Preparation of materials

1 Preparation of materials
Nutrient medium
Medium1:MS+1.0 mg/L 2, 4-D+20 g/L sucrose+7g/L Agar
Medium2:MS+1.0 mg/L2, 4-D+0.5 mg/L KT+0.5 mg/L Ag NO₃+20 g/L sucrose MS+2, 4-D+7g/L Agar
Material cultivation
Callus of Longan were subcultured alternately on Medium 1 and Medium 2.

Construction of overexpression vector

2.1 RNA extraction from plant material samples
      1.After weighing the ultra-low temperature frozen RNA extracted samples, they were quickly transferred to a mortar and pestle pre-cooled with liquid nitrogen, and the tissues were ground thoroughly with a mortar and pestle until they were ground into a powder, during which time liquid nitrogen could be added. The yield and quality of the RNA can be affected if it is not ground thoroughly.
      2.The samples ground to powder form were transferred to a centrifuge tube and 1 ml of TransZol Up was added for every 50-100 mg of tissue. homogenization was performed with a homogenizer and repeated blowing with a gun.
      3.Allow to stand at room temperature for 5 minutes.
      4.For each 1 ml of TransZol Up used, add 200 ul of chloroform, shake vigorously for 30 seconds and incubate at room temperature for 3-5 minutes.
      5.The sample was centrifuged at 10,000xg for 15 minutes at 4°C. At this point the sample was divided into three layers, a colorless aqueous phase (top layer), an intermediate layer, and a pink organic phase (bottom layer). At this point the sample was divided into three layers, a colorless aqueous phase (upper layer), an intermediate layer, and a pink organic phase (lower layer.) The RNA was mainly in the aqueous phase, and the volume of the aqueous phase was approximately 50% of the TransZol Up reagent used.
      6.Transfer the colorless aqueous phase to a new centrifuge tube, add 500 ul of isopropanol per 1 ml of TransZolUp used, mix upside down, and incubate in the greenhouse for 10 minutes.
      7.Centrifuge at 10,000 x g for 10 min at 4°C. Remove the supernatant and form a gelatinous precipitate on the side and bottom of the tube.
      8.Add l m1 75% ethanol (DEPC-treated water preparation) and vortex vigorously (add at least 1 ml of 75% ethanol for every 1 ml of TransZol Up used).
      9.Centrifuge at 7500xg for 5 min at 4°C.
      10.Discard the supernatant (for better control of salt ion content in RNA, ethanol should be removed as much as possible), and dry the precipitate at room temperature (1-2 min).
      11.The precipitate is dissolved in 10-15 ul of RNA lysis solution.
      12.The samples were stored at -70°C.

2.2 Reverse transcription cDNA system

2.3 Polymerase Chain Reaction(PCR)

Component	Volume
Green Enzyme Mix	12.5μL
Upstream primers	1μL
Downstream primers	1μL
cDNA	1μL
ddH2O	9.5μL

Temperature (℃)	Time
95	60s
95	20s
57 (adjustable)	20s
72	1min/kb
72	5min
4	∞

2.4 Electrophoresis
①Material
0.17g Agarose + 0.8μL Nucleic acid dye + 20ml 1×TAE
②Method
    Agarose, nucleic acid dye and 1×TAE were added to the conical flask, heated for 2min and poured into the plate, and a comb was inserted. After solidification, add sample, electrophoresis 150V 100A, 20min

2.5 Gum recovery
All centrifugations were performed at room temperature
      1. Cut the target DNA strip from the agarose gel and put it into a clean centrifuge tube to weigh, if the gel weighs 100 mg, it can be regarded as 100 μL, and so on.
      2. Add 3 times the volume of solution GSB, melt the gel in 5SC water bath for 6-10 minutes, intermittently (2-3 minutes) mix to ensure the gel is completely melted, when the gel is completely melted, observe the color of the solution, if the color is purple, add appropriate amount of 3 M sodium acetate (pH5S2), adjust the color to be the same as GSB color (yellow). (To increase the amount of DNA recovery, add 1x volume of isopropanol to the melted gel solution, e.g. 100 mg of gel weight, add 100 μl of isopropanol).
      3. to be melted gel solution to room temperature (high temperature centrifugation column binding DNA ability is weak), added to the centrifugation column to stand for 1 minute, 1000xg centrifugation for 1 minute, discard the effluent.
      4. Add 650μl of solution WB, centrifuge at 10.000xg for 1 minute, discard the effluent.
      5. Centrifuge at 10,000*g for 1-2 minutes to completely remove the residual WB.
      6. Place the centrifugal column in a clean centrifuge tube, open the cap and let it stand for 1 minute to make the residual ethanol evaporate cleanly, add 30-50μl of EB or deionized water (pH>7.0) in the center of the column (EB or deionized water preheated in a water bath at 60-70C is better for use) and let it stand for 1 minute at room temperature.
      7. Centrifuge at 10000xg for 1 min, elute DNA, and store the eluted DNA at -20°C.

