CN109706104B

CN109706104B - Application of sll0528 gene in improvement of ethanol tolerance of synechocystis PCC6803

Info

Publication number: CN109706104B
Application number: CN201811591708.3A
Authority: CN
Inventors: 陈谷; 许白雪
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-12-25
Filing date: 2018-12-25
Publication date: 2020-12-22
Anticipated expiration: 2038-12-25
Also published as: CN109706104A; WO2020134427A1

Abstract

The invention discloses an application of an sll0528 gene in improving ethanol tolerance of synechocystis PCC6803, and belongs to the field of industrial microorganisms. According to the invention, the sll0528 gene in synechocystis PCC6803 is knocked out and over-expressed by a homologous recombination method, so that synechocystis PCC6803 strain Osll0528 with remarkably improved ethanol tolerance is obtained. The growth state of the strain in BG11 medium added with ethanol of different concentrations (1.5%, 2.0%, 2.5%, 3.0% v/v) is obviously better than that of wild strain. The ethanol-tolerant algal strain obtained by the invention has important theoretical and practical significance for constructing genetic engineering bacteria for producing fuel ethanol, and has wide application prospect.

Description

Application of sll0528 gene in improvement of ethanol tolerance of synechocystis PCC6803

Technical Field

The invention belongs to the field of industrial microorganisms, and particularly relates to application of an sll0528 gene in improvement of ethanol tolerance of synechocystis PCC 6803.

Background

The energy crisis and the increasing environmental pollution and greenhouse effect caused by fossil fuels have stimulated interest in renewable green biofuels. Bioethanol is a green renewable energy source, and not only the dependence on petroleum resources is reduced, but also the emission of carbon dioxide is greatly reduced. Blue algae can directly synthesize bioethanol by carbon dioxide through photosynthesis, and has shown great potential in the future production of green fuels. Synechocystis PCC6803, a photosynthetic cyanobacteria, has been widely used as a genetically engineered bacterium for the development of bioethanol because of its ease of molecular biological manipulation.

However, the yield of bioethanol is far from meeting the requirements of industrial production, and one important reason is the poor tolerance of Synechocystis PCC6803 to ethanol. Therefore, the tolerance of the synechocystis PCC6803 to ethanol is improved, and the method plays an important role in industrially producing bioethanol and solving energy crisis in the future.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide the application of an sll0528 gene in improving ethanol tolerance of synechocystis PCC 6803.

The invention also aims to provide a method for constructing synechocystis PCC6803 strain with obviously improved ethanol tolerance.

The invention also aims to provide the synechocystis PCC6803 strain which is constructed by the construction method and has obviously improved ethanol tolerance. The strain can be used for constructing genetic engineering bacteria for producing bioethanol.

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

an application of sll0528 gene in improving ethanol tolerance of synechocystis PCC 6803.

The ethanol tolerance of synechocystis PCC6803 is improved by over-expressing the sll0528 gene, and the ethanol tolerance of the synechocystis PCC6803 is reduced by knocking out the sll0528 gene.

Further, the sll0528 gene is applied to construction of synechocystis PCC6803 strain with remarkably improved ethanol tolerance.

The nucleotide sequence of the sll0528 gene is shown as (a), (b) or (c):

(a) the cDNA sequence of sll0528 gene, shown as SEQ ID NO: 17 is shown;

(b) genomic DNA sequence of sll0528 gene;

(c) a DNA sequence which has more than 90% homology with (a) or (b) and encodes the same functional protein.

The invention identifies a gene sll0528 related to ethanol tolerance by comparing a wild type with a knock-out strain delta sll0528 of sll0528 gene in constructed synechocystis PCC6803 and an overexpression strain Osll0528, wherein the sequence number of the gene sll0528 is shown as SEQ ID NO: 17, and successfully obtaining an alga strain capable of obviously improving the ethanol tolerance.

