iGEM 2025 — Team rains-eco

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{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your daily activities, experiments, discussions, and decisions.{% endblock %}
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### Content

[Overview](#a1)
[Schedule](#a2)

Overview

We come from all over the country and come together to form the Rains-ECO team. Due to our dispersed locations, online meetings at least once a week are our daily routine, and we hold weekly meetings. Everyone counts their time and brainstorm and discuss projects online together. During this process, all of our team members worked together to advance the tasks of the dry and wet teams. Despite facing various challenges during the experiment, with everyone's concerted efforts and love for scientific research, we ultimately achieved a successful conclusion.

Schedule

1. 2025.07.05-07.08

Topic: Familiarize yourself with the laboratory environment, prepare reagents and consumables in advance before the experiment, and learn how to configure LB culture medium.

Recorder：DING YUNQING/LIU LUJIA

Process：Accurately weigh pancreatic peptone, yeast extract, and sodium chloride in proportion and place them in a conical flask with distilled water. For the preparation of solid LB medium, agar needs to be added to the liquid base, while liquid medium does not. Dissolve the ingredients, seal, sterilize with high-pressure steam (121 ℃, 103kPa (1atm) for 20 minutes), remove after sterilization, and store for later use after cooling.

In the ultra clean workbench, add antibiotics such as ampicillin, pour the culture medium into a sterile culture dish, cover the dish lid, and avoid the formation of bubbles. Let it stand for 30 minutes, wait for the agar to completely solidify, seal and invert the plate to prevent condensation from contaminating the colony. Store in a refrigerator at 4 ℃.

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Laboratory Safety Guidelines

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Topic：Sequence acquisition and structural analysis of NbCER1 and NbHY5 genes

Recorder：DING YUNQING

Process：Login to Arabidopsis official website address（ https://www.arabidopsis.org/ ）When selecting the gene option, search for CER1, click on the annotation of the gene ID corresponding to CER1, AT1G02205 (CER1), select Protein from the Sequence, and copy the protein sequence. Log in to the tobacco database（ https://solgenomics.net/organism/Nicotiana\_benthamiana/genome ）Click on the Blast option, select blastp (protein to protein), and choose N. benthamiana Genome V1.0.1 predicted proteins for Database. Enter the protein sequence of Arabidopsis CER1 for alignment.

Result: Based on the comparison results, select the gene with the highest Score value, an evaluation value of 0, and features of the Wax2 domain. The gene ID is Niben101Scf17024g00002.1. Click on the ID, select View in genome context, right-click and select View details to obtain the gene DNA sequence and CDS sequence. The same method is used to obtain the HY5 gene. The Arabidopsis HY5 gene ID is AT5G11260 (HY5). After Blast alignment, the gene ID for HY5 in Nicotiana benthamiana is Niben101Scf15744g00003.1.

1. 2025.07.09-07.11

Topic：Extraction of DNA from Nicotiana benthamiana

Recorder：SONG YIMING

Process：

1. Take about 50 to 100 mg of fresh tobacco leaf tissue, add liquid nitrogen and grind thoroughly.
2. Collect the ground powder into a centrifuge tube (self provided), add 700 μ l of 65 ℃ preheated solution A (add β - mercaptoethanol to the preheated solution A before use to make the final concentration 0.1%), quickly invert and mix, then place the centrifuge tube in a 65 ℃ water bath for 20 minutes. During the water bath process, invert the centrifuge tube and mix the sample several times.
3. Attention: To remove RNA, after completing the above steps, add 4 μ l of RNase A solution with a concentration of 100 mg/ml, shake well, and let it stand at room temperature for 5-10 minutes.
4. Add 700 μ l chloroform, mix well, centrifuge at 12000 rpm for 5 minutes.

Carefully transfer the upper aqueous phase into a new centrifuge tube (provided), add 700 μ l of solution B, and mix thoroughly.

