Experiments

This experimental page is about all the experimental protocols used in our dry lab and the breakdown of each specific experimental step, where the target gene fragment is transformed into E. coli to extract the plasmid, and then the plasmid is used to construct the gene overexpression vector, after which Agrobacterium-mediated methods are used to obtain the overexpression plants, and finally the flavonoid content is detected by EC-MS.

1、Sample handling:

① A 50-100 mg plant sample was taken from a centrifuge tube treated with RNAase scavenger using RNAase scavenger and loaded into steel beads burned with alcohol;

② Liquid nitrogen grinding. Add 1000ul of cell lysis solution A and place in a vortex shaker for 30s to fully lysed.

2、Add 300ul of Deproteinized Liquid B and 200ul of chloroform to the centrifuge tube and mix well with rapid shaking, at this time the solution is uniformly turbid, and leave it at room temperature for 2 minutes.

3、Centrifuge for 5-10 min at room temperature using a centrifuge at 12,000 rpm, and aspirate the supernatant (no more than 700ul) into another clean 1.5 ml centrifuge tube, in which the lower and middle layers contain mainly DNA, proteins, and other impurities, so be careful not to aspirate them.

4、Add an equal volume of rinse solution C, mix well by inversion, and then the resulting mixture was divided into two times if the same adsorption column, centrifuged at 12000 rpm for 1 min, and poured off the waste liquid.

5、Then add 500ul of column wash solution D to the adsorption column, centrifuge at 12000rpm for 1min at room temperature, pour off the waste liquid, add another 500ul of column wash solution, repeat once, and finally leave the column in vacuo for 1min to remove the residual liquid.

6、RNA elution: transfer the adsorption column into RNase-free 1.5ml centrifuge tube, which can be divided into two times by adding 30ul of RNA eluent F each time, leaving it at room temperature for 3-5min, and centrifuging it at 12000rpm for 1min, then total RNA of plant body can be obtained.

1、To calculate the volume of RNA required, since a typical experiment requires 2ug of RNA, divide the RNA concentration by 2 to obtain the volume of RNA required.

2、Sequentially add RNase free ultrapure water, 1ul gDNA Remove, 4ul 5×TURE RT Mix, RNA.

3、PCR program: 42°C 15min , 85°C 5s , 12°C ∞ .

4、The cDNA obtained after reversal was placed in a 4°C refrigerator for backup.

1、Open the DNAMAN software, select the Primer option Load Primer, and then select From input, at this time, a dialog box will appear, and then in our coding region of the sequence of the most upstream in accordance with the principles of primer design to pull the 20-30bp fragment sequence, imported into the dialog box, click ok.

2、Then in the selection of Primer options in the Melting Temperature, at this time you can observe the annealing temperature, GC content of the fragment we pull the value.

3、According to the principle of primer design, we keep testing the sequence fragments until we find a compatible one that can be used as our F primer.

4、R primer design steps and F primer consistent, the final choice of primer fragments for reverse complementation can be.

1、First of all, prepare the cDNA template, primers, droplet water, and M5-Taq enzyme.

2、According to the following indicators in the PCR tube to add the prepared materials: 1.25 μl F primer, 1.25 μl R primer, 2.5 μl cDNA, 25 μl M5-Taq enzyme, 20 μl droplet water.

3、After adding the PCR system, set up the PCR program according to the following parameters: (1) pre-denaturation: 95℃ for 3 minutes.

(2) Denaturation: 94℃ 25s (3) Annealing: 58℃ 25s (4) Extension: 72℃ 1min (5) End extension: 72℃ 5min (6) ∞: 4℃. (In which steps 2-4 set 34 cycles, the annealing time can be adjusted by the transcription speed of the enzyme used and the maximum length of the fragment to be copied.)

4. At the end of PCR, use gel electrophoresis to verify that the length of the sample is correct.

1、Preparation of TAE buffer: according to the ratio, dilute 50×TAE solution into 1×TAE solution.

2、Preparation of agarose gel mixture: mix 0.9g agarose with 60ml 1×TAE solution in a conical flask.

3、Dissolve agarose: Heat the agarose gel mixture in a high-power microwave oven. When the liquid begins to boil, remove the conical flask and gently stir with gloves on. Then return the conical flask to the microwave and repeat heating and stirring until the agarose powder is completely dissolved.

