Transgenic synechocystis algal strain with enhanced stress resistance and application of transgenic synechocystis algal strain in sewage treatment

Abstract

The invention discloses a transgenic synechocystis strain with enhanced stress resistance and application of the transgenic synechocystis strain in sewage treatment, and belongs to the technical field of microorganisms. According to the invention, the MntH gene is integrated into the genome of the synechocystis PCC6803, so that the transgenic synechocystis PCC6803 is constructed. Through stress of Cr < 6 + > metal ions and hydrogen peroxide, the difference of stress resistance between algae strains before and after transformation is verified. The result shows that the stress resistance of the synechocystis strain is effectively improved by inserting the functional element gene MntH into the genome of the synechocystis PCC6803. Under stress culture of hydrogen peroxide or Cr < 6 + > metal ions, the transgenic algal strain shows better stress resistance, the growth condition is better than that of a wild algal strain, and meanwhile, the content of photosynthetic pigment, protein and the like related to the growth of the transgenic algal strain is higher than that of the wild algal strain. The invention provides a technical means for constructing transgenic algal strains with enhanced stress resistance and researching physiological and metabolic functions of microalgae.

Description

Technical Field

[0001] The present invention relates to a strain of Synechocystis with enhanced stress resistance transformed by synthetic biology transgenic technology, and also relates to the application of the strain of Synechocystis in sewage treatment. The present invention belongs to the field of microbial technology. Background Art

[0002] Synechocystis sp. PCC6803 ( Synechocystis sp PCC6803) is a photosynthetic prokaryotic organism that can be autotrophic or heterotrophic, and has the characteristics of fast growth and easy operation, making it an ideal receptor for cyanobacterial genetic engineering. In the past few decades, the research progress on cyanobacteria has shifted from gene expression to genome editing and metabolic regulation or synthetic biology. The further development of cyanobacterial strains under the analysis of multi-combination data such as bioinformatics and genomics will build a powerful genetic engineering platform to obtain strains with higher growth rates, better carbon fixation capacity, more high-value compounds, better carbon dioxide weather resistance and high temperature resistance, and promote their application in environmental remediation, synthetic biology, etc.

[0003] In the past decade, efficient photosynthetic microalgae have been widely used in synthetic biology, such as the production of biofuels and chemical products. The research and development of cyanobacteria synthetic biology technology has become increasingly rapid and mature, and has applications in sewage treatment, biosynthesis, agriculture, etc. Synechocystis PCC6803 has broad application prospects in synthetic biology. In the long-term evolution process, in order to adapt to the ever-changing living environment, Synechocystis PCC6803 has evolved a variety of regulatory mechanisms, and cells initiate different metabolic regulation to cope with different stress conditions. Strengthening the development and optimization of cyanobacteria synthetic biology toolboxes, selecting and improving cyanobacteria with chassis performance are of great significance for production and application, and have important significance for promoting the development of cyanobacteria light-driven carbon fixation synthesis technology. Cyanobacteria will face different environmental stresses in applications such as environmental remediation, biosynthesis and agriculture, and the improvement of their stress resistance is very important for their application.

[0004] Microalgae have great potential in developing sustainable technologies for removing inorganic and organic pollutants from the environment. This potential is attributed to various mechanisms such as biosorption, bioaccumulation and biodegradation, involving different functional groups, proteins and peptides, responsible for the binding and detoxification of pollutants. Heavy metal pollution in sewage has become the main aspect of heavy metal pollution, among which cadmium pollution is the most common. In recent years, the application of biosorption technology in the treatment of heavy metal contaminated water has attracted widespread attention from researchers. Biosorption refers to the non-specific binding of heavy metal ions to cell surface or extracellular proteins or polysaccharides. Among these microorganisms, algae have unique advantages such as high affinity, multiple binding sites and large surface area, and can efficiently adsorb heavy metals. The use of microalgae for bioremediation of heavy metals has become a hot topic in recent years. For example, the fungus-algae symbiotic system (FASS) is often used to assist in the immobilization of algae and enhance the adsorption of heavy metals. When adsorbing heavy metal ions, they are usually poisoned by heavy metals. By modifying microalgae and using synthetic biology and omics to create engineered microalgae species with greater tolerance to toxic pollutants, it is beneficial to purify the environment. Compared with wild-type microalgae species, they absorb or convert pollutants at a higher rate, which can further expand the application of microalgae in sewage treatment.

