Experiment on microbiology

1.Medium formulation

LB medium (for E. coli and Agrobacterium)

Yeast extract 5 g/L
Sodium chloride 10 g/L
Tryptone 10 g/L
Agar 20g/L (solid）.

PDA/PDB medium (for Rhizoctonia solanum and Trichoderma)

PDA medium (potato glucose water AGAR medium, PDA) :
ddH2O was added to 46 g PDA medium powder until the total volume was 1 L.
PDB medium (potato glucose water medium, PDB) :
26 g of PDB medium powder was added with ddH2O until the total volume was 1 L.

Induction Medium（For the transformation of Agrobacterium Trichoderma）

MM salt for Induction Medium

1M MES stock solution

19.25g MES was dissolved in 80mL of distilled water, then the pH was adjusted to 5.3 with 5M KOH and the volume was constant to 100mL. The resulting solution was filtered with 0.45um filter paper and then divided into 9mL and stored at -20℃. Tips：If this salt precipitates when used, it can be dissolved by shaking in a water bath at 65℃.

10mM Acetosyringone

0.0192g of AS was added to 10mL sterile water and dissolved for 1h with stirring. Then, the pH was adjusted to 8.0 with 5M KOH, the bacteria was removed by filtration, and the tubes were divided into 5mL each and stored at -20℃.

Mandels medium（For protoplasts）

Mandels Nutrient salt concentrated solution：

(NH4)2SO4：14 g/L
Urea：3 g/L
KH2PO4：20 g/L
CaCl2·2H2O：4 g/L（or CaCl2：3 g/L）
MgSO4·7H2O：3 g/L，Add ddH2O until 1000 mL.

Mandels Trace element concentrated solution：

FeSO4·7H2O：5 g/L
ZnSO4·7H2O：1.7 g/L(or ZnCl2：0.7 g/L)
CoCl2·6H2O：3.7 g/L（or CoCl2：2 g/L）
MnSO4·H2O：1.6 g/L（or 23 MnCl2：1.67 g/L，or MnSO4 7H2O：2.6 g/L）
Add ddH2O until 1000 mL.

1 mol/L Citrate buffer solution：

Dissolve about 78 g NaOH(96% purity) and 210g citric acid with 750mL water, cool and add water to 1000 mL.

2.Strain culture

Culture of E. coli

Plate culture: the preserved/cultured strains were inoculated onto the pre-prepared LB solid medium and incubated for 24h in a 37℃ incubator.

Liquid culture: the preserved strain/culture medium was inoculated into 5ml of pre-prepared LB liquid medium. The samples were placed on a constant temperature shaker and incubated at 37℃ and 185rpm for 24h.

Culture of Trichoderma atroviride

Plate culture: the preserved/cultured strains were inoculated onto pre-prepared PDA medium and incubated in a 28℃ incubator for 2 days in the dark.

Liquid culture: The preserved strain/culture medium was inoculated into 5mL pre-prepared PDB medium, placed on a constant temperature shaker, and incubated at 28℃ and 180rpm for 2 days in the dark.

Induction and resuscitation of Trichoderma protoplasts

1. Spores of Trichoderma atroviride were inoculated into PDA solid plates and cultured at 28 ° C for 3-5 days (cellophane can be added to facilitate resuspended spores).
2. Inoculate the mature spores into sterile water (add 1ml sterile water to the medium and mix it with a flat gun to fully separate the spores and avoid spore clumping).
3. The 1mL suspension containing 1.2×108 spores was inoculated into 30mL Mandels medium, incubated at 28℃ and 160rpm for 10 hours, and then the germination of spores was examined by microscopy (most of the spores were germinated).
4. 5 mL spore germination solution (50ml centrifuge tube) was centrifuged at 5000 RPM for 10min. After the supernatant was discarded, the precipitated hyphae were collected.
5. The obtained mycelia were washed with 10mL 1 mol/L MgSO4, centrifuged at 6000rpm for 10min, and the supernatant was removed. Repeat this operation once.
6. Precipitate (spores) was resuspended with 10ml of enzymolysis solution (prepared and used now; 100mg of lysozyme was prepared with 10ml of 1M MgSO4 and then filtered to remove bacteria). The cells were incubated at 28℃ and 70rpm for 2 hours, and the enzymatic hydrolysis of spore protoplasts was observed with a microscope.
7. 20ml of STC solution was added to the enzymatic hydrolysate and thoroughly mixed.The supernatant was discarded after centrifugation at 5000rpm for 10min in a centrifuge at 4°C.
8. The precipitated protoplasts were washed with 20ml of STC solution and the supernatant was removed by centrifugation at 5000rpm for 10min at 4°C. Repeat once.
9. Finally, the washed protoplasts were suspended in 500ul 1X STC solution to obtain protoplasts.

