An engineered strain overexpressing sulfite reductase, its construction method and application

Abstract

The present invention discloses an engineered strain overexpressing sulfite reductase, and its construction method and application. This strain is obtained by inserting the strong erythromycin promoter PermE in front of the cysJ gene expressing sulfite reductase SiR, starting from the Citrobacter XT1-2-2 strain, and the original Citrobacter is Citrobacter sp. XT1-2-2. The engineered strain with cadmium fixation activity obtained by the construction method of the present invention is taxonomically named Citrobacter XT1-2-2-::SiR, and its deposit number is: CCTCC NO. M 20241947. It can be used as an ecological restoration strain for cadmium-polluted paddy fields of rice, laying a foundation for subsequent research on ecological restoration in cadmium-polluted paddy field soil.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to an engineered strain overexpressing sulfite reductase, a construction method thereof, and an application thereof. Background Art

[0002] With the development and utilization of mining resources, the discharge of industrial waste, and the excessive and unreasonable use of agricultural fertilizers, etc., heavy metal pollution in farmland in China has become serious. Among them, the exceeding standard rate of cadmium element site pollution still ranks first among inorganic pollutants. Hunan is a land of non-ferrous metals and also a region with relatively serious cadmium pollution in the country. In addition, cadmium has strong biological toxicity to the human body, is difficult to degrade in the soil and has biological accumulation, and is easily accumulated in animals, plants, and the human body through the food chain, directly threatening human health. Commonly used physical and chemical remediation methods are relatively costly, and the use of reagents in crop soil is likely to cause secondary pollution of the soil and changes in the soil structure. Using plants for soil heavy metal remediation is easily limited by the growth cycle and biomass of the plants, resulting in less obvious and timely effects. Therefore, using microorganisms for soil heavy metal remediation is not only efficient, economical, and environmentally friendly, but also the use of microorganisms improves the microbial flora in the soil and enhances the biological activity of the soil.

[0003] Microorganisms in the soil can survive naturally in soil with high cadmium pollution, and regulate the content and existing form of Cd in the soil through complex enzymatic reactions in the cells of the bacteria and functions such as cell metabolism, redox, adsorption, and absorption of heavy metals by the strains, reducing the content of Cd in the soil. In recent years, a large number of scientific researchers have isolated and screened functional microorganisms with strong cadmium tolerance and growth-promoting effects on crop growth, and applied them in the field to reduce the toxicity of Cd in the soil and the absorption of cadmium by plants. Some studies have shown that the addition and use of bacterial agents can not only promote the growth of rice, but also have a significant alleviating effect on cadmium pollution in rice seedlings at the seedling stage. The cadmium content in the above-ground parts of rice treated with the addition of bacterial agents decreased by 59.9%, the cadmium translocation coefficient decreased by 33.3%, the cadmium enrichment coefficient decreased by 58.8%, and the cadmium accumulation per rice plant decreased by 24.3% compared with before the treatment. Summary of the Invention

[0004] The primary object of the present invention is to provide an engineered Citrobacter strain into which a strong erythromycin promoter is inserted to overexpress the cysJ gene, with an increased expression level of sulfite reductase (SiR) protein, promoting the conversion of SO3 2- to S 2- in the sulfate metabolic pathway, so that the engineered strain obtained in the present invention has high cadmium fixation activity and great potential in the ecological restoration function of cadmium-polluted paddy fields.

[0005] The original strain Citrobacter sp. XT1-2-2 with cadmium tolerance and cadmium fixation activity isolated and screened by the present invention has obtained all the genomic data of this bacterium, and has preliminarily carried out functional enrichment analysis and KEGG metabolic pathway analysis on this bacterium. Since the genetic operating system of this strain is relatively clear, it can be genetically modified and transformed efficiently.

[0006] The engineered strain of the present invention overexpressing sulfite reductase is named Citrobacter XT1-2-2-::SiR, and its deposit number is: CCTCC NO: M 20241947.

[0007] The second object of the present invention is to provide a construction method of the above-mentioned genetically engineered strain. This construction method uses the original Citrobacter sp. XT1-2-2 as the starting strain, inserts the strong erythromycin promoter in front of the cysJ gene, and transforms to obtain an engineered strain with cadmium fixation ability.

