CN113817752B - Application of slr0681 gene in synthesis of synechocystis carotene - Google Patents

Abstract

The invention relates to the application of slr0681 gene in synthesis of synechocystis carotene, the invention discloses for the first time that the slr0681 gene of synechocystis PCC6803 has obvious influence on carotenoid synthesis in a high-salt environment, the invention constructs a slr0681 gene knockout mutant strain delta slr0681 by utilizing a homologous recombination method, the result shows that the growth condition of delta slr0681 has obvious advantages under the high-salt stress condition, the total carotenoid content and the chlorophyll a content are increased, and HPLC analysis shows that the contents of cyanobacteria lutein, zeaxanthin, echinoctone and beta-carotene are respectively increased by 1.2 times, 1.1 times, 1.7 times and 1.05 times compared with the wild type, and the expression of psbB and psbD genes in PSI psaA, psaB, psaL and PSII is increased.

Description

Application of slr0681 gene in synthesis of synechocystis carotene

Technical Field

The invention relates to an application of slr0681 gene in synthesis of synechocystis carotene, in particular to an application of slr0681 gene in synechocystis PCC6803 in synthesis of synechocystis carotene under high salt stress condition, and belongs to the technical field of genetic engineering.

Background

Blue algae (cyanobacteria) is one of the most important primary producers of the earliest photoautotrophic prokaryotes on earth. The blue algae cells can carry out autotrophic growth, can survive independently of external organic matters, and can also provide energy for biological activities by utilizing simple inorganic matters to synthesize organic matters. And most blue algae and extracts thereof are nontoxic to human beings and animals, and are good receptors for transgenic research. The Synechocystis sp.PCC 6803 is a single-cell blue algae, has a natural exogenous DNA conversion system, has the advantages of high growth speed, simple culture condition, no toxin, clear genetic background, no inclusion body formed by expressing exogenous products and the like, is suitable for large-scale production by utilizing a bioreactor, and is an ideal blue algae genetic engineering receptor.

Carotenoids play an important role in animals and humans, such as preventing diseases, enhancing immunity, maintaining normal growth and reproduction of animals, and the like. Is a class of lipid-soluble isoprenoid compounds, is a bio-produced lipophilic pigment that performs photosynthesis, plays a central role in many organisms, prevents oxidation by quenching active oxygen, and collects light energy in photosynthetic organisms.

In recent years, carotenoids have been widely used in the pharmaceutical, food, health care, cosmetic and feed industries, and with the increasing health consciousness of people, carotenoids obtained from natural raw materials are welcome by more and more consumers. At present, the production modes of carotenoid mainly comprise three modes of chemical synthesis, natural extraction and biosynthesis. Compared with the former two modes, the microbial synthesis mode is safe and natural, and is a healthy and feasible mode for producing carotenoid.

The accumulation of pigments in blue algae is affected by external environment, such as light, nutrient salts, temperature, acidity, etc. Synechocystis is a moderate salt tolerance cyanobacteria that can tolerate up to 1.2mol/L sodium chloride in a short period of time. After the addition of high salt to the culture medium, free water in the cells is reduced, and the increased concentration of inorganic ions produces toxic effects on the metabolism of the cells. Blue algae cells use a "salt efflux strategy" to accommodate high salt environments. First, blue algae cells rapidly lose water and shrink. Then Na ⁺ And Cl ^- Entry into the cell reduces the water potential and water returns to the cell. High Na in cells ⁺ The amount inhibits various physiological reactions in the cells, particularly photosynthesis and translation. Will be poisonous Na ⁺ Exchange for less toxic K ⁺ Photosynthesis can be re-activated and synthesis of compatible solutes can begin. The main compatible solute of blue algae is glucosyl glycerol, and accumulation of glucosyl glycerol is allowedExcess ions flow out and then gene expression is activated again, and finally the change in gene expression results in complete adaptation to high salt.

The water resources on earth are limited, so that large-scale cultivation of cyanobacteria transformed in future biotechnology must rely on seawater, and thus ideal production strains must be able to grow at least in seawater and preferably be tolerant to higher concentrations of salts.

Photosystem I complex (PSI) is a pigment protein complex on photosynthetic membranes that catalyzes the transfer of electrons from plastid blue (PC) to Fd (iron redox protein) via a series of electron transfer bodies. PSI consists of a series of proteins required for light energy absorption, conversion and transmission, and dye molecules bound to the proteins, and electron transfer chains. PSI reaction center complex in blue algae basically comprises PsaA-F and PsaI-L. Among these subunits, psaA-F mainly participates in electron transfer reactions, and the central core subunits PsaA and PsaB bind in a heterodimeric state on the thylakoid membrane, and deletion of the PsaA or PsaB gene results in deletion of PSI complexes in the thylakoid. PSI primarily mediates Linear Electron Transport (LET). Meanwhile, other forms of electron transfer pathways exist on photosynthetic cyst membranes, including cyclic electron transfer around PSI (PSI-CET), and under certain conditions, photosynthetic hydrogen production pathways also exist in Chlamydomonas or in certain prokaryotic algae. The alternate electron transfer paths and the linear electron transfer are cooperated to ensure the efficient photosynthesis of plants under the stress condition. The relationship between the slr0681 gene and carotenoid synthesis in synechocystis PCC6803 has not been reported.

Disclosure of Invention

The invention provides an application of slr0681 gene in synthesis of synechocystis carotene aiming at the defects of the prior art.

The invention constructs the slr0681 gene knockout mutant strain delta slr0681 by using a homologous recombination technology. The mutant strain was studied for chlorophyll and carotenoid component changes and light system I (PSI) and light system II (PSII) related gene expression and growth conditions with the wild type under high salt stress (3% NaCl). The results show that under normal conditions, the culture temperature is 30 ℃ and the illumination is 40 mu mol photon.m ^-2 ·s ^-1 Under the condition ofCompared with the wild type, the expression level of psaA, psaB, psaL gene in delta slr0681 is reduced by 40%, 40% and 70% respectively; under the high salt stress treatment condition of 3% NaCl, the total carotenoid content in Deltaslr 0681 is increased by 0.356 mug/mL compared with the wild type, and HPLC analysis shows that the cyanobacteria lutein, zeaxanthin, echinone and beta-carotene content are respectively increased by 1.2 times, 1.1 times, 1.7 times and 1.05 times compared with the wild type, and the expression level of psbB and psbD genes in PSI psaA, psaB, psaL and PSII is respectively increased by 3.4 times, 1.9 times, 1.5 times and 2.1 times and 1.6 times.

