CN109022285B - A kind of method and application of improving ammonium salt tolerance of Synechocystis PCC6803 - Google Patents

Abstract

The invention discloses a method and application for improving the ammonium salt tolerance of Synechocystis sp. PCC6803, and belongs to the field of industrial microorganisms. By applying this method, the Synechocystis PCC6803 overexpressing algal strain OE0528 with significantly improved ammonium salt tolerance was obtained, and the algal strain can be used to construct a genetically engineered strain for purification of high-concentration ammonium salt wastewater. The method of the present invention is to overexpress the sll0528 gene in Synechocystis PCC6803, and the obtained Synechocystis PCC6803 overexpressed algal strain OE0528 has significantly improved tolerance to ammonium salts. Under the stress of 180-210 mM ammonium salt, the growth state of the overexpressed algal strain was obviously better than that of the wild type. The ammonium salt-tolerant overexpression algae strain obtained by the invention has important theoretical and practical significance for further construction of genetically engineered bacteria utilizing high-concentration ammonium salts, and has broad application prospects.

Description

Method for improving tolerance capacity of Synechocystis PCC6803 ammonium salt and application thereof

Technical Field

The invention belongs to the field of industrial microorganisms, and particularly relates to a method for improving tolerance capacity of synechocystis PCC6803 to high-concentration ammonium salt and application thereof.

Background

Ammonium salts are the main nutritional elements of microalgae and are also metabolic intermediates that are ubiquitous in microalgae. However, the ammonium salt with high concentration has toxic effect on most microalgae, which limits the survival range and application prospect of the microalgae, such as wastewater purification with higher concentration of ammonium salt. Therefore, the research on the method and the application for improving the tolerance of the synechocystis PCC6803 to the high-concentration ammonium salt has important significance for developing the industrial production and the application of the synechocystis.

The genetic modification of synechococcus PCC6301 of cyanobacteria improves the tolerance of the synechococcus PCC6301 ammonium salt by introducing escherichia coli glutamate dehydrogenase. However, the literature and the patent for improving the tolerance capability of the synechocystis PCC6803 ammonium salt are not reported. Meanwhile, the function and mechanism of the S2P (site 2) protease Sll0528 in synechocystis PCC6803 participating in ammonium salt stress adaptation are not reported.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a method for improving the tolerance of Synechocystis PCC6803 to high-concentration ammonium salt. The synechocystis PCC6803 overexpression algal strain OE0528 with obviously improved ammonium salt tolerance is obtained by the method and can be used for constructing a genetic engineering strain for purifying high-concentration ammonium salt wastewater.

The purpose of the invention is realized by the following technical scheme:

a method for improving tolerance of Synechocystis PCC6803 to high-concentration ammonium salts comprises the following steps:

the invention constructs an synechocystis PCC6803 over-expression algal strain OE0528 tolerant to high-concentration ammonium salt by solving the technical problems of constructing recombinant plasmids, carrying out homologous double exchange on the recombinant plasmids and synechocystis PCC6803 Wild Type (WT) genome, screening over-expression algal strains, culturing the synechocystis high-concentration ammonium salt and the like, and aims to provide a method for improving the tolerance of microalgae to the high-concentration ammonium salt, so that the method is applied to constructing engineering bacteria tolerant to the high-concentration ammonium salt. The method specifically comprises the following steps:

(1) construction of recombinant plasmid P3031P 0K: using wild genomic DNA of synechocystis PCC6803 as a template, and carrying out PCR amplification by using primer pairs of SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 to obtain target fragments of slr2030, psbA2 promoter, sll0528 and slr2031, wherein the target fragment gene sequences are shown as SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO. 19; using plasmid pET-30b (+) as template, using primer pair SEQ ID NO.3 and SEQ ID NO.4, PCR amplifying to obtain kanamycin-km resistance^rThe fragment has a gene sequence shown as SEQ ID NO. 16; sequentially shearing the obtained target fragment by using restriction endonucleases Hind III and Pst I, Pst I and Xba I, Xba I and Sma I, Sma I and EcoRI, Pst I and Pst I, simultaneously sequentially shearing a vector plasmid pUC118 by using the same restriction endonucleases, and sequentially connecting the target fragment and the vector plasmid by using T4DNA Ligase to construct a recombinant plasmid P3031P 0K; finally, the PCR amplification technology and the sequencing technology are used for identifying that the 5 target fragments are all inserted into the vector plasmid pUC118 at the DNA level;