[0008] Furthermore, it is a genetic engineering method of electrotransformation in the competent cells of Citrobacter XT1-2-2 to introduce a recombinant plasmid carrying a strong erythromycin promoter and an SiR fragment.

[0009] Specifically, it includes the following steps:

[0010] 1) Using the genomic DNA of Citrobacter XT1-2-2 strain as a template, design upstream and downstream primers of cysJ with restriction enzyme sites, and PCR amplify the cysJ gene fragment with restriction enzyme sites. At the same time, using the vector containing the strong erythromycin promoter PermE as a template, design upstream and downstream primers of PermE with restriction enzyme sites, and amplify the PermE fragment with restriction enzyme sites; fuse the PermE and cysJ fragments by the method of fusion PCR to obtain the PermE-cysJ recombinant fragment;

[0011] 2) Double-digest the plasmid and the PermE-cysJ recombinant fragment by double digestion, and ligate the linearized plasmid and the PermE-cysJ recombinant fragment by a ligase to obtain a recombinant vector;

[0012] 3) Introduce the constructed recombinant vector into the competent cells of Citrobacter XT1-2-2 by electrotransformation, and obtain positive transformants, namely Citrobacter XT1-2-2-::SiR, through resistance screening.

[0013] Further, in step 1), using the genomic DNA of Citrobacter sp. XT1-2-2 strain as a template, upstream and downstream primers of cysJ with XbaI restriction sites were designed, and the cysJ gene fragment with restriction sites was amplified by PCR. Meanwhile, using pOJ260-PremE containing the strong erythromycin promoter PermE as a template, upstream and downstream primers of PermE with EcoRI restriction sites were designed, and the PermE fragment with restriction sites was amplified. The PermE and cysJ fragments were fused by fusion PCR to obtain the PermE-cysJ recombinant fragment.

[0014] In step 2), the plasmid pBBR1MCS-2 and the PermE-cysJ recombinant fragment were double-digested with XbaI and EcoRI, and the linearized pBBR1MCS-2 plasmid and the PermE-cysJ recombinant fragment were ligated by T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysJ.

[0015] In step 3), the constructed recombinant vector pBBR1MCS-PermE-cysJ was introduced into the competent cells of Citrobacter sp. XT1-2-2 by electroporation, and positive transformants, namely Citrobacter XT1-2-2-::SiR, were obtained by screening with 50 μg / mL kanamycin resistance.

[0016] The third object of the present invention lies in the application of the above genetic engineering strain in repairing and reducing cadmium pollution in soil.

[0017] The verification of the above application method mainly includes the improvement of cadmium removal ability in liquid medium, the obvious enhancement of cadmium dynamic adsorption ability, the obvious increase in the expression level of SiR protein, the enhancement of the enzyme activity of SiR protease detected by a kit, and the obvious enhancement of the cadmium adsorption ability of the strain observed by transmission electron microscopy.

[0018] (1) Improvement of cadmium removal ability in liquid medium:

[0019] The starting strain and the genetic engineering strain of the present invention were cultured in 100 mL blue-capped bottles. The medium of the engineering strain of the present invention was added with kanamycin resistance at a final concentration of 50 μg / mL and was respectively transferred to Cd 2+In a 100 mL Erlenmeyer flask containing 100 mL of liquid SRB medium with a content of 50 mg / L, an experiment was conducted to detect the cadmium removal ability in the liquid medium: Treatment 1: Citrobacter sp. XT1-2-2; Treatment 2: Citrobacter sp. XT1-2-2-::SiR. The cadmium content in the supernatant was measured at different culture times of 0 h, 20 h, 40 h, 60 h, 80 h, 100 h, and 120 h. The concentration of Cd in the collected supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). 2+ concentration.