3. Inoculation, recovery and preservation of strains

From solid to liquid: Single colonies were picked with a pipettor tip into a shaker tube containing 5 ml of medium, the pipettor tip was rinsed and discarded.

From liquid to liquid: 50 μl of medium was aspirated into a shaker tube containing 5ml of medium (diluted 1:100), the tip of the pipette was rinsed, and discarded.

Strain preservation: 50% glycerol solution and culture bacteria solution were added to the same storage tube at a volume of 1:1, absorbed and transferred evenly, and stored in the refrigerator at -80 °C.

4. Transformation of plasmid genes

Chemical transformation of Escherichia coli

1. Thawing competent cells on ice.
2. 2uL of recombinant plasmid was added to 100uL of competent cells, and then injected upward with pipetting gun from the bottom of the tube while turning and standing on ice for 30min.
3. Heat shock at 42℃ water bath for 45s (put and take at the same time), immediately put on ice to cool for 2-3min.
4. 500-700ul LB liquid medium (without resistance) was added to the transformed cells, and the bacteria were shaken for 1h at 30℃. Some of the supernatant was then removed by centrifugation for 1-2min at 12,000 rpm.
5. The solid medium of corresponding resistance was preheated in the incubator.
6. After the culture was completed, 500-700ul of supernatant was removed at 5000rpm for 5 min, and the remaining bacteria and medium were blown up and down with a pipetting gun to suspend the bacteria.
7. Prepare solid LB medium (containing resistance), applicator, pipetting gun, sterilization tip, and marker (sterilization tip prepared the day before) in advance.
8. Operate on the ultra-clean table, open the medium, lightly transfer the transformed bacteria solution to the medium, and then evenly spread with a disposable coater, slightly air dry, cover and seal glue, and finally invert and write the date, name and number.
9. Transformants on plates were obtained after incubation for 12-16h (bacteria at 37°C).

Transformation of protoplasts from Trichoderma atroviride

1. The prepared protoplasts were diluted appropriately to a final concentration of 103 cells /mL, and then 50 to 150 μl of the diluent was aspirated and coated in protoplast regeneration medium plates for accurate calculation of regeneration rates.
2. The final concentration of protoplasts was adjusted to 108 cells /mL, and then 200 μl of each was taken, 20μg was added, and mixed for later use.
3. The prepared sample was heat shocked at 48 ° c for 2 min, and then 50 μl of 60% PEG 4000 was added and left for 20min at room temperature. The solution was transferred to a 15ml centrifuge tube, 2ml of 60% PEG was added, and the mixture was still for 5min.
4. Finally, 8mL of STC solution was added at 5000 r/min, and the supernatant was removed after centrifugation for 15min.
5. The protoplast precipitates were resuspended in 1 mL STC solution, then added to 10ml protoplast regeneration medium and incubated at 28 °C at 50 r/min for 1 day (20h).
6. The culture medium was centrifuged at 5000 rpm for 10 min, the supernatant was removed, 1mL was left, and the tip of the gun was gently blown and resuspended.(After regeneration, microscopic examination, and coating plate after sprouting.
7. The bacterial solution was coated on PDA plates containing 200ug/ml hygromycin B at 28°C for 2 days. 100ul per board (to ensure that the protoplast concentration is not too high).
8. After mycelium had grown on the plate, the mycelium was transferred to a new PDA solid screening plate containing hygromycin and incubated at 28°C for 2 days.
9. After spore production, the original plate was transferred to PDA medium without antibiotics and cultured for 4 days.

