Construction method and application of carbon sequestration engineering bacteria

Abstract

The invention discloses a construction method and application of a carbon sequestration engineering bacterium, Rhodospirillum rubrum is transformed by using a genetic engineering technology to obtain the carbon sequestration engineering bacterium, the carbon sequestration engineering bacterium is applied to a water body of a freshwater aquaculture pond, and the carbon sequestration efficiency and the biological safety of the carbon sequestration engineering bacterium are inspected and determined. The invention provides a simple, feasible, green and efficient method and application product for reducing carbon emission of a fresh water pond culture system.

Description

Technical Field

[0001] The present invention relates to a construction method and application of carbon fixation engineering bacteria, and specifically to a construction method of Rhodospirilla rubrum carbon fixation engineering bacteria and application of the same in reducing carbon emissions in a freshwater pond aquaculture system, belonging to the technical field of aquaculture. Background Art

[0003] Freshwater pond farming is the main production method of aquaculture in my country and an important source of aquatic products in my country. At present, the farming process and waste treatment are the main sources of carbon emissions in the freshwater aquaculture industry. The high-density, high-input freshwater pond farming model has problems such as excessive pollutants in the aquaculture tailwater and large-scale greenhouse gas emissions. Therefore, reducing the emission of pollutants such as carbon and nitrogen in the farming process is an important issue facing the healthy development of the freshwater aquaculture industry.

[0004] At present, in addition to optimizing aquaculture management processes, such as controlling stocking density, establishing a precise feeding system, digitalizing aquaculture intelligent management, and building a multi-trophic level farming model, freshwater aquaculture is the main way to reduce carbon emissions. In addition, the use of biological filters and the construction of water biofilms have also shown good application effects in freshwater pond aquaculture. It can be seen that microbial technology has great application potential in freshwater pond aquaculture carbon fixation and emission reduction. However, in the current aquaculture industry, there are no reports on the use of genetic engineering technology to transform microorganisms and apply the modified microorganisms to aquaculture practice. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a method for constructing carbon fixation engineering bacteria and its application in reducing carbon emissions in freshwater pond aquaculture systems.

[0006] Technical solution: The present invention provides a method for constructing carbon fixation engineering bacteria, comprising the following steps:

[0007] (1) connecting the double-digested pET-32a plasmid and the oorA gene fragment, and transferring them into competent Escherichia coli cells, screening positive bacteria and extracting the recombinant expression plasmid pET-32a-oorA; the sequence of the oorA gene is shown in SEQ NO.3 or SEQ NO.4;

[0008] (2) After expanding the culture of Rhodospirilla crimsonii, the bacterial cells were collected and prepared into competent cells, and the recombinant expression plasmid pET-32a-oorA was transformed into the competent cells of Rhodospirilla crimsonii to obtain the carbon fixation engineering bacteria of Rhodospirilla crimsonii.

[0009] Furthermore, the restriction sites of the pET-32a plasmid in step (1) are EcoR I and Xho I.

[0010] Furthermore, when screening positive bacteria in step (1), the primer sequences used for amplifying the pET-32a plasmid are shown in SEQ NOs. 9 to 10, and the primer sequences used for amplifying the oorA gene are shown in SEQ NOs. 11 to 12.

[0011] Furthermore, the conditions for the expansion culture of Rhodospirillum rubrum in step (2) are 25-32° C., 200 rpm shaking, and 30° C. inverted culture, and the components of the culture medium used are as follows:

[0012]

[0013] Among them, each 1 mL of the multivitamin solution in Formula 1 contains 20 mg of vitamin B2, 200 mg of niacin, 80 mg of biotin and 400 mg of thiamine; Formula 2 controls the pH of the culture medium at 6.8-7.0.

[0014] Furthermore, in step (2), the Rhodospirilla rubrum competent cells are treated with CaCl 2 Preparation by infiltration method.

[0015] Furthermore, when the Rhodospirilla rubrum carbon fixation engineering bacteria are prepared in step (2), the volume of the recombinant expression plasmid pET-32a-oorA does not exceed 10% of the competent cells of Rhodospirilla rubrum.

[0016] Furthermore, the concentration of the recombinant expression plasmid pET-32a-oorA is 10pg to 1ng.