1. Add all the solution obtained in step 4 to the Spin Column DM that has been loaded into the Collection Tube. If the solution cannot be added at once, it can be transferred in multiple times. Centrifuge at 10000 rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the recovery manifold.
2. Add 500 μ l of solution C to the adsorption column (check if anhydrous ethanol has been added before use), centrifuge at 10000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the recovery header.
3. Add 500 μ l of solution D to the adsorption column (check if anhydrous ethanol has been added before use), centrifuge at 10000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the recovery header. (Note: To further improve DNA purity, repeat step 7)
4. Centrifuge at 12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly dry. (Note: The purpose of this step is to remove residual ethanol from the adsorption column, which can affect subsequent enzymatic reactions such as digestion and PCR).
5. Place the adsorption column in a new centrifuge tube (self provided), suspend 50-100 μ l of solution E or sterile water in the middle of the adsorption column, let it stand at room temperature for 2-5 minutes, centrifuge at 10000 rpm for 1 minute, collect the DNA solution, and store the DNA at -20 ℃.

Topic：Experimental cultivation of Nicotiana benthamiana and extraction and quality testing of tobacco leaf RNA

Recorder：HU RUOYU

Process：Experimental plant cultivation of Nicotiana benthamiana. As a commonly used model organism in botanical research, Nicotiana benthamiana has the characteristics of short growth cycle, high genetic transformation efficiency, and easy cultivation, providing material basis for gene function verification, protein expression, etc.

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Extraction of RNA

1. Take 50-100 mg of tender tobacco leaves (approximately 2.0cm \* 2.0cm in size) from scissors treated with RNAse scavenger, transfer them into a 2.0 ml RNase free centrifuge tube, add steel balls burned with alcohol, and quickly place them in liquid nitrogen. Note: Place the material in the dark for 24 hours before the experiment to reduce the starch content in the material.
2. Place the centrifuge tube containing the sample into a plant tissue grinder, grind for 30 seconds, and repeat the grinding process twice.
3. Take out the sample and add 1ml of Trizol reagent. Place it in a vortex oscillator and shake for 30 seconds to mix thoroughly. Leave it at room temperature for 5 minutes to allow it to fully lyse. Centrifuge at 12000 rpm for 5 minutes.
4. Transfer the supernatant (not exceeding 700ul) to another clean 1.5ml RNase free centrifuge tube, add 200ul of chloroform, quickly shake and mix well. At this point, the solution is in a uniform milky state and left at room temperature for 15 minutes.
5. Centrifuge at 12000 rpm for 10 minutes using a low-temperature centrifuge (4 ℃), transfer the supernatant to another centrifuge tube, add 500 μ l of isopropanol and mix well (precipitate RNA), and let it stand at room temperature for 5-10 minutes.
6. Centrifuge at 12000 RPM at 4 ℃ for 10 minutes, discard the supernatant, and allow RNA to settle at the bottom of the tube. Add 1ml of 75% ethanol, gently shake the centrifuge tube, and suspend the precipitate. Centrifuge at 4 ℃ and 8000RPM for 5 minutes, and discard the supernatant as much as possible.
7. Dry at room temperature or vacuum dry for 5-10 minutes. Note: RNA samples should not be too dry, otherwise they will be difficult to dissolve. Dissolve the RNA sample in 50ul RNase free ultrapure water at 55-65 ℃ for 5-10 minutes to obtain the total RNA of the plant body. Store the extracted RNA at -80 ℃.

Determination of RNA concentration

Take 2 μ l of the extracted solution with a pipette, open the Nanodrop software, use the elution buffer as a blank control, and measure the RNA concentration and relative absorbance of A260/A280 on RNA Nanodrop 2000 Spectropppmeter. The pure RNA A260/A280 should be between 1.8-2.2.

1. 2025.07.12-07.13

Topic: Detection of RNA integrity by agarose gel electrophoresis

Recorder：HU RUOYU

Process：

1. Gel making: Weigh 0.9g of agarose and pour it into a 200ml triangular flask. Use a measuring cylinder to weigh 60ml of 1 × TAE solution and heat it in the microwave for 2 minutes. Wrap the bottle mouth with a towel and rinse the bottle body with water for 10 seconds. Add 6 μ l of nucleic acid dye, mix well, pour it into a gel making plate, remove bubbles, insert a comb, and wait for cooling.
2. RNA processing: in clean RNase free PCR tubes.
3. Sampling: Take the liquid from the PCR tube and add it to the sampling wells in sequence; Put the gelled gel into the electrophoresis tank, load the sample, and the voltage is about 50V~70V. After electrophoresis for 10 minutes, put it into the gel imaging system to observe and analyze the results.