4、 Add Nucleic Acid Dye: After cooling slightly, add nucleic acid dye to the gel mixture at a ratio of 1:10,000, stirring or gently rotating to mix completely.

5、 Pour into gel mold: Pour the liquid gel into the gel mold and wait for about 20 minutes until the gel is completely dry, then take out the gel from the mold (make a gel plate, choose a large hole or a small hole comb as needed).

6、Prepare the electrophoresis tank: Pour TAE solution into the electrophoresis tank and put in the gel. Add more TAE solution until the gel is completely covered by TAE solution.

7、Add PCR product: Add PCR product and Marker into the wells of the gel. The amount added can be determined according to the size of the gel.

8、Start running the gel: run the gel at 150V for 15 minutes. If needed, increase the running time until the product runs at least to the middle of the gel. For clearer results, reduce the voltage and extend the run time.

1、Under the long-wave ultraviolet light or blue light, use a clean blade to cut off the DNA bands to be recovered, try to excise the gel without DNA, and get the gel volume as small as possible.

2、Put the cut off gel containing DNA bands into a 1.5ml centrifuge tube and weigh it.

3、For every 100mg of 1% agarose gel, add 100μl of lysis/binding solution HB (high concentration). For high concentration agarose gels, the amount of lysate/binding solution HB (high concentration) added should be increased proportionally. In order to simplify the experiment, it is recommended that the amount of lysate/binding solution HB (high concentration) added is uniformly 400 μl.

4、Place in a water bath at 56°C for 5-10 minutes (or until the gel is completely dissolved). Vortex shaking every 2-3 minutes to help accelerate dissolution.

Column equilibration: Take a new silica gel membrane adsorption column EC in a collection tube, draw 100μl of equilibration buffer into the column. centrifuge at 12000rpm for 1 minute, pour out the waste liquid in the collection tube and put the adsorption column back into the collection tube. At this time, the equilibrium solution pretreatment column is completed.

5、Add the solution obtained in the previous step to the adsorbent column EC (the adsorbent column is put into the collection tube), leave it at room temperature for 1 minute, centrifuge at 12,000 rpm for 30-60 seconds, and pour out the waste liquid in the collection tube.

6、Add 600μl of rinsing solution WB (please check if you have added anhydrous ethanol first!) Centrifuge at 12,000pm for 30 seconds and discard the waste liquid.

7、Repeat step 6 again.

8、Place the column EC back into the empty collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much rinse solution as possible.

9、Take out the adsorbent column EC and put it into a clean centrifuge tube, add 50 μl of elution buffer EB to the middle of the adsorbent membrane, leave it at room temperature for 2 minutes, and centrifuge it at 12,000 rpm for 1 minute. The final liquid in the centrifuge tube is the PCR purified product.

1、Room temperature (25 ℃ -37 ℃) set up 10 μl ligation system. The system is: purified PCR product 8μl, pTOPO Vector 1μl, 10 × Enhancer 1μl.

2、After adding the reagents, use a pipette to gently blow and mix or flick the bottom of the tube to mix, and then centrifuge at low speed to collect all the liquid at the bottom of the tube.

3、Room temperature (25℃-37℃) ligation 5-10 minutes (recommended to put on the PCR instrument temperature control). Place the ligation product on ice and set aside.

4、Take out the E. coli receptor cells from -80℃, quickly insert them into the ice bath, thaw and melt (about 3 hours).

5、Immediately add 10 μl of ligation product, gently mix by hand by dialing the bottom of the centrifuge tube (avoid sucking and beating with a gun), and place in the ice bath for 30 min. The 30-minute ice bath will help the effective binding between the ligation product and the E. coli cells.

6、Warm up the centrifuge tube at 42℃ for 90 seconds, then put it back to the ice bath and leave it for 2-3 minutes, do not shake the tube during the process.

7、Add 300μl LB medium (without antibiotics), 37 ℃ 200rpm shaking culture 1h.

8、Take 100-200μl of bacterial solution with a coater plate (ampicillin plate). When coating the plate must make the bacterial liquid evenly distributed in the ampicillin plate, and must be blown dry before sealing the plate, the plate inverted culture in 37 ℃ biochemical incubator.

1、Generally in the bacterial solution coated ampicillin plate into the biochemical incubator 12-16h, the plate will grow single colonies, at this time you can carry out the operation of picking bacteria.

