The nucleotide sequence of the target gene containing the upstream and downstream fragments was selected from the genome sequence of Phaeodactylum tricornutum, and then the upstream and downstream homologous arm primers (upstream and downstream homologous arm forward and reverse primers) were designed by using PrimerPremier5.0 at about 500bp of the upstream and downstream fragments. At the same time, the forward and reverse primer pairs of the target gene were designed, and the primers were designed at the left and right of the front end of the 100bp of the upstream homologous arm, respectively. And the reverse primers of the downstream homologous arm were designed at the left and right of the back end of 100bp. The primer sequence is shown in the attachment.
1. Place 10g Tnypxone, 5g Yeast extract, 10g NaCl, 15g Ager, in 1L of dd-water.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room temperature, mix and pour into disposable sterile Petri dishes and store at 4℃.
1. 75 mg NaNO3, 5 mg NaH2PO4-H2O, 4.36 mg Na2EDTA-2H2O, 3.16 mg FeCl3-6H2O, 0.01 mg CuSO4-5H2O, 0.023 mg ZnSO4-7H2 O, 0.012 mg COCl2-6H2O, 0.018 mg MnCl2- 4H2O, 0.07mg NaMoO4-2H2O, 0.1×10-3mg vitamin B1, 0.5×10-6mg biotin (vit. H), 0.5×10-6mg vitamin B12, 15g Ager placed in 1L of seawater.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room temperature, mix and pour into disposable sterile Petri dishes and store at 4℃.
1. Place 10g Tnypxone, 5g Yeast extract, 10g NaCl, in 1L of dd-water.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room temperature, mix and pour into disposable sterile Petri dishes and store at 4℃.
1.75 mg NaNO3, 5 mg NaH2PO4-H2O, 4.36 mg Na2EDTA-2H2O, 3.16 mg FeCl3-6H2O, 0.01 mg CuSO4-5H2O, 0.023 mg ZnSO4-7H2O, 0.012 mg COCl2-6H2O, 0.018 mg MnCl2- 4H2O, 0.07mg NaMoO4-2H2O, 0.1×10-3mg vitamin B1, 0.5×10-6mg biotin (vit. H), 0.5×10-6mg vitamin B12, placed in 1L of seawater.
2. Autoclave at 121℃ for 20min.
3. Add the required antibiotics after cooling at room temperature, mix and pour into disposable sterile Petri dishes and store at 4℃.
Formula (1 ×): NaAC 0.385g/L NH4Cl 0.115g/L MgSO4·7H20 0.066g/L KH2PO4 0.0130g/L CaCl2 0.006g/L FeSO4·7H2O 0.00055g/L
Statistical analysis was performed with the Student's paired t-test. All assays were performed independently for a minimum of three biological replications. Data are represented as mean ± SD.
Phaeodactylum tricornutum（P. tricornutum） is provided by the CCMP algae repository in the United States, under the species label CCMP2561.
The marine diatoms P. tricornutum were grown in batches in triangular conical flasks containing f/2-Si medium and incubated in an artificial climate chamber. The parameters of the artificial climate chamber were: temperature 21 ± 1°C, photoperiod 12 h/12 h (L/D), and light intensity 150 μmol photon m-2s-1.
The 100 mL of algal solution was incubated in 250 mL triangular conical flasks and the mouths of the flasks were sealed with a sealing film containing 0.22 μm filter membrane to isolate airborne bacteria and other materials. The algal solution was manually shaken three times a day during the incubation period to slow down the aggregation of cells settling in the triangular conical flask.
The cell density of P. tricornutum was observed and counted by hemocytometer plate and light
The density formula was CD = (N/80) × 400 × 10^4, where N is the total number of cells in 80 small compartments of the hemocytometer plate .
At least 400 cells were counted by light microscopy with a hemocytometer plate and cell growth curves were plotted for each treatment group at regular intervals daily during the Brown Finger Algae triangle culture cycle.
Enrichment of 500 ml of algal solution was performed using a sterile 50 ml centrifuge tube at 4400 rpm, 4°C for 10 min. The supernatant was discarded, the precipitate was rinsed with RO water, the supernatant was discarded by centrifugation, and the procedure was repeated three times. The collected algal precipitate was transferred to 1.5 ml EP tubes and snap-frozen in liquid nitrogen, followed by freeze-drying for 12 h or longer until all water evaporated. Finally, after snap-freezing in liquid nitrogen, the algal precipitate was immediately stored at -80°C.
