Small molecule RNA AsrR9 and application thereof in regulation and control of ammonium salt stress tolerance of synechocystis PCC6803

Abstract

The invention discloses a micromolecule RNA AsrR9 and application thereof in regulation and control of ammonium salt stress tolerance of synechocystis PCC6803, and belongs to the field of industrial microorganisms. According to the invention, AsrR9 is subjected to overexpression, such that an overexpressed algae strain OEAsrR9 is obtained. In a BG11 solid culture medium, the growth state of the OEAsrR9 in the range of 30 mM to 120 mM is obviously superior to that of WT. In a BG11 liquid culture medium added with 90 mM to 150 mM of ammonium salt, illumination culture is carried out for five days, the growth condition of the OEAsrR9 on the third day is obviously superior to that of WT, and the growth condition of the WT is obviously superior to that of an inhibiting algae strain SPAsrR9. The OEAsrR9 obtained by the invention provides a genetic engineering algal strain chassis for constructing algal strains tolerant to various environmental stresses, provides important theoretical support for high-concentration and high-ammonia wastewater treatment and biological energy production by utilizing microalgae in the future, and has practical significance and wide application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of industrial microorganisms, and particularly relates to a small molecule RNA - Ammonium stress - respond RNA 9 (AsrR9) and its application in regulating the ammonium stress tolerance of Synechocystis sp. PCC6803. Background Art

[0002] Cyanobacteria are the ancestors of plant chloroplasts and are ancient photosynthetic autotrophic organisms on earth. As an engineered model organism, Synechocystis sp. PCC6803 can perform efficient homologous recombination between its own genome and foreign DNA, enabling targeted gene regulation and effective functional modification, and is an important chassis organism for the production of high - value compounds.

[0003] Nitrogen sources are essential elements during the growth of cyanobacteria. The most commonly used nitrogen sources are ionic nitrogens such as nitrates, ammonium salts, and urea. Among them, ammonium salts are the preferred nitrogen sources for microalgae. The energy required for cells to assimilate ammonium is relatively low. Ammonium salts can be converted into amino acids such as glutamate, glutamine, and arginine in cyanobacteria through the GS / GOGAT cycle and N - acetylglutamate kinase (NAGK). However, high concentrations of ammonia nitrogen are toxic to microalgae to a certain extent and will inhibit their normal growth and reproduction, which limits the application of microalgae in the treatment of high - concentration ammonium - containing wastewater and the prospects of industrial production.

[0004] There are a large number of regulatory small molecule RNAs in prokaryotes. They were once considered non - coding RNAs of transcriptional noise. With the development of technology, small molecule RNAs have been proven to play a key role in the response and regulation of prokaryotic cells to environmental stress. It has been reported that small molecule RNAs can significantly improve the tolerance of Synechocystis sp. PCC6803 to ethanol - butanol stress. However, the relevant literature and patents on using small molecule RNAs to improve the tolerance of Synechocystis sp. PCC6803 to high - concentration ammonium salts are very limited. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the low ammonium stress tolerance of Synechocystis sp. PCC6803 in the prior art, the primary object of the present invention is to provide a small molecule RNA AsrR9 and its application in regulating the ammonium stress tolerance of Synechocystis sp. PCC6803.

[0006] The second object of the present invention is to provide a method for constructing a Synechocystis sp. PCC6803 strain with improved ammonium stress tolerance.

[0007] The third object of the present invention is to obtain a Synechocystis sp. PCC6803 engineered strain with significantly improved ammonium stress tolerance through the above - mentioned construction method, and this strain can be used as the chassis for constructing a genetically engineered algal strain for purifying high - concentration ammonia wastewater.

[0008] The object of the present invention is achieved by the following technical solutions: A small molecule RNA AsrR9, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0009] Furthermore, the biological material related to the above small molecule RNA AsrR9 is any one or a combination of the following biological materials: (1) An expression cassette containing the above small molecule RNA AsrR9; (2) A recombinant expression vector containing the above small molecule RNA AsrR9; (3) A recombinant expression vector containing the expression cassette described in (1); (4) A recombinant bacterium containing the above small molecule RNA AsrR9; (5) A recombinant bacterium containing the expression cassette described in (1); (6) A recombinant bacterium containing the recombinant expression vector described in (2) or (3); Preferably, the expression cassette described in (1) further contains a promoter and a terminator; the promoter includes the psbA2 promoter, etc.; the terminator includes terminator T biobrickB0015 etc.; Preferably, the nucleotide sequence of the psbA2 promoter is shown in SEQ ID NO: 2; the terminator T biobrickB0015 has a nucleotide sequence shown in SEQ ID NO: 17.