[0005] Therefore, the present invention obtains Synechocystis PCC6803 with enhanced stress resistance by inserting the exogenous gene MntH into the genome of Synechocystis PCC6803, which provides an effective technical means for constructing transgenic algae strains with strong stress resistance and sewage treatment. Summary of the invention

[0006] One of the purposes of the present invention is to provide a strain of Synechocystis with enhanced stress resistance transformed by synthetic biology transgenic technology.

[0007] The second object of the present invention is to provide the application of the Synechocystis strain with enhanced stress resistance in studying the physiological and metabolic functions of microalgae, constructing transgenic algae strains with strong stress resistance, and sewage treatment.

[0008] In order to achieve the above object, the present invention adopts the following technical means: The present invention is carried out by S. Typhimurium ) The manganese transporter MntH gene of SL1344 was integrated into the genome of Synechocystis PCC6803, thereby constructing MntH transgenic Synechocystis PCC6803. The growth curves of the newly constructed MntH transgenic strain and the wild-type Synechocystis PCC6803 were measured to determine their growth differences. 6+Metal ion and hydrogen peroxide stress were used to stress wild-type and MntH transgenic algae strains, and physiological indicators such as chlorophyll between wild-type and MntH transgenic algae strains were measured to verify the difference in stress resistance of the algae strains before and after transformation. The results showed that inserting the functional element gene MntH into the genome of Synechocystis PCC6803 effectively improved the stress resistance of the algae strain. Under normal culture conditions, there was no difference in growth between the wild-type and MntH transgenic algae strains; however, under hydrogen peroxide or Cr 6+ When cultured under metal ion stress, the MntH transgenic algae strains showed better stress resistance and better growth than the wild-type algae strains. At the same time, the contents of various growth-related photosynthetic pigments, proteins, etc. in the MntH transgenic algae strains were higher than those in the wild-type algae strains.

[0009] On the basis of the above research, the present invention proposes the use of the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 in constructing a transgenic Synechocystis PCC6803 with enhanced stress resistance.

[0010] Among them, preferably, the stress resistance includes resistance to heavy metal ion stress and resistance to oxidative stress.

[0011] Among them, preferably, the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 is inserted into the genome of Synechocystis PCC6803, so as to improve the stress resistance of Synechocystis PCC6803.

[0012] Preferably, the nucleotide sequence of the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 is as shown in SEQ ID NO: 1.

[0013] Among them, preferably, the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 is regulated by the promoter PpsbA2.

[0014] Furthermore, the present invention also proposes a transgenic Synechocystis PCC6803 with enhanced stress resistance, wherein the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 is inserted into the genome of the transgenic Synechocystis PCC6803, and the nucleotide sequence of the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 is shown in SEQ ID NO: 1.

[0015] Preferably, the transgenic Synechocystis sp. PCC6803 is prepared by psbA , MntH gene , Kan The resistance gene and the recombinant fragment of the downstream fragment are obtained by inserting the recombinant fragment into the genome of Synechocystis PCC6803 by homologous recombination.

[0016] Wherein, preferably, the upstream fragment, the promoter P psbA , MntH gene , Kan The nucleotide sequence of the resistance gene and the recombinant fragment of the downstream fragment is shown in SEQ ID NO:2.