A method for constructing an sll0528 gene knockout strain delta sll0528 comprises the following steps:

(1) taking a wild synechocystis PCC6803 genome as a template, and taking the nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 2 as upstream and downstream primers of sll0528 gene to be knocked out, SEQ ID NO: 3 and SEQ ID NO: and 4, upstream and downstream primers of the sll0528 downstream of the gene to be knocked out are obtained through PCR amplification, and an upstream segment sll0528-up and a downstream segment sll0528-down of the gene to be knocked out are obtained. Plasmid pACYC184 was used as a template, and SEQ ID NO: 5 and SEQ ID NO: 6 is an upstream primer and a downstream primer, and a fragment Cm containing a chloramphenicol resistance gene is obtained by PCR amplification^r；

(2) The target fragments sll0528-up, sll0528-down, Cm obtained by cutting BamHI and NdeI, XhoI and SacI, BamHI and SacI in turn^rAnd inserting the recombinant plasmid into a vector plasmid PET-30b to obtain a homologous recombination double-exchange plasmid PET-Cm^r；

(3) Homologous recombination double exchange plasmid PET-Cm^rThe strain is transferred into synechocystis PCC6803 in a natural transformation mode, obtained transformants are screened by chloramphenicol with different concentrations and are identified on a DNA level, and finally a monoclonal algae strain with the sll0528 gene completely knocked out is obtained and is named as delta sll 0528.

A method for constructing an sll0528 gene over-expression strain Osll0528 comprises the following steps:

(1) taking a wild synechocystis PCC6803 genome as a template, and taking the nucleotide sequence shown as SEQ ID NO: 7 and SEQ ID NO: 8 as upstream and downstream primers of target fragment slr2030, SEQ ID NO: 11 and SEQ ID NO: 12 are the upstream and downstream primers of the psbA2 promoter, SEQ ID NO: 13 and SEQ ID NO: 14 is an upstream primer and a downstream primer of sll0528, SEQ ID NO: 15 and SEQ ID NO: 16 are upstream and downstream primers of slr2031, and slr2030, psbA2 promoter, sll0528 and slr2031 are obtained by PCR amplification. Taking plasmid pET-30b as a template and SEQ ID NO.9 and SEQ ID NO.10 as primers, and carrying out PCR amplification to obtain kanamycin resistance gene km^rA fragment;

(2) restriction enzymes Hind III and Pst I, Pst I and Pst I,slr2030, km obtained by sequentially shearing Pst I, Xba I, Sma I and EcoR I^rpsbA2 promoter, sll0528, slr2031 target fragment and insertion into the vector plasmid pUC118 resulted in the homologous recombination double-crossover plasmid P3031.

(3) The homologous recombination double-exchange plasmid P3031 is transferred into synechocystis PCC6803 in a natural transformation mode, obtained transformants are screened by kanamycin with different concentrations and are identified on a DNA level, and finally an overexpression strain of sll0528 gene is obtained and named as Osll 0528.

According to the invention, by the construction method, a knock-out strain delta sll0528 and an overexpression strain delta sll0528 of sll0528 in synechocystis PCC6803 are obtained, a gene sll0528 related to ethanol tolerance is identified, and an ethanol-tolerant algal strain Osll0528 is obtained.

The tolerance of the algal strain Osll0528 constructed by the method to ethanol is remarkably improved, and the growth state of the algal strain Osll0528 in a BG11 culture medium containing 1.5-3.0% (v/v) (preferably 1.5-2.0% (v/v)) ethanol is obviously better than that of a wild type algal strain.

The strain Osll0528 can be used for constructing genetic engineering bacteria for producing fuel ethanol.

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

according to the invention, the sll0528 gene in synechocystis PCC6803 is knocked out and over-expressed by a homologous recombination method, so that synechocystis PCC6803 strain Osll0528 with remarkably improved ethanol tolerance is obtained. In BG11 medium added with ethanol of different concentrations, the growth state of the strain is obviously better than that of the wild strain. The ethanol-tolerant algal strain obtained by the invention has important theoretical and practical significance for constructing genetic engineering bacteria for producing fuel ethanol, and has wide application prospect.

Drawings

FIG. 1 is a diagram of the homologous recombination double-crossover plasmid PET-Cm^rSchematic structural diagram of (1).

FIG. 2 is a schematic diagram of the structure of the homologous recombination double-crossover plasmid P3031.

