Citrobacter citrobacter genetic engineering strain as well as construction method and application thereof

Abstract

The invention discloses a citrobacter gene engineering strain as well as a construction method and application thereof. The strain is obtained by taking citrobacter XT1-2-2 as a starting strain and inserting an erythromycin strong promoter PermE at the front end of a cysH gene for expressing sulfate reductase APS for transformation, and the original citrobacter is Citrobacter sp. XT1-2-2. The invention further discloses a preparation method of the strain. The engineering strain is obtained by overexpressing a cysH gene by using an erythromycin strong promoter on the basis of the original strain, the preservation number is CCTCC NO: M 20241948, and the engineering strain has remarkable cadmium reduction activity, can be used as an ecological restoration strain of the cadmium-polluted rice field, and lays a foundation for subsequent research on ecological restoration in the cadmium-polluted rice field.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and relates to a high-efficiency cadmium-fixing engineering strain and a construction method and application thereof, specifically refers to obtaining an engineering strain through the construction of a high-efficiency cadmium-fixing engineering strain, and also relates to the application of the strain's cadmium tolerance and cadmium adsorption functions. Background Art

[0002] Cadmium is also highly biotoxic to humans. It is difficult to degrade in the soil and is bioaccumulative. It is easily accumulated in animals, plants and humans 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 soil pollution 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 the use of microorganisms also improves the soil's microbial flora 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 cysH gene, which increases the expression level of sulfate reductase (APS) protein and promotes SO4 2- To SO3 2- The transformation makes the engineered strain obtained by the present invention have efficient cadmium reduction activity and has great potential in the heavy metal cadmium repair function in rice fields.

[0005] The original strain Citrobacter XT1-2-2 with cadmium resistance and cadmium fixation activity separated 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 genetically modified and transformed efficiently. The genetically engineered strain of Citrobacter of the present invention is named Citrobacter sp. XT1-2-2-::APS, and the deposit number is: CCTCC NO: M20241948.

[0006] 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 PermE in front of the cysH gene to obtain an engineered strain with cadmium fixation ability.

[0007] Furthermore, the invention relates to a genetic engineering method for introducing a recombinant plasmid carrying an erythromycin strong promoter and an APS fragment into a Citrobacter acidobacterium XT1-2-2 competent cell through electroporation.

[0008] The specific steps include:

[0009] 1) Using the genomic DNA of Citrobacter rodentium XT1-2-2 strain as a template, the upstream and downstream primers of cysH with restriction sites were designed, and the cysH gene fragment with restriction sites was amplified by PCR. At the same time, using the vector containing the erythromycin strong promoter PermE as a template, the upstream and downstream primers of PermE with restriction sites were designed to amplify the PermE fragment with restriction sites; the PermE and cysH fragments were fused by fusion PCR to obtain the PermE-cysJ recombinant fragment;

[0010] 2) Double-digest the plasmid and the PermE-cysH recombinant fragment by double enzyme digestion, and connect the linearized plasmid and the PermE-cysH recombinant fragment by ligase to obtain a recombinant vector;

[0011] 3) The constructed recombinant vector was introduced into the competent cells of Citrobacter rodentium XT1-2-2 by electroporation, and positive transformants, namely Citrobacter sp. XT1-2-2-::APS, were obtained by resistance screening.

[0012] Furthermore, step 1) using the genomic DNA of the Citrobacter rodentium XT1-2-2 strain as a template, designing cysH upstream and downstream primers with XbaI restriction site, PCR amplifying the cysH 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 cysH fragments by fusion PCR to obtain the PermE-cysJ recombinant fragment.

[0013] Step 2) The pBBR1MCS-2 plasmid and the PermE-cysH recombinant fragment were double-digested with XbaI and EcoRI, and the linearized pBBR1MCS-2 plasmid and the PermE-cysH recombinant fragment were ligated with T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysH.

[0014] Step 3) The constructed recombinant vector pBBR1MCS-PermE-cysH was introduced into the competent cells of Citrobacter rodentium XT1-2-2 by electroporation, and the positive transformants, namely Citrobacter sp. XT1-2-2-::APS, were obtained by screening for resistance to 50 μg / mL kanamycin.

[0015] The third object of the present invention is to provide the use of the above-mentioned genetically engineered strain in repairing and reducing heavy metal cadmium pollution in soil.

[0016] 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 APS protein expression, the enhancement of APS protease activity detected by the kit and the significant enhancement of cadmium adsorption ability of the strain under transmission electron microscopy observation.

[0017] (1) Improved cadmium removal capacity in liquid culture medium:

[0018] 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-::APS. 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 in the collected supernatant was determined by inductively coupled plasma mass spectrometry (ICP-MS, Agilent-7500cx). 2+ concentration.