[0010] Preferably, the starting vector of the recombinant expression vectors described in (2) and (3) is a pUC series vector, etc.; pUC118 is used in the present invention.

[0011] Preferably, the recombinant expression vectors described in (2) and (3) further contain a resistance gene; further, the resistance gene is the kanamycin resistance gene Km r and its nucleotide sequence is shown in SEQ ID NO: 19.

[0012] Preferably, the host bacterium of the recombinant bacteria described in (4), (5), and (6) is Synechocystis sp. PCC6803, etc.

[0013] Furthermore, the biological material related to the above small molecule RNA AsrR9 is any one or a combination of the following biological materials: (a) A sequence that inhibits or blocks the expression of the above small molecule RNA AsrR9; (b) A vector that inhibits or blocks the expression of the above small molecule RNA AsrR9 prepared using the sequence described in (a); (c)A Synechocystis sp. PCC6803 strain that inhibits or blocks the expression of the small molecule RNA AsrR9, prepared using the sequence described in (a) or the carrier described in (b).

[0014] Preferably, the sequence described in (a) is an antisense RNA or sgRNA of the small molecule RNA AsrR9.

[0015] Application of the above-mentioned small molecule RNA AsrR9 or its related biological materials in regulating the ammonium salt stress tolerance of Synechocystis sp. PCC6803.

[0016] Overexpression of the small molecule RNA AsrR9 improves the ammonium salt stress tolerance of Synechocystis sp. PCC6803, and inhibition of the effective expression of the small molecule RNA AsrR9 significantly reduces the ammonium salt stress tolerance of Synechocystis sp. PCC6803.

[0017] The ammonium stress mentioned above refers to the stress of high-concentration ammonium salt ions.

[0018] Furthermore, application of the above-mentioned small molecule RNA AsrR9 in constructing a Synechocystis sp. PCC6803 strain with improved ammonium salt stress tolerance.

[0019] Overexpression of the small molecule RNA AsrR9 is regulated by the light-inducible strong promoter psbA2, and the nucleotide sequence is as shown in SEQ ID NO: 2.

[0020] In the present invention, by constructing a recombinant plasmid, integrating the target gene on the recombinant plasmid into the genome of the wild-type (WT) Synechocystis sp. PCC6803 through homologous recombination, screening for stably inherited strains and other techniques, an overexpression strain OEAsrR9 of the small molecule RNA AsrR9 and an inhibitory strain SPAsrR9 of the small molecule RNA AsrR9 are constructed, and the ammonium salt stress tolerances of these two strains are compared with that of the wild-type WT. A small molecule RNA AsrR9 related to ammonium salt stress tolerance is identified, and its sequence is as shown in SEQ ID NO: 1. Overexpression of the small molecule RNA AsrR9 is regulated by the psbA2 promoter, and its sequence is as shown in SEQ ID NO: 2. A strain that can significantly improve ammonium stress tolerance is successfully obtained and named OEAsrR9.

[0021] A method for constructing an overexpression strain OEAsrR9 of a small molecule RNA AsrR9, comprising the following steps: (1) Amplification of the target fragment: Using the genome of wild-type Synechocystis sp. PCC6803 as a template, and using SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing as amplification sll1354Primers for the upstream fragment, SEQ ID NO: 7 and SEQ ID NO: 8 were used as primers for amplifying the small molecule RNA AsrR9, and SEQ ID NO: 11 and SEQ ID NO: 12 were for amplifying sll1354 the downstream fragment, and the amplification yielded sll1354 the upstream fragment, the small molecule RNA AsrR9, and sll1354 the downstream fragment. Using the pJA2 plasmid as a template, SEQ ID NO: 5 and SEQ ID NO: 6 were used as primers for amplifying the promoter psbA2, and SEQ ID NO: 9 and SEQ ID NO: 10 were for amplifying the terminator T biobrickB0015 ; the amplification yielded the promoter psbA2 and the terminator T biobrickB0015 fragment; using the plasmid pET-30b(+) as a template and SEQ ID NO: 13 and SEQ ID NO: 14 as primers, the kanamycin resistance gene Km r was amplified; using the plasmid pUC118 as a template and SEQ ID NO: 15 and SEQ ID NO: 16 as primers, the vector fragment vector-puc118 was amplified; (2) Construction of the recombinant plasmid: The obtained target fragments sll1354 the upstream fragment, the kanamycin resistance gene Km r , the psbA2 promoter, the small molecule RNA AsrR9, the terminator T biobrickB0015 and sll1354 the downstream fragment were ligated to the vector fragment vector-puc118 using seamless cloning enzyme, and all 6 target fragments were identified to be inserted into the vector plasmid pUC118 at the DNA level by PCR amplification technology and sequencing technology, and the recombinant plasmid pUC-OEAsrR9 was successfully constructed; (3) Construction of an overexpression algal strain of the small molecule RNA AsrR9: The recombinant plasmid pUC-OEAsrR9 was introduced into the wild type of Synechocystis PCC6803 by natural transformation, so that the inserted fragment carrying the kanamycin resistance gene km r , the psbA2 promoter, the small molecule RNA AsrR9, and the terminator T biobrickB0015 was integrated into the genome of Synechocystis PCC6803 by homologous double crossover sll1354 gene ( sll1354At the position of the nucleotide sequence of the gene shown in SEQ ID NO: 18, an overexpression algal strain was obtained. Transformants grew on a solid medium containing low-concentration kanamycin sulfate, and the transformants were inoculated into a liquid medium containing low-concentration kanamycin sulfate for culture. The antibiotic concentration in the medium was gradually increased and the overexpression algal strain was subcultured. The transformed algal strain and the wild-type algal strain were amplified and compared using the primers SEQ ID NO: 13 and SEQ ID NO: 10, and it was verified at the DNA level that the inserted fragment was completely integrated into the Synechocystis genome. Finally, an overexpression algal strain that could be stably inherited and the inserted fragment was completely integrated into the Synechocystis PCC6803 genome was obtained and named OEAsrR9.