The invention has important practical significance in researching the physiological change of the synechocystis slr0681 gene and the knockout mutant strain thereof under the condition of high salt stress.

The technical scheme of the invention is as follows:

a gene sequence slr0681 with a function of regulating salt tolerance of synechocystis is provided, and the nucleotide sequence of the slr0681 gene is shown as SEQ ID NO. 1.

According to a preferred embodiment of the invention, the synechocystis is synechocystis PCC6803.

The application of slr0681 gene in synthesis of synechocystis carotene is disclosed, wherein the nucleotide sequence of slr0681 gene is shown as SEQ ID NO. 1.

In the application, the amino acid sequence of the slr0681 gene is shown as SEQ ID NO. 2.

According to a preferred aspect of the invention, the above-mentioned use is by knocking out the slr0681 gene in synechocystis to obtain a mutant strain.

According to a preferred aspect of the invention, the above-mentioned application, the synechocystis is synechocystis PCC6803.

According to a preferred aspect of the invention, the above application comprises the steps of:

(1) Respectively amplifying upstream and downstream fragments of the slr0681 gene from the genomic DNA of synechocystis PCC6803 by using sequences shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as upstream primers and sequences shown as SEQ ID NO.5 and SEQ ID NO.6 as downstream primers;

(2) Connecting the two gene fragments to a T3 cloning vector, then converting the two gene fragments to escherichia coli DH5 alpha, screening positive clones, and sequencing to obtain slr0681-T3 plasmid;

(3) Cutting slr0681-T3 plasmid with BamHI, recovering carrier fragment;

(4) The pBluescript-Kan vector was cut with BamHI single enzyme to recover kanamycin resistant fragment;

(5) Ligating the vector fragment and the kanamycin resistance fragment with T4 ligase and then transforming into E.coli DH5 alpha;

(6) Obtaining a synechocystis slr0681 gene knockout vector named p delta slr0681 by screening positive clones;

(7) Converting the recombinant vector p delta slr0681 prepared in the step (6) into synechocystis PCC6803, and screening to prepare synechocystis mutant strain with the slr0681 gene knocked out;

(8) Culturing the mutant strain obtained in the step (7), and extracting to obtain carotenoid.

The synechocystis mutant strain from which the slr0681 gene is knocked out is hereinafter referred to as 'delta slr 0681'.

According to a preferred embodiment of the present invention, in the step (1), the PCR amplification system is as follows:

2X Trans Taq HiFi PCR SuperMix. Mu.L, 1. Mu.L of template DNA, 1. Mu.L of SEQ ID NO.3 primer in the sequence Listing, 1. Mu.L of SEQ ID NO.5 primer in the sequence Listing, ddH ₂ O7. Mu.L, 20. Mu.L in total;

2X Trans Taq HiFi PCR SuperMix. Mu.L, 1. Mu.L of template DNA, 1. Mu.L of primer SEQ ID NO.4 in the sequence Listing, 1. Mu.L of primer SEQ ID NO.6 in the sequence Listing, ddH ₂ O7. Mu.L, 20. Mu.L in total;

the PCR amplification procedure was as follows:

pre-denaturation at 94℃for 2min, denaturation at 94℃for 30s, renaturation at 60℃for 30s, extension at 72℃for lmin, final extension at 72℃for 5min after 35 cycles, and storage at 4 ℃.

According to the present invention, preferably, the mutant strain in step (8) is cultured under high salt conditions.

Still more preferably, the high salt condition is stationary culture in BG-11 liquid medium containing 0.51mol/LNaCl (3%, w/v);

more preferably, the mutant strain in step (8) is at a temperature of 28 to 32℃and 35 to 45. Mu. Mol of photon.m ^-2 ·s ^-1 Under continuous light conditions, the mixture was irradiated with light containing 0.51mol/LNaCl (3%Stationary culturing in BG-11 liquid culture medium.

A synechocystis mutant strain knocks out the slr0681 gene, and the nucleotide sequence of the slr0681 gene is shown as SEQ ID NO. 1.

According to a preferred embodiment of the invention, the synechocystis is synechocystis PCC6803.

The application of the synechocystis mutant strain in synthesis of synechocystis carotene.

According to the invention, preferably, the mutant strain of synechocystis is used for synthesizing synechocystis carotene under high-salt condition.

Further preferably, the high salt condition is 0.51mol/LNaCl.

The invention discloses a method for constructing a directed knockout carrier of an slr0681 gene, which comprises the steps of adding an enzyme cutting site of BamHI into the slr0681 gene in synechocystis PCC6803, and connecting kanamycin resistance fragments.

An application of slr0681 gene in improving the expression level of psaA, psaB, psaL, psbD, psbB gene in synechocystis, wherein the nucleotide sequence of slr0681 gene is shown in SEQ ID NO. 1.

According to a preferred aspect of the invention, the above-mentioned use is by knocking out the slr0681 gene in synechocystis to obtain a mutant strain.

Further preferably, the mutant strain is cultured under high salt conditions.

More preferably, the high salt condition is 0.51mol/LNaCl.

According to a preferred aspect of the invention, the above-mentioned application, the synechocystis is synechocystis PCC6803.

An application of slr0681 gene in enhancing salt tolerance of synechocystis, wherein the nucleotide sequence of slr0681 gene is shown as SEQ ID NO. 1.

According to the invention, the application is that the growth of the synechocystis mutant strain with the slr0681 gene knocked out is promoted under the condition of high salt stress by knocking out the slr0681 gene in synechocystis.