[0020] (2) The cadmium dynamic adsorption ability was significantly enhanced:

[0021] The starting strain and the genetically engineered strain described in the present invention were cultured in a 100 mL blue-capped bottle. Kan resistance with a final concentration of 50 μg / mL was added to the medium of the genetically engineered strain described in the present invention. The strain was cultured until OD 600nm = 0.5, and the strain activity remained stable. The starting strain and the genetically engineered strain described in the present invention were added to the prepared solution containing 40 mg / L of Cd 2+ solution. The cadmium content in the supernatant of the solution was measured at 15 min, 30 min, 60 min, 120 min, and 240 min. The concentration of Cd in the collected supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). 2+ concentration.

[0022] (3) The expression level of SiR protein was significantly increased:

[0023] The starting strain and the genetically engineered strain described in the present invention were cultured in a 100 mL blue-capped bottle. Kan resistance with a final concentration of 50 μg / mL was added to the medium of the genetically engineered strain described in the present invention. They were respectively transferred to Erlenmeyer flasks containing 100 mL of liquid SRB medium for culture and washed 4 times with PBS (12 mM, pH = 7.6, pre-cooled at 4°C). The cell pellet was resuspended in 100 μL of lysozyme (200 mg / mL), and 500 μL of lysis buffer was added to each tube. The whole cell protein was extracted by ultrasonic disruption (JY92 ultrasonic cell disruptor, Ningbo Xinzhi Biotechnology Co., Ltd.). It was found by SDS-PAGE that the expression level of SiR protein was significantly increased.

[0024] (4) The enzyme activity of SiR protease was enhanced:

[0025] The protein samples of the starting strain obtained above and the genetically engineered strain of the present invention were subjected to enzyme activity detection of sulfite reductase (SiR). A color reaction was carried out using the Quickcheck SRB™ kit, and then a 96-well plate was analyzed and detected using a microplate reader.

[0026] (5)The cadmium adsorption capacity of the strain was significantly enhanced under transmission electron microscope observation:

[0027] The starting strain and the genetically engineered strain of the present invention were cultured in a 100 mL blue-capped bottle containing the corresponding resistant seed liquid, and were respectively transferred to a 100 mL liquid SRB medium with a Cd 2+ content of 50 mg / L and a conical flask containing a liquid SRB medium without Cd 2+ The images of the bacterial cells were scanned using a transmission electron microscope (TEM) (H-7650, Hitachi, Japan).

[0028] The beneficial effects of the present invention are as follows:

[0029] In the present invention, the sulfite reductase gene cysJ derived from Citrobacter XT1-2-2 was successfully overexpressed under the action of the strong erythromycin promoter PermE by genetic engineering methods, and the engineering strain Citrobactersp. XT1-2-2-::SiR with cadmium repair potential was successfully constructed, which can effectively improve the cadmium tolerance and fixation ability in paddy fields. It can be clearly seen through transmission electron microscope observation that the cadmium adsorption effect of the engineering strain is significantly enhanced. For the starting strain, this is the first time that the sulfite reductase (SiR) gene cysJ has been successfully overexpressed in Citrobacter XT1-2-2 using the broad-host vector pBBR1MCS-2, and to a certain extent, the cadmium tolerance of the host bacterium, the enzyme activity of sulfite reductase, and the cadmium enrichment amount have been improved. The invention also provides more application possibilities for the ecological restoration of cadmium-polluted paddy fields using Citrobacter XT1-2-2-::SiR as a functional strain.

[0030] The strain described in the present invention is classified and named as Citrobacter sp. XT1-2-2-::SiR; its depository unit is called the China Center for Type Culture Collection, abbreviated as CCTCC, with the address of Wuhan University, Wuhan, China. The deposit date is September 9, 2024, and the deposit number is CCTCC NO: M 20241947. Description of the Drawings

[0031] Figure 1 . PCR electrophoresis map of the PCR amplification products of the sulfite reductase (SiR) gene cysJ and the strong erythromycin promoter PermE;

[0032] In the figure, A: 1. Detection result of PCR amplification of PermE fragment; 2. Detection result of PCR amplification of cysJ fragment; B: 1-2. Fusion PCR product of PermE and cysJ fragments;

[0033] Figure 2 . Construction map of broad-host expression vector pBBR1MCS-PermE-cysJ;

[0034] Figure 3 . Diagram of the results of double digestion detection and positive transformant screening experiment of expression vector pBBR1MCS-PermE-cysJ;