[0017] Furthermore, the present invention also proposes a method for constructing the transgenic Synechocystis PCC6803, comprising the following steps: (i) pMD19T- MnH Construction of recombinant plasmid: (1) Using the genome of Synechocystis PCC6803 as a template, the primer pairs up-F / up-R and down-F / down-R were used to amplify the upstream homology arm up and the downstream homology arm down fragments respectively; (2) pET-28a was used as template and primer pair Kan -F / Kan -R was used to amplify the Kan resistance fragment, and the genome of Synechocystis PCC6803 was used as a template. Ppsb -F / Ppsb -R amplified promoter ppbA2 Then use the primer pair up-F / down-R to fragment up, ppbA2 , kan , down and other proportions as a template for overlapping PCR to amplify up- ppsbA-kan-down The fragment was then connected to the pMD19T vector using T4 ligase and transformed into Escherichia coli TG1; the up-F / down-R primers were then used for PCR detection and verification, and the plasmid was extracted after the correct strain was identified and cultured. The correctly constructed plasmid was named pMD19T- ppabA-kan ; (3) Using a single colony of Salmonella typhimurium SL1344 as a template, the primer pair MntH-F / MntH-R was used to amplify the MntH fragment; the plasmid pMD19T- ppabA-kan and MntH fragments using restriction endonucleases Bg lII and Spe I was digested and recovered, and then ligated and transformed into Escherichia coli TG1, and primer pair Ppsb -F / MntH-R was tested by PCR. After the correct strain was cultured, the plasmid was extracted and sequenced. The correctly constructed plasmid was named pMD19T- MnH ; The sequences of the primer pairs described above are as follows: ; (II) Obtaining MntH transgenic Synechocystis: (1) Preparation of mixed cellulose ester membrane: Rinse the mixed cellulose ester membrane with distilled water three times, boil it in a glass dish for 5 min, and sterilize it for later use; (2) Preparation of algae: scrape a small amount of Synechocystis PCC6803 from the BG11 plate and culture it in BG-11 liquid culture medium. When the algae liquid grows to the logarithmic phase, take 10 mL of the algae liquid into a 15 mL centrifuge tube and centrifuge it at 1800 g for 5 min. (3) Wash the algal cell pellet three times with fresh BG-11 liquid medium, and add 400 μL of BG-11 liquid medium to resuspend the algal cells; (4) Add pMD19T- HkDJ , mix well and incubate at 30°C, 1400 lux for 6-8h; (5) Place a treated mixed cellulose ester membrane on the non-resistant BG-11 solid culture medium, making sure there are no bubbles between the membrane and the plate, and evenly spread 200 μL of incubated algal cells on the surface of the mixed cellulose ester membrane; (6) After 18-24 h of light culture, the membrane was transferred to BG-11 solid medium containing kanamycin and continued to be cultured under light; (7) After 10-14 days, pick a single algae colony and streak it on BG-11 solid medium containing kanamycin and continue culturing; (8) Inoculate the streaked algae colony in BG-11 liquid medium containing kanamycin and grow it. Extract its DNA and use specific primers to HkDJ -F / R PCR test, if amplified MnH The algae strain with the gene band indicated that the exogenous target gene was correctly transferred, and it was the MntH transgenic Synechocystis obtained by screening.

[0018] Finally, the present invention also proposes the application of the transgenic Synechocystis PCC6803 in the following aspects: (1) Use of the transgenic Synechocystis PCC6803 in constructing a transgenic algae strain with enhanced stress resistance; (2) Application of the transgenic Synechocystis PCC6803 as a model strain in studying the physiological and metabolic functions of microalgae; (3) Application of the transgenic Synechocystis PCC6803 in sewage treatment.

[0019] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention identifies the functional element MntH that can be used in Synechocystis PCC6803. By inserting the exogenous gene MntH into the genome of Synechocystis PCC6803, the stress resistance of Synechocystis PCC6803 can be significantly enhanced. The differences in physiological index data and stress resistance between different algae strains were analyzed by heavy metal ion stress and hydrogen peroxide stress. The results showed that the stress resistance of Cr 6+ Under stress, the transgenic strains had better growth than the wild-type strains, and the contents of total protein, phycobiliprotein, chlorophyll a, carotenoids, SOD and CAT were significantly higher than those of the wild-type strains. Under hydrogen peroxide stress, the ROS content of the wild-type strains gradually increased with the increase of hydrogen peroxide concentration, while the ROS content of the MntH strains changed little, and the growth and survival rate of the MntH strains were better than those of the wild-type strains.