Liquid nitrogen is poured into the mortar to pre-cool the mortar and mortar rod. The enriched algal masses were then poured into the mortar and quickly ground to a powder, while liquid nitrogen was continuously added to ensure that the mortar and sample were kept at a low temperature. Finally, the powder was poured into 1.5 ml RNAse free EP tubes and placed back into liquid nitrogen for freezing. The powder was freeze-dried in a freeze-dryer until all the water was evaporated, then frozen in liquid nitrogen and stored at -80°C.
Take 11 test tubes, number them, add various solutions according to Table 2, and then measure the absorbance value at 445nm with the mixture of test tube No.0 as reference. Taking fucoxanthin as abscissa and absorbance as ordinate, the regression equation was obtained.
Fig7.Folding graph of the remaining content of nitrogen and phosphorus elements
The extraction and content determination of fucoxanthin from P.tricornutum were carried out by organic solvent extraction method. 80mL of P. tricornutumwas accurately extracted and centrifuged at 5000r·min-14 for 10min. The supernatant was discarded, freeze-dried for 2d, weighed and ground into powder about 0.1g with anhydrous ethanol, alga powder: anhydrous ethanol =1:40(g·mL1),60 The supernatant was taken and the absorbance was measured at 445nm wavelength (OD445) by uv spectrophotometer. The content of fucoxanthin was calculated by regression equation
The BCA Protein Assay Kit is based on the BCA method, which is one of the most commonly used protein concentration assays in the world, and offers simplicity, stability, sensitivity and compatibility. The lower limit of detection is 10 μg/ml, and the minimum amount of protein to be detected is 0.2 μg. In this experiment, total protein was extracted and measured using the BCA Protein Quantification Kit BCA protein concentration assay kit (enhanced version 20201es76).
1.Take an enzyme plate and add reagents as shown below
1. Formulation of BSA standard system
Standard dilutions are solubilizations of protein samples, in principle in what solution the protein sample is in, and in what solution the standard is also diluted appropriately. But also available in 0.9% NaCl or 1 × PBS for dilution.
2. Formulation of BCA working solution
1) Calculate the total BCA working fluid volume required.
Total BCA working fluid volume = (Standard + sample to be tested) × Number of repeats × BCA working solution required for each sample.
2) Prepare BCA working solution: 50 volumes of BCA reagent a add 1 volume of BCA reagent B (A: B = 50:1) and mix well.
1. Cuvette detection method (sample: BCA working solution = 1:20)
1) Of 100 each μ L standard and sample to be tested added to
the
reaction tube.
2) Add 2.0 ml of BCA working solution to each tube and mix.
Incubate at 37 ° C for
30min.
3) Cool to room temperature. Spectrophotometer detection with a
set wavelength of 562
nm. Correct the instrument
with a cuvette filled with water. Readings were taken within 10 min for all samples.
4) A standard curve (X-protein concentration UG / ml; y-final od562nm) was
generated by
subtracting the OD value
of the blank well in the standard from the absorbance of the BSA standard (i.e., the final reading). Sample
protein concentrations were calculated according to the standard curve and dilution factor of the samples.
2. Microplate assay method (sample: BCA working solution = 1:8)
1) G of each 25.0 μ L standard and the sample to be tested
added to the microplate.
2) 200 μ l was added to each well μ L BCA working solution and
mix thoroughly by shaking for
30s. Close the
microplate and incubate at 37 ° C for 30min.
3) Cooled to room temperature, the absorbance was detected at the wavelength
range of 540-595 nm on a microplate
reader with 562 nm wavelength as optimal.
4) A standard curve (X-protein concentration UG / ml; y-final od562nm) was
generated by subtracting the OD value
of the blank well in the standard from the absorbance of the BSA standard (i.e., the final reading). Sample
protein concentrations were calculated according to the standard curve and dilution factor of the samples.
Total carbohydrate content was quantified using the phenol-sulphuric acid method . About 10 mg of lyophilized algae was resuspended in 1 mL of deionized water. This algal suspension was added with 1 mL of 5% phenol solution (w/v) was rapidly added directly to be mixture. The mixture was incubated for 10 min at room temperature and then 30°C for 20 min. The content of carbohydrates was detected at 483 nm. Glucose was used as standard at different concentrations (up to 1 g L−1).
Fig7.Folding graph of the remaining content of nitrogen and phosphorus elements
Total lipid content was also determined by gravimetric analysis. About 20 mg of lyophilized algae was mixed with 2 mL of chloroform, 2 mL of methanol and 1 mL of 5% NaCl by vortex for 2 min, then centrifuged at 8000g for 4 min at 10 °C. The chloroform phase was collected and kept for subsequent analysis. The residual extract was extracted three more times. All the collected chloroform phase were mixed together and dried under nitrogen flow. The dried lipid residue was further dried in oven at 60 °C and the total lipid content was quantified as a percentage of the dried weight of algae.
