Engineering strain for overexpressing sulfite reductase as well as construction method and application of engineering strain

Abstract

The invention discloses an engineering strain for overexpressing sulfite reductase as well as a construction method and application of the engineering strain. The strain is obtained by taking citrobacter XT1-2-2 as an original strain and inserting an erythromycin strong promoter PermE at the front end of a cysJ gene for expressing sulfite reductase SiR for transformation, and the original citrobacter is Citrobacter sp.XT1-2-2. The engineering strain with cadmium immobilization activity, which is obtained by the construction method disclosed by the invention, is classified and named as Citrobacter XT1-2-2-:: SiR, and the preservation number of the engineering strain is CCTCC (China Center For Type Culture Collection) NO.M 20241947. The strain can be used as an ecological restoration strain for the cadmium-polluted rice field, and a foundation is laid for subsequent research on ecological restoration in the cadmium-polluted rice field soil.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and relates to an engineering strain over-expressing sulfite reductase, a construction method and 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, my country's farmland has been seriously polluted by heavy metals. Among them, the rate of excessive pollution of cadmium elements still ranks first among inorganic pollutants. Hunan is the hometown of non-ferrous metals and is 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 bioaccumulation. It is easy to accumulate in animals, plants and the human body through the food chain, directly threatening human health. Commonly used physical and chemical remediation methods are relatively expensive, and the use of reagents in crop soils can easily cause secondary pollution of the soil and changes in soil structure. The use of plants for soil heavy metal remediation is easily limited by the growth cycle and biomass of plants, resulting in insufficient and timely effects. Therefore, the use of microorganisms for soil heavy metal remediation is not only efficient, economical, and environmentally friendly, but also the use of microorganisms improves the microbial flora of the soil and increases the biological activity of the soil.

[0003] Microorganisms in the soil can survive naturally in highly cadmium-contaminated soils, and regulate the content and form of Cd in the soil through complex enzymatic reactions in the bacteria and the cell metabolism, oxidation-reduction, adsorption and absorption of heavy metals by the strains, thereby reducing the content of Cd in the soil. In recent years, a large number of scientific researchers have isolated and screened functional microorganisms that are highly resistant to cadmium and have a growth-promoting effect 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. Studies have shown that the addition and use of microbial agents can not only promote the growth of rice, but also have a significant mitigation effect on cadmium pollution in rice seedlings. The cadmium content in the aboveground part of rice treated with microbial agents decreased by 59.9% compared with before treatment, the cadmium migration coefficient decreased by 33.3%, the cadmium enrichment coefficient decreased by 58.8%, and the cadmium accumulation of a single rice plant decreased by 24.3%. Summary of the invention

[0004] The primary purpose of the present invention is to provide an engineered strain of Citrobacter acidophilus with an erythromycin strong promoter inserted to overexpress the cysJ gene, the expression level of sulfite reductase (SiR) protein is increased, and the SO3 2- To S 2- The transformation makes the engineered strain obtained by the present invention have efficient cadmium fixation activity and has great potential in the ecological restoration function of cadmium-contaminated rice fields.

[0005] The original strain Citrobacter barbadensis XT1-2-2 with cadmium resistance and cadmium fixation activity isolated and screened by the present invention has obtained the complete genome data of the bacterium, and preliminarily performed functional enrichment analysis and KEGG metabolic pathway analysis on the bacterium. Since the genetic operating system of the strain is relatively clear, it can be efficiently genetically modified.

[0006] The engineered strain for overexpressing sulfite reductase is named as 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 method for constructing the above-mentioned genetically engineered strain, which uses the original Citrobacter sp. XT1-2-2 as the starting bacterium, inserts the erythromycin strong promoter in front of the cysJ gene, and transforms the engineered strain with cadmium fixation ability.

[0008] Furthermore, the invention relates to a genetic engineering method for introducing a recombinant plasmid carrying an erythromycin strong promoter and a SiR fragment into a Citrobacter rodentium XT1-2-2 competent cell through electroporation.