[0020] 2. Using the transgenic Synechocystis PCC6803 with application potential proposed in the present invention as a platform, we systematically apply synthetic biology technology to develop new "editable, controllable, and scalable" algae species, conduct in-depth understanding, redesign and targeted transformation of the physiological and metabolic functions of Synechocystis PCC6803, and lay the foundation for the research of synthetic biology of microalgae. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To construct and identify transgenic algae strains (MntH); In the figure: A is a single clone screened during the transformation of Synechocystis PCC6803; B is the PCR test result of the transgenic algae colony, where 1-6 are sample numbers, M is a DNA marker, and as shown in the figure, sample No. 3 is positive; Figure 2 The growth diagram of wild-type algae strain (WT) and transgenic algae strain (MntH) under normal and different concentrations of chromium ion stress culture conditions; In the figure: A is the growth curve of normal culture; B is the growth curve of 1 mg / L Cr 6+ Growth curve under stress; C is 1.75 mg / LCr 6+ Growth curve under stress; D is 2 mg / L Cr 6+ Growth curve under stress; E is 4 mg / L Cr 6+ Growth curve under stress, F is different concentrations of Cr 6+ It affected the growth of the two algae strains to different degrees; Figure 3 Cr 6+ Physiological indicators of wild-type algae strain (WT) and transgenic algae strain (MntH) under stress; In the figure: A is Cr 6+The total protein content of algae under stress; B is Cr 6+ Comparison of total protein content of algae strains under stress; C is Cr 6+ The content of phycobiliprotein (PBP) under stress; D is Cr 6+ The content of extracellular polymers (EPS) under stress; P (Probability value) is the probability of the sample observation result or more extreme results when the original hypothesis is true. Indicates P < 0.05 (lowest significant difference), Indicates P < 0.001 (highest significance); Figure 4 Cr 6+ Carotenoid and chlorophyll a contents of wild-type algae strain (WT) and transgenic algae strain (MntH) under stress; In the figure: A is the determination of chlorophyll a; B is the determination of carotenoids; Indicates P < 0.05, indicates P < 0.001; Figure 5 Cr 6+ Figure 2. Reactive oxygen and other related oxidative physiological indicators of wild-type algae (WT) and transgenic algae (MntH) under stress; In the figure: A is Cr 6+ Stress affects the content of reactive oxygen species (ROS) in algae; B is Cr 6+ Comparison of superoxide dismutase (SOD) of algae strains under stress; C is Cr 6+ The content of catalase (CAT) in algae strains under stress; Indicates P < 0.05, Indicates P < 0.01 (moderate significance); Figure 6 The growth difference diagram of wild-type algae strain (WT) and transgenic algae strain (MntH) under hydrogen peroxide stress; In the figure: A is the content of reactive oxygen species (ROS) in algae strains affected by hydrogen peroxide stress; B is the growth curve of algae strains under hydrogen peroxide stress; C is the survival rate of algae strains under hydrogen peroxide stress; D is the intracellular ion content of algae strains under hydrogen peroxide stress. Indicates P < 0.05, Indicates P < 0.001. DETAILED DESCRIPTION

[0022] The present invention is further described below by way of examples, which are intended only to provide a better understanding of the present invention and are not intended to limit the scope of the present invention. Except for special examples, the following examples are all conventional experimental methods and operating procedures in the art.

[0023] The main sources of experimental materials are as follows: The Synechocystis sp. PCC6800 ( Synechocystis sp. PCC6803) was purchased from the Freshwater Algae Collection of Type Culture Collection of the Chinese Academy of Sciences.

[0024] The Salmonella typhimurium used in this study S. Typhimurium ) SL1344 was preserved and provided by the Agricultural and Environmental Microbiology Laboratory of the College of Life Sciences, Northwest Agriculture and Forestry University. The strain has been disclosed in many patent documents and non-patent literature, such as the patent application with publication number CN113444739A and invention name "Construction of a broad-host plasmid for inducible expression of green fluorescent protein and its application in fluorescent tracing", and non-patent literature: 1) Wang Wenjie, Du Fuxi, Yu Chuan, et al. Construction and characterization of recombinant attenuated Salmonella typhimurium SL1344ΔcrpΔasd (pYA3493-VP2) expressing feline panleukopenia virus VP2 protein [J]. Chinese Journal of Veterinary Science, 2023, 53 (02): 189-197. DOI:10.16656 / j.issn.1673-4696.2023.0029. and 2) Li Rongxu. Construction of crp and hfq gene deletion strains of goose-derived Salmonella typhimurium and determination of its biological and immune characteristics[D]. Foshan University of Science and Technology, 2020. DOI:10.27960 / d.cnki.gfskj.2020.000054. Escherichia coli TG1 was used for DNA cloning and plasmid construction and was purchased from Beijing Solebow Technology Co., Ltd.

[0025] The pMD19T plasmid was purchased from TAKARA Biotechnology (Beijing) Co., Ltd.

[0026] Restriction endonucleases were purchased from TAKARA Biotechnology (Beijing) Co., Ltd.

[0027] The universal DNA purification and recovery kit (DP210), plasmid mini-extraction kit (DP103), and bacterial genomic DNA extraction kit (DP302) were all purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0028] DNA ligation kit (AG11801) was purchased from Hunan Aikerui Bioengineering Co., Ltd.

[0029] The total SOD activity detection kit (S0109), reactive oxygen species detection kit (S0033S), and lipid oxidation (MDA) detection kit (S0131S) were all purchased from Biotech Biotechnology Co., Ltd.

[0030] Catalase (CAT) activity detection kit (D799598-0100) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0031] Cyanobacteria BG-11 freshwater liquid culture medium (referred to as "BG-11 liquid culture medium") was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., product number C3061. Adding an appropriate amount of agar (e.g., 15-20 g / L) to the BG-11 liquid culture medium gives a BG-11 solid culture medium.