Result：The main components in the total RNA sample are 28s rRNA, 18s rRNA, and 5s rRNA. After electrophoresis, there were three distinct bands on the gel. And the brightness of the 28srRNA band is twice that of the 18srRNA band, indicating that the results are relatively complete.

Topic：Synthesis of cDNA

Recorder：WANG CHUNQI

Process：

Reagent kit used: In this experiment, the AMV first strand cDNA synthesis kit [Biotech (Shanghai) Co., Ltd.], product number B532445, was used. Add 2 μ g of total RNA, 1 μ l of Oligo dT, and RNase free ddH2O to the RNase free PCR tube in an ice bath to a volume of 12 μ l. Gently mix and centrifuge for 3-5 seconds. The reaction mixture is then immersed in a 65 ℃ water bath for 5 minutes, followed by an ice bath for 30 seconds, and then centrifuged for 3-5 seconds. Add the components to a clean RNase free PCR tube and perform reverse transcription reaction on a PCR instrument under the following conditions: 42 ℃, 30-60 minutes; Terminate the reaction at 85 ℃ for 5 minutes (enzyme inactivation). After treatment, cDNA is obtained and stored in a refrigerator at 4 ℃ for future use.

1. 2025.07.14-07.15

Topic：Cloning Primer Design

Recorder：WANG CHUNQI

Process：Open Snapgene software, select the file, enter the new DNA or RNA software, and enter the CDS sequences of NbCER1 and NbHY5 respectively. According to the principle of primer design, the primer length is 15-35 bp. It is advisable to avoid a string arrangement of more than 5 purine or pyrimidine nucleotide sequences with a G+C content of 40% -60% to avoid complementarity between primers and prevent the formation of primer dimers. Select a sequence from ATG, click on 'Add Primer', choose the top or bottom chain, and you will see the values of annealing temperature, binding sites, and GC content. Ensure that the Tm value difference between upstream and downstream primers is less than 5 ℃. Send the primer name and the sequence from the 5 'end to the 3' end of the primer to the biotech company for primer synthesis.

Topic：PCR amplification of CDS sequences of NbCER1 and NbHY5 genes

Recorder：WANG CHUNQI

Process：

Firstly, prepare the cDNA template, primers ddH2O、 And M5 Taq enzyme. Establish a reaction system in a sterile and clean PCR tube as required. The annealing time can be adjusted by the transcription rate of the enzyme used and the maximum length of the fragment to be replicated.

Topic：Agarose gel electrophoresis

Recorder：TONG YANBO

Process：

TAE buffer preparation: Dilute 50 x TAE solution to 1 x TAE solution according to the ratio. Prepare agarose gel mixture: according to the size of DNA/RNA fragments to be separated, prepare agarose solution of appropriate concentration with electrophoresis buffer. The concentration of agarose gel prepared in this experiment is 1.5% (suitable for separating 0.2-3Kb linear DNA fragments). Weigh 0.9 g of agarose and mix it with 60 ml of 1 × TAE solution in a triangular flask. Dissolve agarose: Gently cover the neck of the triangular flask with KimWipes wiping paper and heat it in the microwave to melt. If not completely dissolved, it is necessary to wear insulated gloves and carefully rotate the triangular flask to ensure that the unmelted agarose particles stuck to the wall enter the solution, and repeat heating until completely melted. Transfer the triangular flask to a 55 ℃ water bath using insulated gloves. Add 6 μ l GelRed nucleic acid dye after the melted gel cools slightly. Gently rotate to fully mix the gel solution. Pour into the gel mold: pour the warm liquid gel into the gel mold, drive away the bubbles, insert the comb, wait for about 20 minutes until the gel is completely dry, gently pull out the comb, and then take out the gel from the mold (glue making plate, select large or small hole comb as required). Prepare electrophoresis tank: pour TAE solution into electrophoresis tank and put it into gel. Continue to add electrophoretic buffer, which is about 1mm above the gel. Sample loading: add PCR product and Marker into the hole of gel. The amount added can be determined according to the size of gel.(The extracted RNA needs to be added to a 6 × loading buffer with a volume of 0.2 times.) Glue running: Close the electrophoresis tank cover and connect the electrode plug. DNA swims sideways towards the sun level (red plug). The sample well should be located at the negative electrode. When the power is turned on, dense small bubbles can be seen emerging from the electrophoresis tank, indicating that the program is in progress. Electrophoresis should be carried out at a constant voltage of 150V for 15 minutes. If necessary, increase the running time until the product reaches at least the middle of the gel. To obtain clearer results, the voltage can be reduced and the operating time can be extended. Observation results: The band position was observed by gel imager.