Agrobacterium transformation of Trichoderma atroviride

1. GV3101 competent cells stored at -80°C were thawed on ice.
2. Under sterile conditions, 1ug of the plasmid to be transformed (epl1, prb1) was added to the thawed competent state and placed on ice for 5min.
3. The centrifuge tube was transferred to liquid nitrogen for flash freezing for 5min and quickly transferred to 28°C for 5min.
4. Return to the ice bath for 5min.
5. Under sterile conditions, 700uL of LB medium without antibiotics was added and cultured at 28°C for 3h to revive the bacteria.
6. The cells were centrifuged at 6000rpm for 1min, resuspended in about 100uL, coated on LB medium with cana resistance, and grown for 2-3 days.
7. The single colony of agrobacterium transformant was selected for small-scale expansion culture: 180rpm, 28°C, 16h, and cultured to about OD600=0.6-0.8.
8. After centrifugation at 5000rpm for 10min, the supernatant was discarded, the bacterial solution was resuspended in 2 ml Induction medium, the OD600 was measured, and the bacterial solution was diluted to OD600=0.6-0.8 with Induction medium.
9. Culture was induced at 28°C for 4-6 hours.
10.The induced Agrobacterium tumefaciens and Trichoderma spores germinated in induction medium for 3 hours were mixed at a ratio of 1: 1, evenly coated on induction medium plate covered with wet cellophene, and inverted cultured in the dark for 2 days.
11.Transfer cellophene to PDA medium containing hygromycin B and cephalosporin, and incubate inverted in the dark for 2-3 days to observe whether the proposed transformants appear. If so, pick the mycelium of the proposed transformants to a new plate containing the two resistance strains for further verification.

Transformation of Trichoderma atroviride by magnesium-silicate nanoclay

1. Preparation of conidia suspension

Conidia of Trichoderma atroviride HB20111 were washed from PDA plates cultured for 5 days with 5 mL sterilized distilled water, collected by centrifugation at 3000 rpm for 2 min, and the concentration of spores was adjusted to 1×108 conidia /mL suspension with sterilized distilled water. The suspension was added in PDB medium at 10% (volume fraction) inoculum and incubated overnight (12h) at 28 °C and 120 r/min with shaking, and the germination of spores was observed under a microscope.

2. Preparation of nanoclay

4.2g MgCl2·6H2O was dissolved in 100g ethanol, and then 6.5 mL of 3-aminopropyl triethoxysilane (APTES) was dropped into the solution, and the solution became white slurry after 5 minutes of addition and continued to stir overnight. Then the precipitate was separated by centrifugation at 6000 r/min for 10 min, rinsed twice with 250 mL ethanol, and dried at 40 °C to obtain magnesium-silicate nanoclay. 0.1 g of clay was weighed and dissolved in 100 g of sterile ultrapure water to make a 1 g/L suspension for later use.

3. Plasmid complex preparation (using DEPC water).

5ug of plasmid was added to 100ul of 100mg/L nanoclay solution, vortexed thoroughly, and incubated for 5min at room temperature.

4. 100ul plasmid complex + 100ul trichoderma spore suspension (12h of germination).