Caution
      1.To ensure the recovery effect, use fresh electrophoresis buffer when electrophoresis.
      2.When cutting the gel, make sure the gel is as small as possible; when melting the gel, make sure the gel is completely melted.
      3.To avoid damage to DNA caused by UV irradiation, which may affect downstream experiments (e.g. cloning, ligation), keep the UV irradiation time as short as possible.

2.6 Connection and Transformation
①LB Formulation
      LB broth 25g (preparation of liquid LB)
      LB nutrient agar 40g (prepared solid LB)
      Distilled water 1000ml
      Dissolve LB in distilled water, dispense, sterilize at 121℃ and set aside.
②Connection System

Component	Volume
T4 DNA ligase Buffer	2 μL
T4 DNA ligase	1 μL
1301/1302 Gel recovery product after emptying enzyme digestion	4 μL
The target gene fragment after enzyme digestion	13 μL

③Transformation
      a. DH5α competent cells were taken out from -80℃, quickly inserted into ice, and after 5 minutes, the bacterial block was thawed, the target DNA (plasmid or ligation product) was added, and the bottom of the EP tube was gently mixed by hand to avoid using a gun Suck and let stand in ice for 25 minutes.
      b. Heat shock in a 42℃ water bath for 45 seconds, quickly put back on ice and let stand for 2 minutes, shaking will reduce the transformation efficiency.
      c. Add 700μL of antibiotic-free sterile medium (2YT or LB) to the centrifuge tube , mix well, and recover at 37℃ and 200rpm for 60 minutes.
      d. Centrifuge at 5000 rpm for 1 minute to collect the bacteria, and take about 100 μL of the supernatant to resuspend the bacteria by gently pipetting and spread on the 2YT or LB medium containing the corresponding antibiotics.
      e. Invert the plate and incubate overnight in a 37℃ incubator.

Precautions
      1. Competent cells are best thawed slowly in ice. Add the target DNA within 8 minutes of inserting it into ice. Do not keep it in ice for too long, as long-term storage will reduce the transformation efficiency.
      2. Be gentle when mixing the target DNA.
      3. Transformation of high concentrations of plasmids or high-efficiency ligation products can correspondingly reduce the amount of bacteria that are ultimately used for plating.

2.7 Identify positive clones
(1) Pick single colonies
①600μL of L B liquid medium to 1.5 ml centrifuge tubes respectively
②Pick a single colony on the medium into a centrifuge tube
③shaking at 37℃ for 1 h on a shaker, the bacterial liquid PCR was performed
(2) Bacterial liquid PCR

Component	Volume
2 XHiefft Tm PCR Master Mix	7.5μL
Bacteria liquid	5.7μL
Forward primer	0.6μL
Reverse primer	0.6μL
ddH2O	0.6μL

15μL system(34x)

Temperature (℃)	Time
95	3min
95	30s
55	30s
72	1kb/1 min
72	10min
4	∞

2.8 Plasmid Extraction
      1.Culture for 12h until OD600 or so.
      2.Centrifuge leveling, 7800r 5min at room temperature, supernatant removal.
      3.Add Resuspension Solution 250μL and mix well by pipetting, transfer to 1.5mL centrifuge tube.
      4.Add 250μL of Lysis Solution and digest, mix well.
      5.Add 350μL Neutralization Solution and mix well (13000r, 5min)
      6.Take about 800μL of supernatant and add it to the special plasmid centrifuge tube (13000r, 5min)
      7.Remove the waste solution, add 500μL Wash Solution (13000r,1min) and repeat 2 times
      8.13000r,1min centrifugal
      9.Put the special centrifuge column into an empty 1.5mL centrifuge tube, add 35μL Elution (preheated at 65℃), cover, and after 2min at room temperature, 13000r, 1min
      10.Store in -20℃ refrigerator

2.9 Enzymatic digestion
1.Essential digestion (digestion system 20μL)
①Endonuclease I (1μL)
②Endonuclease II (1 μL )
③FD Buffer (green) (2μL)
④1301/plasmid containing the target fragment 1000/plasmid concentration
⑤ddH₂O Complement
2.PCR setup (20μL system)