2、Fifteen minutes in advance, the gun head, pipette gun, LB liquid medium, 2ml centrifuge tube, ampicillin antibiotics, etc. into the ultra-clean bench for ultraviolet sterilization (not less than 15min).

3、After spraying the plate full of single colonies with alcohol, take it into the ultra-clean bench.

4、Add one thousandth of ampicillin antibiotic into LB culture medium (Example: 50ml culture medium + 50μl ampicillin antibiotic).

5、Add the mixed medium into 2ml centrifuge tube for spare (each tube + 1ml).

6、Take a small gun tip to pick the single colony on the plate, and then hit the gun tip into the prepared centrifuge tube.

7、Followed by the centrifugal tube into the 37 ℃ shaker for shaking bacteria, 4-5h or so.

8、At this time, the bacterial liquid has been shaken mixed, you can use the bacterial liquid as a template for PCR amplification, and then run the gel to initially determine whether it is consistent with the length of our target fragment.

9、According to the following indicators in the PCR tube in order to add the prepared materials: 0.5 μl F primer, 0.5 μl R primer, 1 μl of bacterial liquid, 5 μl M5-Taq enzyme, 3 μl of droplet water (of which the F, R primer is the primer used in the gene cloning).

10、After adding the PCR system, set up the PCR program according to the following parameters: (1) pre-denaturation: 95℃ for 3min

(2) denaturation: 94 ℃ 25s (3) annealing: 58 ℃ 25s (4) extension: 72 ℃ 1min (5) end extension: 72 ℃ 5min (6) ∞: 4 ℃.

11、After the PCR program is finished, start to run the gel. If the gel electrophoresis results show a roughly similar bp number to that of our target fragment, then the bacterial fluid can be sent to the company for sequencing.

12、Compare the sequencing results of the company, the sequence is correct, let the company return to the sample, keep bacteria. That is, take the same volume of glycerol and E. coli bacterial fluid in the ultra-clean bench, mix well, and put it into -80℃ refrigerator for storage.

1、Take 1.5-4.5ml of overnight culture, centrifuge at 12,000rpm for 30s, pour dry supernatant as much as possible and collect the bacterial bodies. Collect more than 1.5 ml of bacterial fluid, you can centrifuge and discard the supernatant, then add more bacterial fluid in the same 1.5 ml tube and repeat step 1 until enough organisms are collected.

2、Resuspend the bacterial body precipitate with 250 μl of solution P1 and vortex and shake until thoroughly suspended.

3、Add 250μl of solution P2, gently turn up and down 6-8 times to make the bacterial body fully lysed, and leave it at room temperature for 4min. mix gently, do not shake vigorously to avoid the plasmid being damaged.

4、Add 350 μl of solution P3, immediately turn up and down gently 6-8 times, white flocculent precipitate will appear when mixing thoroughly, centrifuge at 12,000rpm for 10min, take the supernatant carefully. The supernatant should be removed carefully. Solution P3 should be mixed immediately after addition to avoid localized precipitation of SDS.

Column equilibration: Take a new silica membrane adsorption column in a collection tube, draw 100 μl of equilibrium solution into the column. centrifuge at 13,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube. At this point, the equilibrium solution pretreatment of the column is completed. Then proceed to the next step.

5、Add the supernatant obtained in the previous step to the column (the column is put into the collection tube), centrifuge at 12,000rpm for 30-60s, and pour out the waste liquid in the collection tube.

6. Add 600μl of rinse solution WB (please check if anhydrous ethanol has been added first!) ), centrifuge at 12,000rpm for 30s, discard the waste liquid.

7. Repeat step 6.

8、Place the adsorption column back into the empty collection tube and centrifuge at 12,000rpm for 2min to remove as much rinse solution as possible.

9、Take out the adsorption column AC, put it into a clean centrifuge tube, add 50-100 μl of elution buffer EB to the middle part of the adsorption membrane (the elution buffer can be heated up in a 65-70℃ water bath for better effect), leave it at room temperature for 2 minutes, and then centrifuge at 12,000 rpm for 1 minute, and the liquid obtained in the centrifuge tube is the desired plasmid.

1、According to the mapping of different vectors to determine the required endonuclease, and then according to the nature of the endonuclease for enzymatic operation.