(2) homologous double exchange of recombinant plasmid P3031P0K and synechocystis PCC6803 wild type genome, and screening of over-expression algaeStrain: introducing the constructed recombinant plasmid P3031P0K into Synechocystis PCC6803 wild type body, and performing homologous double-crossover to allow overexpression component km^rInserting the promoter + sll0528 of the psbA2 into a neutral site between slr2030 and slr2031 of a wild type gene of synechocystis PCC6803 to obtain an over-expression strain; after a transformant grows on a solid culture medium containing low-concentration kanamycin sulfate, transferring the transformant to a liquid culture medium containing low-concentration kanamycin sulfate for culture, gradually increasing the concentration of the antibiotic in the culture medium and carrying out passage on an over-expressed strain; and identifying at DNA level, using primer pair SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, using wild type and over-expression strain genomes as templates, PCR amplifying over-expression elements inserted into the genome or neutral sites between slr2030 and slr2031, and identifying whether the neutral sites are completely replaced by the over-expression elements; finally obtaining an over-expression strain with neutral sites completely replaced by over-expression elements and capable of stably inheriting, and the over-expression strain is named as OE 0528;

(3) and (3) carrying out a high-concentration ammonium salt stress experiment on the constructed over-expression strain OE 0528: starting OD₇₃₀Synechocystis wild type and overexpressed algal strain OE0528, 0.1, were inoculated in media containing various concentrations of ammonium chloride, cultured continuously for 5 days, and the absorbance value at 730nm was measured (A)₇₃₀/OD₇₃₀) Or performing full-wavelength scanning, drawing a growth curve and a whole-cell absorption graph, observing the growth speed of the wild type and the over-expressed algal strain OE0528 under high-concentration ammonium salt and the peak values of pigments phycobiliprotein and chlorophyll of the photosynthetic system, and comparing the growth trends of the wild type and the over-expressed algal strain OE 0528.

Synechocystis PCC6803 overexpression strain OE0528 with remarkably improved tolerance to high-concentration ammonium salt is obtained by the method.

The synechocystis PCC6803 over-expression strain OE0528 constructed by the method has obviously improved tolerance to ammonium salt, and the growth state of the synechocystis PCC6803 over-expression strain OE0528 in a BG11 culture medium containing ammonium salt (ammonium chloride) of 180-210 mM is obviously superior to that of the wild type synechocystis PCC 6803.

The synechocystis PCC6803 overexpression strain OE0528 with obviously improved tolerance to high-concentration ammonium salt can be applied to construction of microalgae engineering bacteria tolerant to high-concentration ammonium salt.

Compared with the prior art, the invention has the following advantages and effects:

according to the method, the tolerance of the obtained synechocystis PCC6803 over-expression algal strain OE0528 to ammonium salt is remarkably improved through over-expression of sll0528 genes in the synechocystis PCC 6803. Under the stress of 180-210 mM ammonium salt, the growth state of the over-expression strain is obviously superior to that of a wild type. The ammonium salt tolerance overexpression strain obtained by the invention has important theoretical and practical significance for further constructing genetic engineering bacteria utilizing high-concentration ammonium salt, and has wide application prospect.

Drawings

FIG. 1 is a schematic diagram of the structure of the P3031P0K plasmid.

FIG. 2 is a schematic representation of the homologous double crossover of the overexpression elements with the Synechocystis PCC6803 wild-type genome, along with the respective primer action sites.

FIG. 3 is an agarose electrophoresis picture of the PCR-technical identification of recombinant plasmid P3031P0K by the primer pairs SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO. 10; wherein, lane M: marker; lanes 1, 3, 5, 9: synechocystis PCC6803 wild type genome is taken as a template; lane 7: pET-30b (+) is taken as a template; lanes 2, 4, 6, 8, 10: the recombinant plasmid P3031P0K was used as a template.