FIG. 3 is a graph of the growth of Synechocystis PCC6803 wild-type WT, Δ sll0528 and Osll0528 algal strains under 1.5% (v/v) ethanol stress, where E represents ethanol.

FIG. 4 is a graph of the growth of Synechocystis PCC6803 wild-type WT, Δ sll0528 and Osll0528 algal strains under 2.0% (v/v) ethanol stress, where E represents ethanol.

FIG. 5 is a graph of the growth of Synechocystis PCC6803 wild-type WT, Δ sll0528 and Osll0528 algal strains under 2.5% (v/v) ethanol stress, where E represents ethanol.

FIG. 6 is a graph of the growth of Synechocystis PCC6803 wild-type WT, Δ sll0528 and Osll0528 algal strains under 3.0% (v/v) ethanol stress, where E represents ethanol.

FIG. 7 is a graph of the trend of Synechocystis PCC6803 wild type WT, Δ sll0528 and Osll0528 algal strains at ethanol concentrations of 0%, 1.5%, 2.0%, 2.5%, 3.0% (v/v), respectively, on day four.

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.

The synechocystis PCC6803 wild type strain used in the examples of the present invention was isolated and purified from ATCC27184, which is an abbreviation for American type culture Collection and 27184 is a strain number. Plasmids pUC118, pACYC184 were purchased from Takara and pET-30b was purchased from Novagen.

Example 1

Homologous recombination double-exchange plasmid PET-Cm^rAnd construction of P3031:

(1) amplification of the insert:

taking a wild synechocystis PCC6803 genome as a template, and taking the nucleotide sequence shown as SEQ ID NO: 1 and SEQ ID NO: 2 as upstream and downstream primers of sll0528 gene to be knocked out, SEQ ID NO: 3 and SEQ ID NO: and 4, upstream and downstream primers of the sll0528 downstream of the gene to be knocked out are obtained through PCR amplification, and an upstream segment sll0528-up and a downstream segment sll0528-down of the gene to be knocked out are obtained. Plasmid pACYC184 was used as a template, and SEQ ID NO: 5 and SEQ ID NO: 6 is an upstream primer and a downstream primer, and a fragment Cm containing a chloramphenicol resistance gene is obtained by PCR amplification^r(ii) a Using wild type synechocystis PCC6803 genome as a template toSEQ ID NO: 7 and SEQ ID NO: 8 as upstream and downstream primers for target fragment slr2030, SEQ ID NO: 11 and SEQ ID NO: 12 are the upstream and downstream primers of the psbA2 promoter, SEQ ID NO: 13 and SEQ ID NO: 14 is an upstream primer and a downstream primer of sll0528, SEQ ID NO: 15 and SEQ ID NO: 16 are upstream and downstream primers of slr2031, and slr2030, psbA2 promoter, sll0528 and slr2031 are obtained by PCR amplification. Taking plasmid pET-30b as a template and SEQ ID NO.9 and SEQ ID NO.10 as primers, and carrying out PCR amplification to obtain kanamycin resistance gene km^rAnd (3) fragment. The genome extraction of the synechocystis PCC6803 adopts a plant genome DNA rapid extraction kit (Guangzhou Dongsheng biological technology limited company, the product number N1191), and the plasmid extraction adopts a high-purity plasmid small-quantity extraction kit (Guangzhou Dongsheng biological technology limited company, the product number N1011).

The PCR reaction used a 20. mu.L system: template 1. mu.L, 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. And adding each reaction component into the PCR tube, centrifuging for a short time, and placing on a PCR instrument for amplification reaction. The amplification procedure was: 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 amplifying 1kb DNA; cycle, denaturation-annealing-extension cycle 38; 72 ℃ for 5 min; 16 ℃ for 10 min.

The size of the PCR product was verified by agarose electrophoresis to be consistent with the theoretical length. Before carrying out subsequent experiments, each PCR product needs to be recovered and purified by glue.