Further preferably, the high salt condition is 0.51mol/LNaCl.

According to a preferred aspect of the invention, the above-mentioned application, the synechocystis is synechocystis PCC6803.

Advantageous effects

1. The invention discloses that the slr0681 gene of synechocystis PCC6803 has obvious influence on growth of synechocystis and carotenoid synthesis in synechocystis for the first time, the invention constructs a slr0681 gene knockout mutant strain delta slr0681 by utilizing a homologous recombination method, and the result shows that the wild type growth condition has obvious advantages by comparing and culturing with the wild type under the normal culture condition, the total carotenoid content is increased, the expression of psaA, psaB, psaL genes in PSI is increased, and related genes of PSII are not influenced. The knockout of slr0681 gene affects the growth of algae strain, the synthesis of carotenoid and the expression of related genes of optical system. Because of the intricate complexity of algae growth and carotenoid metabolic gene regulation, the invention cannot judge the specific action position of the slr0681 gene, but lays a foundation for the subsequent research of specific functions.

2. The invention researches the influence of physiological and biochemical indexes of wild synechocystis PCC6803 and slr0681 gene knockout mutant strain delta slr0681 under the condition of high salt stress to determine whether the slr0681 gene is related to salt tolerance. The research result shows that under the condition of high salt stress, the growth condition of Deltaslr 0681 has obvious advantages, the contents of total carotenoid and chlorophyll a are increased, and HPLC analysis shows that the contents of cyanobacteria lutein, zeaxanthin, echinone and beta-carotene are respectively increased by 1.2 times, 1.1 times, 1.7 times and 1.05 times compared with the wild type, and the expression of psbB and psbD genes in PSI psaA, psaB, psaL and PSII are increased. The slr0681 gene is shown to affect the salt tolerance of synechocystis.

3. The synthetic biological strategy utilized by the invention lays a foundation for improving the carotene metabolism of the microalgae, and provides a theoretical basis for promoting the application of the microalgae on biological energy sources.

Drawings

FIG. 1 is a schematic representation of the carotenoid synthesis pathway in Synechocystis PCC 6803;

FIG. 2 is a schematic diagram of construction of a directed knockout vector of the synechocystis PCC6803slr0681 gene;

FIG. 3 is a PCR amplification detection chart of the synechocystis PCC6803slr0681 gene knockout mutant;

FIG. 4 is a graph of growth of wild-type Synechocystis PCC6803 and Deltaslr 0681 under different salt concentration treatment conditions;

in the figure: (A) growth curve graph under normal BG-11 culture condition; (B) A graph of growth under BG-11 stress conditions containing 3% NaCl;

FIG. 5 shows growth of wild-type Synechocystis PCC6803 and Deltaslr 0681 under different salt concentration treatment conditions;

in the figure: (A) Growth conditions of wild-type synechocystis PCC6803 under normal BG-11 culture conditions; (B) Growth conditions of Δslr0681 under normal BG-11 culture conditions; (C) Growth conditions of wild-type Synechocystis PCC6803 under BG-11 stress conditions containing 3% NaCl; (D) Growth conditions under BG-11 stress conditions with Δslr0681 containing 3% NaCl;

FIG. 6 is a graph showing the variation of total carotenoid content under treatment conditions of different salt concentrations of wild-type Synechocystis PCC6803 and Deltaslr 0681;

in the figure: (A) A graph of the total carotenoid content under normal BG-11 culture conditions; (B) A graph of total carotenoid content under BG-11 stress conditions containing 3% NaCl;

FIG. 7 is a graph of carotenoid components of wild-type Synechocystis PCC6803 and Deltaslr 0681 under high salt concentration treatment conditions;

in the figure: (A) A carotenoid component profile for wild-type synechocystis PCC6803 under high salt concentration treatment conditions; (B) A carotenoid composition map under high salt concentration treatment conditions for Δslr0681; 1: blue algae lutein; 2: zeaxanthin; 3: chlorophyll; 4: echinones; 5: beta-carotene;

FIG. 8 is a graph showing the variation of chlorophyll a content of wild-type Synechocystis PCC6803 and Deltaslr 0681 under different salt concentration treatment conditions;

in the figure: (A) A change chart of chlorophyll a content under normal BG-11 culture conditions; (B) A change chart of chlorophyll a content under BG-11 stress condition containing 3% NaCl;

FIG. 9 is a fluorescent quantitative PCR plot of wild-type Synechocystis PCC6803 and Deltaslr 0681 under different salt concentration treatment conditions;

in the figure: (A) The relative expression of different genes under the normal BG-11 culture condition; (B) Relative expression levels of different genes under BG-11 stress conditions containing 3% NaCl.

Detailed Description

The following examples describe the application of the invention in constructing the slr0681 gene directed knockout vector of synechocystis PCC6803, in the method of transforming synechocystis PCC6803 and in regulating and controlling synechocystis PSI-related genes. The invention is further described below with reference to the drawings and examples, but the scope of the invention is not limited thereto.

What is not described in detail in the examples is known in the art.

FIG. 1 is a schematic representation of the carotenoid synthesis pathway in Synechocystis PCC 6803;

fig. 2 is a schematic diagram of construction of a synechocystis PCC6803slr0681 gene targeting knockout vector.

The experimental materials used in the examples were derived as follows:

coli strain (Escherichia coli) DH5 alpha and quick-connect vector T ₃ Purchased from beijing all gold biotechnology limited;

wild type synechocystis PCC6803 is purchased from the institute of science, china, typical culture collection committee, freshwater algae seed stock;

the plasmid pBluescript is purchased from the China center for type culture collection of plasmid vector strain cell genes;

DNA gel recovery kit, plasmid extraction kit, evo M-ML V reverse transcription kit are all purchased from AG company;

restriction endonucleases, T4 DNA ligase were purchased from TaKaRa;

ChamQ Universal SYBR Master Mixes from Vazyme;

other enzymes, reagents, kits, etc. are commercially available.