FIG. 4 is an agarose electrophoresis picture of the identification of over-expressed algal strain OE0528 at the DNA level; wherein, the A picture is an agarose electrophoresis picture of the genome of wild type and over-expressed alga strain OE0528 by PCR amplification of the primer pair SEQ ID NO.11 and SEQ ID NO. 12; lane M: marker, lane B: blank, lane W: wild-type genome as template, lane 1: the genome of an over-expressed algal strain OE0528 is taken as a template. Panel B is an agarose electrophoresis picture of the PCR amplification of wild type and over-expressed algal strain OE0528 genomes with primer pair SEQ ID NO.13 and SEQ ID NO. 14; lane M: marker, lane B: blank, lane W: wild-type genome as template, lane 1: the genome of an over-expressed algal strain OE0528 is taken as a template.

FIG. 5 is a graph of the growth of Synechocystis PCC6803 wild-type and over-expressed algal strain OE0528 under multiple concentrations of ammonium chloride stress.

FIG. 6 is a photograph of algal fluid from synechocystis PCC6803 wild type and over-expressed algal strain OE0528 at day 5 under stress of 180mM ammonium chloride.

FIG. 7 is a graph of whole cell uptake of synechocystis PCC6803 wild type and over-expressed algal strain OE0528 at day 5 under stress of 180mM ammonium chloride.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Synechocystis PCC6803 wild-type strain used in the examples of the present invention was isolated and purified from ATCC27184(ATCC is an abbreviation for American type culture Collection, 27184 is the strain number), Escherichia coli DH10B was obtained from Takara, vector plasmid pUC118 was obtained from Takara, and pET-30b (+) was obtained from Novagen.

Example 1

The construction of the recombinant plasmid P3031P0K comprises the amplification of a target gene segment, the restriction enzyme digestion linkage of the target gene segment and a carrier plasmid, and the identification of the recombinant plasmid.

PCR amplification is carried out by taking primer pair SEQ ID NO.1 and SEQ ID NO.2 in the sequence table and synechocystis PCC6803 wild type genome DNA (bacterial genome extraction kit) as a template to obtain slr2030 fragment, wherein the sequence of the fragment is shown as SEQ ID NO. 15. Meanwhile, restriction endonucleases Hind III and Pst I are used for double digestion of the target fragment slr2030 and the vector plasmid pUC118, and then the target fragment slr2030 and the vector plasmid pUC118 are connected by using a ligase to form a recombinant plasmid pUC 118-2030. The recombinant plasmid pUC118-2030 is introduced into competent Escherichia coli DH10B for replication, the Escherichia coli grows in a solid culture medium and a liquid culture medium for 16h and 8h in sequence, and then a bacterium solution is collected to extract plasmids (a plasmid miniextraction kit) for subsequent experiments. According to the method, the remaining target segments are sequentially km^rThe psbA2 promoter, sll0528, slr2031 was inserted into recombinant plasmid pUC 118-2030. PCR amplification of target fragment and target fragment sequence by using primer pairs SEQ ID NO.3 and SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 in sequence and using plasmid pET-30b (+) and Synechocystis PCC6803 wild type genome as templateThe recombinant plasmid P3031P0K is prepared by sequentially digesting each target fragment and plasmid with restriction enzymes Pst I and Pst I, Pst I and Xba I, Xba I and Sma I, Sma I and EcoRI respectively, and ligating the target fragments into plasmid pUC118 by ligase, as shown in SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO. 19. The structure of the P3031P0K plasmid is shown in FIG. 1.