[0009] The specific steps include:

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

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

[0012] 3) The constructed recombinant vector was introduced into the competent cells of Citrobacter rodentium XT1-2-2 by electroporation, and positive transformants, namely Citrobacter XT1-2-2-::SiR, were obtained by resistance screening.

[0013] Furthermore, step 1) using the genomic DNA of the Citrobacter rodentium XT1-2-2 strain as a template, designing cysJ upstream and downstream primers with XbaI restriction site, PCR amplifying the cysJ gene fragment with restriction site, and simultaneously using pOJ260-PremE containing the erythromycin strong promoter PermE as a template, designing PermE upstream and downstream primers with EcoRI restriction site, amplifying the PermE fragment with restriction site; and fusing the PermE and cysJ fragments by fusion PCR to obtain the PermE-cysJ recombinant fragment.

[0014] Step 2) The pBBR1MCS-2 plasmid 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 with T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysJ.

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

[0016] The third object of the present invention is to use the above genetically engineered strains to repair and reduce cadmium pollution in soil.

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

[0018] (1) Improved cadmium removal capacity in liquid culture medium:

[0019] The starting strain and the genetically engineered strain of the present invention were cultured in 100 mL blue-capped bottles, wherein the culture medium of the engineered strain of the present invention was added with kan resistance at a final concentration of 50 μg / mL, and the strains were transferred to Cd 2+The cadmium removal capacity test in liquid culture medium was carried out in a triangular flask containing 100 mL liquid SRB culture medium with a content of 50 mg / L: 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 Cd content in the collected supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). 2+ concentration.

[0020] (2) The dynamic adsorption capacity of cadmium is significantly enhanced:

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

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

[0023] The starting strain and the genetically engineered strain of the present invention were cultured in 100 mL blue-capped bottles, wherein the engineered strain culture medium of the present invention was added with kan resistance at a final concentration of 50 μg / mL, and transferred to 100 mL of liquid SRB medium in a triangular flask for culture, and washed 4 times with PBS (12 mM, pH=7.6, precooled 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 whole cell protein was extracted by ultrasonic disruption (JY92 ultrasonic cell grinder, Ningbo Xinzhi Biotechnology Co., Ltd.), and the expression of SiR protein was significantly increased by SDS-PAGE observation.

[0024] (4) Enhanced SiR protease activity:

[0025] The protein samples of the starting strain obtained above and the genetically engineered strain of the present invention were tested for sulfite reductase (SiR) enzyme activity, a color reaction was performed using the Quickcheck SRB™ kit, and then the 96-well plate was analyzed and tested using an ELISA reader.

[0026] (5) Transmission electron microscopy showed that the strain's cadmium adsorption capacity was significantly enhanced:

[0027] The starting strain and the genetically engineered strain of the present invention were cultured in 100 mL blue-capped bottles containing corresponding resistant seed solution and transferred to Cd 2+ 100 mL of liquid SRB medium containing 50 mg / L Cd and 100 mL of liquid SRB medium containing 50 mg / L Cd 2+ Images of bacteria grown in a Erlenmeyer flask in liquid SRB medium were scanned using a transmission electron microscope (TEM) (H-7650, Hitachi, Japan).

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

[0029] The present invention successfully overexpressed the sulfite reductase gene cysJ derived from Citrobacter XT1-2-2 under the action of the erythromycin strong promoter PermE through a genetic engineering method, and successfully constructed Citrobacter sp. XT1-2-2-::SiR, and the engineered strain with cadmium repair potential can effectively improve the tolerance and fixation ability of cadmium in rice fields. Through transmission electron microscopy observation, it can be clearly seen that the adsorption effect of cadmium by the engineered strain is significantly enhanced. For the starting strain, this is the first time that the sulfite reductase (SiR) gene cysJ is successfully overexpressed in Citrobacter XT1-2-2 through the wide host vector pBBR1MCS-2, and the host bacteria cadmium tolerance, sulfite reductase enzyme activity and cadmium enrichment are improved to a certain extent. The invention also provides more application possibilities for the ecological restoration of cadmium-contaminated rice fields using Citrobacter XT1-2-2-::SiR as a functional strain.

