--Add liquid nitrogen to a container and freeze poplar leaves in it

--Grind the frozen leaves into powder and keep it frozen with liquid nitrogen

-- Use DNAsecure Plant Kit to extract DNA from powder

--Prepare the PCR reaction system

Content of the solution (50 μL):

--Divide the solution into eight

--Add the solution into a test tube and add cDNA

--Place the 8 tubes in the PCR apparatus for 1.5 hour

--Prepare the agarose gel

Mix all the ingredients in a beaker

Heat the solution in a microwave oven for 1 minute (dissolve the agarose)

Place the beaker in the air to cool it

Pour the solution into the mold and place the cataphoresis to create holes

Wait for the gel to congeal

--Running agarose gel electrophoresis

Place the gel in the flume

Add the PCR product into the hole (each tube for on hole)

Add the marker into one hole

Run the gel for 30 min

--Cut the gel

Check the fluorescence on the gel under ultraviolet

--Dissolve the gel

Add pw (3 times the volume of the gel) into a tube

Add the gel in

Keep the tube in the thermostat water bath at 55 Celsius degrees for 5 minutes

Place the absorbing column into the tube

Place the tube in the Hydro-extractor 12000rpm 5min

Take out the column and add it to a new tube

Add pw 600μL into the tube

Rest it for 2-5min

Place the tube in the Hydro-extractor 12000rpm 1min

1)pEasy vector (Trans-T1)

With antibody Kana and AMP

Binding Sites: (T)CCCTT

2)LDNA (gene of interest)

3)Blunt Vector

4)Topo enzyme (binding sites: (T)CCCT)

2.2 transformation of competent cells

--Melt the LDNA on ice for 30 minutes

--Keep the tube in the thermostat water bath at 42 Celsius degrees for 30 seconds

--Place the tube on the ice for 2 minutes

--Add 500ul liquid LB medium into the tube

--Keep the tube in the thermostat water bath at 37 Celsius degrees for 1 hour

--Place the tube in the Hydro-extractor 200rpm 4min

--Pour out the liquid

--Rub the remain on the culture medium which contain the antibody Kana

--Foster the cell

--Content of the solution (50 μL):

--Divide the solution into eight

--Add the solution into a test tube and add cDNA

--Place the 8 tubes in the PCR apparatus for 1.5 hour

--Prepare the agarose gel

Mix all the ingredients in a beaker

Heat the solution in a microwave oven for 1 minute (dissolve the agarose)

Place the beaker in the air to cool it

Pour the solution into the mold and place the cataphoresis to create holes

Wait for the gel to congeal

--Running agarose gel electrophoresis

Place the gel in the flume

Add the PCR product into the hole (each tube for on hole)

Add the marker into one hole

Run the gel for 30 min

--Cut the gel

Check the fluorescence on the gel under ultraviolet

Cut down the part which is glittering

Add pw (3 times the volume of the gel) into a tube

Add the gel in

Keep the tube in the thermostat water bath at 55 Celsius degrees for 5 minutes

Place the absorbing column into the tube

Place the tube in the Hydro-extractor 12000rpm 5min

Take out the column and add it to a new tube

Add pw 600μL into the tube

Rest it for 2-5min

Place the tube in the Hydro-extractor 12000rpm 1min

-- Keep the tube in the thermostat water bath at 50 Celsius degrees for 2 minutes

--Melt the LDNA on ice for 30 minutes

--Keep the tube in the thermostat water bath at 42 Celsius degrees for 30 seconds

--Place the tube on the ice for 2 minutes

--Add 500ul liquid LB medium into the tube

--Keep the tube in the thermostat water bath at 37 Celsius degrees for 1 hour

--Place the tube in the Hydro-extractor 200rpm 4min

--Pour out the liquid

--Rub the remain on the culture medium which contain the antibody Kana

--Foster the cell

--The pBI121-PtoMYB46 vector was mobilized into Agrobacterium tumefaciens strain GV3101, which was used for poplar transformation.

--The pYES2-PtoMYB46 vector was mobilized into Yeasts tumefaciens strain BY4741 and ycf1, which was used to check the ability of bearing and absorbing the lead.

--Pick leaves from poplar grown in asepsis environment

--Cut the leaves into squares

--Use knife to slice the leaves

--Use a stick to get the agrobacterium vector on the culture medium

--Put the sticks into a container of LB

--Put the prepared leaves into the container

--Take them out and dry them with tissues after 15minuts

--Put the leaves in a dark environment for 2 days

--Grow it in the common place

--The correctly identified yeast mutant transformants ycf1-pYES2-PtMYB46, ycf1-pYES2, BY474-pYES2-PtMYB46 and BY474-pYES2 were transferred to liquid medium and cultured at 30 °C at 300rpm, OD = 2.0.

--Use 50ml centrifuge tube at 4 ℃, 8000rpm for 5min, and wash once aseptically.

--The cells were centrifuged with sterile water and resuscitated until OD was 1.0.

--After gradient dilution to OD 0.1,0.01,0.01,0.0001, 10ul was added to the medium with low lead (SD-ura containing 2% galactose, 0, 10, 20,30, mM (CH3COO)2Pb).

--After upside-down culture for 3 days, observe and take pictures.

--The correct yeast transformants ycf1-pYES2-PtMYB46, ycf1-pYES2, BY474-pYES2-PtMYB46 and BY474-pYES2 were transferred into liquid SD/Ura.

--100 uL yeast liquid was inoculated into SD/Ura (2% galactose + CH3COO)2Pb 20mmol·L-1) medium containing different concentrations of heavy metals in 50mL conical flask, and OD was adjusted to 1.0.

--30℃, 250r·min-1, foster the yeast cell.

-- Samples were taken at different time points (0,6, 12, 24, 30 h), and the OD600 value was measured to make the growth curve.

--MYB46 protein sequences of multiple species were collected from NCBI, and then MAGE was used to perform multiple sequence alignment and draw phylogenetic evolutionary trees.

--Positive selected amino acid sites were analyzed by EasyCodeML. And the library of potential mutants was constructed according to the positive selected amino acid sites.

--We used alphafold to predict protein structure.