[0032] Example 1 Construction of MntH transgenic Synechocystis PCC6803 1. Methods (I) pMD19T- HkDJ Construction of recombinant plasmid: Synechocystis Synechocystis sp.) PCC6803 genome (GCF_000009725.1) was used as a template, and the primer pairs up-F / up-R and down-F / down-R were used to amplify the upstream homology arm up and the downstream homology arm down fragments, respectively; the resistance plasmid pET-28a was used as a template, and the primer pairs Kan -F / Kan -R amplification Kan Resistance fragment, using the genome of Synechocystis PCC6803 as a template, primer pair Ppsb -F / Ppsb -R amplified promoter ppbA2 Then use the primer pair up-F / down-R to fragment up, ppbA2 , kan , down and other proportions as a template for overlapping PCR to amplify up-ppsbA- kan-down The fragment (sequence as shown in SEQ ID NO.2) was then connected to the pMD19T vector using T4 ligase and transformed into E. coli TG1. The primers up-F / down-R were then used for PCR detection and verification. After the correct strain was identified and cultured, the plasmid was extracted and the correctly constructed plasmid was named pMD19T- ppabA-kan . With Salmonella typhimurium ( S. Typhimurium )SL1344 single colony was used as template and primer pair HkDJ -F / HkDJ -R amplification HkDJ fragment; pMD19T- ppabA-kan Plasmid and HkDJ Restriction enzymes Bg lII and Spe I was digested and recovered, and then ligated and transformed into Escherichia coli TG1, and the primers ppsb-F / HkDJ -R was used for PCR verification. After the correct strain culture was verified, the plasmid was extracted and used Ppsb seq F sequencing, and the correctly constructed plasmid was named pMD19T- HkDJ The primer sequences are shown in Table 1.

[0033] ; The PCR amplification system is as follows: 2×Ape Mix 25 μL, template DNA 2 μL, primer F 2 μL, primer R 2 μL, ddH2O 19 μL, total 50 μL; The PCR amplification procedure is as follows: Pre-denaturation at 95 °C for 5 min, denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 1 min. After 31 cycles, final extension was performed at 72 °C for 10 min.

[0034] (II) Obtaining MntH transgenic algae strains: (1) Preparation of mixed cellulose ester membrane: Rinse the mixed cellulose ester membrane with distilled water three times, boil it in a glass dish for 5 min, and sterilize it for later use.

[0035] (2) Preparation of algae: Scrape a small amount of Synechocystis PCC6803 from the BG11 plate and culture it in BG-11 liquid culture medium. 730 =0.6-0.8), take 10 mL of algae solution into a 15 mL centrifuge tube and centrifuge at 1800g for 5 min.

[0036] (3) Wash the algal cell pellet three times with fresh BG-11 liquid culture medium and resuspend the algal cells in 400 µL of BG-11 liquid culture medium.

[0037] (4) Add plasmid pMD19T- HkDJ , mix well and incubate at 30°C, 1400 lux for 6-8 h.

[0038] (5) Place a treated mixed cellulose ester membrane on the non-resistant BG-11 solid culture medium, making sure there are no bubbles between the membrane and the plate, and evenly spread 200 µL of incubated algal cells on the surface of the mixed cellulose ester membrane.

[0039] (6) After 18-24 h of light culture, the membrane was transferred to BG-11 solid culture medium containing 50 μg / mL kanamycin and continued to be cultured under light.

[0040] (7) After 10-14 days, pick a single algae colony, streak it on BG-11 solid culture medium containing 50 μg / mL kanamycin and continue culturing.

[0041] (8) Inoculate the streaked algae colony in BG-11 liquid medium containing kanamycin and grow it, extract its DNA, and use specific primers to HkDJ -F / R for PCR testing, such as Figure 1 , amplified HkDJ The algae strain with the gene band indicates that the exogenous target gene has been correctly transferred, and is the MntH transgenic algae strain obtained by screening.