[0035] In the figure, A. 1-2. Detection of recombinant expression vector plasmid extraction; 3. Detection result of double digestion control of blank pBBR1MCS-2 plasmid; 4. Diagram of double digestion detection and identification of expression vector pBBR1MCS-PermE-cysJ; B. Results of positive transformant screening experiment of Citrobactersp. XT1-2-2-::SiR;

[0036] Figure 4 . Removal ability determination chart of Cd in wild-type strain and engineered strain in liquid medium at different culture times 2+ ;

[0037] Figure 5 . Comparison chart of cadmium dynamic adsorption capacity between wild-type strain and engineered strain;

[0038] Figure 6 . SDS-PAGE electrophoresis expression difference chart of sulfite reductase protein in wild-type strain and engineered strain;

[0039] Figure 7 . Enzyme activity detection chart of sulfite reductase (SiR) in wild-type strain and engineered strain;

[0040] Figure 8 . Transmission electron microscopy observation chart of wild-type strain and engineered strain;

[0041] Figure 9 . Schematic diagram of the overexpression of cysJ by the strong PermE erythromycin promoter;

[0042] In the figure, A. Diagram of the overexpression of cysJ by the strong PermE erythromycin promoter; B. Schematic diagram of the sulfate metabolic pathway. Detailed implementation method

[0043] Description of the source of biological materials of the present invention:

[0044] 1. Source of plasmid: Plasmid pBBR1MCS-2 is a broad-host shuttle vector, purchased from the global plasmid sharing platform of Addgene and preserved, with the plasmid number #85168.

[0045] 2. Source of plasmid: Plasmid pOJ260-PermE is a gene knockout expression vector previously purchased and preserved in the laboratory. The plasmid was purchased from .

[0046] 3. Source of the starting strain Citrobacter XT1-2-2: In previous work, the research team where the applicant is located screened a strain with good cadmium fixation function in cadmium-polluted paddy fields in Hunan Province. Through genome sequencing and 16S rRNA alignment, this bacterium was identified as Citrobacter and named XT1-2-2.

[0047] 4. Design and synthesis of primers: Self-designed and synthesized by Shanghai Bioengineering Technology Company.

[0048] Promoters are cis-acting elements necessary for gene expression and play an important role in the regulation of gene transcription level. The strength of promoter activity directly affects the gene expression level. In this invention, a study on the screening of sulfate metabolic pathway promoters was carried out in Citrobacter XT1-2-2. We summarized the publicly available promoter sequences and screened the promoters that can be used in Citrobacter XT1-2-2. The endogenous promoter PSACE_2101, the exogenous strong promoter PermE, and the constitutive promoter Psf were selected for overexpression experiments of genes related to the sulfate metabolic pathway in Citrobacter. After the PSACE_2101 fragment and the Psf fragment were respectively fused with the target gene and electrotransformed into Citrobacter XT1-2-2, compared with the wild strain, the mutant strain did not produce H2S during cultivation, and thus the medium containing Fe 2+ was not turned black during the cultivation process. Therefore, subsequent experiments mainly carried out fusion and overexpression experiments of the exogenous strong promoter PermE with the target gene respectively.

[0049] Example 1

[0050] This example illustrates the method for constructing the recombinant vector pBBR1MCS-PermE-cysJ containing the PermE-SiR fragment. The process includes:

[0051] 1. Design upstream and downstream primers for the erythromycin strong promoter fragment and the SiR fragment with restriction enzyme sites:

[0052] PermE-F: 5’-CCGGAATTCCTGGACTTCTAGAGCTAGCC-3’ (EcoRI)

[0053] PermE-R: 5'-GCATGCCGGTCGACTCTA-3'

[0054] PermE-SiR-F: 5'-GATCCTCTAGAGTCGACCGGCATGCATGACGACACAGGCCCCACC-3'

[0055] PermE-SiR-R: 5'-TGCTCTAGATTAGTAGACATCTCGCTGATAA-3' (XbaI)

