for future human space migration. If we want to march to Mars, we need to solve the problem of planting on Martian soil. However, the toxic heavy metals such as cadmium and mercury in the Martian soil are very high, which will enter the human body through the planting of crops and will become an obstacle to the planting of edible plants on Mars. The surface of Mars has high radiation and cytotoxic substances such as oxidants, so the cultivation of radiation-resistant biosorbents that can reduce or even eliminate the toxicity of heavy metals is of great significance to the development of future space biology. Summary of the invention

[0005] In view of the current lack of radiation-resistant bacteria that can efficiently adsorb heavy metals, and the potential demand for such bacteria in the future, the present invention deeply explores the characteristics of heavy metal adsorption gene elements and radiation-resistant bacteria, and provides a cadmium-lead heavy metal adsorption recombinant bacteria and its construction method and application. The present invention uses the most radiation-resistant bacteria with strong antioxidant ability found on the earth so far as the parent body to construct an enhanced recombinant strain for heavy metal adsorption, thereby providing a radiation-resistant genetically engineered extreme microorganism, which has application value in reducing the toxicity of heavy metals such as cadmium and lead in crops in special strong oxidizing and high radiation areas.

[0006] A cadmium and lead heavy metal adsorption recombinant bacterium, Deinococcus radiodurans D. radiodurans It contains the recombinant plasmid pRAD-P8-LPP-OmpA-PbBD-MTT5, thus having both heavy metal adsorption and radiation resistance capabilities; the P8-LPP-OmpA-PbBD-MTT5 is an expression module, and the DNA sequence is shown in SEQ ID NO.13.

[0007] The method for constructing the cadmium and lead heavy metal adsorption recombinant bacteria comprises the following steps: 1) Target strain Deinococcus radiodurans D. radiodurans Deinococcus deserticola ( Deinococcus deserti ) promoter sequence was used as a prototype, and the transcription and expression levels of the reporter gene mCherry were used to screen for a super strong constitutive promoter element P8 suitable for expression in Deinococcus radiodurans, and the DNA sequence is shown in SEQ ID NO.12.

[0008] 2) Escherichia coli Escherichia coli , copper-resistant bacteria Cupriavidus metallidurans The genomic DNA of Tetrahymena thermophila was used as a template to clone the corresponding inner membrane localized peptide LPP-OmpA and the gene fragment of the specific heavy metal lead binding protein PbBD, and synthesize the Tetrahymena thermophila The gene fragment of cadmium metallothionein MTT5 was then connected to the promoter element P8 to form the P8-LPP-OmpA-PbBD-MTT5 expression module; 3) Directly integrate the expression module into the shuttle vector pRADK to obtain the recombinant plasmid pRAD-8-LPP-OmpA- PbBD -MTT5, and then transform it into Deinococcus radiodurans D. radiodurans R1, obtained the cadmium and lead heavy metal adsorption recombinant bacteria LOPM.

[0009] The cadmium and lead heavy metal adsorption recombinant bacteria are used to treat rice roots with cells and co-cultivate to reduce the cadmium and lead content in the roots and above-ground parts.

[0010] The application of the cadmium and lead heavy metal adsorption recombinant bacteria is used to adsorb high-concentration heavy metal ions in high-radiation areas or strong oxidation areas.

[0011] Excess heavy metals used to improve the surface soil of alien planets.

[0012] The alien planet is Mars.

[0013] Compared with the prior art, the present invention has the following beneficial effects: Most heavy metal adsorbing bacteria such as cadmium and lead are not resistant to radiation and strong oxidants, and cannot survive and adsorb heavy metals in radiation and / or strong oxidation environments. However, the strong cadmium and lead heavy metal adsorbent prepared by the present invention can adsorb heavy metals such as cadmium and lead in radiation and / or strong oxidation environments, and treat rice roots. After co-cultivation, the cadmium content in the root system and the aboveground part can be reduced to 47.0% and 43.4%, and the lead content can be reduced to 55.4% and 26.9%, respectively. The present invention provides a possible option for improving soils in high-radiation / strong oxidation areas and the surface of Mars containing high concentrations of heavy metals in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a schematic diagram of the construction of the promoter strength test vector pRAD-mCherry.

[0015] Figure 2 This is a comparative analysis of the expression levels of the reporter gene mCherry protein in the strains transformed with various promoter elements; PG is the control (marked in red), which is constructed from the classic strong promoter PG of Radiodurans deinococci.