[0042] Example 2 Determination of growth curves of wild-type Synechocystis PCC6803 (WT) and MntH transgenic strain (MntH) under different concentrations of chromium ion stress The MntH transgenic algae strain (MntH) constructed in Example 1 and the wild-type Synechocystis sp. PCC6803 (WT) were inoculated into 10 mL of BG-11 liquid culture medium and then cultured in a light incubator. OD was measured after 10 days. 730 Value, adjust OD 730 The values ​​were consistent and then transferred to 10 mL BG-11 liquid culture medium. 0, 1, 1.75, 2, and 4 mg / L of metal ion Cr were added to the BG-11 liquid culture medium. 6+ , and cultured in a light shaker, with three replicates per group. Ensure that the initial OD 730 The OD was measured every 12 h. 730 The values ​​were calculated and the culture was continued for 10 days to draw the growth curve.

[0043] In order to determine the different concentrations of Cr 6+ Effects on the growth of algae strains were firstly measured at 0, 1, 2, 4, and 8 mg / L Cr 6+ The growth of algae was measured under stress. 6+ The stress had no significant effect on the growth of algae, but 2 mg / L Cr 6+ The stress had a strong inhibitory effect on the growth of algae, so the 6+ The growth of algae strains was measured under stress and it was found that 1.75 mg / L Cr 6+ The stress caused significant growth differences in the algae strains, but the growth inhibition was not too strong, so this concentration was selected for the subsequent measurement of physiological indicators.

[0044] Depend on Figure 2 As can be seen, with Cr 6+ With the increase of Cr concentration, the growth status of wild-type algae and MntH transgenic algae gradually deteriorated. 6+With the increase of concentration, the growth state of MntH transgenic algae strain was better than that of wild-type algae strain. It should be that the expression of exogenous gene MntH helped the algae strain to alleviate the accumulation of ROS caused by heavy metal ion stress.

[0045] Example 3 Wild-type Synechocystis sp. PCC6803 (WT) and MntH transgenic strain (MntH) under 1.75 mg / L Cr 6+ Determination of physiological indicators under stress The contents of total protein, phycobiliproteins (PBP), extracellular polymers (EPS), chlorophyll a (Chlorophyll a), carotenoids (Carotenoids), reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) in the wild-type algae strain and the MntH transgenic algae strain were measured respectively.

[0046] Depend on Figure 3 A and Figure 3 B shows that the total protein content of the wild-type algae is 0.78 mg / L, and the total protein content of the MntH transgenic algae is 0.96 mg / L. The total protein content of the MntH transgenic algae is higher than that of the wild-type algae. It is speculated that the insertion of the exogenous gene MntH has a certain effect on the stress resistance and protein expression of the algae.

[0047] Depend on Figure 3 C shows that the PBP content of the wild-type algae strain is lower, while the PBP content of the MntH transgenic algae strain is higher, indicating that the phycobilisomes of the wild-type algae strain are severely damaged and the PBP content is affected; while the phycobilisomes in the MntH transgenic algae strain are less damaged and the PBP content is higher than that of the wild-type algae strain, which corresponds to the growth condition.

[0048] Depend on Figure 3 D shows that in the heavy metal Cr 6+ Under stress, the difference in extracellular polymers between the wild-type algae and the MntH transgenic algae was small, which led to the speculation that the algae did not alleviate the stress by secreting extracellular polymers, but rather regulated growth by transporting heavy metal ions into the cells and binding to them or changing the intracellular redox state.

[0049] Depend on Figure 4A shows that compared with the MntH transgenic algae, the wild-type algae is in a weaker state of growth and contains less photosynthetic pigments, with a chlorophyll content of 7.29 µg / mL. The photosynthetic pigment content of the MntH transgenic algae is higher, with a chlorophyll content of 9.88 µg / mL, which is 1.36 times that of the wild-type algae.

[0050] Depend on Figure 4 B shows that the carotenoid content of the wild-type algae is 2.43 µg / mL. The carotenoid content of the MntH transgenic algae is 2.87 µg / mL, which is 1.18 times that of the wild-type algae. Under the same culture conditions, the wild-type algae grows slower and the color of the wild-type algae culture fluid is lighter; the MntH transgenic algae has better stress resistance than the wild-type algae, and its photosynthetic pigment content is also higher than that of the wild-type algae.

[0051] Depend on Figure 5 A shows that compared with the wild-type algae, the ROS content of the MntH transgenic algae is significantly reduced, indicating that its stress resistance is significantly improved. When Synechocystis PCC6803 encounters excessive ROS, it activates enzymatic and non-enzymatic protection mechanisms to protect the body from oxidative damage.