5. The ultrasonic output power was 100 W/cm2 and the transmission frequency was 50 kHz for 30 s.

6. Coated and incubated at 28°C for 5 days in the dark.

Experiment on molecular biology

1. Plasmid /DNA extraction

Extraction of plasmids from Escherichia coli

Small extraction: See TIANGEN plasmid small extraction kit.
Large-scale extraction based on TIANGEN plasmid large-scale extraction kit.
1. Take 100 mL (choose the appropriate amount according to the concentration of the cultured cells, 200 mL is recommended for a soft copy) overnight cultured bacteria liquid into the centrifuge tube, room temperature 8000 rpm (~8228×g) centrifugation for 3 minutes to collect the bacteria liquid, try to suck Except the supernatant. Note When there is much bacterial liquid, the bacterial pellet can be collected in a centrifuge tube by centrifugation. The amount of bacterial liquid should be sufficiently lysed. Too much bacterial liquid will lead to insufficient lysis and reduce the efficiency of plasmid extraction.
2. Try to absorb the supernatant as much as possible. To ensure that the supernatant is completely absorbed, please use clean absorbent paper to absorb the water droplets on the bottle wall.
3. Add 8 mL of solution P1 (please check whether RNaseA has been added) to the centrifuge tube with bacterial pellet, and use a pipette or vortex to lyse the suspended bacterial cell pellet completely. Note: Be sure to suspend the bacterial sediment thoroughly. If some bacteria are clumps that are not thoroughly mixed, it will affect the lysis effect, resulting in low extraction volume and purity. For low-copy plasmids, increase the number of bacteria while increasing the proportion of P1, P2. The amount of P4.
4. Add 8 mL of solution P2 to the centrifuge tube, turn it upside down gently 6-8 times, and leave it at room temperature for 5 minutes. Note: Mix gently, do not shake vigorously to avoid contamination of genomic DNA. At this time, the bacterial liquid should become transparent and viscous. If it does not become apparent, it may be due to too much bacteria and incomplete lysis, so fewer bacteria should be reduced.
5. Add 8 mL of solution P4 to the centrifuge tube, and immediately turn it upside down gently 6-8 times, mix well, until the solution appears white dispersed flocculent precipitate. Then leave it at room temperature for about 10 minutes. Centrifuge at 8000 rpm (~8228×g) for 5-10 min to allow the white precipitate to separate to the bottom of the tube (the centrifugation time can be increased appropriately), and carefully pour all the solution into the filter CS1 (please avoid pouring a large amount of precipitate to block the filter ), slowly push the push handle to filter, and collect the filtrate in a clean 50 mL tube (provided by yourself). Note: Mix the solution immediately after adding solution P4 to avoid local precipitation. If the solution poured into the filter CS1 after centrifugation has white precipitation, it would not affect the filtration. If there are too many bacteria (>100 mL), it is recommended to extend the centrifugation time to 20-30min.
6. Add isopropanol equal to the filtrate volume to the filtrate, mix upside down, and centrifuge at 8000 rpm (~8228×g) for 5 min.
7. Pour and discard the supernatant. Take care to prevent the precipitate from being poured out. Add 6 mL of 70% ethanol to wash the precipitate. Centrifuge at 8000 rpm (~8228×g) at room temperature for 5 min. Carefully discard the ethanol. Repeat once.
8. Leave the lid open in the air for 5-10 minutes, and dissolve the precipitate with an appropriate volume of ddH2O as needed.

2.Genome extraction of Trichoderma atroviride and Rhizoctonia orbicularis

CTAB method

CTAB extraction buffer formula: The preparation must be heated to dissolve at 50°C and store at room temperature.

1. Tissue crushing

Genomic DNA was extracted using a modified CTAB protocol. The obtained mycelium was placed in a sterilized mortar and ground with liquid nitrogen, followed by DNA extraction using a modified CTAB method.

2. Cell lysis

About 600 mg of frozen pileus tissue was cut and transferred to a 1.5-ml Eppendorf microcentrifuge tube. Containing 700 μl preheated (60°C) 2×CTAB extraction buffer [2% (w/v) CTAB, 100 mM Tris-hcl, 1.4 M NaCl, 20 mM EDTA, pH 8.0]

3. The purification of DNA

Seven hundred microliters of phenol: chloroform: isoamyl alcohol (25:24:1) were added to each tube and mixed thoroughly to form an emulsion. [Chloroform accelerates the stratification of organic phase and aqueous phase to remove residual phenol; isoamyl alcohol reduces bubbles generated during protein denaturation. Chloroform isoamyl alcohol is toxic, so it is necessary to wear a mask and gloves and operate in the fhood]

The mixture was spun at 13,000 RPM in a microcentrifuge for 15 min at room temperature, and 600 μl of the upper aqueous phase was transferred to a new 1.5-ml tube. The aqueous phase containing DNA was re-extracted with an equal volume of chloroform: isoamyl alcohol (24:1) until the interface was not visible.