[0016] Figure 3 is a schematic diagram of the fusion protein module.

[0017] Figure 4 It is a schematic diagram of the construction of fusion expression vector.

[0018] Figure 5 It is the sequencing map constructed by the fusion expression vector.

[0019] Figure 6 It is the fluorescence localization expression of the expression module in the recombinant strain LOPM.

[0020] Figure 7 are the antioxidant capacity and radiation resistance of the recombinant strain LOPM.

[0021] Figure 8 The effects of cadmium and lead on the growth of wild type R1 and recombinant strain LOPM of Radiodurans deinococcus.

[0022] Fig. 9 This is the adsorption of heavy metal cadmium and lead (100 μM Cd) by the recombinant strain LOPM.

[0023] Fig.10 This is the adsorption of heavy metal cadmium and lead (200 μM Pb) by the recombinant strain LOPM.

[0024] Fig.11 This is the adsorption of heavy metal cadmium and lead (100 μM Cd & 200 μM Pb) by the recombinant strain LOPM.

[0025] Fig.12 It is the content of cadmium and lead in rice planting medium with different treatments under cadmium and lead stress. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0027] Example 1: Screening of strong promoter elements (1) Using the NCBI database of Deinococcus deserticola ( Deinococcus deserti , numbered SRX2611096), analyzed the expression intensity of each gene under physiological conditions (reported as FPKM), and ranked the expression intensity. The top 24 were selected as candidate promoters and named P1 to P24. Then, the sequences of the regions where the 24 candidate promoters were located (500 bp upstream of the gene) were intercepted, and the -35 box, -10 box regions and the inferred ribosome binding sites (RBS) were included as much as possible.

[0028] (2) Using the genome of Deinococcus deserticola as a template, Primer Premier 5.0 was used to design 24 primers for promoter sequences, which were amplified in vitro and cloned into a promoter strength test vector. The promoter strength test vector pRAD-mCherry was constructed as follows: Figure 1 As shown, the fragment was inserted using the double digestion method with SacⅠ / HindⅢ and SpeⅠ restriction endonucleases, and the mCherry sequence was specifically added as a biomarker expressed by the promoter. The strength test vector of the promoter fragment was successfully inserted, and the correctness of each sequence was verified by sequencing.

[0029] (3) Each vector was then transformed into a radioresistant Deinococcus that is easy to genetically manipulate, and the fluorescence intensity of the reporter protein mCherry in each vector was detected in vivo. Each recombinant strain was cultured to OD 600The value was 1.0, and 300 μL was taken to a 96-well black ELISA plate (the strain pRAD-PG without fragments was used as the blank control). The mCherry fluorescence signal intensity was collected using a SpectraMax M5 multi-function microplate reader (488 nm excitation wavelength, 588 nm absorption wavelength) to obtain the reporter protein expression level of each transformed strain. It was found that the strain with the highest abundance of reporter gene expression was pRAD-P8, and its fluorescence intensity was 5 times that of the control strain pRAD-PG ( Figure 2 ), thus determining that the promoter with the strongest expression intensity is P8.

[0030] Example 2: Construction of genetically engineered strain LOPM (1) Using a bacterial genomic DNA extraction kit (Tiangen Biochemical Company), Escherichia coli Escherichia coli (laboratory storage) and copper-tolerant bacteria Cupriavidus metallidurans The genomic DNA was purchased from German Microbiological Collection Center DSMZ, No. DSM 2839, and its concentration and purity were determined using NanoDrop 1000 (Thermo Fisher).