DNA level and sequencing level identification were performed on recombinant plasmid P3031P 0K. DNA level identification means that a primer pair SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are used, recombinant plasmid P3031P0K and WT genome are respectively used as templates, and target fragments slr2030, psbA2 promoter, sll0528 and slr2031 are amplified; amplifying a target fragment km by using primer pairs SEQ ID NO.3 and SEQ ID NO.4 and recombinant plasmids P3031P0K and pET-30b (+) as templates respectively^r. The products were subjected to gel electrophoresis and analyzed and compared, as shown in FIG. 3, and the results showed that the positions of the bands of the PCR products of the respective fragments were identical to those of the PCR products of the respective fragments after gel electrophoresis using the recombinant plasmid P3031P0K as a template and the WT genome and PET-30b (+) as a template. The sequencing level verification means that DNA sequencing and analysis comparison are respectively carried out on each fragment PCR product by taking the recombinant plasmid P3031P0K as a template and each fragment PCR product by taking the WT genome and the PET-30b (+) as a template, and the results show that the sequencing comparison results of the fragments are the same. All the above shows that 5 target fragments are successfully inserted into the vector plasmid pUC118 to form the recombinant plasmid P3031P 0K.

PCR amplification reaction: sample adding system: mu.L of template, 10 XPCR Buffer (Mg)²⁺plus) 2. mu.L, dNTP mix (2.5 mM each) 1.6. mu.L, forward primer 1. mu.L (10. mu.M), reverse primer 1. mu.L (10. mu.M), rTaq enzyme 0.2. mu.L, ddH₂O13.2. mu.L was added to a 200. mu.L PCR tube, centrifuged briefly, and then subjected to amplification reaction on a PCR instrument. And (3) amplification procedure: pre-denaturation at 94 ℃ for 3 min; denaturation, at 98 ℃ for 10 s; annealing at 5-10 deg.c below the Tm value of the primer for 15 sec; extension, at 72 ℃ for 1min for each amplification of 1kb of DNA; cycle, denaturation-annealing-extension cycle 38; 72 ℃ for 5 min; 16 ℃ for 10 min.

Double enzyme digestion reaction: sample adding system: target geneThe double enzyme digestion of the fragment adopts a 30 mu L system, 10 mu L DNA and 10 XBuffer 3 mu L, 1 mu L each of two rapid enzyme digestion enzymes and ddH₂O15 mu L; the plasmid is double digested with 20. mu.L system, DNA 10. mu.L, 10 XBuffer 2. mu.L, two fast digesting enzymes 1. mu.L each, ddH₂O6. mu.L. Reaction conditions are as follows: water bath at 37 deg.C for 1 h. After the reaction is finished, the enzyme is inactivated by warm bath for 5min at 80 ℃.

And (3) connection reaction: sample adding system: mu.L of the target gene fragment DNA, 5. mu.L of plasmid DNA, 2. mu.L of 10 XBuffer and 1. mu.L of enzyme were added to a 200. mu.L PCR reaction tube. Reaction conditions are as follows: the temperature is 16 ℃, and the reaction time is 8 h.

Example 2

Obtaining an over-expressed algal strain OE0528, wherein the recombinant plasmid P3031P0K and synechocystis PCC6803 wild type genome are homologously and doubly exchanged, and screening the over-expressed algal strain OE 0528.

Mixing the constructed recombinant plasmid P3031P0K and synechocystis PCC6803 wild type, culturing for 6 hours in a shaking table, then coating the mixture on a solid BG11 culture medium paved with a mixed fiber filter membrane, transferring the mixed fiber membrane to a solid BG11 culture medium containing 10 mu g/mL kanamycin after 24 hours, placing the solid BG11 culture medium in a light incubator, and obtaining a transformant after several weeks when a single algal colony grows out from the culture medium. In the process, the recombinant plasmid P3031P0K enters the wild type Synechocystis PCC6803, homologous double exchange is carried out by virtue of homologous arms slr2030 and slr2031, and the component km is overexpressed^rThe + psbA2 promoter + sll0528 was inserted into the wild-type genome. The transformant was transferred to liquid BG11 medium containing 10. mu.g/mL kanamycin sulfate, and the algal solution was grown to OD₇₃₀About 1.0, the suspension was centrifuged, inoculated into fresh liquid BG11 medium containing 15 μ g/mL kanamycin sulfate, and verified at the DNA level whether the fragment between the neutral sites-slr 2030 and slr2031 was replaced. In this way, the kanamycin sulfate content in the culture medium is increased step by step until finally verifying that the over-expressed algal strain OE0528 with completely replaced neutral sites is obtained. A schematic representation of the homologous double crossover of the overexpression elements with the Synechocystis PCC6803 wild-type genome, and the respective primer action sites, is shown in FIG. 2.