For fat analysis, total fat was extracted from groups of three independent biological replicates with reference to the method of Lepage and Roy 1984.
Fatty acids were applied at 30 μ M × 0.25 mm × .The determination was performed by GC-MS of a 0.25 um DB-5 quartz capillary column. The thin tube column was held at 60 ° C for 1 min, then ramped to 160 ° C at a rate of 10 ° C Min 'and raised again to a final 250 ° C at a rate of 2.5 ° C min 1. The injection temperature was 280 ° C, and the injection volume was 1 UL. The mass spectrometry transfer line temperature was 200 ° C, and fatty acids were identified with an NBS equipped spectral library (National Bureau of Standards, Gaithersburg, MD, USA) and quantitatively analyzed by determining the comprehensive peak area (Yang et al. 2013, 2014). Finally, the relative amounts of the detected fatty acids were calculated by normalization.
1. Divide one sample into three tubes, each filled with 100 μ L stock solution
2. Add 790 μ L PBS buffer
3. Add 100 μ Dmso, close nozzle and start shaking, count for 20 min
4. Open one tube at a time, add 10 μ L Nile red and shaken every 2-3 min
until the end
of 20
min.
5. Measure at 590 nm wavelength
1. Digestion: Take a certain amount of the alga like centrifugation. Pipette 400 μl of the supernatant after centrifugation into 10ml colorimetric tube. Then add 4.6 ml of ultrapure water and 5 ml of digestion agent to the colorimetric tube. Cover the colorimetric tube and shake it well, tie the lid tightly with tape, put it in a high pressure steam sterilizer, digest it under high pressure at 120 ℃ for 30 minutes, and take it out after cooling. Aspirate the clear liquid of the digestive juice for the determination of NO3--N.
In addition, 0, 1, 2, 3, 4, 5, and 6 ml of nitrogen mixed standard solution of 10 μg/ml were respectively drawn into 7 digestion containers, that is, the nitrogen content in each tube was 0, 10, 20, 30, 40, 50 , 60 μg, to form a standard series. Then digest simultaneously with the sample according to the above method.
2. Determination of NO3-—N: Since the final pH of the digestate is 2, it exactly meets the pH requirements of the UV assay, so the supernatant of the digestate can be directly measured at wavelengths of 220 nm and 276 nm using a 1 cm quartz cuvette. The difference in absorption (A220-2A270) corresponds to the working curve to find out the corresponding micrograms of nitrogen (μg), divided by the water sample (mg) to get the total nitrogen content (μg/mg), which is reduced by 10 times to obtain total N %.
1. Digestion: Take a certain amount of the alga like centrifugation. Pipette 160 μl of the supernatant after centrifugation into 10ml colorimetric tube. Then add 1.84 ml of ultrapure water and 2 ml of digestion agent to the colorimetric tube. Cover the colorimetric tube and shake it well, tie the lid tightly with tape, put it in a high pressure steam sterilizer, digest it under high pressure at 120 ℃ for 30 minutes, and take it out after cooling. Aspirate the clear liquid of the digestive juice for the determination of PO 4 3- —P.
In addition, 0, 1, 2, 3, 4, 5, and 6 ml of phosphorus mixed standard solution of 10 μg/ml were respectively drawn into 7 digestion containers, that is, the phosphorus content in each tube was 0, 10, 20, 30, 40, 50 , 60 μg, to form a standard series. Then digest simultaneously with the sample according to the above method.
2. Determination of PO43-—P: Add 1ml molybdenum antimony anti-mixing developer and 5ml ultrapure water into the colorimetric tube and shake well. After 30 minutes of color development at a temperature of 20-40 ℃, at 700 nm, use an enzyme plate to measure phosphorus on a microplate reader. The measured value A700 of the sample corresponds to the corresponding phosphorus content (μg) found in the working curve, and divided by the water sample (mg) to obtain the total phosphorus content (μg/mg), which is reduced by 10 times to obtain the total P%.
1. Prepare Phaeodactylum tricornutum growing to the exponential stage of 1.0E+07~1.0E+08. Sorbitol (375 mm / L) (i.e. 34.1568 g / 500 ml) RO water configuration, autoclaved. Salmon DNA, heated at 100 ℃ for 1 min. The electric shock cup is ultrasonically cleaned with RO water, soaked in absolute ethanol, and pre-cooled at -20 ℃. Filter paper and tweezers are autoclaved.
2. Pour the algal liquid into a 50 ml centrifuge tube on the ultra-clean workbench, 4400 rpm at room temperature for 5 minutes, take it back to the ultra-clean, pour off the supernatant, and repeat several times until all the algal liquid is collected. Add 50 ml of sorbitol, suspend with slight shaking, centrifuge at 4400 rpm, discard the supernatant, and repeat three times.