Temperature (℃)	Time
37℃	25min
65℃	20min
4℃	∞

2.10 Shaking bacteria
    LB liquid medium into the ultra clean bench, ultraviolet 20min, each 50ml centrifuge tube add 20mL of LB liquid medium and 20μL of AMP, mark well and pour all the bacteria into the corresponding large tube, if the pour is not exhausted, use the gun to inhale the large tube, after that put into the shaking E. coli shaker (37℃) for 1 day.

pGW3 vector construction

3.1 Target primer annealing
①Material
target primer
0.5×TE ( pH8.0 ) 40 μL
②Method
Dissolve the target primers in water into a 10 μM stock solution, add 5 μl of each to 40 μl of 0.5 × TE (pH 8.0), the final concentration is 1μM; heat at 98°C for 3 min in the PCR machine, immediately take out the PCR tube, cool to room temperature naturally, and use for connection.

3.2 Attachment: Preparation of TE
      (1) Preparation of 50ml of 1 M Tris- HCl (pH8.0)
Weigh 6.06 g of Tris base , add 40 ml of ultrapure water to dissolve, add about 2.1 ml of concentrated HCl dropwise to adjust the pH to 8.0 , and dilute to 50 mL.
      (2) 0.5M EDTA ( pH8.0 ) 50 ml preparation
Weigh 9.306 g of EDTA-Na2·2H2O , add 35 mL of ultrapure water , stir vigorously, adjust the pH to 8.0 with about 1 g of NaOH particles , and dilute to 50 ml EDTA (The sodium salt will dissolve only when NaOH is added to adjust the pH to close to 8.0 )
      (3) 1×TE ( 10 m MT ris- HC l , 1mM EDTA ) preparation

Component	Volume
1 MT ris -HC l	1ml
0.5M EDTA	0.2 mL
Ultra-pure water	Make up to 100 ml

(4) Preparation of 0.5 × TE

Component	Volume
1 MT ris -HC l	100 μL
0.5M EDTA	20μL
Ultra-pure waterr	Make up to 20 mL

Adjust pH to 8.0 with NaOH solution

3.3 The pGW3 plasmid was digested with BsaI
①Material

Component	Volume
BsaI enzyme	1μL
plasmid	1μg
10× NEB buffer	5μL
water	Make up to 50 μL

②Method
Take 1μg pGW 3 plasmid, digest it with 10 U BsaI-HF at 37℃ for 60 min in a 50 μL reaction , use a DNA purification kit, aliquot, and store it in a freezer (reusable).

Component	Volume
BsaI enzyme	1μL
plasmid	1μg
10× NEB buffer	5μL
water	Make up to 50 μL
at 37 ℃ for 1h

Stop by adding 10 μL of 6× loading buffer to the 50 μL reaction. After enzyme digestion, the gel was run and the gel was recovered.

3.4 Target ligation reaction
①Material

Component	Volume
Material	concentration
10 × T4 DNA ligase buffer	2 μL
The digested pGW3 vector	About 40 ng (use the measured concentration to calculate the amount of sample added)
Annealed target primer	1 μL
T4 DNA ligase 40 U	0.2μL (Takara ligase 350U/ μL add about 0.11μL , add at the end)
ddH2O	to 10 μL

②Method
Connect at room temperature (20 -25℃) for 30 min.

3.5 Convert
①Material
Ligation product 5 μL
Escherichia coli DH5α competent 50 μL
1 bottle of LB solid medium
1 bottle of LB liquid medium
Kana kana :LB solid medium = 1:1 000 ratio
②Method
      a. Take out the DH5a competent cells from -80°C and insert them into ice quickly. After 5 minutes, wait for the bacterial block to melt, add the target DNA (plasmid or ligation product) and mix gently by hand to the bottom of the EP tube to avoid using it. Suck it with a gun and let it sit in ice for 25 minutes.
      b. Heat shock in a 42°C water bath for 45 seconds, quickly put it back on ice and let it stand for 2 minutes, shaking will reduce the transformation efficiency.
      c. Add 700ul L B liquid medium without antibiotics to the centrifuge tube, shake at 37 ℃ for 1h
      d. Centrifuge at 5000rpm for 1 minute to collect the bacteria, and take about 100ul of the supernatant to resuspend the bacteria by gently pipetting and spread it on the LB solid medium containing kanamycin antibiotic .
      e. Place the plate upside down in a 37°C incubator overnight for 10-12 h .