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

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

[0032] Figure A: 1. PermE fragment PCR amplification test results; 2. CysJ fragment PCR amplification test results; B: 1-2. PermE and cysJ fragment fusion PCR product;

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

[0034] Figure 3 . Results of double enzyme digestion detection and positive transformant screening of expression vector pBBR1MCS-PermE-cysJ;

[0035] In the figure, A. 1-2. Recombinant expression vector plasmid extraction test; 3. Blank pBBR1MCS-2 plasmid double enzyme digestion control test results; 4. Double enzyme digestion detection and identification map of expression vector pBBR1MCS-PermE-cysJ; B. Citrobacter sp. XT1-2-2-::SiR positive transformant screening experiment results;

[0036] Figure 4 . Wild-type strain and engineered strain, Cd at different culture times in liquid culture medium 2+ Removal capacity determination diagram;

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

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

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

[0040] Figure 8 . Transmission electron microscopy observations of wild-type strains and engineered strains;

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

[0042] Figure A. Schematic diagram of overexpression of cysJ by the PermE erythromycin strong promoter; B. Schematic diagram of the sulfate metabolism pathway. DETAILED DESCRIPTION

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

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

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

[0046] 3. The origin of the starting strain Citrobacter rodentium XT1-2-2: In the previous work, the applicant's research team screened a strain with good cadmium fixation function in cadmium-contaminated rice fields in Hunan Province. Through genome sequencing and 16S rRNA comparison, the bacteria were identified as Citrobacter rodentium and named XT1-2-2.

[0047] 4. Primer design and synthesis: designed by ourselves and synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0048] The promoter is a cis-acting element necessary for gene expression and plays an important role in regulating the gene transcription level. The strength of the promoter activity directly affects the gene expression level. The present invention carried out a screening study on the promoter of the sulfate metabolic pathway in Citrobacter XT1-2-2. We summarized the existing promoter sequences disclosed and screened the promoters that can be used in Citrobacter XT1-2-2. We selected the endogenous promoter PSACE_2101, the exogenous strong promoter PermE and the constitutive promoter Psf to conduct overexpression experiments on genes related to the sulfate metabolic pathway of Citrobacter. After the PSACE_2101 fragment and the Psf fragment were fused with the target gene and electroporated into Citrobacter XT1-2-2, compared with the wild strain, the mutant strain did not produce H2S during culture, and thus failed to make the Fe-containing strain overexpress during the culture process. 2+ The culture medium turned black, therefore, subsequent experiments mainly carried out the fusion and overexpression experiments of the exogenous strong promoter PermE with the target gene.

[0049] Example 1

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

[0051] 1. Design upstream and downstream primers of the erythromycin strong promoter fragment and SiR fragment with restriction 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'-TGCTCTAGATTAGTAGACATCTCCGTGATAA-3' (XbaI)

[0056] 2. Using pOJ260-PermE as a template, PCR amplification of PermE fragment, PCR reaction conditions are: 94℃ pre-denaturation for 5min, 94℃ 30s, 58.5℃ 30s, 72℃ 50s, 72℃ 5 min, 30 cycles. After completion, PCR products were purified. Using the genome of Citrobacter rodentium XT1-2-2 as a template, PCR amplification of SiR fragment, PCR reaction conditions are: 94℃ pre-denaturation for 5min, 94℃ 30s, 58.5℃ 30s, 72℃ 1 min 50s, 72℃ 5 min, 30 cycles. After completion, PCR products were purified. Then, PermE and SiR fragments were subjected to fusion PCR. First, two fragments, high-fidelity primer STAR enzyme, dNTP, ddH2O were added, and the PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ 30 s, 58.5℃ 30 s, 72℃ 1 min 50 s, 72℃ 5 min, 13 cycles. Then primers PermE-F and PermE-SiR-R were added to perform the second step of fusion PCR. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min, 94℃ 30 s, 58.5℃ 30 s, 72℃ 2 min, 72℃ 5 min, 30 cycles.