[0056] 2. Using pOJ260-PermE as a template, PCR amplify the PermE fragment. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s, 58.5°C for 30 s, 72°C for 50 s, 72°C for 5 min, for 30 cycles. After completion, purify the PCR product. Using the Citrobacter XT1-2-2 genomic DNA as a template, PCR amplify the SiR fragment. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s, 58.5°C for 30 s, 72°C for 1 min 50 s, 72°C for 5 min, for 30 cycles. After completion, purify the PCR product. Then perform fusion PCR on the PermE and SiR fragments. First, add the two fragments, high-fidelity primer STAR enzyme, dNTP, ddH2O. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s, 58.5°C for 30 s, 72°C for 1 min 50 s, 72°C for 5 min, for 13 cycles. Then add primers PermE-F and PermE-SiR-R to perform the second-step fusion PCR. The PCR reaction conditions are as follows: pre-denaturation at 94°C for 5 min, 94°C for 30 s, 58.5°C for 30 s, 72°C for 2 min, 72°C for 5 min, for 30 cycles.

[0057] 3. Double-digest the pBBR1MCS-2 plasmid and the PermE-cysJ recombinant fragment with XbaI and EcoRI, and ligate the linearized pBBR1MCS-2 plasmid and the PermE-cysJ recombinant fragment with T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysJ; the recombinant vector is double-digested with XbaI and EcoRI, and the results are as Figure 3 shown, demonstrating the successful construction of the recombinant vector pBBR1MCS-PermE-cysJ;

[0058] Example 2

[0059] This Example 2 describes the method of introducing the constructed recombinant vector pBBR1MCS-PermE-cysJ into the competent cells of Citrobacter sp. XT1-2-2 by electroporation and screening for positive transformants through kanamycin resistance of 50 μg / mL, namely Citrobacter sp. XT1-2-2-::SiR.

[0060] (1) Preparation of competent cells of Citrobacter sp. XT1-2-2: Inoculate Citrobacter sp. XT1-2-2 into 10 mL of SRB liquid medium and culture at 37 °C until the logarithmic phase; Take 1 mL (2%) of the pre-cultured bacterial solution and inoculate it into a 50 mL conical flask of SRB liquid medium, and statically culture at 37 °C for 24 h until the OD 600nm reaches 0.4 - 0.5; Transfer the bacterial solution into two 50 mL centrifuge tubes, place them on ice for 10 min, and pre-cool the centrifuge, centrifuge tubes, tips and CaCl2 solution at the same time; Centrifuge to collect the cells (9000 rpm, 4 °C, 5 min), pour out the supernatant in the sterile workbench, invert the centrifuge tube for a moment to drain the supernatant; Add 25 mL of pre-cooled 0.1 mol / L CaCl2, gently blow and suspend the cells, and place them on ice for 5 - 10 min. Centrifuge at 9000 rpm, 4 °C, 5 min, discard the supernatant; Repeat the washing once with 25 mL of pre-cooled 0.1 mol / L CaCl2, and freeze-centrifuge to discard the supernatant; Resuspend the cells with 2 mL of ice-pre-cooled 0.1 mol / L CaCl2 containing 15% glycerol, and gently blow with a pipette to suspend the cells; Aliquot the competent cells at 100 μL / tube and store at -80 °C.

[0061] Electroporation conditions: Take 50 ng of the recombinant vector pBBR1MCS-PermE-cysJ and perform electroporation according to the following conditions: 1250 V, 10 μF, 3.3 kΩ, 1 mm. After suspending with 1 mL of SRB liquid medium, transfer it into a 15 mL centrifuge tube and culture at 30 °C for 12 h. Screen for positive transformants, namely the target strain Citrobacter XT1-2-2-::SiR, on solid SRB medium through kanamycin resistance of 50 μg / mL. The results of the positive transformants on the plate are as Figure 3 shown in B.

[0062] Example 3

[0063] This Example 3 describes the differences in cadmium removal ability, cadmium dynamic adsorption ability and sulfite reductase activity between the successfully constructed engineered strain Citrobacter sp. XT1-2-2-::SiR and the wild-type strain in liquid medium.