[0031] (2) Using the genomic DNA of Escherichia coli and copper-resistant bacteria as templates, we cloned the gene fragments of the inner membrane-localized peptide LPP-OmpA and the specific heavy metal binding protein PbBD, and synthesized the gene fragments of the thermophilic Tetrahymena Tetrahymena thermophilaThe partial gene sequence of cadmium metallothionein MTT5 was then connected to the strong promoter element P8 obtained by previous screening to form the P8-LPP-OmpA-PbBD-MTT5 expression module. The upstream primer P8-F used to fuse the pRAD vector module is: 5´-acccgggagctcgaattctagaagctt CTAAGGAAAGCCTCCACATGCCG-3´ (SEQ ID NO.1, lowercase letters are the homology arm sequence connected to the pRAD vector), and the downstream primer P8-R is: 5´-tttggtggctttcatCAGAATAGTGATAATCCAACCCATGTTC-3´ (SEQ ID NO.2, lowercase letters are the homology arm of LPP-OmpA). The upstream primer LPP -OmpA-F used to amplify the LPP -OmpA fragment is 5´-gattatcactattctgATGAAAGCCACCAAACTGGT-3´ (SEQ ID NO.3, lowercase letters are the homology arms of P8), and the downstream primer LPP -OmpA-R is 5´-gctgccgccgccgccACGCGTTGCAATTTCCGG-3´ (SEQ ID NO.4, lowercase letters are the flexible linker sequence connected to the PbBD fragment). The upstream primer PbBD-F used to amplify the PbBD fragment is 5´-ggcggcggcggcagcCAGTTCATTCGTCACTGCCGG-3´ (SEQ ID NO.5, the lowercase letters are the flexible linker sequence connected to the upstream PbBD fragment), and the downstream primer PbBD-R is 5´-gctgccgccgccgccgctgccgccgccgccCGACAGTCCCTGCAGAATCC-3´ (SEQ ID NO.6, the lowercase letters are the flexible linker sequence connected to the downstream MTT5 fragment). The upstream primer MTT5-F used to amplify the MTT5 fragment is 5´- ggcggcggcggcagcggcggcggcggcagcGATAAAATTTCTGGTGAAAGCACT-3´ (SEQ ID NO.7, the lowercase letters are the flexible linker sequence connected to the upstream PbBD fragment), and the downstream primer MTT5-R is 5´-cctgcaggtcgaatcggatccTCAGCAACTACCTCCAGG-3´ (SEQ ID NO.8, the lowercase letters are the homology arm sequence connected to the pRAD vector).

[0032] (3) The four PCR product fragments were purified and recovered using Promega's Wizard® SV Gel and PCR Clean-Up System kit, and then overlapping PCR was performed on the three target fragments using TaKaRa's PrimeSTAR® HS DNA Polymerase with P8-F as the upstream primer and MTT5-R as the downstream primer. Finally, three ligated products with pRADK vector fragments on both sides were obtained, and the three modules were sequentially integrated into the shuttle vector pRADK to obtain the recombinant plasmid P8-LPP-OmpA-PbBD-MTT5. The sequence of each module is as follows: Figure 3 As shown. Lpp-OmpA is Escherichia coli Escherichia coli The inner membrane localization peptide, PbBD, is a metal-grabbing copper bacterium Cupriavidus metallidurans The lead ion binding sequence of MTT5 is a Tetrahymena thermophila Tetrahymena thermophila Partial sequences of cadmium metallothionein together form the LOMP fusion protein. Figure 4 The schematic diagram of the construction of the fusion expression vector is shown. Figure 5 The sequencing result of successful plasmid construction is shown in SEQ ID NO.9, the amino acid sequence of LPP-OmpA is shown in SEQ ID NO.10, the amino acid sequence of PbBD is shown in SEQ ID NO.11, and the DNA sequence of P8 is shown in SEQ ID NO.12; the P8-LPP-OmpA-PbBD-MTT5 is an expression module, and its DNA sequence is shown in SEQ ID NO.13.

[0033] (4) Transfer the recombinant plasmid into Deinococcus radiodurans D. radiodurans R1 (purchased from the American Type Culture Collection, number: ATCC 13939) was screened using TGY resistance medium (0.5% trypsin, 0.1% glucose and 0.3% yeast extract) containing 3.4 μg / mL chloramphenicol and cultured inverted at 30°C for 4-5 days to obtain the recombinant bacteria LOPM.

[0034] (5) Protein localization characterization experiment using fusion green fluorescent protein (GFP) Figure 6 ), the fusion protein was well expressed and localized on the cell membrane, realizing the use of Lpp-OmpA signal peptide to express the chimeric protein of MTT5 and PbBD in radioresistant cocci and localize it on the bacterial surface.