Verification of Synechocystis genome neutral sites at the DNA level-slr 2030 and slr2031, using primers shown in the sequence table of SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, and WT and over-expressed algal strain OE0528 genomes as templates to amplify sequences between the over-expression element fragment inserted into the neutral site and the neutral site respectively, and observing whether the position of the gel electrophoresis pattern strip of the product is consistent with the theoretical value. The wild type and over-expressed algal strain OE0528 genomes were first PCR amplified with primers to SEQ ID NO.11 and SEQ ID NO. 12. The wild type amplification result is partial fragments of slr2030 and slr2031 and fragments among genes, and the length is 1001 bp; over-expression of algal strain OE0528, when km^rWhen the three fragments of the promoter psbA2 and the sll0528 are completely inserted into neutral sites, the amplification result is three fragments, and the length is 3350 bp; when the three fragments do not completely replace neutral site fragments, two bands of 3350bp and 1001bp appear as the amplification result. As shown in FIG. 4A, the wild type PCR amplified band is around 1001bp, and the over-expressed algal strain OE0528PCR amplified band is only around 3350bp, and the length is consistent with the theoretical value, which primarily indicates that the over-expression element is completely inserted into the over-expressed algal strain OE 0528. Then, PCR amplification is carried out on the primers SEQ ID NO.13 and SEQ ID NO. 14. The wild type result is the middle part of slr2030 and slr2031, and the length is 610 bp; when three fragments of the overexpression element completely replace the middle parts of the slr2030 and slr2031 of neutral sites in the overexpression algal strain OE0528, the amplification result should be no band; when the three fragments do not completely replace neutral sites, the amplification result has a band at 610 bp. As a result, as shown in FIG. 4B, the band after electrophoresis was around 610bp in the wild-type PCR amplification result, and no band was observed in the amplification result of the over-expressed algal strain OE 0528. Further illustrating the overexpression element km^rThe three fragments, + psbA2 promoter + sll0528 completely replaced the neutral site between wild type slr2030 and slr2031 of Synechocystis PCC 6803.

BG11 liquid culture medium preparation: measuring 1L of deionized water by using a measuring cylinder, and sequentially adding K₂HPO₄1000 Xstock solution, MgSO₄·7H₂O1000 Xstock solution, CaCl₂·2H₂O1000 stock solution, citric acid 1000 stock solution, ferric ammonium citrate 1000 stock solution, ethylenediaminetetraacetic acid (EDTA)1000 stock solution, Na₂CO₃1000 Xstock, Trace MetaL A5Mix1000 Xstock solutions 1mL each, 1.5g of NaNO added₃And (5) solid and uniform mixing. Each 50mL of BG11 liquid medium was dispensed into 250mL Erlenmeyer flasks. Sealing with sealing film, and sterilizing at 121 deg.C under 1.1MPa for 30 min.

BG11 solid medium: subpackaging 100mL BG11 liquid culture medium into 250mL Erlenmeyer flask, adding 2g agar powder, sealing, sterilizing at 121 deg.C under 1.1MPa for 30 min.

And (3) cultivating synechocystis: by OD₇₃₀Synechocystis PCC6803 was inoculated into BG11 broth at 0.1 starting concentration, 1mL HEPES (4-hydroxyethylpiperazineethanesulfonic acid) was added per 50mL broth, and the inoculated medium was irradiated with light at 30. mu. mol.m^-2·s^-1Continuously culturing for 5 days in a multifunctional combined shaking table at the temperature of 29 ℃ and the rotating speed of 150 r/min. Synechocystis PCC6803 colonies were grown in BG11 solid medium supplemented with 2mL HEPES, 0.3% NaS₂SO₃And TES (trimethylol methylaminoethanesulfonic acid) at a final concentration of 8 mM. The solid culture medium is irradiated by light at the temperature of 29 ℃ and the light intensity of 60 mu mol.m^-2·s^-1Culturing in an illumination incubator. Corresponding antibiotics are added in the culture process.