1. 2025.07.16-07.20

Topic: Purification and recovery of PCR products

Recorder：TONG YANBO

Process：

Column balancing steps: Add 500 μ l of balancing solution BL to adsorption column CA2 (place the adsorption column into the collection tube), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the recovery collection tube. (Please use the column that has been processed on the same day). Cut a single target DNA strip from the agarose gel (try to remove the excess) and put it into a clean centrifuge tube, and weigh it. Add 3 times the volume of sol solution PN to the gel block; when the recovered target fragment is<150 bp or the concentration of agarose gel is>2%, 6 times the volume of sol solution PN is required (for example, if the weight of gel is 0.1 g, its volume can be regarded as 100 μ l, and so on). Place in a 50 ℃ water bath for 10 minutes, during which gently flip the centrifuge tube up and down to ensure that the rubber block is fully dissolved. If there are still undissolved glue blocks, you can add some sol solution or continue to let it sit for a few minutes until the glue blocks are completely dissolved (if the volume of the glue blocks is too large, you can cut them into pieces in advance).(Note: After the gel block is completely dissolved, the temperature of the gel solution needs to be lowered to room temperature before being loaded onto the column, as the adsorption column has a weaker ability to bind DNA at higher temperatures.). ）Add the solution obtained in the previous step into an adsorption column CA2 (place the adsorption column in the collection tube) and let it stand at room temperature for 2 minutes. Centrifuge at 12000 rpm for 30-60 seconds, discard the waste liquid from the collection tube, and place the adsorption column CA2 into the collection tube. Attention: The adsorption column has a volume of 800 μ l. If the sample volume is greater than 800 μ l, it can be added in batches. Add 600 μ l of rinsing solution PW to the adsorption column CA2 (please check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA2 into the collection tube. Repeat the previous step. Place adsorption column CA2 in the recovery manifold, centrifuge at 12000 rpm for 2 minutes, and try to remove the rinse solution as much as possible.Place the adsorption column CA2 at room temperature for several minutes and thoroughly air dry it to prevent residual rinsing solution from affecting the next step of the experiment. Attention: The residual ethanol in the rinse solution can affect subsequent enzyme reactions (such as enzyme digestion, PCR, etc.) experiments. Place the adsorption column CA2 in a clean centrifuge tube and suspend an appropriate amount of elution buffer E in the middle of the adsorption membrane. (If the recovered target fragment is>10 kb, the elution buffer EB should be preheated in a water bath at 65-70 ℃.) Let it stand at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 2 minutes.

Topic：Construction of pTopo-CER1 Vector

Recorder：LIU LUJIA

Process：Preparation before the connection reaction, establish a 10 μ l reaction system at room temperature (20-30 ℃), insert different fragments of different sizes, connect the reaction and transform the competent state of Escherichia coli. Connect at room temperature (20-30 ℃) for 5 minutes, and the connecting product can be directly transformed into competent cells or stored at -20 ℃. Transforming Escherichia coli competent DH5 α: Take out 100 μ l of Escherichia coli competent cells from the -80 ℃ refrigerator, quickly insert them into an ice bath, thaw and melt (about 3 minutes).

Immediately add 5-10 μ l of the connecting product, gently mix by hand at the bottom of the centrifuge tube (avoid using a gun to suck), and ice bath for 30 minutes. A 30 minute ice bath helps to effectively bind the product to Escherichia coli cells. Heat shock in a 42 ℃ water bath for 90 seconds, then quickly ice bath for 2-3 minutes. Do not shake the centrifuge tube during this process. Add 400 μ l of LB free medium and shake the transformed Escherichia coli at 37 ℃ and 200 rpm for 45-60 minutes. Coat the board and collect monoclonal colonies in the ultra clean workbench.

Result：Comparing sequencing results, open DNAMAN software, select Alignment in Sequence, click Mutiple Sequence Alignment, click File to enter the corresponding file, select DNA for alignment, and determine if there are sequence differences. If there is no difference from the CDS in the database, the CDS in the database will be used as the final gene result for the subsequent construction of other vectors. If there are differences, there may be natural evolutionary mutations. Repeat the above steps multiple times to ensure the reliability of the experimental results, and the final sequencing results shall prevail.