[0052] Depend on Figure 5 B shows that there are obvious differences in the superoxide dismutase (SOD) activity between the algae strains. The SOD activity of the wild-type algae strain is 3.48 U / mg, and the SOD activity of the MntH transgenic algae strain is 4.09 U / mL, which is 1.17 times that of the wild-type algae strain. Under heavy metal ion stress, intracellular heavy metal ions accumulate, leading to an increase in ROS in the body, which in turn induces an increase in SOD expression. The expression of the MntH exogenous gene causes heavy metal ions to accumulate faster in the body, thereby promoting the expression of SOD, so the activity of SOD is higher than that of the wild-type algae strain.

[0053] Depend on Figure 5 C shows that the CAT activity of the wild-type strain is lower than that of the MntH transgenic strain. The CAT activity of the wild-type strain is 0.89 U / mg, and the CAT activity of the MntH transgenic strain is 1.11 U / mg, which is 1.25 times that of the wild-type strain. Heavy metal ions cause an increase in ROS content, and the increase in ROS content promotes the expression of SOD, which can convert O 2- Disproportionately converted into hydrogen peroxide; the increase in hydrogen peroxide content causes the increase in CAT expression, and CAT can remove hydrogen peroxide from the body.

[0054] Example 4 Determination of physiological parameters of wild-type Synechocystis PCC6803 and MntH transgenic strains under hydrogen peroxide stress The constructed MntH transgenic algae strain and wild-type Synechocystis PCC6803 were inoculated into 10 mL BG-11 liquid medium at the same time. 0 mmol / L, 1 mmol / L, 2 mmol / L, and 4 mmol / L hydrogen peroxide were added to the BG-11 liquid medium, respectively, and cultured in a light shaker, with three replicates per group. The reactive oxygen species, growth curve, survival rate, and intracellular ion content of wild-type Synechocystis PCC6803 and MntH transgenic algae strains under hydrogen peroxide stress were determined.

[0055] Depend on Figure 6 A shows that with the increase of hydrogen peroxide concentration, the difference in ROS content between the two algae strains was small at first, and then increased; the ROS content of the wild-type algae strain gradually increased with the increase of hydrogen peroxide concentration, while the ROS content of the MntH algae strain changed little, which should be the role of exogenous genes in resisting oxidative stress. Under 4 mM hydrogen peroxide stress, the difference in ROS content between the two algae strains was more obvious, so the subsequent experiments were carried out under 4 mM hydrogen peroxide stress.

[0056] Depend on Figure 6 B shows that under 4 mM hydrogen peroxide stress, there is a slight difference in the growth between the wild-type algae strain and the MntH transgenic algae strain. The growth of the MntH algae strain is better than that of the wild-type algae strain.

[0057] Depend on Figure 6 C shows that under 4 mM hydrogen peroxide stress, the growth of wild-type algae and MntH transgenic algae was inhibited. The survival rate of wild-type algae and MntH transgenic algae was measured after 12 hours of stress. The survival rate of MntH transgenic algae was higher than that of wild-type algae, and its resistance to oxidative stress was better.

[0058] Depend on Figure 6 D shows that the Mn ion content in the MntH transgenic algae is lower than that in the wild-type algae, indicating that MntH helps bacteria transport manganese ions under oxidative stress.

[0059] By inserting foreign genes into the genome of Synechocystis sp. PCC6803 HkDJ , causing changes in the oxidative stress capacity of algal cells, thereby enhancing their ability to resist oxidative stress.

Claims

1. Salmonella Typhimurium ( S. Typhimurium ) The manganese transporter encoding gene MntH of SL1344 was used in the construction of transgenic Synechocystis PCC6803 with enhanced stress resistance ( Synechocystis sp PCC6803).

2. The use according to claim 1, characterized in that The stress resistance includes resistance to heavy metal ion stress and resistance to oxidative stress.

3. The use according to claim 1 or 2, characterized in that: The manganese transporter encoding gene MntH of the Salmonella typhimurium SL1344 is inserted into the genome of Synechocystis PCC6803, thereby improving the stress resistance of Synechocystis PCC6803.

4. The use according to claim 1, characterized in that The nucleotide sequence of the manganese transporter encoding gene MntH of Salmonella typhimurium SL1344 is shown in SEQ ID NO:

1.

5. The use according to claim 1, characterized in that The manganese transporter encoding gene MntH of the Salmonella typhimurium SL1344 is regulated by the promoter PpsbA2.

6. A transgenic Synechocystis sp. PCC6803 with enhanced stress resistance, characterized in that: The gene MntH encoding the manganese transporter of Salmonella typhimurium SL1344 is inserted into the genome of the transgenic Synechocystis PCC6803. The nucleotide sequence of the gene MntH encoding the manganese transporter of Salmonella typhimurium SL1344 is shown in SEQ ID NO:

1.