Culture medium:

the BG-11 liquid medium comprises the following components per liter:

100 XBG-11 (Fe, phosphate, carbonate free) 10mL, ferric ammonium citrate 1mL at 0.02mol/L, and LNa at 0.19mol/L ₂ CO ₃ 1mL, denseDegree of 0.18mol/LK ₂ HPO ₄ 1mL, balance water.

The BG-11 solid culture medium is prepared by adding the following components in each liter based on the BG-11 liquid culture medium:

3g of sodium thiosulfate, 10mL of trimethylol methylaminoethane sulfonic acid with the pH value adjusted by 1mol/L NaOH, and 15g of agar powder.

Wherein, the 100 XBG-11 (without Fe, phosphate and carbonate) culture medium comprises the following components per liter:

NaNO ₃ 149.6g，MgSO ₄ ·7H ₂ O 7.5g，CaCl·2H ₂ O3.6g，Citric acid0.6g，Na ₂ EDTA (pH 8.0, 0.25M) 1.12mL,Trace Minerals100mL, the balance water.

Example 1

Construction of slr0681 knock-out (insertional inactivation) plasmid:

(1) Respectively amplifying upstream and downstream fragments of the slr0681 gene from the genomic DNA of synechocystis PCC6803 by using sequences shown as SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as upstream primers and sequences shown as SEQ ID NO.5 and SEQ ID NO.6 as downstream primers;

(2) Connecting the two gene fragments to a T3 cloning vector, then converting the two gene fragments to escherichia coli DH5 alpha, screening positive clones, and sequencing to obtain slr0681-T3 plasmid;

(3) Cutting slr0681-T3 plasmid with BamHI to recover carrier fragment;

(4) The pBluescript-Kan vector was cut with BamHI single enzyme to recover kanamycin resistant fragment;

(5) Ligating the vector fragment and the kanamycin resistance fragment with T4 ligase and then transforming into E.coli DH5 alpha;

(6) Obtaining a synechocystis slr0681 gene knockout vector named p delta slr0681 by screening positive clones;

the PCR amplification system was as follows:

2X Trans Taq HiFi PCR SuperMix. Mu.L, 1. Mu.L of template DNA, 1. Mu.L of SEQ ID NO.3 primer in the sequence Listing, 1. Mu.L of SEQ ID NO.5 primer in the sequence Listing, ddH ₂ O7. Mu.L, 20. Mu.L in total;

2X Trans Taq HiFi PCR SuperMix. Mu.L, 1. Mu.L template DNA, sequence1 mu L of primer SEQ ID NO.4 in the table and 1 mu L of primer SEQ ID NO.6 in the sequence table as ddH ₂ O7. Mu.L, 20. Mu.L in total;

the PCR amplification procedure was as follows:

pre-denaturation at 94℃for 2min, denaturation at 94℃for 30s, renaturation at 60℃for 30s, extension at 72℃for lmin, final extension at 72℃for 5min after 35 cycles, and storage at 4 ℃.

Example 2

Obtaining of slr0681 knockout (insertional inactivation) mutant:

take logarithmic phase (OD) ₇₃₀ =0.6) (culture conditions: BG-11 liquid medium, light conditions of 30℃and 40. Mu. Mol photon.m ^-2 ·s ^-1 ) 4ml of the culture solution of wild type Synechocystis PCC6803, centrifuging for 10min at 4,000rmp under the condition of room temperature, and discarding the supernatant; after washing once with fresh BG-11 liquid medium, the culture solution was centrifuged to remove the culture solution, the algal cell pellet was resuspended in 1mLBG-11 medium, 15. Mu.L of the recombinant plasmid (pDeltaslr 0681) prepared in example 1 was added, and incubated under light conditions for 6 hours (light conditions: 10. Mu. Mol photon. M) ^-2 ·s ^-1 The temperature was 30 c) and the mixture was gently inverted and mixed once. Then, the mixed culture of the synechocystis and the plasmid was coated on a nitrocellulose filter of a BG-11 solid plate, and cultured in an illumination incubator for 24 hours (culture conditions: 30 ℃ C., illumination conditions: 40. Mu. Mol photon. M) ^-2 ·s ^-1 ) The membrane was transferred to BG-11 (containing 50. Mu.g/mL of kanamycin) for cultivation on solid medium, and the kanamycin concentration was gradually increased to 200. Mu.g/mL. Further separating and purifying, adding into BG-11 liquid culture medium, and shaking culturing on shaking table (culture condition: 30deg.C, 180rpm, light condition: 40. Mu. Mol photon.m) ^-2 ·s ^-1 ). And (3) identifying the insertion of the kanamycin resistance gene by PCR until all wild-type copies completely disappear, and obtaining the slr0681 gene knockout (insertion inactivation) mutant strain algae liquid delta slr0681.

Example 3

PCR detection of slr0681 knockout mutant:

the wild type synechocystis PCC6803 and Deltaslr 0681 are taken as materials, and the total DNA is extracted for PCR detection analysis. The specific method comprises the following steps: DNA extraction of phenol reagentPurchased from Solarbio company) and DNA was extracted from wild-type synechocystis PCC6803 and Δslr0681 using a glass bead shaking method. The specific operation steps are as follows: take 2mL OD ₇₃₀ Blue algae=0.6, algae cells were collected by centrifugation at 12,000rpm for 1min at room temperature, and 0.4mL of 1×te Buffer and an appropriate amount of glass beads (available from sigma company) having a diameter of about 0.17mm were added to a suspension of 0.5mL above the interface of the glass beads. The phenol reagent was extracted by vortexing at maximum rate for 1min followed by the addition of 0.4mL DNA. Centrifuge at 12,000rpm for 10min, take supernatant into a new 1.5mL centrifuge tube, add 0.4mL Tris saturated phenol/chloroform (volume ratio 1:1), centrifuge at 12,000rpm for 2min. The supernatant was taken into a new 1.5mL centrifuge tube, 1mL of isopropyl alcohol was added, and after mixing was reversed, it was settled for 1h at-20 ℃. Centrifugation at 13,000rpm for 10min, removal of supernatant, addition of 1mL of 75% ethanol (v/v), shaking upside down several times, centrifugation at 12,000rpm for 1min. The supernatant is discarded, the mixture is dried in the open air at room temperature until the sediment is transparent, and a proper amount of 1 xTE Buffer is added to dissolve the sediment, thus obtaining the total DNA of the wild type synechocystis PCC6803 and Deltaslr 0681.