3. According to the actual amount, add an appropriate volume of sorbitol and suspend.
4. To a sterile EP tube (1.5 ml), add 4 μg of linearized plasmid, 4-6 μl (1 μg/μl) of salmon sperm DNA heated at 100°C for 1 min, and 200 μl of the above-mentioned algal solution. Ice bath for 10min.
5. Use tweezers to remove the electric shock cup from the absolute ethanol, place it on the sterilized filter paper, and air-dry until the absolute ethanol is completely evaporated.
6. Add the above solution to the shock cup.
7. During electroporation, the metal contacts of the electric shock cup should be dry and wiped clean with paper. Electroporator parameters: voltage (U): 1500; Capacitance (uF): 25; resistance (Ω): 400; Cuvette (mm): 4.
8. At the end of the electric shock, Pour the algal liquid into a 50 ml conical flask, add 10 ml f/2 seawater (protect from light). Wrap the conical flask tightly with tin foil and place it in the incubator, dark for 24 hours, and then light again for 24 hours.
9. Coating: Take 5 ml of algae liquid and centrifuge, discard the supernatant, leave a small amount of liquid, suck it up with a pipette, and evenly point it to the f/2 agarose medium with 85 mg/L bleomycin.
MolPure ® Plant DNA Kit(18800ES50) adopts MolPure ® DNA Column P1 and a new solution system are suitable for rapid and simple extraction of genomic DNA from plant tissues (including plants containing phenols, polysaccharides and enzyme inhibitors), cells and fungal samples.The specific experimental procedures were as follows.
1. Sampling: grind 20mg dry weight tissue in liquid nitrogen environment
2. Splitting: add 400ul LB buffer P1 and 4ul RNase A
3. Extraction: add 130ul BD BufferP1
4. Transfer: suck the supernatant into a new centrifuge tube
5. Settlement: add 1.5 times the volume of PL Buffer P1
6. Adsorption: transfer to the adsorption column and centrifuge
7. Washing: add 600ul wash buffer and centrifuge
8. Residue removal: empty column centrifugation
9. Elution: add 100ul Elution Buffer, centrifuge
10. Add water
TRIeasyTM Total RNA Extraction Reagent(10606ES60) is a total RNA extraction reagent suitable for various animals and plants, bacterial tissues and cells, with strong lysis ability, which can lyse cells and tissue samples in a short period of time and effectively inhibit the degradation of RNA in the sample.The specific experimental procedures were as follows.
1) Add 1/5 volume of chloroform to the above lysate
2) Centrifuge at 4 °C, 12000 g for 10-15 min.
3) Carefully pipette the upper aqueous phase into a new centrifuge tube and add
1/2 volume of isopropanol
4) Centrifuge at 4 °C, 12,000 g for 10 min.
5) Carefully discard the supernatant and add an equal volume of 75% ethanol
Vortex to wash well and flick the bottom of the tube to allow the pellet to levitate.
6) Centrifuge at 4 °C, 7500 g for 5 min, discard the supernatant, take care not
to lose the RNA pellet.
7) Place air dry at room temperature for 5-10 min. Add 30-100 μL of RNase-free
water to dissolve RNA
1) RNA integrity testing
2) RNA purity assay
1. Extraction of total protein from monolayer adherent cells
2. Extraction of total protein from tissues
3. Extraction of total protein from adherent cells treated with drugs
1. Extraction of total protein from monolayer adherent cells
2. Extraction of total protein from tissues
3. Extraction of total protein from adherent cells treated with drugs
1. Wash glass plates
2. Sizing and loading
3. Electrophoresis
1. Place the clips used for the transfer, two sponge pads, a glass rod, filter paper, and the
soaked membrane in the enamel dish with transfer fluid.
2. Open the clamp so that the black side remains horizontal. Pad a sponge pad on
top
3. Glue the glass plate first by prying it out
4. Place the clamp in the transfer tank and transfer it with 60 V for 2 h
5. After turning, the membrane is washed with 1 × Ponceau dye solution was
stained for 5 min (shaking on a destaining shaker). The proteins on the membrane were then visualized by rinsing
away the dye free from the stain with water. Leave the membrane to dry for later use.
1. Take a certain volume of algal cell concentrate and pre-sterilized SA (sodium alginate) solution 1:1 and mix well to form the mixture. Shaker 180rpm 21℃ 30min
2. Inject it into a 1ml syringe and drop the mixture at a distance of 20cm from the surface of the pre-cooled cacl2 solution to form a 4mm diameter algal sphere, fix it at room temperature for 2h, then rinse it with sterile water three times and place it in seawater spiked with mother liquor for cultivation.