[0017] Furthermore, the specific steps of transforming the recombinant expression plasmid pET-32a-oorA into Rhodospirillum rubrum competent cells in step (2) include: placing the mixture of the recombinant expression plasmid pET-32a-oorA and the Rhodospirillum rubrum competent cells in an ice water bath for 30 minutes; heat shocking for 2 minutes in a 42°C water bath; immediately placing in an ice water bath after the heat shock for 2 minutes; adding 900 μL of carbon source-free liquid culture medium and culturing overnight at 30°C and 200 rpm.

[0018] The present invention also provides the use of the carbon fixation engineering bacteria prepared according to the above construction method in reducing carbon emissions in a freshwater pond aquaculture system.

[0019] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the present invention uses genetic engineering technology to isolate and transform a carbon fixation engineering bacterium in a freshwater pond aquaculture environment, and applies it in the aquaculture pond to test and determine its carbon fixation efficiency and biosafety. The present invention provides a simple, feasible, green and efficient method and application product for reducing carbon emissions in freshwater pond aquaculture systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1.Single colony of carbon-fixing bacteria;

[0021] Figure 2 .Carbon-fixing bacteria culture;

[0022] Figure 3 .Comparison results of NT library of carbon-fixing bacteria sequences;

[0023] Figure 4 .Amplify the full-length gene sequence of the target gene oorA;

[0024] Figure 5 .pET-32a vector restriction enzyme electrophoresis results;

[0025] Figure 6 .Construct the E. coli culture plate transformed with pET-32a-oorA;

[0026] Figure 7 .Construction of PCR gel electrophoresis results of E. coli colonies transformed with pET-32a-oorA;

[0027] Figure 8 . Rhodospirilla rubrum competent cells;

[0028] Fig. 9 .Constructed carbon fixation engineering bacteria;

[0029] Fig.10 .Identification of carbon fixation engineering bacteria 16s;

[0030] Fig.11 .Scanning electron microscope image of Rhodospirillum crimsonii;

[0031] Fig.12 .Scanning electron microscope image of the modified engineered bacteria;

[0032] Fig.13 .CO at the water-air interface 2 Emission flux determination;

[0033] Fig.14 .The effect of bacteria addition to aquaculture water on ACP and AKP of zebrafish;

[0034] Fig.15 .Effects of bacteria addition to aquaculture water on antioxidant enzymes in zebrafish. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0036] Example 1 Acquisition and analysis of carbon-fixing strains

[0037] (1) Obtaining the strain: The carbon-fixing strain used is Rhodospirillum rubrum preserved in the National Microbial Resource Center (NMRC), with strain number CGMCC1.3369. Restore the culture of the vacuum freeze-dried strain. Under a sterile environment, aspirate 0.3 mL of liquid culture medium and drop it into an ampoule. Gently shake it to dissolve the freeze-dried bacteria into a suspended state. Aspirate all the bacterial suspension and transplant it into a 5 mL liquid culture medium (formula one) test tube, and culture it statically at 30°C. Aspirate 200 μL of the sample suspension and spread it on the solid culture medium (formula two), invert and culture it at 30°C for 10 to 14 days, separate and purify it through multiple streaking, and store it at 4°C to obtain colonies such as Figure 1 The culture medium formula is shown in Table 1.

[0038] Table 1. Medium formula for isolating carbon-fixing bacteria

[0039]

[0040] Note: In formula 1, each 1 mL of multivitamin solution contains 20 mg vitamin B2, 200 mg niacin, 80 mg biotin and 400 mg thiamine; in formula 2, the pH of the culture medium is controlled at 6.8-7.0.

[0041] (2) Cultivation and identification of strains: Pick a single colony and inoculate it into a carbon-free liquid culture medium. Cultivate it at 30°C and 200 rpm for 48 h. When the strain reaches the logarithmic growth phase, pipette 5 mL of the bacterial solution into a sterile centrifuge tube (such as Figure 2 The strain was centrifuged at 3,000 rpm to collect the bacteria and store them in a 4°C refrigerator. The genomic DNA of the strain was extracted according to the steps of the Ezup column bacterial genomic DNA extraction kit (Shanghai Shenggong Company) and stored in a -20°C refrigerator for future use.