Example 3

Synechocystis PCC6803 wild type and over-expressed algal strain OE0528 were cultured under high concentration ammonium salt stress.

By OD₇₃₀Synechocystis PCC6803 wild type and over-expressed algal strain OE0528 were inoculated into BG11 liquid medium containing ammonium chloride at a concentration of 0mM, 120mM, 150mM, 180mM, 210mM, respectively at an initial concentration of 0.1, and irradiated with light at 30. mu. mol. m.^-2·s^-1Continuously culturing at 29 deg.C and 150r/min for 5 days, and measuring OD every 24h₇₃₀And drawing a growth curve, measuring the scanning absorption value of the wavelength of 800 nm-400 nm from the culture to the 5 th day, and drawing a whole cell absorption graph.

FIG. 5 is a graph of growth curves, in which it can be observed that the WT growth rate gradually slows down as the concentration of ammonium chloride is gradually increased, and especially WT growth is severely limited under the conditions of 180mM and 210mM ammonium chloride. Whereas the growth rate of OE0528 was not much affected by 120mM, 150mM ammonium chloride, the growth rate was only slightly retarded by 180mM and 210mM ammonium chloride, and was clearly different from wild-type WT. Indicating that the OE0528 mutant is more tolerant to high concentrations of ammonium chloride relative to WT.

FIG. 6 is a picture of algal fluid cultured at 180mM ammonium chloride until day 5, where it can be seen that under 180mM ammonium chloride, WT was pale green and white, and clear, whereas the over-expressed algal strain OE0528, which was much darker than WT, showed normal dark green color, suggesting vigorous growth, indicating that OE0528 is more tolerant to high concentrations of ammonium chloride relative to WT.

FIG. 7 is a graph of whole cell uptake by culturing at 180mM ammonium chloride until day 5, where it can be seen that the chlorophyll and phycobiliprotein peaks of WT were significantly lower than the over-expressed algal strain OE0528 at 180mM ammonium chloride, indicating that the photosynthesis system of WT was severely damaged and the photosynthesis was greatly affected while the photosynthesis system damage of OE0528 was not significant and the photosynthesis was less affected under 180mM ammonium chloride. It was revealed that the over-expressed algal strain OE0528 is more adaptable to high concentration ammonium chloride stress than WT.

Taken together, it was demonstrated that the over-expressed algal strain OE0528 is more tolerant to high concentrations of ammonium chloride than the wild type.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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gacggaattt atgcctcttc cgaccatcaa gcattttatc cgtactcctg atgatgcatg 780