[0022] (4)High-concentration ammonium salt stress experiment was carried out on the constructed overexpression algal strain OEAsrR9: Wild-type and overexpression algal strain OEAsrR9 with an initial OD 730 of 0.1 were inoculated into a medium containing 90 mM - 240 mM ammonium chloride and continuously cultured for 5 days. The absorbance value (A 730 / OD 730 ) at 730 nm was measured and a full-wavelength scan was performed to plot the growth curve and the whole-cell absorption map, observing the growth rate of the wild-type and overexpression algal strain OEAsrR9 under high-concentration ammonium salt and the peak values of the photosynthetic system pigments phycobiliprotein and chlorophyll, and comparing the growth trends of the two. Synechocystis wild-type and overexpression algal strain OEAsrR9 with an initial OD 730 of 1.0 were inoculated into a solid medium containing various concentrations of ammonium chloride and continuously cultured for 10 days, comparing the growth of the two under different ammonium salt concentrations.

[0023] Through the above construction method and phenotypic experiment comparison, the present invention obtained an overexpression algal strain OEAsrR9 of the small molecule RNA AsrR9 that is tolerant to ammonium salt stress.

[0024] The overexpression algal strain OEAsrR9 of Synechocystis PCC6803 with significantly improved tolerance to high-concentration ammonium salt, its growth and chlorophyll synthesis at an ammonium salt concentration of 90 mM - 150 mM are significantly better than those of the wild-type. This algal strain can be applied to construct a microalgae engineering bacterium tolerant to high-concentration ammonium salt; further, it can be applied to construct a microalgae engineering bacterium for purifying high-concentration ammonia wastewater.

[0025] The present invention has the following advantages and effects compared with the prior art: (1) The present invention uses the newly discovered small molecule RNA AsrR9 in Synechocystis PCC6803 to improve the ammonium salt stress tolerance of Synechocystis at the small molecule RNA level.

[0026] (2) By means of homologous recombination, the present invention overexpresses the small molecule RNA AsrR9 in Synechocystis sp. PCC6803 to obtain an overexpressing algal strain OEAsrR9 with significantly improved tolerance to ammonium stress.

[0027] (3) In BG11 solid medium supplemented with different concentrations (30 mM, 60 mM, 90 mM, 120 mM) of ammonium salts, the growth state of OEAsrR9 is significantly better than that of WT, and the growth state of WT is significantly better than that of the inhibitory algal strain SPAsrR9 of AsrR9.

[0028] (4) In BG11 liquid medium supplemented with 90 mM and 150 mM ammonium salts, under light culture for five days, the growth of the overexpressing algal strain OEAsrR9 is better than that of WT starting from the third day, and it is also better than WT in terms of chlorophyll content, while the growth state of WT is significantly better than that of the inhibitory algal strain SPAsrR9.

[0029] (5) The high ammonium stress-tolerant algal strain obtained by the present invention and its construction method provide a chassis of genetically engineered algal strains for constructing algal strains tolerant to environmental stress, and provide important theoretical support for future treatment of high-concentration high-ammonia wastewater and production of bioenergy using microalgae, having practical significance and broad application prospects. Description of the Drawings

[0030] Figure 1 is a schematic structural diagram of the pUC-OEAsrR9 plasmid.

[0031] Figure 2 is a schematic diagram of homologous double exchange between the overexpression element of the small molecule RNA AsrR9 and the wild-type genome of Synechocystis sp. PCC6803.