DNA of wild synechocystis PCC6803 and Deltaslr 0681 is used as a template, sequences shown as SEQ ID NO.7 and SEQ ID NO.8 in a sequence table are respectively used as upstream and downstream primers, and PCR amplification is carried out, wherein the specific amplification procedure is as follows:

pre-denaturation at 94℃for 5min, denaturation at 94℃for 30s, renaturation at 60℃for 30s, extension at 72℃for 1min, after 35 cycles, 10min at 72℃and storage at 4 ℃.

The amplification system is as follows:

2X Trans Taq HiFi PCR SuperMix. Mu.L, 1. Mu.L of wild-type Synechocystis genome, 1. Mu.L of SEQ ID NO.7 primer in the sequence Listing, 1. Mu.L of SEQ ID NO.8 primer in the sequence Listing, ddH ₂ O7. Mu.L, 20. Mu.L in total;

2X Trans Taq HiFi PCR SuperMix. Mu.L, Δslr 06811. Mu.L, 1. Mu.L of SEQ ID NO.7 primer in the sequence Listing, 1. Mu.L of SEQ ID NO.8 primer in the sequence Listing, ddH ₂ O7. Mu.L, 20. Mu.L in total.

The electrophoresis detection PCR products are shown in FIG. 3. In the figure, M is 5K plusDNA Marker,WT and 1 is Deltaslr 0681PCR amplification result.

Example 4

Growth curve determination under different salt treatment conditions of wild-type synechocystis PCC6803 and Δslr0681:

wild-type Synechocystis PCC6803 and Deltaslr 0681 were inoculated into BG-11 liquid medium containing 0.51mol/LNaCl (3%, w/v), respectively, and OD was adjusted ₇₃₀ To 0.2, 40. Mu. Mol of photons.m at 30 DEG C ^-2 ·s ^-1 Culturing under continuous illumination, sampling at 0, 1, 2, 3, 4, 5, 6, 7, 8 days, and measuring OD every day ₇₃₀ To monitor the growth and the results are shown in fig. 4.

As can be seen from fig. 4 (a), under normal culture conditions, Δslr0681 growth rates were lower than that of the wild-type synechocystis PCC6803, and the concentration difference was maximized on day 5, but the concentration difference was gradually decreased after day 6, and the difference was comparable on day 7. The slr0681 gene knockout affects the growth of the algae strain to a certain extent, but the difference between the slr0681 gene knockout and the algae strain is gradually reduced after the algae strain is cultured for a period of time.

As can be seen from fig. 4 (B), the concentrations of the mutant strains were higher than that of the wild-type synechocystis PCC6803 (WT) under high salt stress conditions, starting from day 5. The slr0681 gene knockout affects the growth of algal strains to a certain extent, and high salt stress can also promote the growth of Δslr0681.

Example 5

Phenotypic changes under different salt treatment conditions of wild-type synechocystis PCC6803 and Δslr0681:

wild-type Synechocystis PCC6803 and Deltaslr 0681 were cultured in BG-11 liquid medium containing 0.51mol/LNaCl (3%, w/v) when grown to OD ₇₃₀ At 0.2, 100mL was dispensed into 250mL Erlenmeyer flasks. Photographs were taken every 24h to observe phenotypic changes between the different treatments.

As can be seen from FIG. 5, the color difference between wild-type Synechocystis PCC6803 (WT) and Δslr0681 was most pronounced by day 6 of culture under stress conditions of 0.51mol/LNaCl (3%, w/v). WT and Δslr0681 are in a weak state under high salt stress compared to growth under normal culture conditions. There was no significant difference in color between WT and Δslr0681 cultured under normal conditions, whereas Δslr0681 cultured under high salt stress was more viable than WT. Indicating that slr0681 gene knockout has an effect on the growth of algal strains.

Example 6

Total carotenoid, chlorophyll a content determination and HPLC analysis under different salt treatment conditions of wild type synechocystis PCC6803 and Δslr0681:

periodic sampling to determine the growth curve of algal cells in example 4, taking 2mL of each sample, centrifuging at 13,000rpm for 10min, suspending the algae precipitate with 1mL of N, N-dimethylformamide solution and 1mL of methanol solution, sucking and mixing to extract cytochromes, centrifuging at 13,000rpm for 10min, collecting supernatant, and measuring OD with spectrophotometer ₄₆₁ 、OD ₆₂₅ 、OD ₆₆₄ Calculating the total carotenoid and chlorophyll a content according to a formula; and analyzing the carotenoid component by High Performance Liquid Chromatography (HPLC).

Carotenoids (μg/mL) = (OD) ₄₆₁ -0.046×OD ₆₆₄ )×4

Chlorophyll a (μg/mL) =12.1×od ₆₆₄ -0.17×OD ₆₂₅

The carotenoids were separated by HPLC using a column model Sphermsorb ODS 2.0 mm X250 mm C18 column, mobile phase comprising A, B two phases, phase A: methanol/ethyl acetate (68:32); and B phase: acetonitrile/methanol/0.1M Tris-HCl, pH8.0 (84:2:14). Setting the detection wavelength to 450nm; setting the column temperature to 25 ℃; the sample injection amount is set to 20 mu l, and the flow rate is set to 1.2ml/min; eluting with 100% mobile phase B for 20min, and gradient eluting from 100% B to 100% A for 15min; a 100% a rinse for 12min, from 100% a gradient elution to 100% b, for 1min,100% b mobile phase rinse for 10min, starting the next sample; standards for related different types of carotenoids were purchased from sigma and standard curves were plotted using the same conditions. Carotenoids are identified by absorption spectra and typical retention times.