Measurement of GABA content

4.1 Sample preparation.
     Weigh about 0.1g of tissue sample into the mortar, add 1mL of extraction solution, homogenize on ice, centrifuge at 12000rpm, 4℃ or room temperature for 10min, and take the supernatant for measurement.


4.2 Detection on the machine
①Preheat the visible spectrophotometer for 30 min, adjust the wavelength to 645 nm, and zero it with distilled water.
②Thaw all reagents to room temperature (25℃).
③Add to the EP tube in order.

Reagent name ( μL )	Assay tube	control tube
sample	1 00	1 00
Reagent one	6 0	6 60
Reagent two	2 00
Reagent two	4 00
Mix well, take a boiling water bath (95-100℃) for 10min, ice bath to room temperature, showing blue-green color, if it is turbid, centrifuge at 12000rpm for 5min, take all the clear supernatantand transfer it to a 1mL glass cuvette ( optical path 1cm), Read the A value of each tube at 645nm, △A=A determination-A control (each sample needs to be a self-control).

【Note】
1. If the A value of the measurement tube is greater than 1, the sample should be diluted (diluted with distilled water), and the dilution factor D should be recalculated by substituting the calculation formula.
If the difference between A measurement and A control hovers around zero, increase the sample size (e.g. to 0.2g) and recalculate the sample mass W by substituting the formula.

4.3 Calculation of results
①Standard curve: y = 0.0086x - 0.0033, x is the mass of the standard (μg), y is the absorbance value ΔA.

②Calculated by sample mass:
GABA content (μg/g weight) = [(ΔA+0.0033) ÷ 0.0086] ÷ (W×V1÷V) × D = 1162.8 × (ΔA + 0.0033) ÷ W × D
V-extraction volume, 1 mL; D-dilution, undiluted is 1; W- sample sampling quality

Agrobacterium infestation(transient transformation for 6d)

      1. Resuscitate Agrobacterium first LB+Kan+Rif plate at -80°C by scratching and place in dark culture at 28°C for 48h
      2. Pick single clone, inoculate it in LB liquid medium containing Kan+Rif, 28℃, 48h, 200r
      3. Centrifuge the bacterial solution at 4500rpm for 10min, remove the supernatant to collect the bacterium, add MS (i.e. 30g/L sucrose, 4.43g/L MS, PH5.8, 10mmol/LMgCl₂, 200umol/L AS (not high pressure) to resuspend the bacterium, OD adjusted to 0.6-0.9.
      4. The healings cultured for 10-15 d were added to Agrobacterium infestation solution and infested for 30 min with shaking and mixing every 10 min during the period.
      5. Filter the longan healing tissue material at the end of the infestation, blot it slightly and inoculate it in co-culture medium (normal plus 2,4D medium + 1:1000 cephalosporins, PH 5.8). wash the bacteria after 5-6 d, you can leave some for Gus staining. 1301 empty should be colorless (positive control); 1301 + target gene (stained), Agrobacterium (no 1301, colorless ).
      6. Transfer the co-cultured longan healings to a well pressurized 50 ml centrifuge tube in an ultra clean bench, rinse with MS medium or water after pressure, and finally rinse with sterile water + cephalosporin, let stand and shake gently.
      7. Filter and aspirate the water on the surface (filter paper tweezers should be high-pressure) received into normal 2,4D + 1:1000 cephalosporin medium, 20d succession.
Attachment.
      1. Cephalosporin: 0.3g+1ml high pressure water to 1.5ml centrifuge tube, filter and remove bacteria (green filter, filter once), ready to use
      2. AS: 0.02g acetosyringone (molecular room 4 ℃ refrigerator) + 500ul dimethyl sulfoxide (drug room under the cabinet) to 1.5ml centrifuge tube, put -20 ℃ can be reused.
      3. Step 3 specific practices.
1:1000 add magnesium chloride to MS suspension, shake well and add 1mL to 50mL centrifuge tube to aspirate and mix, then pour 25ml of MS suspension, take some liquid to measure OD value, use the previous MS suspension to adjust the zero. If the OD value is too high, continue to add MS suspension diluted to OD 0.8 or so. 1:1000 add AS to the already adjusted OD in the bacterial solution. Put the bacterial solution into the 28 ℃ shaker for half an hour, during which the table continues to turn on the UV.
      4. Step 4 specific practice.
Strain the material through filter paper until loose, then add the material to the bacterial solution, add the material to about 20ml. Put it into the shaker of the culture room for 30min.
      5. Step 5.
After shaking, filter with filter paper folded into a funnel shape. After filtering, receive the co-culture medium and put a clump in the middle.