[0032] Figure 3 is a spot plate picture of the overexpressing algal strain OEAsrR9 of the small molecule RNA AsrR9, the inhibitory algal strain SPAsrR9, and the wild-type algal strain WT on the 10th day of solid culture under different concentrations of ammonium stress.

[0033] Figure 4 is a picture of the algal liquid of the overexpressing algal strain OEAsrR9 of the small molecule RNA AsrR9, the inhibitory algal strain SPAsrR9, and the wild-type algal strain WT under continuous five-day liquid culture with 90 mM, 150 mM, and 240 mM ammonium stress.

[0034] Figure 5 is a whole-cell absorption map of the overexpressing algal strain OEAsrR9 of the small molecule RNA AsrR9, the inhibitory algal strain SPAsrR9, and the wild-type algal strain WT on the fifth day under 90 mM, 150 mM, and 240 mM ammonium stress. Detailed Embodiments

[0035] The present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0036] The wild-type strain of Synechocystis sp. PCC6803 used in the embodiments of the present invention was isolated and purified from ATCC27184. ATCC is the abbreviation of the American Type Culture Collection, and 27184 is the strain number. The plasmid pUC118 was purchased from Takara, pET-30b(+) was purchased from Novagen, and pJA2 was derived from the laboratory's own collection. pJA2 was disclosed in the literature "Sun T, Chen L, Zhang WW. 2017. Quantitative Proteomics Reveals Potential Crosstalk between a Small RNA CoaR and a Two-Component Regulator Slr1037 in Synechocystis sp PCC6803. Journal of Proteome Research 16:2954-2963."

[0037] Example 1 Construction of the recombinant plasmid pUC-OEAsrR9, including amplification of the target fragment, ligation of the fragments by seamless cloning method, transformation, and identification and verification of the recombinant plasmid.

[0038] Using the genome of wild-type Synechocystis sp. PCC6803 as a template, SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing were used as sll1354 primers for the upstream fragment, SEQ ID NO: 7 and SEQ ID NO: 8 were used as primers for the small molecule RNA AsrR9, and SEQ ID NO: 11 and SEQ ID NO: 12 were sll1354 primers for the downstream fragment, and the upstream fragment, small molecule RNA AsrR9, and sll1354 downstream fragment were amplified. Using the pJA2 plasmid as a template, SEQ ID NO: 5 and SEQ ID NO: 6 were used as primers for the promoter psbA2, and SEQ ID NO: 9 and SEQ ID NO: 10 were primers for the terminator T sll1354 fragment, and the promoter PpsbA2 and terminator T biobrickB0015 fragment were amplified. Using the plasmid pET-30b(+) as a template, and using SEQ ID NO: 13 and SEQ ID NO: 14 as primers, the kanamycin resistance gene km biobrickB0015 was amplified. rUsing plasmid pUC118 as a template and SEQ ID NO: 15 and SEQ ID NO: 16 as primers, the vector fragment vector-puc118 was amplified.

[0039] The PCR product was electrophoresed on a 1.5% agarose gel. After cutting out the target band with the same theoretical length, a DNA purification kit was used for collection and purification to obtain the DNA product of the target band.

[0040] Construct a recombinant plasmid by using the purified fragment DNA product obtained above: sll1354 The upstream fragment, kanamycin resistance gene km r , psbA2 promoter, small molecule RNA AsrR9, terminator T biobrickB0015 and sll1354 The downstream fragment was ligated to the vector fragment vector-puc118 using seamless cloning enzyme (Jiangsu Kangwei Century Biotechnology Co., Ltd.). After the seamless cloning reaction was completed, the recombinant product was transferred into competent Escherichia coli DH5α and cultured in LB liquid medium. The culture product was spread on LB solid medium containing 50 µg / mL kanamycin sulfate to screen out the transformants with resistance. This transformant carried the successfully ligated recombinant plasmid.

[0041] Identify the ligation of the target fragment at the DNA level and sequencing level of the recombinant plasmid. The transformants with resistance were individually picked into 1 mL of LB liquid medium containing 50 µg / mL kanamycin sulfate and cultured at 37 °C for 4 h. Take 1 µL as the PCR bacterial liquid DNA template, and identify the transformants in which all 6 target fragments were inserted into the vector plasmid pUC118 at the DNA level by PCR amplification technology and sequencing technology to construct the recombinant plasmid pUC-OEAsrR9. The structural schematic diagram of the plasmid is as Figure 1 shown. Collect the transformant bacterial liquid for plasmid extraction and use it for subsequent steps.