As can be seen from FIG. 6, under normal culture conditions, the total carotenoid content of the wild-type Synechocystis PCC6803 was higher than Δslr0681, the difference was apparent at day 5, the difference was 1.23. Mu.g/mL, and the difference was gradually decreased from day 6 to day 8. Under high salt stress conditions Δslr0681 was significantly higher than wild type Synechocystis PCC6803 in total carotenoid content after day 5, with a maximum difference of 0.356 μg/mL at day 7.

As can be seen from fig. 7, HPLC analysis shows that the cyanobacterial lutein, zeaxanthin, echinoctone, and beta-carotene content in Δslr0681 are increased by 1.2-fold, 1.1-fold, 1.7-fold, and 1.05-fold, respectively, compared to wild type under high salt stress conditions.

As can be seen from fig. 8, the chlorophyll a content differences of the wild-type synechocystis PCC6803 and Δslr0681 were not apparent under normal culture conditions. Under high salt stress conditions, the chlorophyll a content of wild-type synechocystis PCC6803 and Δslr0681 differ by 1.40 μg/mL at day 4. This is probably because knockout of slr0681 gene affects salt tolerance of wild type synechocystis, and promotes growth of Δslr0681 under high salt stress, thereby increasing total carotenoid and chlorophyll a content.

Example 7

RNA isolation and cDNA synthesis:

taking logarithmic growth phase (OD) ₇₃₀ To 0.6) 50mL of algae cells, 7,000rmp centrifuging for 10min, collecting the precipitate, grinding in liquid nitrogen, extracting RNA by Trizol method, and detecting the integrity of RNA fragment by electrophoresis; and detecting the concentration and purity of the RNA by using a nucleic acid protein tester. cDNA was synthesized using Evo M-ML V reverse transcription kit. The cDNA obtained above is used as a template, the sequences shown as SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13, SEQ ID NO.15, SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.21 in the sequence table are respectively used as upstream primers, the sequences shown as SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.20 and SEQ ID NO.22 are respectively used as downstream primers, and the sargassum rnpB genes are used as internal reference genes to analyze the expression quantity of the psaA, psaB, psaL genes in PSI and the psbA2, psbD and psbB genes in PSII. The fluorescent dye is ChamQ Universal SYBR Master Mixes, and 2 is used ^-△△Ct The method analyzes real-time quantitative PCR data. The specific method comprises the following steps:

RNA was extracted from wild synechocystis and Δslr0681 using Trizol method:

and (3) cooling the mortar by liquid nitrogen, namely respectively placing wild synechocystis and Deltaslr 0681 into the mortar for grinding, transferring the ground powder into a 1.5mL centrifuge tube without RNase, adding 1mL Trizol, mixing the mixture upside down, and standing for 5min. 0.2mL of chloroform was added to each tube, and after shaking for 20s, the tubes were allowed to stand for 3min. Centrifuging at 12,000rpm for 10min, collecting supernatant, adding equal volume of isopropanol into a new RNase-free centrifuge tube, mixing, and standing for 10min. Centrifuge at 12,000rpm for 10min, discard supernatant, add 0.5ml of 75% ethanol, flick the bottom of the tube, suspend the pellet and invert it up and down several times. Centrifugation at 8,000rpm for 3min, the supernatant was discarded and the procedure was repeated. Air-dried at room temperature, and 50. Mu.L of DEPC was added to dissolve the RNA precipitate sufficiently.

Detection of changes in light system-related gene expression of wild-type synechocystis PCC6803 and Δslr0681:

cDNA was synthesized using Evo M-MLV reverse transcription kit. Using the cDNA obtained as described above as a template and the synechocystis rnpB gene as a reference gene, expression level analysis was performed on the psaA, psaB, psaL gene in PSI and the psbA2, psbD and psbB genes in PSII. The reaction was prepared according to the instructions of ChamQ Universal SYBR Master Mixes (Vazyme) manufacturer and qRT-PCR was performed using ABI 7500.

The reaction system is as follows:

each PCR was performed in a total volume of 20. Mu.L containing 2X ChamQ SYBR qPCR Master Mix (Vazyme), 0.4. Mu.L of each primer at a concentration of 100nM and 1. Mu.L (diluted 1:20 by volume) of template cDNA, 3 replicates per sample.

The reaction procedure was as follows:

pre-denaturing at 95 ℃ for 1min, denaturing at 95 ℃ for 10s, renaturating at 60 ℃ for 30s, and extending at 72 ℃ for 30s,42 cycles; an additional cycle: denaturation at 95℃for 30s, renaturation at 58℃for 30s, extension at 72℃for 5min, and extension at 95℃for 10s.

As can be seen from FIG. 9, under normal culture conditions, the psaA, psaB, psaL gene expression level in Δslr0681PSI was lower than that of WT, and the psbA2, psbD, and psbB gene expression levels in PSII were not much different from that of WT. Under the condition of high salt stress, the expression quantity of psaA, psaB, psaL, psbD, psbB genes in Deltaslr 0681 is higher than that of WT, and the expression quantity of psbB and psbD genes in psaA, psaB, psaL and PSII in PSI are respectively increased by 3.4 times, 1.9 times, 1.5 times, 2.1 times and 1.6 times. This is probably because the slr0681 gene affects the expression of one or more genes in the strain, and changes the expression of the related genes in PSI.