[0019] (2) The dynamic adsorption capacity of cadmium is significantly enhanced:

[0020] 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 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.

[0021] (3) APS protein expression increased significantly:

[0022] 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 APS protein was significantly increased by SDS-PAGE observation.

[0023] (4) Enhanced APS protease activity:

[0024] The protein samples of the starting strain obtained above and the genetically engineered strain of the present invention were tested for the enzyme activity of sulfate reductase (APS). A color reaction was performed using the Quickcheck SRB™ kit, and then the 96-well plate was analyzed and tested using an ELISA reader.

[0025] (5) Transmission electron microscopy showed that the strain's cadmium adsorption capacity was significantly enhanced:

[0026] 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 the bacteria were taken in Erlenmeyer flasks containing liquid SRB medium using a transmission electron microscope (TEM) (H-7650, Hitachi, Japan).

[0027] The beneficial effects of the present invention are:

[0028] The present invention successfully overexpressed the sulfate reductase gene cysH derived from Citrobacter acidobacter 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-::APS, and an engineered strain with a certain heavy metal cadmium repair potential, which can effectively improve the tolerance to heavy metal cadmium in rice fields. Through transmission electron microscopy observation, it can be clearly seen that the adsorption effect of the engineered strain on metal cadmium is significantly enhanced. For the starting strain, this is the first time that the sulfate reductase (APS) gene cysH is successfully overexpressed in Citrobacter acidobacter XT1-2-2 through the broad host vector pBBR1MCS-2, and the host bacteria cadmium tolerance, sulfate reductase enzyme activity and cadmium enrichment are improved to a certain extent. The invention also provides more application possibilities for using Citrobacter sp. XT1-2-2-::APS as a functional strain in reducing rice cadmium absorption.

[0029] The strain described in the present invention is classified and named Citrobacter sp. XT1-2-2-::APS; 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 20241948. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 . Erythromycin strong promoter PermE, sulfate reductase (APS) gene cysH and PermE+cysH fragment fusion PCR product;

[0031] In the figure, 1. Electrophoresis diagram of PCR amplification of PermE erythromycin strong promoter fragment; 2. Electrophoresis diagram of PCR amplification of cysH fragment; 3. Electrophoresis diagram of PCR amplification of PermE+cysH fragment fusion;

[0032] Figure 2 . Construction map of the broad-host expression vector pBBR1MCS-PermE-cysH;

[0033] Figure 3 . Double enzyme digestion detection electrophoresis and positive transformant screening experiment results of expression vector pBBR1MCS-PermE-cysH;

[0034] Figure A. 1.2 are double enzyme digestion detection bands; B. Citrobacter sp. XT1-2-2-::APS positive transformant SRB solid culture medium screening results

[0035] Figure 4 . Wild-type strain and engineered strain, Cd at different culture times in liquid culture medium 2+ Removal capacity determination diagram;

[0036] Figure 5 . Comparison of the dynamic cadmium adsorption capacity of wild-type strains and engineered strains;

[0037] Figure 6 . SDS-PAGE electrophoresis expression difference of sulfate reductase protein between wild-type strain and engineered strain;

[0038] Figure 7 . Detection diagram of sulfate reductase (APS) enzyme activity of wild-type strain and engineered strain;

[0039] Figure 8 . Transmission electron microscopy observations of wild-type strains and engineered strains;

[0040] Fig. 9 . Schematic diagram of overexpression of cysH and sulfate metabolic pathway by PermE erythromycin strong promoter;

[0041] In the figure, A. Schematic diagram of overexpression of cysJ by the strong PermE erythromycin promoter; B. Schematic diagram of the sulfate metabolic pathway. DETAILED DESCRIPTION

[0042] Description of the source of biological materials of the present invention:

[0043] 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.

[0044] 2. Plasmid source: Plasmid pOJ260-PermE is a gene knockout expression vector purchased and preserved by the laboratory. .

[0045] 3. The origin of the starting strain Citrobacter 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 bacterium was identified as Citrobacter and named XT1-2-2. Design and synthesis of primers: designed by ourselves and synthesized by Shanghai Bioengineering Technology Co., Ltd.

[0046] 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.