[0042] The PCR reaction system for the target fragment used in the present invention is: 1 µL of DNA template, 25 µL of super PCR enzyme mix, 2.5 µL of 10 µM forward primer, 2.5 µL of 10 µM reverse primer, and 22 µL of ddH2O to make up 50 µL.

[0043] The PCR reaction conditions for the target fragment used in the present invention are: pre-denaturation at 94 °C for 3 min; denaturation at 98 °C for 10 s; annealing for 15 s, and the temperature is generally 5-10 °C lower than the primer Tm value; extension at 72 °C, 1 min is required for every 1 kb of DNA amplification, 38 cycles; extension at 72 °C for 5 min.

[0044] The seamless cloning system used in the present invention is: sll1354Upstream fragment, kanamycin resistance gene Km r , psbA2 promoter, small molecule RNA AsrR9, terminator T biobrickB0015 and sll1354 1 μL of each of the downstream fragment, 1 μL of pUC118 vector, 5 μL of seamless cloning enzyme, and 8 μL of ddH2O were added to make up a reaction system of 20 μL.

[0045] The reaction conditions of the seamless cloning system used in the present invention were: seamless cloning at 55°C for 30 min and termination of the reaction at 8°C for 5 min.

[0046] Example 2 Obtaining the overexpressing algal strain OEAsrR9 included homologous double exchange between the recombinant plasmid pUC-OEAsrR9 and the wild-type genome of Synechocystis PCC6803, and screening of the overexpressing algal strain OEAsrR9.

[0047] Take 30 mL of wild-type Synechocystis PCC6803 in the logarithmic growth phase, centrifuge at 6000 rpm for 5 min, remove the supernatant, wash the centrifuged product with fresh BG11 liquid medium, repeat once, and then resuspend the algal sludge in 1 mL of BG11 liquid medium in a sterile 2 mL centrifuge tube. Add 200 μL of the recombinant plasmid pUC-OEAsrR9 filtered through a 0.22 μm filter membrane, mix well, and place it in a light incubator (30°C, 150 rpm, 1400 Lux) for 12 h. The recombinant plasmid pUC-OEAsrR9 was integrated into the genome of Synechocystis PCC6803 by means of Figure 2 the method with the help of homologous arms sll1354 upstream fragment and downstream fragment. Then spread the algal solution on a mixed fiber filter membrane on BG11 solid medium without antibiotics. The fiber filter membrane needs to be sterilized before use. After solid culture for 24 h, transfer the mixed fiber membrane to BG11 solid medium containing 10 μg / mL kanamycin sulfate, place it upright in a light incubator, and after several weeks, when single algal colonies grow on the medium, they are transformants. Inoculate the transformants into a new BG11 liquid medium containing 10 μg / mL kanamycin sulfate and culture until OD 730 = about 1.0, and then inoculate into a new BG11 liquid medium containing 20 μg / mL kanamycin sulfate, and so on until the antibiotic concentration in the medium is 50 μg / mL. Each time the antibiotic is increased, take 50 μL of the algal solution into a PCR tube, heat at 85°C for 30 s, freeze in liquid nitrogen for 5 s, repeat 5 times, briefly centrifuge and take 1 μL of the supernatant as a template for PCR to verify at the DNA level whether the target fragments Km r , psbA2, AsrR9, T biobrickB0015 were completely homologously recombined and inserted into the target position of Synechocystis PCC6803.

[0048] The algal solution with correct DNA - level verification and antibiotic concentration screened to 50 µg / mL was streaked onto BG11 solid medium containing 50 µg / mL kanamycin. After single colonies grew out, single colonies were picked and cultured in BG11 liquid medium containing 50 µg / mL kanamycin to obtain the over - expression strain OEAsrR9 of the small - molecule RNA AsrR9.

[0049] Preparation of BG11 liquid medium: Measure 1 L of deionized water with a graduated cylinder, and successively add 1 mL of each of the 1000× stock solutions of K2HPO4, MgSO4·7H2O, CaCl2·2H2O, citric acid, ammonium ferric citrate, ethylenediaminetetraacetic acid (EDTA), Na2CO3, and Trace MetaL A5 Mix. Then add 1.5 g of solid NaNO3 and mix well. Dispense every 100 mL of BG11 liquid medium into 250 - mL conical flasks. Seal with sealing film and sterilize at 121℃, 1.1 MPa for 30 min.

[0050] BG11 solid medium: Dispense every 100 mL of BG11 liquid medium into 250 - mL conical flasks, add 1.5 g of agar powder, seal, and sterilize at 121℃, 1.1 MPa for 30 min.