(2) Restriction ligation of the insert with the plasmid

The resulting sll0528-up fragment and PET-30b vector were double digested with restriction enzymes BamHI and NdeI. The insert was double digested using a 30. mu.L system: 10 μ L of DNA, 10. mu.L of Buffer 3 μ L, 1 μ L of each of the two fast-acting restriction enzymes, ddH₂O15. mu.L. The plasmid was double digested using a 20 μ L system: 10 μ L of DNA, 10. mu.L of Buffer 2 μ L, 1 μ L each of two fast-acting restriction enzymes, ddH₂O6. mu.L. The temperature of the enzyme digestion reaction is 37 ℃ and the time is 1 h. After the reaction is finished, the enzyme is inactivated by warm bath for 5min at 80 ℃.And recovering and purifying the enzyme digestion product by using glue, and then carrying out ligation reaction by using T4DNA ligase. The ligation reaction used a 20 μ L system: the insert DNA was 12. mu.L, plasmid DNA 5. mu.L, 10 XBuffer 2. mu.L, enzyme 1. mu.L. The ligation temperature was 16 ℃ and after 8h the ligation product was transformed into E.coli DH5 alpha. And (4) identifying whether the grown transformant is successfully connected or not, and confirming the successfully connected plasmid by Sanger sequencing to obtain the plasmid PET-30 b-up. The sll0528-down fragment and Cm are subjected to the same enzyme digestion reaction and ligation reaction conditions^rConnecting the fragment with the plasmid PET-30b-up, wherein the restriction sites are XhoI and SacI, and SacI and BamHI, and obtaining the homologous recombination double-exchange plasmid PET-Cm^rThe schematic structure is shown in fig. 1. Sequentially dividing the target fragment slr2030 km^rThe psbA2 promoter, sll0528 and slr2031 are ligated to plasmid pUC118 at HindIII and pstI, pstI and XbaI, XbaI and SmaI, SmaI and EcoRI, respectively, to give a homorecombinant double-crossover plasmid P3031, the structure of which is shown in FIG. 2.

Example 2

Acquisition of sll0528 gene knockout strain delta sll0528 and sll0528 gene overexpression strain Osll0528

(1) Plasmid transformation

Homologous recombination double exchange plasmid PET-Cm^rAfter filtration sterilization through a 0.22 μm microporous membrane, the cells were placed in a 2mL sterile centrifuge tube. BG11 medium (HEPES buffer has been added) was added to the medium in an amount to give a final plasmid concentration of about 10 ng/. mu.L. 30mL of wild type PCC6803 at log phase was centrifuged at 6000rpm for 7min and the supernatant was removed. The algal mud was resuspended in 20mL fresh BG11 medium, centrifuged at 6000rpm for 7min, and the supernatant removed. The algal mash was resuspended in plasmid-containing medium. The resuspended algal solution was incubated at 29 ℃ at 150rpm at 1400Lux for 6h with continuous light. Spreading the algae liquid in a solid culture medium paved with a mixed fiber filter membrane, culturing for 1 day by illumination (upright culture), transferring the membrane into a solid culture medium containing 10 mu g/mL chloramphenicol, and culturing for several days by illumination until single algae colony grows on the surface of the membrane. Finally, the grown algal colonies were transferred to 20mL BG11 vial medium containing the same concentration of chloramphenicol for culture, and transferred after growing to a logarithmic phase. Transformation method of homologous recombination double-exchange plasmid P3031The antibiotic added was changed from chloramphenicol to kanamycin as described above.

(2) Screening of algal strains

And (2) carrying out transfer culture on the alga liquid obtained in the step (1), wherein the culture conditions are 29 ℃, 150rpm and 1400Lux continuous illumination. The concentration of the antibiotics in BG11 medium was increased to 20. mu.g/mL during the transfer. The medium was then transferred until log phase, after which the concentration of the antibiotic in the medium was increased by 10. mu.g/mL for each transfer. When the antibiotic concentration in the medium reached 50. mu.g/mL, the algal solution plates were streaked. After single algae grows on the plate, single algae is picked to fall into BG11 culture medium with corresponding antibiotic concentration for culture, and finally gene knockout strain delta sll0528 and gene over-expression strain Osll0528 are obtained.