[0057] 3. Double digest the pBBR1MCS-2 plasmid and the PermE-cysJ recombinant fragment with XbaI and EcoRI, connect 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 was double digested with XbaI and EcoRI, and the result was as follows: Figure 3 As shown, it was proved that the recombinant vector pBBR1MCS-PermE-cysJ was successfully constructed;

[0058] Example 2

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

[0060] (1) Preparation of Citrobacter sp. XT1-2-2 competent cells: Inoculate Citrobacter sp. XT1-2-2 into 10 mL 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 SRB liquid medium and culture it at 37°C for 24 h until the OD 600nm Reach 0.4-0.5; transfer the bacterial solution to two 50mL centrifuge tubes and place on ice for 10 min, while precooling the centrifuge, centrifuge tubes, tip heads and CaCl2 solution; centrifuge to collect the bacteria (9000 rpm, 4℃, 5 min), pour out the supernatant on a sterile workbench, and invert the centrifuge tube for a moment to allow the supernatant to flow out; add 25 mL of precooled 0.1 mol / L CaCl2, blow gently and suspend the cells, and place on ice for 5-10 min. Centrifuge at 9000 rpm, 4℃, 5min, and remove the supernatant; repeat washing with 25 mL of precooled 0.1 mol / L CaCl2, freeze centrifuge and remove the supernatant; resuspend the bacteria with 2 mL of ice-cold 0.1 mol / L CaCl2 containing 15% glycerol, and blow gently with a gun to suspend the bacteria; distribute the competent cells at 100 μL / tube and freeze at -80℃.

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

[0062] Example 3

[0063] This Example 3 illustrates 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 culture medium.

[0064] (1) Comparison of cadmium removal capacity in liquid culture medium: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::SiR were inoculated into 10 mL SRB liquid culture medium and cultured at 37°C until the logarithmic phase; 1 mL (2%) of the pre-cultured bacterial solution was inoculated into a conical flask containing 50 mL SRB liquid culture medium and cultured at 37°C for 48 h until the OD 600nm Reach 0.4-0.5; transfer to Cd respectively 2+ The cadmium removal capacity test in the liquid culture medium was carried out in a triangular flask containing 100 mL of liquid SRB culture medium with a content of 50 mg / L: Treatment 1: Citrobacter sp. XT1-2-2; Treatment 2: Citrobacter sp. XT1-2-2-::SiR. The supernatants were collected at different culture times of 0 h, 20 h, 40 h, 60 h, 80 h, 100 h and 120 h, and the Cd content in the collected supernatants was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). 2+ The results are as follows Figure 4 As shown in the figure, we can see that over time, the cadmium content in the liquid culture medium of the genetically engineered strain decreased significantly more than that of the wild-type strain, indicating that the engineered strain has a stronger ability to remove cadmium in liquid culture medium than the wild-type strain.

[0065] (2) Comparison of dynamic adsorption capacity of cadmium: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::SiR were inoculated into 10 mL SRB liquid culture medium and cultured at 37°C until the logarithmic phase; 1 mL (2%) of the pre-cultured bacterial solution was inoculated into a conical flask containing 50 mL SRB liquid culture medium and cultured at 37°C for 48 h until the OD 600nm The concentration of cadmium in the supernatant was 0.4-0.5, and a 100 mg / L cadmium solution was prepared and added to the cultured strain. The supernatant was collected at 15 min, 30 min, 60 min, 120 min, and 240 min, and the Cd content in the collected supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). 2+ The results are as follows Figure 5 As shown, the dynamic adsorption capacity of cadmium by the genetically engineered strain was significantly improved.