Topic：Construction of NbCER1 gene knockout vector

Recorder：LIULUJIA

Process:

Firstly, log in to the website http://crispr.hzau.edu.cn/CRISPR2/ Click on Start Dessin; Select U6 for snoRNA promoter, 19 for Guide Sequence Length, Nicotiana benthamiana (v0.4.4) for Target Genome, and default for other values. After setting the parameters, input the CDS sequence or segmented exon sequence of NbCER1 in Sequence, and then select two suitable targets with a hit rate greater than 0.6 and a miss rate less than 0.1, mainly in the non coding region. Generally, the target not far behind ATG is chosen because editing often leads to premature termination of translation. At the same time, the score is relatively high, there is no off target, and the GC content is moderate. For example, we selected two sgRNAs and confirmed ATAGCATTGCTGCACGCTGG (sg1)

The two sequences, GTTAGCACTGTCACAATGG (sg2), are completely consistent with the above sequencing results and can proceed to the next step

DT1-BsF: ATATATGGTCTCGATTGATAGCAGCATTGCTGCACGCGTT

DT1-F0: TGATAGCAGCATTGCTGCACGCGTTTTAGAGCTAGAAATAGC

DT2-R0: AACTTGTTGGTGACAGTGCTAACCAATCTCTTAGTCGACTCTAC

DT2-BsR: ATTATTGGTCTCGAAACTTGTTGGTGACAGTGCTAACCAA

In addition, we also performed four primer amplification, purified and recovered PCR products, established an enzyme cleavage ligation system, and the ligation products can be directly transformed into E. coli competent cells or stored at -20 degrees Celsius. Afterwards, the following experiments were conducted in sequence: transforming E. coli competence, coating with kanamycin resistance, selecting monoclonal antibodies, and using U626-IDF and U629-IDR primers for colony PCR identification. Use U626-IDF forward sequencing (around 700bp to detect 2 targets). Preserving bacteria (Escherichia coli/Agrobacterium), extracting plasmids from Escherichia coli, and transforming Agrobacterium GV3101.

1. 2025.07.21-07.25

Topic： Construction of pGreenII 62-SK-HY5 Vector

Recorder：YU QIYAO

Process：Cloning of the CDS fragment of HY5 gene, with a CDS fragment size of 477bp, an extension time of 20s, and a cycle number of 32. Then the CDS fragment of HY5 was purified by agarose gel electrophoresis and gel recovery. (Using a marker of 1000). Connect the HY5 gene to pGreenII 62 SK homologous B using Snapgene software, open the pGreenII 62 SK vector map, and select two unique endonuclease sites PstI and HindIII. Select the left PstI enzyme cleavage site 6bp+the left 15bp on the carrier skeleton as the left homologous B, and select the right HindIII enzyme cleavage site 6bp+the right 15bp on the carrier skeleton as the right homologous B. Add them to the 5 'end of the HY5 cloning primer F/R for primer synthesis. The primer name sequence is as follows:

62-SK-HY5-F:AGTGGATCCCCCGGGCTGCAGATGCAGGAGCAAGCGA (Tm:69℃)

62-SK-HY5-R:GTCGACGGTATCGATAAGCTTTCACTCACCGCTTCTCCT (Tm:68℃)

Continue PCR reaction with annealing temperature set to 62 ℃ and extension time set to 30s. Plasmid digestion (pGreenII 62-SK and pGreenII 0800-LUC). Determine the required endonuclease based on different carrier maps, and then perform enzyme cleavage operations according to the properties of the endonuclease. The required endonucleases for pGreenII 62-SK are PstI and HindIII, while the required endonucleases for pGreenII 0800-LUC are HindIII and PstI. Take a clean PCR tube and add the enzyme digestion system. Place the PCR tube in the PCR machine and perform enzyme digestion at 37 ℃ for 2 hours. Separation and purification of enzyme digestion products. Use a pipette to aspirate 6 μ l of plasmid before enzyme digestion and 1 μ l of 6 × Loading Buffer. Use the pipette to beat and mix well as a control sample. At the same time, 8.8 μ l of 6 × Loading Buffer was added to the PCR tube after enzyme digestion, and the mixture was shaken and mixed with a pipette, followed by agarose electrophoresis. After electrophoresis, place the gel in the gel imager to observe whether the enzyme is cut. The cut vector fragment is linear, and during electrophoresis, the migration rate is slower than that of the circular plasmid. Then, the target band is cut and purified by gel recovery. Take a clean PCR tube and add homologous recombination system.