2、The three vectors to be cut in this experiment are the overexpression vector pCY-35S, yeast monohybrid PJG and PlacZi vector.

3、The endonucleases required for pCY-35S are BamHI and PmlI, and the endonucleases required for PJG and PlacZi are XhoI and EcoRI-HF.

4、Add 50ul of digestion system, 1ul of endonuclease 1, 1ul of endonuclease 2, 5ul of rcutSmart, 8ul of uncut empty plasmid, 35ul of ddH2O.

5、37 ℃ reaction 2h, add 6 × loading buff running gel to see the band to determine whether the cut.

Recover the purified plasmid after cleavage.

1、Before transforming the yeast, the plasmids of PJG+ , PlacZi +, PJG-, PlacZi -, PlacZi (carrying cis-acting regulatory elements of downstream target genes), PJG (carrying transcription factors) and other bacterial fluids should be extracted out in advance.

2、Take 100 μl of EGY48 receptor cells melted on ice, and add 2-5 μg of pre-cooled target plasmid, 10 μl of Carrier DNA (95-100 degrees for 5 min, rapid ice bath, repeat once), and 500 μl of PEG/LiAc according to the combinations of the negative control, the positive control and the experimental group.

3、Pipetting and mixing several times, 30 degrees water bath for 30 minutes (turn 6-8 times at 15 minutes to mix).

4、Water bath at 42 degrees for 15 min (7.5 min, turn 6-8 times to mix).

5、Centrifuge at 5000 rpm for 40s and discard the supernatant. ddH2O 400μl resuspension, centrifuge for 30s and discard the supernatant.

6、ddH2O 50μl resuspension, applied to the SD plate (also known as two lack of plate), put into 28 ℃ biochemical incubator culture 48-96h. After culture, will grow white yeast cells.

1、the preparation of chromogenic medium: in a conical flask add 3.35g YNB, 0.36g D0/-Trp-Ura, 412.5ml H2O, and then adjust the PH to 5.8, after the completion of the conical flask add 10g agar.

2、Put the configured mixture into the sterilizer, 115 ℃ sterilization for 15 min. after the end of sterilization, lower the temperature to 55 ℃ in the ultra-clean bench to add the following reagents:

10×BU salt (55℃ preheating) 50ml

40% galactose (55℃ preheated) 25ml

40% cotton seed sugar (55°C preheated) 12.5ml

X-Gal (mother liquor concentration 20mg/ml) 2ml

3、Prepare 10×BU salt solution (100ml): add 3.71g Na2HPO4 and 3g NaH2PO4 in 100ml water, and then adjust the PH value to 7.0, then put it into the sterilizer and sterilize it at 115℃ for 15min.

4、40% galactose (25ml): take 10g of galactose dissolved in 25ml H2O, then filtered and sterilized.

40% cotton candy (12.5ml): take 10g cotton candy dissolved in 12.5ml H2O and then filtered and sterilized.

5、X-Gal solution: take 40mg of X-Gal powder and dissolve it in 2ml DMSO solution.

6、Spot the yeast grown on the SD plate into the chromogenic medium as needed, and incubate it at 29℃ for 48-96h to develop the color and take pictures.

1、Use SnapGene software to open the sequence of pCY-35S overexpression vector, look for two endonucleases downstream of 35S to cut the plasmid, choose BamHI and PmlI for this experiment, and look for another 15 bases before the BamHI site to act as homology arm F, and look for another 15 bases after PmlI site to act as homology arm R.

2、in order to facilitate the later detection of gene overexpression effect, you can add a piece of flag tag sequence during the amplification process, and divide the amplification process into two steps.

3、Add the gene cloning primer F to the homology arm F to form the overexpression primer F (OE-F), and invert the flag tag sequence to add the gene cloning primer R to form the overexpression primer R1 (OE-R1).

4、Add the reversed and complementary flag tag sequence to the homology arm R to form the overexpression primer R2 (OE-R2).

The first round of PCR amplification was performed using OE-F and OE-R1, and then the second round of amplification was performed using OE-F and OE-R2.

1、Sequencing the correct DH5α bacteriophage with genes and expression vectors were plasmid extracted to obtain plasmid DNA.

2、go to -80 ℃ stored Agrobacterium susceptible cells (GV3101) on ice to melt.