[0064] (1) Comparison of cadmium removal ability in liquid medium: Inoculate Citrobacter sp. XT1-2-2 and the engineered strain Citrobacter sp. XT1-2-2-::SiR into 10 mL of SRB liquid medium and culture at 37 °C until the logarithmic phase; Take 1 mL (2%) of the pre-cultured bacterial solution and inoculate it into a conical flask containing 50 mL of SRB liquid medium, and culture statically at 37 °C for 48 h until the OD 600nm reaches 0.4 - 0.5; Transfer them respectively to Erlenmeyer flasks containing 100 mL of liquid SRB medium with a Cd 2+ content of 50 mg / L for the cadmium removal ability detection experiment in liquid medium: Treatment 1: Citrobacter sp. XT1-2-2; Treatment 2: Citrobacter sp. XT1-2-2-::SiR. Collect the supernatant at different culture times of 0 h, 20 h, 40 h, 60 h, 80 h, 100 h and 120 h respectively, and determine the concentration of Cd 2+ in the collected supernatant by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). The results are as Figure 4 shown. It can be seen from the figure that as time goes by, the degree of decrease in the cadmium content in the liquid medium of the genetically engineered strain is significantly greater than that of the wild-type strain, indicating that the engineered strain has a stronger cadmium removal ability than the wild-type strain in the liquid medium.

[0065] (2) Comparison of dynamic cadmium adsorption ability: Inoculate Citrobacter sp. XT1-2-2 and the engineered strain Citrobacter sp. XT1-2-2-::SiR into 10 mL of SRB liquid medium and culture at 37 °C until the logarithmic phase; Take 1 mL (2%) of the pre-cultured bacterial solution and inoculate it into a conical flask containing 50 mL of SRB liquid medium, and culture statically at 37 °C for 48 h until the OD 600nm reaches 0.4 - 0.5; Prepare a cadmium solution with a concentration of 100 mg / L and add the cultured strains. Take the supernatant at 15 min, 30 min, 60 min, 120 min and 240 min respectively, and determine the concentration of Cd 2+ in the collected supernatant by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). The results are as Figure 5 shown, and the dynamic cadmium adsorption ability of the genetically engineered strain is significantly improved.

[0066] (3) Sulfite reductase activity difference: Citrobacter sp. XT1-2-2 and the engineered strain Citrobacter sp. XT1-2-2-::SiR were inoculated into 10 mL of SRB liquid medium and cultured at 37 °C until the logarithmic phase; 1 mL (2%) of the pre-cultured bacterial liquid was inoculated into a 50 mL conical flask of SRB liquid medium and statically cultured at 37 °C for 48 h until the OD 600nm reached 0.4 - 0.5; the protein samples of the wild-type strain and the genetically engineered strain of the present invention were subjected to enzyme activity detection of sulfite reductase (SiR). Color reaction was carried out using the Quickcheck SRB™ kit, and then the 96-well plate was analyzed and detected with an enzyme-linked immunosorbent assay reader. The results showed that the sulfite reductase enzyme activity of the genetically engineered strain Citrobacter sp. XT1-2-2-::SiR was enhanced, as shown in Figure 7 .

[0067] Example 4

[0068] This Example 4 illustrates the SDS-PAGE expression difference of the sulfite reductase protein between the successfully constructed engineered strain Citrobacter sp. XT1-2-2-::SiR and the wild-type strain, and the morphological difference of the strains observed by transmission electron microscopy

[0069] (1) SDS-PAGE expression difference of sulfite reductase protein: Citrobacter sp. XT1-2-2 and the engineered strain Citrobacter sp. XT1-2-2-::SiR were inoculated into 10 mL of SRB liquid medium and cultured at 37 °C until the logarithmic phase; 1 mL (2%) of the pre-cultured bacterial liquid was inoculated into a 50 mL conical flask of SRB liquid medium and statically cultured at 37 °C for 48 h until the OD 600nm reached 0.4 - 0.5; washed 4 times with PBS (12 mM, pH = 7.6, pre-cooled at 4 °C). The cell pellet was resuspended in 100 μL of lysozyme (200 mg / mL), 500 μL of lysis buffer was added to each tube, and the total protein of the bacterial cells was extracted by ultrasonic disruption (JY92 ultrasonic cell disrupter, Ningbo Xinzhi Biotechnology Co., Ltd.). It was found by SDS-PAGE electrophoresis that the expression level of SiR protein increased significantly, as shown in Figure 6 .