[0051] Culture of Synechocystis: Before each inoculation of the liquid medium, 1 mL of HEPES (4 - hydroxyethylpiperazineethanesulfonic acid) buffer is added per 50 mL. Before each culture of the solid medium, 2 mL of HEPES is added per 100 mL, with a final concentration of 0.3% NaS2SO3 and a final concentration of 8 mM TES (tris - hydroxymethylaminomethaneethanesulfonic acid). The solid medium is cultured in a light incubator at a temperature of 30℃ and a light intensity of 60 µmoL·m -2 ·s -1 and light intensity of 60 µmoL·m·s. During the culture process, the corresponding antibiotics need to be added.

[0052] The PCR reaction system used for verification in the present invention is: 1 µL of DNA template, 12.5 µL of PCR mix, 1 µL of 10 µM forward primer, 1 µL of 10 µM reverse primer, and 9.5 µL of ddH2O to make up 25 µL.

[0053] The PCR reaction conditions used for verification in the present invention are: pre - denaturation at 94℃ for 3 min; denaturation at 98℃ for 10 s; annealing for 15 s, and the temperature is generally 5 - 10℃ lower than the primer Tm value; extension at 72℃, and it takes 30 s for every 1 kb of DNA amplification; 30 cycles; extension at 72℃ for 5 min.

[0054] Example 3 The method comprises the steps of constructing a recombinant plasmid pUC-SPAsrR9, performing homologous double exchange between the recombinant plasmid pUC-SPAsrR and the wild-type genome of Synechocystis sp. PCC6803, and screening the inhibitory algae strain SPAsrR9.

[0055] Construction of recombinant plasmid pUC-SPAsrR9: Using the genome of wild-type Synechocystis PCC6803 as a template and SEQ ID NO: 20 and SEQ ID NO: 21 in the sequence table as primers, PCR amplification was performed to obtain the antisense fragment AsrR9-as of the small molecule RNA AsrR9, which is completely reverse complementary to the small molecule RNA AsrR9. Referring to Example 1, the fragment amplified and purified by PCR: sll1354 Upstream fragment, kanamycin resistance gene km r , psbA2 promoter, AsrR9-as, terminator TbiobrickB0015 and sll1354 The downstream fragment was connected to the vector fragment vector-puc118 using seamless cloning enzyme (Jiangsu Kangwei Century Biotechnology Co., Ltd.). After the seamless cloning reaction was completed, the recombinant product was transformed into competent Escherichia coli DH5α and cultured in LB liquid medium. The culture product was spread on LB solid medium with 50 µg / mL kanamycin sulfate, and the resistant transformants were screened. The successful construction of pUC-SPAsrR9 was confirmed by sequencing.

[0056] pUC-SPAsrR9 was subjected to homologous double exchange with the wild-type genome of Synechocystis PCC6803 to screen for the inhibitory algae strain SPAsrR9. Take 30 mL of wild-type Synechocystis PCC6803 in the logarithmic growth phase, centrifuge at 6000 rpm for 5 min, remove the supernatant, and wash the centrifuged product with fresh BG11 liquid culture medium, repeat once, and then resuspend the algae mud in a sterile 2 mL centrifuge tube with 1 mL of BG11 liquid culture medium, add 200 µL of recombinant plasmid pUC-SPAsrR9 filtered through a 0.22 µm filter membrane, mix well, and place in a light incubator (30 ° C, 150 rpm, 1400 Lux) for 12 hours. pUC-SPAsrR9 uses homology arms sll1354The upstream fragment and the downstream fragment were integrated into the genome of Synechocystis sp. PCC6803. Subsequently, the algal solution was spread on a mixed fiber filter membrane on BG11 solid medium without antibiotics. The fiber filter membrane needs to be sterilized before use. After 24 h of solid culture, the mixed fiber membrane was transferred to BG11 solid medium containing 10 μg / mL kanamycin sulfate and cultured upright in a light incubator. After several weeks, when single algal colonies grew on the medium, they were the transformants. The transformants were inoculated into fresh BG11 liquid medium containing 10 μg / mL kanamycin sulfate and cultured until OD 730 reached about 1.0, and then inoculated into fresh BG11 liquid medium containing 20 μg / mL kanamycin sulfate, and so on until the antibiotic concentration in the medium was 50 μg / mL. Each time the antibiotic concentration was increased, 50 μL of the algal solution was taken into a PCR tube, incubated at 85 °C for 30 s, snap-frozen in liquid nitrogen for 5 s, repeated 5 times, and after brief centrifugation, 1 μL of the supernatant was taken as the template for PCR to verify the target fragments Km r , psbA2, AsrR9-as, T biobrickB0015 at the DNA level to check whether they were completely homologous recombinantly inserted into the target position of Synechocystis sp. PCC6803.