gttactcacc actgcgatcc ccgggaaaac agcattccag gtattagaag aatatcctga 840

ttcaggtgaa aatattgttg atgcgctggc agtgttcctg cgccggttgc attcgattcc 900

tgtttgtaat tgtcctttta acagcgatcg cgtatttcgt ctcgctcagg cgcaatcacg 960

aatgaataac ggtttggttg atgcgagtga ttttgatgac gagcgtaatg gctggcctgt 1020

tgaacaagtc tggaaagaaa tgcataaact tttgccattc tcaccggatt cagtcgtcac 1080

tcatggtgat ttctcacttg ataaccttat ttttgacgag gggaaattaa taggttgtat 1140

tgatgttgga cgagtcggaa tcgcagaccg ataccaggat cttgccatcc tatggaactg 1200

cctcggtgag ttttctcctt cattacagaa acggcttttt caaaaatatg gtattgataa 1260

tcctgatatg aataaattgc agtttcattt gatgctcgat gagtttttct aagaattaat 1320

tcatgagcgg atacatattt gaatg 1345

<210> 17

<211> 562

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> psbA2 promoter

<400> 17

tatcagaatc cttgcccaga tgcaggcctt ctggcgatcg ccatggtgag caacgattgc 60

ggctttagcg ttccagtgga tatttgctgg gggttaatga aacattgtgg cggaacccag 120

ggacaatgtg accaaaaaat tcagggatat caataagtat taggtatatg gatcataatt 180

gtatgcccga ctattgctta aactgactga ccactgacct taagagtaat ggcgtgcaag 240

gcccagtgat caatttcatt atttttcatt atttcatctc cattgtccct gaaaatcagt 300

tgtgtcgccc ctctacacag cccagaacta tggtaaaggc gcacgaaaaa ccgccaggta 360

aactcttctc aacccccaaa acgccctctg tttacccatg gaaaaaacga caattacaag 420

aaagtaaaac ttatgtcatc tataagcttc gtgtatatta acttcctgtt acaaagcttt 480

acaaaactct cattaatcct ttagactaag tttagtcagt tccaatctga acatcgacaa 540

atacataagg aattataacc aa 562

<210> 18

<211> 1269

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> sll0528

<400> 18

atgttaagcc tcagtttagg ggggcagttt atgaacaaca atatccgcgt cggcagtctg 60

tttggcattc ctttttacgt caacccatcc tggtttttaa ttttaggatt ggtgaccctg 120

agctatggcc aagacttagc ccgctttccc caactttccg gtggcacacc ctggattttg 180

gggttaatta cagctttact cctctttgct tccgttgtcg cccacgagtt gggccatagt 240

ttggttgcct tagcccaggg cattgaagtt aaatccatca ctctgttttt gttcggtggt 300

ctagcgagtt tagaaaagga atccaacact ccctggcaag cttttgcggt ggcgatcgcc 360

gggccggcgg tgagtttagt gctctttttg ggtttaacca tagttggtac ccaaatcccc 420

ctacctgtgc cggggcaggc catcattggt ttattgggca tgatcaacct cgccctggca 480

ttgtttaacc tcattcctgg tttacctttg gacggcggca atgtgctcaa atccattgtg 540

tggcaaatca cgggcaatca aaacaaaggt attctcattg ctagtcgggt gggccagggt 600

ttcggttggt tggcgatcgc cattggtagc ttaggtattt taaatattct gcccatcggt 660

agcttctgga ccattttgat cggttggttc ctgttacaaa atgctggttc ctccgcccgc 720

aacgcccagg tcaaagagca aatggaagcc tttactgctg aagatgcggt tattcccaac 780

agccccatta ttcctgccgg gttaaatatt cgggaatttg ctaacgatta tgtgattggt 840

aaaaccccct ggcgacggtt cttggttatt ggtgccgaca atcaactgtt aggtgtactt 900

gctacggaag acatcaaaca cgtccccact tccgattggc cccaggtcac agtggatagc 960

ttgatgcagt atccccaaca gatggtcacc gttaacgcca atcaatcttt gtttgaagtg 1020

gcccagttgt tagatcaaca gaaactgtcg gaacttttgg tggtgcaacc ttcgggagaa 1080

gtggtgggat tattggaaaa agcttccatc atcaaatgtc tgcaaacctc cgccgcctag 1140

agcctgtttt aaaagccccc ctggcccccc aaattttttt gggggggaaa ataagattta 1200

aagtccccca aacttgccgg agctttagtg aggagattta gggggcaagg aaaccataaa 1260

gattgggat 1269

<210> 19

<211> 880

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> slr2031

<400> 19

aggagttggt ggctaagttg taccgggaac aaaataaagt gcaggatctg ttgggggcca 60

tgggctacgc cctgcggagt ttacataatc tgaatcagtt tctggaattg acccctctga 120

tggccaccag ggtcaccgat gccgatggca gtgctttagt tctaatgaga gagggggaaa 180

tatctatctt tgaacaaatc catggccaca aaaatagtct taagggcacc attaaaggcg 240

ctttgcagaa ggcccgccag gttaacctca cattggattc ttccactgtc ctgagttatt 300

ttgaccgtca attgcgccaa gaattaccgg cgatcgcctg ttataacacc cccattttga 360

gtcaccaaga ggaagtgggc cgcttatata tctttagtca agatcgtaac tatagctgga 420

ctcccacccg tcgtaagtta ctacagctaa tttctgatca aaccgctgtg gcgatcgcca 480

atagtgattt aaaccaaaag ttgagatcca gagaaagtca ggatcgggaa ttggaaattg 540

cttcagaaat tcaaaatcaa ctattgcccc gttgttgtcc ccaaattaat ggtttggaca 600

ttgctgccca gtgtaaaacc gccagtcggg taggtgggga ttactacgat ttcattcctg 660

ccaactatga ccaactgcgc cagggggatt ggttatgtcg taacacaagc catttggggg 720

tgccctggag tattgtcatc ggcgatgtca tgggtaaagg agtaccggcg ggtctaatta 780

tgaccatgac tagggggatg ctgcgggcag aagtgctgaa ccgccatagt ccagcccaaa 840

ttcttaacca cctcaaccgg gtaatgtatg ccgacctgga 880

Claims (4)