[0035] Example 3: Extreme resistance detection of genetically engineered strain LOPM (1) Determination of the resistance of recombinant bacteria LOPM to strong oxidants: Wild strains of Radioresistant cocciD. radiodurans R1 and recombinant bacteria LOPM were cultured at 30°C with shaking at 220 rpm until OD 600 The concentration of each cell was about 1.0. 1 mL of bacterial solution was taken and centrifuged at 4000 × g for 5 min. The supernatant was discarded and the cells were resuspended and washed with 1 mL of 1× PB solution. The cells were diluted to 10 with 1× PB solution. 7 , 10 6 , 10 5 , 10 4 , 10 3 cfu / mL, and then treated with 0, 30, and 60 mmol / L H2O2 at 30 ºC for 30 min, then quickly spread on TGY plates and cultured at 30 ºC for 3 days to observe the treatment results. The results showed that the recombinant bacteria LOPM had extremely strong resistance to hydrogen peroxide ( Figure 7 ).

[0036] (2) Determination of the resistance of recombinant bacteria LOPM to ultraviolet light: According to the above-mentioned detection method for strong oxidant resistance, 2.5 μL of bacterial solution was spotted on TGY solid medium, and then 0, 100, and 200 J / m 2 The phenotype was observed after 3 days of culture in a 30°C incubator. The results showed that the recombinant bacteria LOPM had strong resistance to ultraviolet rays and had a high survival rate after high-dose irradiation ( Figure 7 ).

[0037] (3) Determination of the resistance of recombinant bacteria LOPM to ionizing radiation: According to the above resistance detection method, 2.5 μL of bacterial liquid was spotted on TGY solid medium, and then treated with gamma rays at doses of 0, 2 KGy, and 4 KGy. The phenotype was observed after culturing in a 30 ℃ incubator for 3 days. The results showed that the recombinant bacteria LOPM also had extremely strong resistance to ionizing radiation ( Figure 7 ).

[0038] Example 4: Resistance and adsorption capacity of genetically engineered strain LOPM to heavy metals (1) Effects of cadmium and lead on the growth of wild-type R1 and recombinant strain LOPM of Radioresistant cocci: Wild-type strains were selected. D. radiodurans Single clones of R1 and genetically engineered strain LOPM were inoculated into 5 mL of fresh TGY liquid medium and cultured until the exponential growth phase (OD 600 1.0), take 200 μL of bacterial solution and transfer to another bottle of TGY liquid culture medium, shake at 30 °C and 220 rpm, and continue to culture. OD value of bacteria was measured every 2 h. 600 The value was detected. 2+ 、200μM Pb2+ ) and (200 μM Cd 2+ 、400μM Pb 2+ ), without adding heavy metal ions Cd 2+ and Pb 2+ As a control, the results showed that the growth curves of the recombinant strain LOPM under different concentrations of cadmium and lead treatments were significantly better than those of the wild-type strain ( Figure 8 ).

[0039] (2) Adsorption of heavy metals cadmium and lead by the recombinant strain LOPM: The wild type D. radiodurans Cells of R1 and recombinant strain LOPM were cultured in 1 L of TGY liquid medium until OD 600 =1.0, centrifuged at 4000×g for 5 min, and the cell pellet was resuspended in 1 L of 1×PB solution. Then the culture was supplemented with 100 µM CdCl2&200 µM PbCl2 and 100 µM CdCl2&200 µM PbCl2 solutions, respectively. After incubation at different time points (0, 6, 12, 18, 24, 36, 48 h), 5 mL of culture was taken and centrifuged at 4000×g and 4°C for 10 min, and the pellet was washed with 1×PB buffer. The cells were freeze-dried for 48 h. 5 mL HNO3 and 1 mL H2O2 were added to the dried cells and kept at 120°C for 90 min. 5 mL HNO3 was added in a microwave digester (CEM MARS 6, USA) at 140°C for 60 min. The intracellular cadmium and lead contents were detected using an inductively coupled plasma-mass spectrometer (ICP-MS: ELAN DRC-e, PerkinElmer, USA). The results showed that when Cd 2+ or Pb 2 + After treatment, the recombinant strain LOPM showed higher removal efficiency than the wild type R1 ( Fig. 9 , Fig.10 ); and use Cd 2+ and Pb 2+ After 48 h of treatment, the recombinant strain LOPM was able to remove 84.9% Cd from the culture medium. 2+ and 94.1% Pb, which was higher than that of wild type R1 (72.3% Cd 2+ and 82.5% Pb 2+ )( Fig.11 ), indicating that the recombinant strain LOPM has better cadmium and lead absorption efficiency than the wild type R1 and is a strain suitable for cadmium and lead bioaccumulation.