[0057] The algal solution that was verified correctly at the DNA level and had been screened to an antibiotic concentration of 50 μg / mL was streaked onto BG11 solid medium containing 50 μg / mL kanamycin. After single algal colonies grew, single algal colonies were picked and cultured in BG11 liquid medium containing 50 μg / mL kanamycin to obtain the algal strain SPA SrR9.

[0058] Example 4 Phenotypic comparison of wild-type WT of Synechocystis sp. PCC6803, the overexpression algal strain OEAsrR9 of the small molecule RNA AsrR9, and the inhibitory algal strain SPAsrR9 of the small molecule RNA AsrR9 under high-concentration ammonium salt stress.

[0059] Liquid culture under different concentrations of ammonium salt stress: The wild-type WT, the overexpression algal strain OEAsrR9, and the inhibitory algal strain SPAsrR9 that had grown to the logarithmic growth phase were used as the seed solutions and inoculated into 25 mL of BG11 liquid medium containing different concentrations of ammonium chloride. Three bottles were inoculated for each algal strain, and the initial OD 730 of each bottle was 0.1. OD 730 was measured every 24 h of culture to plot the growth curves of the algal strains, and photos were taken every day for record. On the fifth day of culture, the algal solution was scanned at wavelengths from 400 nm to 800 nm to plot the whole-cell absorption spectrum. The above culture conditions were continuous light culture at 30 °C, 150 rpm, and 1400 Lux.

[0060] For solid culture under ammonium salt stress at different concentrations, the wild-type WT, overexpression strain OEAsrR9, and inhibition strain SPAsrR9 that had grown to the logarithmic growth phase were used as seed solutions. The algal solution was diluted to OD 730 of 1.0 with BG11, and then the algal solution was diluted to 10 -1 , 10 -2 , 10 -3 , 10 -4 according to a concentration gradient. The algal solutions at five concentrations were inoculated onto BG11 solid media containing different concentrations (0 mM, 15 mM, 30 mM, 60 mM, 90 mM, 120 mM, and 150 mM) of ammonium chloride. Multiple parallels were set for each concentration on each plate, and 5 μL of the algal solution was spotted at each point on the plate. The plates were cultured in a light incubator at 30 °C with a light intensity of 30 μmol·m -2 ·s -1 for 10 days, cultured in an inverted position, and photographed for recording.

[0061] Figure 3 Figure shows the spotted plate pictures on the tenth day of solid culture under different concentrations of ammonium salt stress. As can be seen from the figure, under ammonium salt stress of 30 mM, 60 mM, 90 mM, and 120 mM, the growth of the overexpression strain OEAsrR9 was significantly better than that of the WT. At 60 mM, OEAsrR9 could still grow when the algal solution was diluted to 10 -3 concentration, while the WT hardly grew after dilution. At 90 mM and 120 mM, the overexpression strain OEAsrR9 still had growth. Although the color was lighter, it still showed better performance compared to the complete non-growth of the WT. The growth of the inhibition strain SPAsrR9 on the solid medium was significantly weaker than that of the WT and OEAsrR9 after adding ammonium salt stress and could not grow on the solid medium under high-concentration ammonium salt stress. This indicates that the overexpression strain OEAsrR9 showed better growth adaptation than the wild-type WT under high-concentration ammonium salt stress.

[0062] Figure 4 Figure shows the pictures of the algal solutions in liquid culture under 90 mM, 150 mM, and 240 mM ammonium chloride. As can be seen from the figure, under high concentrations of ammonium salts of 90 mM and 150 mM, the algal solution concentration and color of the overexpression strain OEAsrR9 were similar to those of the wild-type WT in the first two days, but the color became significantly greener than that of the wild-type WT on the third day, indicating that the overexpression strain OEAsrR9 had less damage to the photosynthetic system caused by high ammonium salt stress. The algal solution of the inhibition strain SPAsrR9 turned white, suggesting serious damage to the photosynthetic system. This shows that under high ammonium salt stress, the overexpression strain OEAsrR9 showed better growth than the wild-type WT, and under long-term high-concentration ammonium salt stress, the overexpression strain OEAsrR9 had more advantages in growth and adaptability than the wild-type WT.

[0063] Figure 5This is the whole-cell absorption spectrum on the fifth day of high ammonium salt stress. As can be seen from the figure, under high-concentration ammonium salt stress of 90 mM, 150 mM, and 240 mM, the chlorophyll peaks of the overexpressing algal strain OEAsrR9 at ammonium salt concentrations of 90 mM and 150 mM are higher than those of the WT. However, the chlorophyll and phycobiliprotein peaks of the inhibitory algal strain SPAsrR9 are lower than those of the wild-type WT and the overexpressing algal strain OEAsrR9, indicating that overexpressing the small molecule RNA AsrR9 can protect the photosynthetic system of cells under high ammonium salt stress, with less damage than the wild-type WT. In contrast, the inhibitory algal strain SPAsrR9 shows obvious and severe damage to the photosynthetic system under high ammonium salt stress, and the photosynthetic pigments are severely damaged.