[0042] The sequence determination for strain identification was completed by General Biotechnology (Anhui) Co., Ltd. The primers used are shown in Table 2, the amplification reaction and procedures are shown in Table 3, and agarose gel electrophoresis was performed for 30 minutes after the amplification. The amplified sequence was sequenced using a sequencer to obtain the abi sequencing peak file. The determined sequence was confirmed by Blast comparison at NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The original sequence of the sequence is shown in SEQ NO.1, the spliced ​​return sequence is shown in SEQ NO.2, and the database comparison result is shown in Figure 3 shown.

[0043] Table 2 Primer sequences for 16s DNA sequencing of carbon-fixing bacteria

[0044]

[0045]

[0046] Example 2 Construction of carbon fixation engineering bacteria

[0047] (1) Selection and purification of target gene: The functional gene in the tricarboxylic acid cycle was determined to be oorA through literature search. Its sequence (SEQ NO.3) was searched in the NCBI database and synthesized by General Biotechnology (Anhui) Co., Ltd. for PCR amplification and identification (the sequence returned by sequencing and splicing after identification was SEQ NO.4, which was used as the target gene sequence). The PCR reaction procedure was the same as in Table 3. The amplified oorA fragment was purified and its concentration was determined using a multifunctional DNA purification and recovery kit (BioTeke, Beijing). Figure 4 ).

[0048] (2) Construction of target gene prokaryotic expression vector and transformation of recombinant plasmid: According to the reaction system shown in Table 4, after 15-20 min in a 37°C water bath, pET-32a plasmid (VT1007, Wuhan Transduction Biological Laboratory) was double-digested, and the restriction endonuclease cleavage effect was detected by nucleic acid gel electrophoresis. The digested samples were recovered and their concentrations were determined ( Figure 5 ). The target gene and linear vector were connected by referring to the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing) for experimental operation. Take 5 μL of the vector and target fragment connection product and add 50 μL of cloning competent cells E. coli DH5a melted on ice, mix well and ice bath for 30 minutes; heat shock for 45-60 seconds in a 42°C water bath, and stand on ice for 2 minutes; add 500 μL of sterile and antibiotic-free LB culture medium to the centrifuge tube, and culture at 37°C and 200 rpm for 1 hour; evenly spread 100 μL of bacterial solution on an LB plate containing 100 ng / mL ampicillin and culture inverted at 37°C overnight ( Figure 6 ).

[0049] Table 4 Enzyme digestion reaction system

[0050]

[0051] (3) Screening and identification of positive monoclones: Randomly pick 10 single colonies from the plate cultured overnight in the previous step, mix thoroughly in 20 μL sterile water and number them; use the universal primers (T7T-F, T7-R) of the pET-32a plasmid to perform PCR amplification with the selected bacterial solution as a template. The amplification system and procedure are the same as those in Table 3. The vector amplification primer sequences are shown in Table 5, and the target gene primers are shown in Table 6. Agarose gel electrophoresis was used to detect the amplification effect ( Figure 7), select the bacterial solution number of the positive band and record it; culture the bacterial solution corresponding to the selected number in LB liquid culture medium containing ampicillin in a shaker; the next day, the cultured bacterial solution was sequenced and identified, and the sequencing results were analyzed (the sequencing results are shown in SEQ NO.5 (pET-32a plasmid) and SEQ NO.6 (target gene), and the consistency with the original sequence comparison results is 99.64% and 96.69%, respectively). The recombinant expression plasmid pET-32a-oorA was extracted from the correctly identified bacterial solution using a high-purity plasmid miniprep kit (BioTeke, Beijing).

[0052] Table 5 Vector amplification primer sequences

[0053]

[0054] Table 6 Target gene oorA amplification primer sequences

[0055]

[0056] (4) Preparation of Rhodospirillum rubrum competent cells: The Rhodospirillum rubrum isolated and identified in Example 1 was cultured in the laboratory. 2 Transformation method was performed. When OD600 reached 0.6-0.7, the Rhodospirilla rubrum bacterial solution was added to a centrifuge tube and cooled in an ice bath for 10 min. 50 mL of bacterial solution was centrifuged at 4,000 g for 15 min at 4°C and the supernatant was discarded. 2 Gently suspend the cells in 10 mL of solution, place on ice for 15-30 min, and centrifuge at 3,000 g for 10 min at 4°C. Discard the supernatant and add 4 mL of pre-cooled 0.05 mol / L CaCl containing 15% glycerol. 2 Solution, gently suspend the cells, and place on ice for 3 minutes to obtain a suspension of competent cells of Rhodospirilla rubrum ( Figure 8 The competent cells were divided into 200 μl aliquots, immediately frozen with liquid nitrogen or dry ice and stored at -70°C.