(3) PCR and sequencing validation

For the knockout strain Δ sll0528, primers 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 left homology arm, right homology arm, and chloramphenicol gene were amplified Sll0528 in wild-type and mutant algal strains. Using the nucleotide sequence of SEQ ID NO: 18 and SEQ ID NO: 19 amplification of the knock-out Sll0528 fragment gene. The wild type strain can not amplify the chloramphenicol gene; the Sll0528 fragment gene could not be amplified in the mutant algal strain (knock-out strain) and the chloramphenicol gene was present. The gene sequence obtained by sequencing and amplification accords with expectations, which indicates that the chloramphenicol gene replaces the gene sll0528 and the knock-out strain delta sll0528 is successfully constructed.

For the over-expressed strain Osll0528, primers SEQ ID NO: 20 and SEQ ID NO: 21 was amplified in wild type algal strain and over-expressed strain Osll0528, the wild type amplification resulted in a partial fragment between neutral sites slr2030 and slr2031 with a length of 1001bp, while the over-expressed strain Osll0528 resulted in Km^rThe promoter psbA2 and the inserted sll0528 are 3350bp in total, and the result is expected after sequencing, thereby proving that the over-expression strain Osll0528 is successfully constructed.

The formula of the BG11 culture medium used in the invention is as follows: 1L of the culture medium contains NaNO₃ 1.5g，K₂HPO₄0.04g，MgSO₄·7H₂O 0.075g，EDTA 0.001g，CaCl₂·2H₂O 0036g, citric acid 0.006g, ferric ammonium citrate 0.006g, Na₂CO₃ 0.02g，H₃BO₃ 0.00286g，MnCl₂·4H₂O 0.00181g，ZnSO₄·7H₂O 0.000222g，Na₂MoO₄·2H₂O 0.00039g，CuSO₄·5H₂O 0.000079g，Co(NO₃)₂·6H₂O0.0000494 g. When used, 1mL of 1mol/L HEPES buffer (pH 7.5) was added to 50mL of the medium. When preparing the solid culture medium, 2% of agar is added.

Example 3

Synechocystis PCC6803 wild type, gene knockout strain Δ sll0528, gene overexpression strain Osll0528, phenotypic analysis under ethanol stress:

wild type, delta sll0528, Osll0528 grown to logarithmic phase were used as seed solutions and inoculated into 50mL BG11 medium containing 0% (v/v), 1.5% (v/v), 2.0% (v/v), 2.5% (v/v), 3.0% (v/v) ethanol, respectively, at the starting OD of each bottle of algae₇₃₀0.1. Continuously culturing for 4 days, sampling daily for measuring OD₇₃₀Values, growth curves were plotted. The culture conditions were 30 ℃, 150rpm, 1800Lux continuous light, and the experimental group and the control group were each in triplicate.

FIGS. 3-6 are graphs of the growth of Synechocystis PCC6803 wild-type WT, Δ sll0528 and Osll0528 algal strains under 1.5%, 2.0%, 2.5%, 3.0% (v/v) ethanol stress, where E represents ethanol. As can be seen from the figure, the growth state of Osll0528 is significantly better than that of wild type, while the growth state of knockout Δ sll0528 is worst when compared under ethanol stress at several concentrations of 1.5%, 2.0%, 2.5%, 3.0% (v/v), respectively.

FIG. 7 is a graph of the trend of Synechocystis PCC6803 wild type WT, Δ sll0528 and Osll0528 algal strains at ethanol concentrations of 0%, 1.5%, 2.0%, 2.5%, 3.0% (v/v), respectively, on day four. As can be seen from the figure, the growth status of Osll0528 is optimal under different concentrations, while the growth status of knockout Δ sll0528 is the worst, and the difference is most obvious when the ethanol concentration is 1.5% and 2.0%.

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.