7. The transgenic Synechocystis PCC6803 according to claim 6, characterized in that: The transgenic Synechocystis PCC6803 is prepared by psbA , MntH gene , Kan The resistance gene and the recombinant fragment of the downstream fragment are obtained by inserting the recombinant fragment into the genome of Synechocystis PCC6803 by homologous recombination.

8. The transgenic Synechocystis PCC6803 according to claim 7, characterized in that: The upstream fragment, promoter P psbA , MntH gene , Kan The nucleotide sequence of the resistance gene and the recombinant fragment of the downstream fragment is shown in SEQ ID NO:

2.

9. A method for constructing the transgenic Synechocystis PCC6803 according to any one of claims 6 to 8, characterized in that: The following steps are involved: (I) Construction of pMD19T-MntH recombinant plasmid: (1) Using the genome of Synechocystis PCC6803 as a template, the primer pairs up-F / up-R and down-F / down-R were used to amplify the upstream homology arm up and the downstream homology arm down fragments respectively; (2) Using plasmid pET-28a as template, use primer pair Kan -F / Kan -R was used to amplify the Kan resistance fragment, and the genome of Synechocystis PCC6803 was used as a template, and the primer pair Ppsb -F / Ppsb -R amplified promoter ppbA2 fragment; then use the primer pair up-F / down-R to fragment up, ppbA2 , kan , down and other proportions as a template for overlapping PCR to amplify up- ppsbA-kan-down The fragment was then connected to the pMD19T vector using T4 ligase and transformed into Escherichia coli TG1; the up-F / down-R primers were then used for PCR detection and verification, and the plasmid was extracted after the correct strain was identified and cultured. The correctly constructed plasmid was named pMD19T- ppabA-kan ; (3) Using a single colony of Salmonella typhimurium SL1344 as a template, the primer pair MntH-F / MntH-R was used to amplify the MntH fragment; the plasmid pMD19T- ppabA-kan and MntH fragments using restriction endonucleases Bg lII and Spe I was digested and recovered, then connected and transformed into Escherichia coli TG1, and primers Ppsb-F / MntH-R were used for PCR detection. After the correct strain culture was detected, the plasmid was extracted and sequenced, and the correctly constructed plasmid was named pMD19T-MntH; The sequences of the primer pairs described above are as follows: ； (II) Obtaining MntH transgenic Synechocystis: (1) Preparation of mixed cellulose ester membrane: Rinse the mixed cellulose ester membrane with distilled water three times, boil it in a glass dish for 5 min, and sterilize it for later use; (2) Preparation of algae: scrape a small amount of Synechocystis PCC6803 from the BG11 plate and culture it in BG-11 liquid culture medium. When the algae liquid grows to the logarithmic phase, take 10 mL of the algae liquid into a 15 mL centrifuge tube and centrifuge it at 1800 g for 5 min. (3) Wash the algal cell pellet three times with fresh BG-11 liquid medium and resuspend the algal cells in 400 µL of BG-11 liquid medium; (4) Add pMD19T- MnH , mix well and incubate at 30°C, 1400 lux for 6-8 h; (5) Place a treated mixed cellulose ester membrane on the non-resistant BG-11 solid culture medium, making sure there are no bubbles between the membrane and the plate, and evenly spread 200 μL of incubated algal cells on the surface of the mixed cellulose ester membrane; (6) After 18-24 h of light culture, the membrane was transferred to BG-11 solid medium containing kanamycin and continued to be cultured under light; (7) After 10-14 days, pick a single algae colony and streak it on BG-11 solid medium containing kanamycin and continue culturing; (8) The streaked single algae colony was inoculated into BG-11 liquid culture medium containing kanamycin for cultivation and growth, and its DNA was extracted. PCR detection was performed using specific primers MntH-F / R. If the algae strain with the MntH gene band was amplified, it indicated that the exogenous target gene was correctly transferred, and the algae was the MntH transgenic Synechocystis obtained by screening.

10. Use of the transgenic Synechocystis PCC6803 according to any one of claims 6 to 8 in the following aspects: (1) Use of the transgenic Synechocystis PCC6803 in constructing a transgenic algae strain with enhanced stress resistance; (2) Application of the transgenic Synechocystis PCC6803 as a model strain in studying the physiological and metabolic functions of microalgae; (3) Application of the transgenic Synechocystis PCC6803 in sewage treatment.

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