4. The precipitation of DNA

A total of 400 μl of supernatant was added to 1000 μl absolute ethanol +50 μl NaAc (NaAc was not added) and frozen in liquid nitrogen for 5min. [DNA is insoluble in ethanol, but CTAB and some proteins are soluble. Low temperature ethanol can also inhibit DNase activity, reduce molecular movement, and easily precipitate DNA]

5. Centrifugation, rinsing and collection

The samples were centrifuged at 12000rpm for 30min at 4℃ and the supernatant was poured off. 200 μl 70% ethanol was added and centrifuged at 12000rpm for 1min. After centrifugation, the residual alcohol was sucked away, and then repeated once to remove the residual alcohol as much as possible. The lid was removed and allowed to dry at room temperature for 5min before the DNA precipitate was re-suspended in 100 µl TE buffer (10 mM tris-HCl，1 mM EDTA).

TPS method

TPS: Tryptone saline solution
EDTA: Ethylenediamine tetraacetic acid, complexing agent, forms stable water-soluble complexes with alkali metals, rare earth elements, and transition metals, and is often used as a nuclease inhibitor
Tris-HCl: nucleic acid, protein solvent
The following EDTA solid dosage: 0.29224g.

1. Tissue crushing: grinding with liquid nitrogen, adding steel beads, grinding into powder.
2. Cell lysis: 500 μl TPS, put into 65℃ oven for 40min, take out and mix every 10min.
3. Purified DNA was centrifuged for 10min at 12000rpm/min, and 300 μl isopropanol was added to the empty tube.
4. Add 300 μl supernatant to isopropanol, mix, put in -20℃ refrigerator, freeze for 20min (can be temporarily stored for one day).
5. Precipitate DNA: centrifugation for 10min, pour the supernatant, add 1000 μl of 75% alcohol, centrifugation for 10min, pour out the remaining liquid, and blot dry with the tip of a gun.
6. Put in the oven for 10min and dry the ethanol; Add 100 μl of water and store in the refrigerator.

3.Enzyme validation

Plasmid DNA: x μl (according to the actual concentration)
Restriction enzyme: 1 μl
10× buffer: 2 μl
ddH₂O:（20-3-x）μl.

Double enzyme digestion system

Plasmid DNA: x μl (according to the actual concentration)
Restriction enzyme: 1 μl
10× buffer: 2 μl
ddH₂O:（20-3-x）μl.

4. Polymerase chain reaction

Reaction system

Reaction process

5. Agarose gel electrophoresis

1. Dissolve 0.3 g of agarose in 30 mL of 1X TAE solution, heat it until the bubbles stop, and the solution becomes transparent, cool slightly and add 3 μL Goldview (10000x)
2. Pour the gel into the gel membrane tool inserted with the comb, and wait for it to set.
3. Add 1/2/3 μL 6× loading buffer to the 5/10/15μL sample and mix by pipetting.
4. Put the gel into the electrophoresis tank and add 1X TAE buffer to completely cover the gel and squeeze out the bubbles in the gel hole.
5. Add the mixed sample and marker to the gel hole. Specific experiments determine the sample loading volume. The marker loading volume is based on the following standards，11 wells gel corresponds to 2 μL, 8 wells correspond to 5 μL, and 6 wells corresponds to 10 μL.
6. Run electrophoresis under 180V voltage until the colour band of loading buffer is electrophoresed to the middle or two-thirds of the gel, stop electrophoresis.
7. Use ultraviolet light to image the gel. If necessary, cut rubber for recycling.