[0057] (5) Transformation of Rhodospirillum rubrum competent cells with recombinant plasmid: Slowly thaw Rhodospirillum rubrum competent cells on ice. The concentration of pET-32a-oorA plasmid extracted in (3) was determined to be 65 ng / μL. Add 5 μL of plasmid solution to a 1.5 mL centrifuge tube, and then add 5 μL of competent cells. Gently flick the centrifuge tube with your fingers to mix the bacteria and plasmid. Place in an ice bath or ice water bath for 30 minutes; heat shock in a 42°C water bath for 2 minutes; immediately place in an ice water bath after heat shock for 2 minutes; add 900 μL of liquid culture medium (Formula 1) and culture overnight at 30°C, 200 rpm ( Fig. 9 ) and then sequenced to identify the conversion sequence ( Fig.10). The carbon fixation engineering bacteria of Rhodospirillum rubrum were obtained. The measured return sequence of 16s DNA of the engineering bacteria was shown in SEQ NO.7, and the measured return sequence of the target gene (oorA) was shown in SEQ NO.8, which was 92% consistent with SEQ NO.3.

[0058] Example 3 Application of Rhodospirilla crimsonii carbon fixation engineering bacteria in carbon fixation in Takifugu obscurus aquaculture water

[0059] (1) In a laboratory environment, the growth of unmodified Rhodospirillum crimsonii and modified Rhodospirillum crimsonii carbon fixation engineering bacteria were compared by electron microscopy. Fig.11 The results show that the unmodified Rhodospirilla rubrum has a spiral or curved rod shape, a rough surface, obvious wrinkles and textures, and a complex cell wall microstructure. The diameter of the bacteria is less than 2 μm, and they aggregate into clusters to form a network structure. The secretion of extracellular polymers (EPS) helps the attachment and stability of the bacterial colony. Fig.12 As shown, the modified bacteria are rod-shaped, with a rough surface and wrinkles, showing strong signs of extracellular polymer (EPS) secretion. Some bacteria are slightly swollen at both ends, and there are filamentous or fibrous structures attached to the cell surface, which may be cellulose or other external secretions formed by the bacteria in the biofilm state. In addition, it can be observed in the image that there is a close stacking between the bacteria, and some cells are in a state of division or irregular thickening, indicating that the engineered bacteria may be in an active growth state. Comparison of the engineered strain and the unmodified R. crimsonii strain is shown in Table 7:

[0060] Table 7 Comparison of electron microscopy structures of the modified strains and Rhodospirilla crimsonii

[0061]

[0062] In summary, the morphological characteristics of the engineered bacteria showed higher secretion of extracellular polymers and intercellular cross-linking, which may be related to changes in their metabolic characteristics or biofilm formation ability after modification. The unmodified R. crimsonii maintained a relatively stable and regular morphology, with smooth bacteria and fewer connections between groups, reflecting a more natural physiological state.

[0063] (2) Evaluation of carbon fixation effect in Takifugu aquaculture water. The water of Takifugu aquaculture pond was taken, and 1 control group (blank) and 2 experimental groups (Rhodospirilla crimsonii and engineered bacteria) were set up. 2L of aquaculture water was added to each of the 3 5L conical flasks. At the beginning of the experiment, 5.81mL of unmodified Rhodospirilla crimsonii liquid and 5.81mL of modified engineered bacteria liquid were added to the water of the two experimental groups respectively, and mixed evenly to maintain the concentration of added bacteria in the water at 3.44×10 ^3CFU / mL, the same amount of sterile culture medium was placed in the control group, and other conditions were the same as those of the experimental group. During the experiment, the natural light environment was maintained, the bottle mouth was closed, and no water replacement or addition was performed. On the 1st to 3rd day of treatment, water-air interface gas samples of the aquaculture water were collected at 10:00 every day and stored in 50ml aluminum foil sealed bags. A gas chromatograph (Agilent, 7890A) was used to measure the CO at the water-air interface of the aquaculture water. 2 The results are as follows Fig.13 As shown, CO 2 The results of emission flux measurement were as follows: modified bacteria group < Rhodospirilla crimsonii group < blank group, indicating that 3.44×10 ^3 The addition of Rhodospirillum crimsonii has a certain carbon fixation effect on the aquaculture water of Takifugu obscurus, and the greenhouse gas CO 2 The emission reduction reached 20%, and the modified carbon fixation engineering bacteria had better carbon fixation ability, further reducing carbon emissions in water bodies, CO 2 Emission reduction reached 40%.