SEQUENCE LISTING

<110> Shandong national academy of agricultural sciences

Application of <120> slr0681 gene in synthesis of synechocystis carotene

<160> 22

<170> PatentIn version 3.5

<210> 1

<211> 1302

<212> DNA

<213> artificial sequence

<400> 1

gtggtctgct ctatagtgac ggcgatattc aaacccaaga agcccagttg cttgcctccc 60

tgcaggaatc agatcctggc catggccacc ccaaatcgtc ccttgataaa tttctcggta 120

aggtccagcg gctttacctt cgggccgtac aaaaggcagg ttaggttttt tcacttgggc 180

ttattgagag caaacatgat gacttggttg accattccgt ttttaattct ggggttaggc 240

attttagtgg cgggggcaga aattttagtc aaaggggctt cccgcattgc cttgatggcg 300

gggctttctc ccttaattat tggtttaacc attgtggcct acggtacgag tatgccggaa 360

atggtggtta gtctccaagc ggcgatcgcc ggccaggccg atatttccat tggcaatgtg 420

gtgggcagta atattttcaa tgtgttgttg atactggggg tctgttccat cattacaccc 480

cttattgtgg cccaacagtt aattcgctta gatattccca ttttgattgg ggtctctgga 540

ctgttgctaa tgtttggttg ggatgggcaa attagtcgag ttgatggcgt aattttagcc 600

agtggagcca ttctctacac aacttttctg atccggcaaa gtaaaaaaga aaataacccc 660

gatgtcaccg aagaatacct caaagaattt ggggaacctg tgcctaaaac cggcaaacag 720

atattcattc aaattgctta tgttgtaggg ggcgttgccc tgcttgtgtt ggggtctagc 780

ttattagtga aaagctccgt ggccattgcc aaaagcctag gcatcagtga attagtaatt 840

ggtttaaccc taattgcggc ggggacttct ttgccagaat tggccacttc tgtagtggct 900

agttaccggg gggagcggga tattgcggtg ggtaatgtgg tgggcagtaa tatttttaat 960

attttggccg tattgggttt tgccgccatt ttttccccca acggtatcca ggtttctaac 1020

tctgctttta actttgatat tcctgtgatg tttgccgttg ccctagtttg tttgcctgtc 1080

tttattaccg gcagattaat tgaccgctgg gagggctttt tattcctgtt ctattacatt 1140

gcctacactg cctatttggt gcttgatgct acccacaatc agaatttaca tatatttaat 1200

aatgtgattc tttttgtggt tattcccgca acggcgatcg ccctaggact atcccttatc 1260

cccgatttag tgagggggaa aaaaaacgat accggaatct aa 1302

<210> 2

<211> 433

<212> PRT

<213> artificial sequence

<400> 2

Met Val Cys Ser Ile Val Thr Ala Ile Phe Lys Pro Lys Lys Pro Ser

1 5 10 15

Cys Leu Pro Pro Cys Arg Asn Gln Ile Leu Ala Met Ala Thr Pro Asn

20 25 30

Arg Pro Leu Ile Asn Phe Ser Val Arg Ser Ser Gly Phe Thr Phe Gly

35 40 45

Pro Tyr Lys Arg Gln Val Arg Phe Phe His Leu Gly Leu Leu Arg Ala

50 55 60

Asn Met Met Thr Trp Leu Thr Ile Pro Phe Leu Ile Leu Gly Leu Gly

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Ile Leu Val Ala Gly Ala Glu Ile Leu Val Lys Gly Ala Ser Arg Ile

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Ala Leu Met Ala Gly Leu Ser Pro Leu Ile Ile Gly Leu Thr Ile Val

100 105 110

Ala Tyr Gly Thr Ser Met Pro Glu Met Val Val Ser Leu Gln Ala Ala

115 120 125

Ile Ala Gly Gln Ala Asp Ile Ser Ile Gly Asn Val Val Gly Ser Asn

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Ile Phe Asn Val Leu Leu Ile Leu Gly Val Cys Ser Ile Ile Thr Pro

145 150 155 160

Leu Ile Val Ala Gln Gln Leu Ile Arg Leu Asp Ile Pro Ile Leu Ile

165 170 175

Gly Val Ser Gly Leu Leu Leu Met Phe Gly Trp Asp Gly Gln Ile Ser

180 185 190

Arg Val Asp Gly Val Ile Leu Ala Ser Gly Ala Ile Leu Tyr Thr Thr

195 200 205

Phe Leu Ile Arg Gln Ser Lys Lys Glu Asn Asn Pro Asp Val Thr Glu

210 215 220

Glu Tyr Leu Lys Glu Phe Gly Glu Pro Val Pro Lys Thr Gly Lys Gln

225 230 235 240

Ile Phe Ile Gln Ile Ala Tyr Val Val Gly Gly Val Ala Leu Leu Val

245 250 255

Leu Gly Ser Ser Leu Leu Val Lys Ser Ser Val Ala Ile Ala Lys Ser

260 265 270

Leu Gly Ile Ser Glu Leu Val Ile Gly Leu Thr Leu Ile Ala Ala Gly

275 280 285

Thr Ser Leu Pro Glu Leu Ala Thr Ser Val Val Ala Ser Tyr Arg Gly

290 295 300

Glu Arg Asp Ile Ala Val Gly Asn Val Val Gly Ser Asn Ile Phe Asn

305 310 315 320

Ile Leu Ala Val Leu Gly Phe Ala Ala Ile Phe Ser Pro Asn Gly Ile

325 330 335

Gln Val Ser Asn Ser Ala Phe Asn Phe Asp Ile Pro Val Met Phe Ala

340 345 350

Val Ala Leu Val Cys Leu Pro Val Phe Ile Thr Gly Arg Leu Ile Asp

355 360 365

Arg Trp Glu Gly Phe Leu Phe Leu Phe Tyr Tyr Ile Ala Tyr Thr Ala

370 375 380

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385 390 395 400

Asn Val Ile Leu Phe Val Val Ile Pro Ala Thr Ala Ile Ala Leu Gly

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Leu Ser Leu Ile Pro Asp Leu Val Arg Gly Lys Lys Asn Asp Thr Gly