Calculate based on the measured concentration and add the corresponding fragments with homologous B and the vector after cleavage. Recombinant at 37 ℃ for 30 minutes. (After homologous recombination, E. coli is transformed, then the plasmid is extracted and transformed into Agrobacterium competent state for preservation and future use.)

Topic：Construction of pGreenII 0800-LUC-CER1 Vector

Recorder：YU QIYAO

Process：Cloning of NbCER1 gene promoter fragment, annealed at 50 ℃, with a promoter sequence fragment size of 2000bp, extension time set to 40s, and cycle number of 32. Then the CDS fragment of NbCER1 was purified by agarose gel electrophoresis and gel recovery. (Using a marker of 5000). The NbCER1 promoter sequence is connected to pGreenII 0800-LUC homology B. Using Snapgene software, open the 0800-LUC vector map and select two unique endonuclease sites, HindIII and PstI. Select the left HindIII enzyme cleavage site 6bp+the left 15bp on the carrier skeleton as the left homologous B, and select the right PstI enzyme cleavage site 6bp+the right 15bp on the carrier skeleton as the right homologous B. Add them to the 5 'end of the CER1 cloning primer F/R for primer synthesis. Continue the PCR reaction, set the annealing temperature to 62 ℃, the extension time to 1 minute, and set other conditions as in process 6; The homologous recombination operation is described in process 11.5. After homologous recombination, transform Escherichia coli in sequence; Coating board; Picking bacteria; Bacterial P; Sequencing; Shaking bacteria; Quality enhancing granules; Transforming Agrobacterium GV3101; Coating board; Picking bacteria; Bacteria P; Protecting bacteria.

1. 2025.07.26-07.29

Topic：Experimental steps for dual luciferase reporter gene detection system

Recorder：YU QIYAO

Process：

Agrobacterium infects tobacco. Add the Agrobacterium that has been transferred into the recombinant plasmid to 1-5ml LB liquid medium containing Rif and Kan, and activate and culture it in advance at 200rpm on a shaker at 28 ℃; Take an appropriate amount of activated Agrobacterium and add it to 30ml of LB liquid medium containing Rif and Kan. Shake at 28 ℃ and incubate at 200rpm until OD600 reaches 1.0; Take an appropriate amount of bacterial solution and centrifuge at 4000rpm for 10 minutes at room temperature. Discard the supernatant and resuspend the bacterial solution in MMG or MES solution until the OD600 reaches 0.8-1.0, then set aside for later use; Mix the recombinant plasmid GENE-PGreenII 62-SK infection solution and PROMOTER-pHreenII 0800-LUC infection solution in a volume ratio of 9:1, and let them stand in the dark at 28 ℃ for at least 3 hours; Select Nicotiana benthamiana during its vigorous growth period (about a month, without flowering), use a 1ml syringe to aspirate the mixed infection solution, remove the needle, and select leaves in good condition for injection from the back. After treating the infected Nicotiana benthamiana under dark conditions for 12-24 hours, continue to culture under normal day night alternation light for 24-48 hours before sampling.

Sampling and luciferase activity detection. Sampling: Use a punch to select 1-2 locations near the injection site of Benshi cigarettes, place them in a 2ml centrifuge tube containing steel balls, freeze them in liquid nitrogen, and shake and grind them in a grinder at 30-40Hz for 30 seconds until they are in powder form. Follow the steps of the Genbiyun Tian dual luciferase assay kit (item number RG027/RG028) for subsequent operations. Add 200-300 μ l of cell lysate and shake well. Centrifuge at room temperature at 15000 × g for 5 minutes, and transfer the supernatant to a new 1.5ml centrifuge tube. The supernatant can be directly used for subsequent testing. If it cannot be measured in a timely manner, it can be stored at -20 ℃ for one month until further testing. The frozen sample needs to be thawed and restored to room temperature before measurement. Prepare the reagents. Dissolve the firefly luciferase detection reagent and sea kidney luciferase detection buffer in advance and allow them to reach room temperature; The substrate for detecting sea kidney luciferase (100x) is stored on ice for future use. Sea kidney luciferase detection working solution: Add 1 volume of 100 x sea kidney luciferase detection substrate to 100 times the volume of sea kidney luciferase detection buffer and mix well. Prepare and use immediately. After preparation, wrap it in tin foil and store it in the dark. If there is any remaining detection working solution, it can be stored at -20 ℃ for one week, but the detection effect will decrease with prolonged storage time. Therefore, it cannot be prepared into a working solution and stored for a long time. Add 100 μ l of the test sample to the enzyme-linked immunosorbent assay (ELISA) plate and mix it evenly with 100 μ l of firefly luciferase detection reagent. Then, measure the firefly luciferase activity in the ELISA reader to obtain the LUC value; After the measurement is completed, add 100 μ l of sea kidney luciferase detection working solution and mix it evenly. Measure the sea kidney luciferase and obtain the REN value; Record data.