[0064] In summary, the overexpressing algal strain OEAsrR9 of the small molecule RNA AsrR9 is more tolerant to high-concentration ammonium salt stress than the wild-type WT and the inhibitory algal strain SPAsrR9, and has better phenotypes and growth rates under long-term high-concentration ammonium salt stress, showing a growth advantage over the WT.

[0065] The above embodiments are preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A small molecule RNA AsrR9, characterized in that, The nucleotide sequence of AsrR9 is shown in SEQ ID NO:

1.

2. The biomaterial related to the small molecule RNA AsrR9 according to claim 1, characterized in that, It is any one or more combinations of the following biological materials: (1) An expression cassette containing the small molecule RNA AsrR9 described in claim 1; (2) A recombinant expression vector containing the small molecule RNA AsrR9 described in claim 1; (3) A recombinant expression vector containing the expression cassette described in (1); (4) A recombinant bacterium containing the small molecule RNA AsrR9 described in claim 1; (5) A recombinant bacterium containing the expression cassette described in (1); (6) A recombinant bacterium containing the recombinant expression vector described in (2) or (3).

3. The biological material according to claim 2, wherein: The expression cassette described in (1) further contains a promoter and a terminator; further, the promoter includes the psbA2 promoter, and its nucleotide sequence is as shown in SEQ ID NO: 2; the terminator includes terminator T biobrick B0015 , and its nucleotide sequence is as shown in SEQ ID NO:

17.

4. The biomaterial related to the small molecule RNA AsrR9 according to claim 1, characterized in that, It is any one or more combinations of the following biological materials: (a) A sequence that inhibits or blocks the expression of the small molecule RNA AsrR9 described in claim 1; (b) A vector that inhibits or blocks the expression of the small molecule RNA AsrR9 described in claim 1 prepared using the sequence described in (a); (c) A Synechocystis PCC6803 strain that inhibits or blocks the expression of the small molecule RNA AsrR9 described in claim 1 prepared using the sequence described in (a) or the vector described in (b).

5. The application of the small molecule RNA AsrR9 described in claim 1 or the biological material related to the small molecule RNA AsrR9 described in any one of claims 2 to 4 in regulating the ammonium stress tolerance of Synechocystis PCC6803, wherein: Overexpressing the small molecule RNA AsrR9 improves the ammonium stress tolerance of Synechocystis PCC6803, and inhibiting the effective expression of the small molecule RNA AsrR9 reduces the ammonium stress tolerance of Synechocystis PCC6803.

6. The application of the small molecule RNA AsrR9 described in claim 1 or the biological material related to the small molecule RNA AsrR9 described in any one of claims 2 to 3 in constructing a Synechocystis PCC6803 strain with improved ammonium stress tolerance.

7. A method for constructing a Synechocystis sp. PCC6803 strain with improved tolerance to ammonium salt stress, characterized in that: Based on Synechocystis PCC6803, overexpressing the small molecule RNA AsrR9 described in claim 1 to obtain an overexpressing strain, that is, a Synechocystis PCC6803 strain with improved ammonium stress tolerance.

8. The construction method according to claim 7, characterized in that, It includes the following steps: Integrate the insertion fragment carrying the kanamycin resistance gene km r , psbA2 promoter, small molecule RNA AsrR9, and terminator T biobrick B0015 into the genomic locus of Synechocystis sp. PCC6803 by means of homologous double crossover and natural transformation sll1354 to finally obtain an overexpression algal strain that can be stably inherited and the insertion fragment is completely integrated into the genome of Synechocystis sp. PCC6803, named OEAsrR9; Among them, the kanamycin resistance gene km r has a nucleotide sequence as shown in SEQ ID NO: 19; the nucleotide sequence of the psbA2 promoter is as shown in SEQ ID NO: 2, and the terminator T biobrick B0015 has a nucleotide sequence as shown in SEQ ID NO:

17.

9. A Synechocystis sp. PCC6803 strain with improved tolerance to ammonium salt stress, characterized in that, It is constructed by the construction method described in claim 7 or 8.

10. Use of the Synechocystis sp. PCC6803 strain with improved tolerance to ammonium salt stress according to claim 9, characterized in that, The Synechocystis PCC6803 strain with improved ammonium stress tolerance is applied to construct a microalgae engineering bacterium tolerant to high-concentration ammonium salts; and further applied to construct a microalgae engineering bacterium for purifying high-concentration ammonia wastewater.

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