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Ile

<210> 3

<211> 19

<212> DNA

<213> artificial sequence

<400> 3

gctttgctgg atgtgccct 19

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<211> 24

<212> DNA

<213> artificial sequence

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<210> 5

<211> 25

<212> DNA

<213> artificial sequence

<400> 5

taggatccgt ttgctctcaa taagc 25

<210> 6

<211> 18

<212> DNA

<213> artificial sequence

<400> 6

ttcggcatgg gctaacaa 18

<210> 7

<211> 18

<212> DNA

<213> artificial sequence

<400> 7

tctcggtaag gtccagcg 18

<210> 8

<211> 18

<212> DNA

<213> artificial sequence

<400> 8

ggtgggagca tatcagtc 18

<210> 9

<211> 20

<212> DNA

<213> artificial sequence

<400> 9

gtgaggacag tgccacagaa 20

<210> 10

<211> 20

<212> DNA

<213> artificial sequence

<400> 10

tgcaccctta cccttttcag 20

<210> 11

<211> 19

<212> DNA

<213> artificial sequence

<400> 11

tggttccact accacgtca 19

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<400> 12

ccccagcatc caaaagttta 20

<210> 13

<211> 20

<212> DNA

<213> artificial sequence

<400> 13

gtcagtttgg gcttggatgt 20

<210> 14

<211> 20

<212> DNA

<213> artificial sequence

<400> 14

gaagtgggct aaaccaacca 20

<210> 15

<211> 20

<212> DNA

<213> artificial sequence

<400> 15

ataacggcga tccttttgtg 20

<210> 16

<211> 20

<212> DNA

<213> artificial sequence

<400> 16

caggggccaa tcaagaagta 20

<210> 17

<211> 20

<212> DNA

<213> artificial sequence

<400> 17

tggtaacctc ctccttggtg 20

<210> 18

<211> 20

<212> DNA

<213> artificial sequence

<400> 18

aaccagatgc cgattacagg 20

<210> 19

<211> 20

<212> DNA

<213> artificial sequence

<400> 19

ttttgttcct gcaagggttc 20

<210> 20

<211> 20

<212> DNA

<213> artificial sequence

<400> 20

gttcaaatgc ccggaaagta 20

<210> 21

<211> 20

<212> DNA

<213> artificial sequence

<400> 21

tgaacagtgg tgatggcatt 20

<210> 22

<211> 20

<212> DNA

<213> artificial sequence

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taacaccggt ttgttccaca 20

Claims (12)

1. The method comprises the following steps ofslr0681Application of gene in synthesis of synechocystis carotene under high salt stress condition, and application of gene in synthesis of synechocystis carotene under high salt stress conditionslr0681The nucleotide sequence of the gene is shown as SEQ ID NO. 1; by knocking out in synechocystisslr0681The gene, and a mutant strain was obtained.

2. The use according to claim 1, wherein the synechocystis is synechocystis PCC6803.

3. The application according to claim 1, wherein the application comprises the steps of:

(1) Respectively using SEQ ID NO.3 and SEQ ID NO.5 in the sequence table as upstream and downstream primers and SEQ ID NO.4 and SEQ ID NO.6 in the sequence table as upstream and downstream primers, respectively amplifying from genomic DNA of synechocystis PCC6803slr0681Upstream and downstream gene segments of the gene;

(2) Connecting the two gene fragments to a T3 cloning vector, then converting the two gene fragments to escherichia coli DH5 alpha, screening positive clones, and sequencing to obtain slr0681-T3 plasmid;

(3) The slr0681-T3 plasmid was usedBamCutting by H I single enzyme, and recovering carrier fragments;

(4) By usingBamH I single-digested pBluescript-Kan vector, recovering kanamycin-resistant fragment;

(5) Ligating the vector fragment and the kanamycin resistance fragment with T4 ligase and then transforming into E.coli DH5 alpha;

(6) Obtaining Synechocystis by screening positive clonesslr0681The gene knockout vector is named p delta slr0681;

(7) Converting the recombinant vector p delta slr0681 prepared in the step (6) into synechocystis PCC6803, and screening to obtain knockoutslr0681A mutant strain of a gene of synechocystis;

(8) Culturing the mutant strain obtained in the step (7), and extracting to obtain carotenoid.

4. The use according to claim 3, wherein in step (1), the PCR amplification system is as follows:

2. x Trans Taq HiFi PCR SuperMix 10. Mu.L, 1. Mu.L template DNA, 1. Mu.L primer of SEQ ID NO.3 in the sequence Listing, 1. Mu.L primer of SEQ ID NO.5 in the sequence Listing, ddH ₂ O7. Mu.L, 20. Mu.L in total;

2. x Trans Taq HiFi PCR SuperMix 10. Mu.L, 1. Mu.L template DNA, 1. Mu.L primer of SEQ ID NO.4 in the sequence Listing, and SEQ ID NO.6 in the sequence Listing as primersSubstance 1. Mu.L, ddH ₂ O7. Mu.L, 20. Mu.L in total;

the PCR amplification procedure was as follows:

pre-denaturation at 94℃for 2min, denaturation at 94℃for 30s, renaturation at 60℃for 30s, extension at 72℃for lmin, final extension at 72℃for 5min after 35 cycles, and storage at 4 ℃.

5. The use according to claim 3, wherein the mutant strain in step (8) is cultivated under high salt conditions.

6. The use according to claim 5, wherein the high salt condition is stationary culture in BG-11 liquid medium containing 0.51mol/LNaCl.

7. The method according to claim 6, wherein the mutant strain in step (8) is grown at a temperature of 28-32deg.C and 35-45. Mu. Mol photon ∙ m ^-2 ∙s ^-1 And (3) carrying out stationary culture in BG-11 liquid medium containing 0.51mol/LNaCl under continuous illumination.

8. Application of synechocystis mutant strain in synthesis of synechocystis carotene under high-salt condition, wherein synechocystis mutant strain is knocked outslr0681Genes of the order ofslr0681The nucleotide sequence of the gene is shown as SEQ ID NO.1, and the synechocystis is synechocystis PCC6803.

9. The use according to claim 8, wherein the high salt conditions are 0.51mol/LNaCl.

10. The method comprises the following steps ofslr0681Gene in improving SynechocystispsaA、psaB、psaL、psbD、psbBUse of the expression level of a gene, saidslr0681The nucleotide sequence of the gene is shown as SEQ ID NO. 1; the application is by knocking out in synechocystisslr0681Genes, obtaining mutant strains; the mutant strain is cultured under high salt conditions.

11. The use according to claim 10, wherein the high salt conditions are 0.51mol/LNaCl.

12. The use according to claim 10, wherein the synechocystis is synechocystis PCC6803.

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