[0070] (2) The wild-type strain and the genetically engineered strain of the present invention were cultured in 100 mL blue-capped bottles containing the corresponding resistant seed liquid, and were respectively transferred to Cd 2+ with a content of 50 mg / L in 100 mL of liquid SRB medium and without Cd 2+In the blue-capped bottle of liquid SRB medium, images of the bacterial cells were taken using a transmission electron microscope (TEM) (H-7650, Hitachi, Japan). The morphology of the strains was observed by TEM, and the results were as follows Figure 8 shown. The engineered strains adsorbed significantly more cadmium, and there were significant changes in the morphology of the bacterial cells.

[0071] The above examples demonstrate that the genetically engineered strain Citrobacter sp. XT1-2-2-::SiR obtained by the construction method of the present invention can further enhance the cadmium adsorption capacity and tolerance of wild-type strains. This invention provides a novel and feasible method for microbial remediation of heavy metal pollution.

Claims

1. An engineered strain overexpressing sulfite reductase, named Citrobacter XT1-2-2-::SiR, with the deposit number: CCTCC NO: M 20241947.

2. The method for constructing an engineered strain overexpressing sulfite reductase according to claim 1, characterized in that: This strain uses the original Citrobacter sp. XT1-2-2 as the starting strain, inserts a strong erythromycin promoter in front of the cysJ gene, and obtains an engineered strain with cadmium fixation ability.

3. The construction method according to claim 2, wherein Electroporation transformation is carried out in Citrobacter XT1-2-2 competent cells to introduce a recombinant plasmid carrying a strong erythromycin promoter and the SiR fragment.

4. The construction method according to claim 3, wherein, It includes the following steps: 1) Using the genomic DNA of Citrobacter XT1-2-2 strain as a template, design upstream and downstream primers for cysJ with restriction enzyme sites, and PCR amplify the cysJ gene fragment with restriction enzyme sites. At the same time, using a vector containing the strong erythromycin promoter PermE as a template, design upstream and downstream primers for PermE with restriction enzyme sites, and amplify the PermE fragment with restriction enzyme sites; fuse the PermE and cysJ fragments by fusion PCR to obtain the PermE-cysJ recombinant fragment; 2) Double-digest the plasmid and the PermE-cysJ recombinant fragment by double digestion, and ligate the linearized plasmid and the PermE-cysJ recombinant fragment by a ligase to obtain a recombinant vector; 3) Introduce the constructed recombinant vector into the competent cells of Citrobacter XT1-2-2 by electroporation transformation, and obtain positive transformants, namely Citrobacter XT1-2-2-::SiR, through resistance screening.

5. According to the construction method described in claim 4, characterized in that In step 1), using the genomic DNA of Citrobacter XT1-2-2 strain as a template, design upstream and downstream primers for cysJ with XbaI restriction enzyme sites, and PCR amplify the cysJ gene fragment with restriction enzyme sites. At the same time, using pOJ260-PremE containing the strong erythromycin promoter PermE as a template, design upstream and downstream primers for PermE with EcoRI restriction enzyme sites, and amplify the PermE fragment with restriction enzyme sites; fuse the PermE and cysJ fragments by fusion PCR to obtain the PermE-cysJ recombinant fragment.

6. According to the construction method described in claim 4, characterized in that In step 2), double-digest the pBBR1MCS-2 plasmid and the PermE-cysJ recombinant fragment by XbaI and EcoRI double digestion, and ligate the linearized pBBR1MCS-2 plasmid and the PermE-cysJ recombinant fragment by T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysJ.

7. According to the construction method described in claim 4, characterized in that Step 3) The constructed recombinant vector pBBR1MCS-PermE-cysJ was introduced into the competent cells of Citrobacter sp. XT1-2-2 by electroporation, and positive transformants, namely Citrobacter XT1-2-2-::SiR, were obtained through screening with 50 μg / mL kanamycin resistance.

8. The application method of the engineered strain overexpressing sulfite reductase according to claim 1, characterized in that: For the remediation of cadmium-contaminated soil.

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