6. Polyacrylamide gel electrophoresis (SDS-PAGE)

1. Prepare running buffer: Dissolve 15.1 g Tris and 94 g glycine in 900 mL deionized water, add 50 ml 10% SDS, and then dilute to 1 L with deionized water.
2. Add 5x SDS loading buffer to the protein sample and control its volume fraction in the mixed solution to 20%.
3. Take out the Meilunbio® Gel protein precast gel, and then fix the precast gel in the electrophoresis tank. Add running buffer solution to check for leaks.
4. Fill the inner tank of the electrophoresis tank with electrophoresis buffer, and add the electrophoresis solution to the corresponding level in the outer tank, and the maximum shall not overflow the inner tank. Take out the comb, use a syringe or other tools to suck the electrophoresis solution, then gently blow the sample well to remove the remaining storage buffer and impurities in the sample well.
5. Add sample: Use conventional 1X loading buffer to process the sample. After aspirating the sample with the pipette, insert the pipette tip into the sample hole vertically to add the sample. Be careful not to pierce the gel with the pipette tip, and do not over-insert the comb hole to deform the rubber plate and cause liquid leakage.
6. Electrophoresis conditions: 110 V. When the bromophenol blue indicator is electrophoresed at the bottom of the gel or a predetermined position in the experiment, the electrophoresis can be ended.
7. After the electrophoresis is over, remove the gel. Use a blade to cut the glue on both sides along the gap between the short glass and the edge strip, open the glass plate to take out the gel.
8. After staining with Coomassie Brilliant Blue for 3 hours, elute with eluent. Eluent configuration 200 mL ethanol 300 mL acetic acid 500 mL water (volumes of ethanol and acetic acid are variable)

7.Western-Blot

1. SDS-PAGE.
2. Prepare electro-transfer solution (prepared during electrophoresis, need to be placed in the refrigerator to pre-cool) or buy directly.
3. Cut the fibre membrane and cut off a corner, put it in methanol for activation, carefully cut the required fragments with a rubber cutting board (compared to maker). This step needs to be soaked in electro transmission fluid.
4. Cut a small piece at the corner of the gel to recognize the order of sample addition.
5. Assemble the sandwich structure: sponge-filter paper-fibre membrane-sample glue-filter paper-sponge (black below).
6. Connect the membrane to the positive electrode and transfer it for 90 minutes at 110 A on ice (4°C).
7. Dilute the 10x blocking solution to 1x (4℃), dilute 10xTBS to 1x and add 2mL Tween (cut off the tip with scissors).
8. Take out the fibre membrane, discard the gel, soak it in the blocking solution and place it on a shaker for 15-20 minutes.
9. Wash with TBST two to three times, once for 10 minutes.
10. The primary antibody (stored in a refrigerator at 4°C) was diluted 1:10000 with TBST, put the membrane into it, and incubated overnight at 4°C on a shaker.
11. Take out the membrane and wash it three times with TBST for 10 minutes each time.
12. Dilute the secondary antibody (stored in a refrigerator at 4°C), dilute TBST 1:10000, put the membrane into it, and incubate at room temperature for 2 hours.
13. Wash three times with TBST and prepare for development.

Note:

1.Electro-hydraulic.

Add 1/4 volume to get methanol when used

2.10xTBS

3.TBST buffer

8. Determination of protein concentration

See BCA protein concentration determination kit produced by Biotime Company
1. Mix Buffer A and Buffer B according to the ratio of 50:1, and mix thoroughly in the vortex mixer.
2. 20uL of the diluted protein solution was added to a 96-well plate, and 200uL of the mixture obtained in step 1 was added to each well in turn.
3.The reaction was carried out at 37°C for 30min.
4. The absorbance value at 562nm of each well was measured in the microplot reader, and the corresponding protein concentration was calculated by the standard curve drawn by BSA standard protein.