[0066] (3) Difference in sulfite reductase activity: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::SiR were inoculated into 10 mL SRB liquid medium and cultured at 37°C until the logarithmic phase; 1 mL (2%) of the pre-cultured bacterial solution was inoculated into a conical flask containing 50 mL SRB liquid medium and cultured at 37°C for 48 h until the OD 600nm The protein samples of the wild-type strain and the genetically engineered strain of the present invention were tested for the enzyme activity of sulfite reductase (SiR). The color reaction was carried out using the Quickcheck SRB™ kit, and then the 96-well plate was analyzed and tested using an ELISA instrument. The results showed that the sulfite reductase enzyme activity of the genetically engineered strain Citrobacter sp. XT1-2-2-::SiR was enhanced. Figure 7 .

[0067] Example 4

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

[0069] (1) SDS-PAGE expression difference of sulfite reductase protein: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::SiR were inoculated into 10 mL SRB liquid medium and cultured at 37°C until the logarithmic phase; 1 mL (2%) of the pre-cultured bacterial solution was inoculated into a conical flask containing 50 mL SRB liquid medium and cultured at 37°C for 48 h until the OD 600nm Reach 0.4-0.5; wash 4 times with PBS (12 mM, pH=7.6, precooled at 4°C). Resuspend the cell pellet in 100 μL lysozyme (200 mg / mL), add 500 μL lysis buffer to each tube, extract the whole bacterial protein by ultrasonic disruption (JY92 ultrasonic cell grinder, Ningbo Xinzhi Biotechnology Co., Ltd.), and observe by SDS-PAGE electrophoresis that the expression of SiR protein is significantly increased, see 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 corresponding resistant seed solution and transferred to Cd 2+ 100 mL of liquid SRB medium containing 50 mg / L Cd and 100 mL of liquid SRB medium containing 50 mg / L Cd 2+The images of the bacteria were taken in a blue-capped bottle of liquid SRB medium using a transmission electron microscope (TEM) (H-7650, Hitachi, Japan). The morphology of the strain was observed by transmission electron microscopy. Figure 8 As shown, the engineered strain adsorbed significantly more cadmium and its bacterial morphology changed significantly.

[0071] The above examples prove 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 and tolerance of the wild-type strain. This invention provides a novel and feasible method for microbial remediation of heavy metal pollution prevention and control.

Claims

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

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

3. The construction method according to claim 2, characterized in that: The recombinant plasmid containing the strong erythromycin promoter and SiR fragment was introduced into the competent cells of Citrobacter rodentium XT1-2-2 by electroporation.

4. The construction method according to claim 3, characterized in that: The following steps are involved: 1) Using the genomic DNA of Citrobacter rodentium XT1-2-2 strain as a template, upstream and downstream primers of cysJ with restriction sites were designed, and PCR was used to amplify the cysJ gene fragment with restriction sites. At the same time, using the vector containing the erythromycin strong promoter PermE as a template, upstream and downstream primers of PermE with restriction sites were designed to amplify the PermE fragment with restriction sites; the PermE and cysJ fragments were fused by fusion PCR to obtain the PermE-cysJ recombinant fragment; 2) Double-digest the plasmid and the PermE-cysJ recombinant fragment by double enzyme digestion, and connect the linearized plasmid and the PermE-cysJ recombinant fragment by ligase to obtain a recombinant vector; 3) The constructed recombinant vector was introduced into the competent cells of Citrobacter rodentium XT1-2-2 by electroporation, and positive transformants, namely Citrobacter XT1-2-2-::SiR, were obtained by resistance screening.

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

6. The construction method according to claim 4, characterized in that: Step 2) The pBBR1MCS-2 plasmid 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 with T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysJ.

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

8. The method for using the engineered strain overexpressing sulfite reductase according to claim 1, characterized in that: Used to repair heavy metal cadmium pollution in soil.

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