3、Add 4-6ul plasmid DNA to each tube of GV3101, gently dial the bottom of the tube by hand to mix well, and then stand on ice for 5min, liquid nitrogen for 5min, 37℃ water bath for 5min, and ice bath for 5min in turn.

4、Add 300ul LB solution, placed in 28 ℃ shaker shaking culture 2-3 hours.

5、Shake the good bacterial solution sucked 150ul coated to Kana-Rif medium, 28 ℃ incubator culture 2-3 days.

6、Pick a single colony to add Kana-Rif culture medium, 28 ℃ shaking incubated for 4 hours, and finally for colony PCR identification, there are bands can be preserved bacteria standby.

1、Soak the seeds of cucumber periwinkle milfoil in warm water at 50℃ for 30 min, dehull and sterilize the seeds.

2、Place the seeds in 75% ethanol for surface disinfection for 45 s, and rinse with sterile water for 3 times.

3、Disinfect the seeds with 4% NaClO for 8 min and rinse with sterile water for 3 times.

4、Seeds were inoculated in MS medium (4.43 g‧L-1MS powder, 30 g‧L-1 sucrose and 3 g‧L-1 gel) and incubated in the dark (28°C) for germination.

5、After 1 d of seed germination, the hypocotyls of cucumber seedlings are carefully removed on an ultra-clean bench, the cotyledons are cut across the cucumber with a scalpel, the distal hypocotyls are carefully removed, and the proximal half of the cotyledons are cut in half to serve as explants for the regeneration of the cucumber.

6、Centrifuge the overexpressed Agrobacterium spp. from overnight incubation, resuspend it to OD 0.2-0.3 by adding infestation solution, infest the explants in two times, each time for 90s, and place the back of the explants upwards in the co-culture medium.

7、Three days later, the exoskeleton was inoculated vertically in the adventitious shoot induction medium with the exoskeleton opening downward.

8、After a period of time, observe whether fluorescent buds are produced under ultraviolet light, if so, it means that Agrobacterium infestation is successful, cut into the rooting medium to root, and then move into the nutrient soil to cultivate into regeneration plants.

9、Take the tissue samples of overexpressed plants for sequence, expression and flavonoid content determination.

1、NCBI website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome) fluorescence quantitative primer search page can get some primers by inputting the information of the gene, and choose the appropriate primers.

2、After the primers are synthesized, the genomic plasmid of the gene should be used for PCR amplification to test the specificity of the primers, if the runners show only one band, it means that the primers have good specificity.

3、Extract RNA from overexpressed plant tissue samples, reverse transcribe it into cDNA, and add 1ul cDNA, 3ul RNAfree H2O, 0.5ul primer F, 0.5ul primer R, and 5ul qRT-PCR Mix to PCR tubes sequentially.

4、PCR amplification was performed using QuantStudio™ Real-Time PCR instrument in the following steps:

Holding Stage

Step 1: 95℃ 30 sec

Cycling Stage

Number of Cycles: 40

Step 1: 95℃ 3 sec

Step 2: 60℃ 30 sec

Melt Curve Stage

5、Finally, the data were analyzed using the 2-ΔΔCt method.

(1) Chromatographic conditions

a. Chromatographic column: ZORBAX SB-C18 (4.6×150 mm, 5-Micron)

b. Mobile phase: acetonitrile: 0.1% (v/v) formic acid, elution gradient see the following table：

c. Detection wavelength: 254 nm

d. Detection column temperature: 25 ℃

e. Flow rate: 1.0 ml/min

f. Injection volume: 10 μl

(2) Standard solution preparation: Dissolve the standard with methanol and dilute with methanol to obtain a series of standard solutions with concentrations of 5.0, 10.0, 25.0, 50.0 and 100.0 μg/mL, respectively.

(3) Preparation of test solution: weigh the sample, add liquid nitrogen into the mortar and grind, add -20 ℃ pre-cooled 70% (v/v) ethanol solution 8 ml, 55 ℃ ultrasonic extraction for 50 min, cooled to room temperature, take the supernatant through a 0.45 μm filter membrane to get 7 ml solution, the solution was placed in vacuum centrifugal concentrator to concentrate to 2 ml of the test solution.

(4) The standard curve was obtained after the standard product was passed through the column, and the peak time was clarified.

(5) The test material was passed through the column, and the concentration of the test material was calculated according to the peak area and the standard curve.