[0047] Example 1

[0048] This example illustrates a method for constructing a recombinant vector pBBR1MCS-PermE-cysH containing a PermE-APS fragment. The process includes:

[0049] 1. Design upstream and downstream primers of the erythromycin strong promoter fragment and APS fragment with restriction sites:

[0050] PermE-F: 5'-CCGGAATTCCTGGACTTCTAGAGCTAGCC-3' (EcoRI)

[0051] PermE-R: 5'-GCATGCCGGTCGACTCTA-3'

[0052] PermE-APS-F: 5'-GATCCTCTAGAGTCGACCGGCATGCATGTCCGTACTTGATCTACA-3'

[0053] PermE-APS-R: 5'-TGCTCTAGATTACCCTTCGTGCAGCCCGC-3' (XbaI)

[0054] 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, purify the PCR product. Using the genome of Citrobacter rodentium XT1-2-2 as a template, PCR amplification of APS 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, purify the PCR product. Then perform fusion PCR on PermE and APS fragments. 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-APS-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.

[0055] 3. Double digest the pBBR1MCS-2 plasmid and the PermE-cysH recombinant fragment with XbaI and EcoRI, connect the linearized pBBR1MCS-2 plasmid and the PermE-cysH recombinant fragment with T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysH; 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-cysH was successfully constructed;

[0056] Example 2

[0057] This Example 2 describes a method for introducing the constructed recombinant vector pBBR1MCS-PermE-cysH into competent cells of Citrobacter rodentium XT1-2-2 by electroporation, and obtaining positive transformants by 50 μg / mL kanamycin resistance screening, namely Citrobacter sp. XT1-2-2-::APS.

[0058] (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℃.

[0059] Electrotransformation conditions: Take 50 ng of the recombinant vector pBBR1MCS-PermE-cysH and electrotransform it according to the following electrotransformation 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 sp. XT1-2-2-::APS. The experimental results of the screened positive transformants on the plate are shown in the following figure. Figure 3 As shown in B.

[0060] Example 3

[0061] This Example 3 illustrates the differences in cadmium removal ability, cadmium dynamic adsorption ability and sulfate reductase activity between the successfully constructed engineered strain Citrobacter sp. XT1-2-2-::APS and the wild-type strain in liquid culture medium.

[0062] (1) Comparison of cadmium removal capacity in liquid culture medium: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::APS 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-::APS. 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.

[0063] (2) Comparison of dynamic adsorption capacity of cadmium: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::APS 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.

[0064] (3) Difference in sulfate reductase activity: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::APS 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 activity of sulfate reductase (APS) in the protein samples of the wild-type strain and the genetically engineered strain of the present invention was detected. The color reaction was carried out by the Quickcheck SRB™ kit, and then the 96-well plate was analyzed and detected by a microplate reader. The results showed that the activity of sulfate reductase in the genetically engineered strain Citrobacter sp. XT1-2-2-::APS was enhanced. Figure 7 .

[0065] Example 4

[0066] This example 4 illustrates the difference in the SDS-PAGE expression of sulfate reductase protein between the successfully constructed engineered strain Citrobacter sp. XT1-2-2-::APS and the wild-type strain and the difference in strain morphology observed by transmission electron microscopy

[0067] (1) SDS-PAGE expression difference of sulfate reductase protein: Citrobacter sp. XT1-2-2 and engineered strain Citrobacter sp. XT1-2-2-::APS 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 the APS protein expression level significantly increased by SDS-PAGE electrophoresis, see Figure 6 .

[0068] (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.

[0069] The above examples prove that the genetically engineered strain Citrobacter sp.XT1-2-2-::APS 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. A genetically engineered strain of Citrobacter sp. XT1-2-2-::APS, with a deposit number of CCTCC NO: M 20241948.

2. The method for constructing the genetically engineered strain of Citrobacter rodentium according to claim 1, Features: The strain uses the original Citrobacter sp. XT1-2-2 as the starting bacterium, inserts the erythromycin strong promoter in front of the cysH gene, and transforms it into an engineered strain with cadmium reduction ability.

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

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

5. The construction method according to claim 4, It is characterized in that Step 1) Using the genomic DNA of the Citrobacter rodentium XT1-2-2 strain as a template, designing cysH upstream and downstream primers with XbaI restriction site, PCR amplifying the cysH 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; fusing the PermE and cysH fragments by fusion PCR to obtain the PermE-cysJ recombinant fragment.

6. The construction method according to claim 4, It is characterized in that Step 2) The pBBR1MCS-2 plasmid and the PermE-cysH recombinant fragment were double-digested with XbaI and EcoRI, and the linearized pBBR1MCS-2 plasmid and the PermE-cysH recombinant fragment were ligated with T4 ligase to obtain the recombinant vector pBBR1MCS-PermE-cysH.

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

8. The application method of the genetically engineered strain of Citrobacter rodentium according to claim 1, It is characterized in that Used to repair heavy metal cadmium pollution in soil.

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