[0064] (3) Evaluation of the biosafety of engineered bacteria on fish. Water from a pond containing dark-striped pufferfish was used to set up a control group (blank) and two experimental groups (Rhodospirilla crimsonii and engineered bacteria). Each group had 4 L of aquaculture water. Ten zebrafish were cultured in three 350*265*120 mm tanks, respectively. The experimental period was 10 days. At the beginning of the experiment, unmodified Rhodospirilla crimsonii liquid and modified engineered bacteria liquid were added to the water of the two experimental groups, respectively, and mixed evenly to maintain the concentration of the added bacteria in the water at 3.44×10 ^3 CFU / mL, the same amount of culture medium was placed in the control group, and other conditions were the same as those of the experimental group. During the experiment, 0.3g (5% of body weight) was fed at 9:00 and 18:00 every day, the culture water temperature was maintained at 28±0.5℃, the dissolved oxygen was 7.0±0.5mg / L, the natural light environment was maintained, and no water replacement or addition was performed. At the end of the treatment on the 10th day, zebrafish liver samples were taken to measure their immune activity and oxidative stress indicators to verify the biosafety of the modified engineered bacteria to fish. The immune indices included alkaline phosphatase (ACP) (A060-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing) and acid phosphatase (AKP) (A059-2-1, Nanjing Jiancheng Bioengineering Institute, Nanjing), and the oxidative stress parameters included catalase (CAT) (A007-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing), glutathione peroxidase (GSH-PX) (A005-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing), total superoxide dismutase (SOD) (A001-3-1, Nanjing Jiancheng Bioengineering Institute, Nanjing), and malondialdehyde (MDA) (A003-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing). Results As shown in Fig.14As shown in the figure, the ACP and AKP of zebrafish liver in the water body with the addition of Rhodospirilla crimsonii were higher than those in the control group, and the addition of engineered bacteria further increased the activity of ACP and AKP, indicating that engineered bacteria can significantly enhance the immune capacity of fish (p<0.05). Fig.15 As shown in the figure, after adding Rhodospirilla crimsonii and engineered bacteria to the water, there was no significant difference in CAT, GSH-PX and SOD in the liver of cultured zebrafish compared with the control group, and the MDA in the engineered bacteria group decreased significantly (p<0.05), indicating that the addition of engineered bacteria to the water does not affect the antioxidant system of fish and is non-toxic to the fish body. On the contrary, it will reduce lipid peroxidation in the fish body. The above experiments show that the addition of modified engineered bacteria can improve the immune capacity of cultured organisms to a certain extent.

[0065] Comparative Example 1

[0066] Comparative Example 1 is basically the same as Example 2, except that the only difference is the difference in the selection of the restriction site of the vector, as follows:

[0067] The expression vector pET-32a was selected, and the restriction sites were MluⅠ and EcoRⅠ. The operation steps of ligation transformation were the same as those in Example 2. The colony growth after ligation transformation was normal, but the agarose gel electrophoresis results after PCR amplification showed only primer dimers and no positive bands. A single colony was selected and cultured overnight in LB liquid medium, and sequencing was performed after 8 to 12 hours.

[0068] The results showed that only 30% of the measured sequence was consistent with the target gene sequence, indicating that the connection between the vector and the target gene was unsuccessful, possibly due to the wrong selection of the restriction site.

[0069] Comparative Example 2

[0070] Comparative Example 2 is substantially the same as Example 2, except that the carrier is selected differently, as follows:

[0071] The wild-type plasmid vector of Rhodospirillum crimsonii was extracted and subjected to second-generation sequencing. The number of plasmid maps obtained from several sequencing runs was different (6, 12, or even 18). The plasmid sequences obtained from several sequencing runs were compared. There were partially similar sequences, but the rest of the plasmid maps were completely different. None of the sequencing results had exactly the same plasmid map.

[0072] The results showed that it was impossible to obtain a wild plasmid that was stably expressed in Rhodospirillum rubrum. Therefore, the transformation plan of extracting the wild plasmid of this strain and introducing exogenous genes was not feasible.