1. a method for improving Synechocystis PCC6803 high concentration ammonium salt tolerance is characterized in that comprising the steps: (1) Construction of recombinant plasmid p3031POK: using the wild-type genomic DNA of Synechocystis PCC6803 as a template, using primer pairs SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.5 and SEQ ID NO.6, SEQ ID NO. 7 and SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, PCR amplification obtains slr2030, psbA2 promoter, s110528 and slr2031 purpose fragment, the target fragment gene sequence such as SEQ ID NO.15, SEQ ID NO. 17, shown in SEQ ID NO.18 and SEQ ID NO.19; taking plasmid pET-30b(+) as a template, using primer pairs SEQ ID NO.3 and SEQ ID NO.4, PCR amplification to obtain anti-kanamycosis Prime-km ^r fragment, the gene sequence is shown in SEQ ID NO. 16; the obtained target fragment was cut with restriction endonucleases HindIII and PstI, PstI and XbaI, XbaI and SmaI, SmaI and EcoRI, PstI and PstI in turn At the same time, use the same restriction endonuclease to cut the vector plasmid pUC118 in turn, and then use T4DNA Ligase to connect the target fragment and the vector plasmid in sequence to construct the recombinant plasmid p3031P0K; finally, PCR amplification technology and sequencing technology are used to identify at the DNA level. All 5 target fragments were inserted into the vector plasmid pUC118; (2) Homologous double exchange between recombinant plasmid p3031POK and Synechocystis PCC6803 wild-type genome, and screening for overexpressing algal strains: The constructed recombinant plasmid p3031POK was introduced into Synechocystis PCC6803 wild-type, and overexpressed by homologous double exchange The element km ^r + psbA2 promoter + sll0528 was inserted into the neutral site between the wild-type genes slr2030 and slr2031 of Synechocystis PCC6803 to obtain an overexpressed algal strain; it grew on solid medium containing low concentration of kanamycin sulfate After the transformant, it was transferred to a liquid medium containing low concentration of kanamycin sulfate for culture, and the antibiotic concentration in the medium was gradually increased and the overexpressed algal strain was passaged; and identified at the DNA level, using primer pair SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14, using wild-type and overexpressed algal strain genomes as templates, PCR amplification of the overexpression elements inserted into the genome or the neutral position between slr2030 and slr2031 point, to identify whether the neutral site was completely replaced by the overexpression element; finally, a stably inherited overexpression algal strain with the neutral site completely replaced by the overexpression element was obtained, named OE0528. 2 . A Synechocystis PCC6803 overexpressing algal strain OE0528 with significantly improved tolerance to high-concentration ammonium salts is characterized in that it is obtained by the method of claim 1 . 3. The Synechocystis PCC6803 overexpressing algal strain OE0528 with significantly improved tolerance to high-concentration ammonium salts according to claim 2, characterized in that: the described Synechocystis with significantly improved tolerance to high-concentration ammonium salts The algae PCC6803 overexpressed the algal strain OE0528, and the growth state in BG11 medium containing up to 180-210 mM ammonium salt was significantly better than that of the wild type Synechocystis. 4. The application of the Synechocystis PCC6803 overexpressing algal strain OE0528 with significantly improved high-concentration ammonium salt tolerance as claimed in claim 2 or 3, characterized in that: the described high-concentration ammonium salt tolerance is significantly improved The Synechocystis PCC6803-overexpressing algal strain OE0528 was used to construct microalgal engineering bacteria that tolerate high concentrations of ammonium salts.

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