Sequence listing

<110> university of southern China's science

Application of <120> sll0528 gene in improvement of ethanol tolerance of synechocystis PCC6803

<160> 21

<170> SIPOSequenceListing 1.0

<210> 1

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggaattccat atgctggtta tggtggttta ctga 34

<210> 2

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cgcggatcca tgttaagaat tgcctgagtg 30

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgagctccta cggaagacat caaacacg 28

<210> 4

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccgctcgagg gcgaaatgtt gaccttgac 29

<210> 5

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgcggatcct catcagtgcc aacatag 27

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgagctcggt aaaccagcaa tagacat 27

<210> 7

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

cccaagctta cactacatta ccggacaaac t 31

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aaaactgcag tcttcctggg gacgaaaacg 30

<210> 9

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aactgcagct cagttcggtg taggtcgtt 29

<210> 10

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aactgcagca ttcaaatatg tatccgctca 30

<210> 11

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaaactgcag tatcagaatc cttgcccaga t 31

<210> 12

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgctctagat tggttataat tccttatgta t 31

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgctctagaa tgttaagcct cagtttaggg 30

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tcccccggga tcccaatctt tatggtttcc 30

<210> 15

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

tcccccggga ggagttggtg gctaagttgt 30

<210> 16

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ccggaattct ccaggtcggc atacattac 29

<210> 17

<211> 1140

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

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

<210> 18

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atgttaagcc tcagtttag 19

<210> 19

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

caagtacacc taacagttga 20

<210> 20

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

agggacttag cccagtttca 20

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

cccaacagat cctgcacttt a 21

Claims

1.sll0528The application of the gene in improving ethanol tolerance of synechocystis PCC6803 is characterized in that:

over-expressionsll0528Gene-enhanced ethanol tolerance of Synechocystis PCC6803, knock-outsll0528The gene reduces ethanol tolerance of synechocystis PCC 6803.

2. Use according to claim 1, characterized in that:

saidsll0528The gene is applied to the construction of synechocystis PCC6803 strain with obviously improved ethanol tolerance.

3. Use according to claim 1 or 2, characterized in that:

saidsll0528The nucleotide sequence of the gene is shown as SEQ ID NO: shown at 17.

4. Use according to claim 1 or 2, characterized in that:

the method for constructing the synechocystis PCC6803 strain with the remarkably improved ethanol tolerance comprises the following steps:

(1) taking a wild synechocystis PCC6803 genome as a template, and taking the nucleotide sequence shown as SEQ ID NO: 7 and SEQ ID NO: 8 as a target fragmentslr2030The upstream and downstream primers of SEQ ID NO: 11 and SEQ ID NO: 12 ispsbA2Upstream and downstream primers of the promoter, SEQ ID NO: 13 and SEQ ID NO: 14 issll0528The upstream and downstream primers of SEQ ID NO: 15 and SEQ ID NO: 16 isslr2031The upstream and downstream primers are obtained by PCR amplificationslr2030，psbA2A promoter,sll0528，slr2031(ii) a Taking plasmid pET-30b as a template and SEQ ID NO.9 and SEQ ID NO.10 as primers, and carrying out PCR amplification to obtain kanamycin resistance gene km^rA fragment;

(2) using restriction endonucleasesHindIII andPstⅠ，Psti andPstⅠ，Psti andXbaⅠ，Xbai andSmaⅠ，Smai andEcoRi obtained by sequential shearingslr2030，km^r，psbA2A promoter,sll0528，slr2031inserting the target fragment into a vector plasmid pUC118 to obtain a homologous recombination double-exchange plasmid P3031;

(3) the homologous recombination double-exchange plasmid P3031 is transferred into Synechocystis PCC6803 in a natural transformation mode, and the obtained transformant is carried out by kanamycin with different concentrationsScreening and identifying at DNA level to obtainsll0528The gene-overexpressed strain was designatedOsll0528。

5. Use according to claim 4, characterized in that: the synechocystis PCC6803 strain with remarkably improved ethanol tolerance has a growth state in a BG11 culture medium containing 1.5-3.0% v/v ethanol which is obviously superior to that of a wild type strain.

6. Use according to claim 5, characterized in that:

the synechocystis PCC6803 strain with remarkably improved ethanol tolerance has a growth state in a BG11 culture medium containing 1.5-2.0% v/v ethanol which is obviously superior to that of a wild type strain.

7. Use of a Synechocystis PCC6803 strain having substantially improved ethanol tolerance as claimed in any one of claims 4 to 6, wherein:

the synechocystis PCC6803 strain with remarkably improved ethanol tolerance is used for constructing genetic engineering bacteria for producing fuel ethanol.