[0040] (3) Cadmium and lead content in rice planting medium under cadmium and lead stress: Rice seeds were sterilized with 2% (W / V) NaClO solution for 20 min, washed three times with ultrapure water, and soaked at 30˚C for 24 h. They were then transferred to a 96-well plastic hydroponic box and planted in a growth chamber with a relative humidity of 75% for 14 days under a light intensity of 30°C for 16 h / dark intensity of 25°C for 18 h. 500 mL of bacterial culture (OD 600 =1.0), let the rice plants grow in the medium containing 100 μM CdCl2 and 200 μM PbCl2 (with or without bacteria), and collect rice seedling samples after 10 days of treatment. The metal ions on the surface of rice roots were washed with 20 mM disodium ethylenediaminetetraacetic acid (EDTA-2Na) for 40 min, the rice seedlings were kept at 105℃ for 3 h, and then dried at 60℃ for 48 h. 200 mg was taken for heavy metal ion analysis, and the cadmium and lead contents of the samples were measured by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the co-cultured wild-type R1 cells could significantly reduce the accumulation of cadmium in the roots and aboveground parts to 71.4% and 60.5%, and the accumulation of lead to 86.2% and 42.5%; while the recombinant strain LOPM cells reduced the accumulation of cadmium to 47.0% (roots) and 43.4% (aerial parts), and reduced the lead level to 55.4% (roots) and 26.9% (aerial parts) ( Fig.12 ), therefore the recombinant strain LOPM cells have a further enhanced ability of rice to absorb cadmium and lead compared to the wild-type strain.

[0041] The technical features of the above-described embodiments can be further combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0042] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, all of which belong to the protection scope of the present invention. The protection scope of the present invention is given by the attached claims and any equivalent technical solutions thereof.

Claims

1. A recombinant bacterium that absorbs heavy metals of cadmium and lead. It is characterized in that Deinococcus radiodurans D. radiodurans It contains the recombinant plasmid pRAD-P8-LPP-OmpA-PbBD-MTT5, thus having both heavy metal adsorption and radiation resistance capabilities; the P8-LPP-OmpA-PbBD-MTT5 is an expression module, and the sequence is shown in SEQ ID NO.

13.

2. The method for constructing the cadmium and lead heavy metal adsorption recombinant bacteria according to claim 1, It is characterized in that Here are the steps: 1) Target strain Deinococcus radiodurans D. radiodurans Deinococcus deserticola ( Deinococcus deserti ) promoter sequence as a prototype, and using the transcription and expression levels of the reporter gene mCherry, a super strong constitutive promoter element P8 suitable for expression in Deinococcus radiodurans was screened, and the sequence is shown in SEQ ID NO.12; 2) Escherichia coli Escherichia coli , copper-resistant bacteria Cupriavidus metallidurans The genomic DNA of Tetrahymena thermophila was used as a template to clone the corresponding inner membrane localized peptide LPP-OmpA and the gene fragment of the specific heavy metal lead binding protein PbBD, and synthesize the Tetrahymena thermophila The gene fragment of cadmium metallothionein MTT5 was then connected to the promoter element P8 to form the P8-LPP-OmpA-PbBD-MTT5 expression module; 3) Directly integrate the expression module into the shuttle vector pRADK to obtain the recombinant plasmid pRAD-8-LPP-OmpA-PbBD-MTT5, and then transform it into Deinococcus radiodurans D. radiodurans R1, obtained the cadmium and lead heavy metal adsorption recombinant bacteria LOPM.

3. The use of the cadmium and lead heavy metal adsorption recombinant bacteria according to claim 1, It is characterized in that Cell treatment of rice roots and co-cultivation reduced cadmium and lead levels in roots and aerial parts.

4. The use of the cadmium and lead heavy metal adsorption recombinant bacteria according to claim 1, It is characterized in that Used to adsorb high concentration heavy metal ions in high radiation or strong oxidation areas.

5. The use of the cadmium and lead heavy metal adsorption recombinant bacteria according to claim 1, It is characterized in that Excess heavy metals used to improve the surface soil of alien planets.

6. The use of the cadmium and lead heavy metal adsorption recombinant bacteria according to claim 5, It is characterized in that The alien planet is Mars.

Images