[0073] Comparative Example 2 shows that it is more feasible to construct the target gene onto an expression vector (plasmid) in Escherichia coli (such as E. coli DH5a) by selecting a suitable plasmid pET-32a.

[0074] Comparative Example 3

[0075] Comparative Example 3 is substantially the same as Example 3, except that different amounts of Rhodospirilla rubrum and engineered bacteria are added, as follows:

[0076] The experimental conditions of the three treatment groups were consistent with those of Example 3. 500 ml of liquid culture medium, Rhodospirilla crimsonii, and engineered bacteria were added to the control group (blank) and the experimental group (Rhodospirilla crimsonii, engineered bacteria) respectively. The bacterial concentration was 10 ^4 CFU / mL, within half an hour after addition, the three groups of zebrafish died to varying degrees, and the survival rate was less than 50% after 12 hours. 4 CFU / mL, excessively high water bacterial concentration will directly cause certain damage to zebrafish.

[0077] Table 8 Effect of high concentration bacteria addition on the survival rate of zebrafish

[0078]

Claims

1. A method for constructing carbon fixation engineering bacteria, characterized in that: The following steps are involved: (1) connecting the double-digested pET-32a plasmid and the oorA gene fragment, and transferring them into competent Escherichia coli cells, screening positive bacteria and extracting the recombinant expression plasmid pET-32a-oorA; the sequence of the oorA gene is shown in SEQ NO.3 or SEQ NO.4; (2) After expanding the culture of Rhodospirilla crimsonii, the bacterial cells were collected and prepared into competent cells, and the recombinant expression plasmid pET-32a-oorA was transformed into the competent cells of Rhodospirilla crimsonii to obtain the carbon fixation engineering bacteria of Rhodospirilla crimsonii.

2. The method for constructing carbon fixation engineering bacteria according to claim 1, characterized in that: The restriction sites of the pET-32a plasmid in step (1) are EcoR I and Xho I.

3. The method for constructing carbon fixation engineering bacteria according to claim 1, characterized in that: When screening positive bacteria in step (1), the primer sequences used for amplifying the pET-32a plasmid are shown in SEQ NOs. 9 to 10, and the primer sequences used for amplifying the oorA gene are shown in SEQ NOs. 11 to 12.

4. The method for constructing carbon fixation engineering bacteria according to claim 1, characterized in that: The conditions for expanding the culture of Rhodospirillum rubrum in step (2) are 25-32° C., shaking on a shaker at 200 rpm, and inverted culture at 30° C. The composition of the culture medium used is as follows: Among them, each 1 mL of the multivitamin solution in Formula 1 contains 20 mg of vitamin B2, 200 mg of niacin, 80 mg of biotin and 400 mg of thiamine; Formula 2 controls the pH of the culture medium at 6.8-7.

0.

5. The method for constructing carbon fixation engineering bacteria according to claim 1, characterized in that: In step (2), the competent cells of Rhodospirillum rubrum are prepared by the CaCl2 infiltration method.

6. The method for constructing carbon fixation engineering bacteria according to claim 1, characterized in that: When the carbon-fixing engineering bacteria of Rhodospirillum rubrum are prepared in step (2), the volume of the recombinant expression plasmid pET-32a-oorA does not exceed 10% of the competent cells of Rhodospirillum rubrum.

7. The method for constructing carbon fixation engineering bacteria according to claim 6, characterized in that: The concentration of the recombinant expression plasmid pET-32a-oorA is 10 pg to 1 ng.

8. The method for constructing carbon fixation engineering bacteria according to claim 1, characterized in that: The specific steps of transforming the recombinant expression plasmid pET-32a-oorA into Rhodospirillum rubrum competent cells in step (2) include: placing the mixture of the recombinant expression plasmid pET-32a-oorA and the Rhodospirillum rubrum competent cells in an ice water bath for 30 minutes; heat shocking in a 42°C water bath for 2 minutes; immediately placing in an ice water bath after the heat shock for 2 minutes; adding 900 μL of carbon source-free liquid culture medium and culturing at 30°C 200rpm overnight.

9. Use of the carbon fixation engineering bacteria prepared by the construction method according to any one of claims 1 to 8 in reducing carbon emissions in freshwater pond aquaculture systems.

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