9. Protein purification

See Filamentous fungal protein extraction Kit（Purchased from Bestbio）and His-tag Protein Purification Kit（Pirchased from Beyotime）
1. Preparation of extract: Add 2ul protease inhibitor mixture to 500ul filamentous fungal protein extract, mix and set aside on ice.
2 Samples of filamentous fungi were collected and washed twice with cold PBS, blotting the supernatant as dry as possible after each wash.
3. Grind the cells in a mortar with liquid nitrogen to powder.
4. Add 300ul-500ul protein extract to every 50-100mg of bacteria, mix and shake for 20-40 minutes at 2-8 °C.
5. Centrifuge for 10 minutes at 12000-14000×g at 4 °C.
6. The supernatant was inhaled into another pre-cooled clean centrifuge tube to obtain the total fungal protein.
7. The above protein extracts were quantified and stored in a refrigerator at -80℃ for later use or directly used for downstream experiments.
8. Take 1mL of evenly mixed 50% BeyoGold his-tag Purification Resin, discard the storage solution by centrifugation at 4°C (1000gX10s), add 0.5mL of non-denaturing lysate to the gel, mix thoroughly to equilibrate the gel, and discard the liquid by centrifugation at 4 °C (1000gX10s). The equalisation was repeated 1-2 more times and the liquid was discarded. About 4 ml of total fungal protein solution was added and slowly shaken on a side shaker or horizontal shaker for 60min at 4°C.
9. The mixture of lysate and BeyoGold His-Tag Purification Resin was loaded into the empty column tube of the affinity chromatography column provided by the kit.
10. The lid at the bottom of the purification column was opened to allow the liquid in the column to flow out under the action of gravity, and about 20uL of the penetrating liquid was collected for subsequent analysis.
11. Wash the column 5 times, adding 0.5-1mL non-denaturing washing solution each time, and collecting about 20uL of washing solution through the column for subsequent analysis and detection each time. The Bradford method can be used to detect the protein content of each wash solution and eluate easily and quickly during the washing column and the next elution process, so as to consider increasing or reducing the number of elution.
12. The target protein was eluted 6-10 times, each time with 0.5mL of non-denaturing eluent. The eluate of each time was collected separately into different centrifuge tubes. The collected eluate was the purified protein sample containing His-tag.

10. Induction of expression

IPTG induces microRNA production in Escherichia coli

1. monoclonal colonies of E.coli HT115 (DE3) on resistant plates were selected and inoculated into 4mL LB medium containing kanamycin (50ug/mL) and cultured overnight at 37℃.
2. 200uL of the bacteria solution cultured overnight were inoculated in 20mL LB medium containing kanamycin (50ug/mL) at 200rpm and 37℃ for 4 hours, so that the OD600 was about 0.6.
3. 100uL of IPTG at a concentration of 0.1M was added and induced at 37 °C for 4 hours at 200rpm.
4. after induction, the bacterial solution was centrifuged at 4000rpm for 10min, the supernatant was discarded, and the bacterial precipitation was used for subsequent operations.

AS induced expression of recombinant plasmid genes in Agrobacterium tumefaciens

11. RNA extraction

1. Add 5mL Trizol reagent to the bacterial precipitate obtained by centrifugation after IPTG induction, vortex and shake for 15s to suspend the bacterial weight, then pack 1mL per tube into 1.5mL EP tube (need to be sterilized by high temperature to remove RNase), and let it stand for 5min at room temperature）.
2. 400ul of chloroform stored in a refrigerator at 4℃ was added to the above samples, and the solution was stratified after standing for 3min.
3. centrifuged at 12000rpm for 15min at 4℃, the supernatant (red part, about 600uL) was sucked into a new EP tube, and the equal volume of isopropanol stored at -20℃ was added, and the EP tube was upside down and mixed for 7-8 times.
4. placed in the refrigerator at -80℃ for 20min, followed by centrifugation at 12000rpm for 10min in a centrifuge at 4℃, and the supernatant was discarded.
5. add 1mL of 75% depc-ethanol (that is, prepare by dilating anhydrous ethanol with DEPC water instead of ddH2O) to wash the precipitate thoroughly (both DEPC water and anhydrous ethanol were stored at 4℃).
6. centrifuge at 7500rpm for 5min at 4℃, discard the supernatant, and dry in a fhood for 15min.
7. add 40uL DEPC water to redissolve the RNA precipitate.
8. Determine the nucleic acid concentration
9. Agarose gel electrophoresis.

12.Preparation of the RNA-LDH complex

MicroRNA samples were mixed with LDH clay nanosheet suspension at a mass ratio of 1:3, and the preparation of rna-ldh complex was completed by incubation at 300rpm in a shaking mixer at 25 ℃ for 30min.