This experiment used the DEVICES multifunctional enzyme-linked immunosorbent assay (ELISA) reader. After opening the SoftMax pro 6.5.1 software, select "Settings" in the Plate Tools menu at the top to set the software parameters. After completing the software parameter settings, prepare the test samples.

Result: Based on the real-time fluorescence intensity values LUC and REN, the LUC/REN ratio was calculated to compare the activation degree of the target reporter gene between different samples, and subsequent data analysis was conducted.

1. 2025.07.30-08.05

Topic：Genetic transformation of Nicotiana benthamiana

Recorder：SONG YIMING

Process：

This experiment used agrobacterium mediated leaf disc method for genetic transformation of tobacco. Plant sterile tobacco seedlings, take healthy and tender leaves, rinse the leaves to remove surface dust, use a punch to make circular leaf discs with a diameter of about 5-10 millimeters, then soak them in 2% NaClO (sterile distilled Tween 20 ddH2O) and gently shake them on a horizontal shaker to sterilize. Remove the agrobacterium previously stored in the -80 ℃ refrigerator, take 100 and add it to liquid LB medium. Incubate overnight at 28 ℃ and 200rpm on a shaker for 16 hours to activate the agrobacterium, and adjust the OD600 of the bacteria to 0.2-0.3 using infection solution. Immerse the leaf disc in the prepared Agrobacterium solution for 15 minutes to allow the Agrobacterium to adhere to the wound site. Then remove the leaf disc, absorb excess bacterial liquid, and place it on a co culture medium (without antibiotics). Incubate in the dark at an appropriate temperature for 2-3 days. During this process, Agrobacterium transfers and integrates T-DNA into the genome of tobacco cells. After 48 hours of dark cultivation at 23 ℃, transfer the leaves to a selected medium (containing kanamycin, 6-BA, NAA, Tim) and continue to observe the cultivation until new shoots grow.

Cut off the callus tissue as much as possible, insert the buds vertically into the rooting medium, with about 3-5 plants per bottle. Continue to cultivate under the conditions of 23 ℃ and light/dark cycle of 16 h/8 h until the root system of the plants develops and matures, and then proceed with seedling refinement. Clean the culture medium of Nicotiana benthamiana and plant it in a soil mixture of peat soil and vermiculite in a 1:1 ratio. Wrap the plants with plastic film, keep the soil moist and ventilated, at 23 ℃, with a light/dark cycle of 16 h/8 h. After 30 days of cultivation, the transgenic Nicotiana benthamiana plants can be identified.

Topic：Treat tobacco plants under different light intensities

Recorder：SONG YIMING

Process：

Phase 1: Cultivation and Light Treatment of Plant Materials

1. Seed disinfection and sowing: After disinfecting tobacco seeds with ethanol and NaClO solution, place them on a culture dish containing MS medium. Spring at 4 ℃ for 2-3 days to break dormancy. Afterwards, transplant to nutrient soil that has been sterilized for growth.
2. Dark cultivation: Place tobacco plants in a completely dark incubator (22 ℃) for 3-4 days. The purpose is to induce dark morphogenesis in seedlings and obtain yellowing seedlings. At this time, the background level of HY5 is extremely low. To detect the expression level of HY5 in multiple tobacco plants at this time, the average is taken as a constant in the regression equation.
3. Light treatment (core step): Randomly divide yellowing seedlings into 6 experimental groups, set up a dark control group (L=0), and set up 5 or more different light intensities (L) for experimental groups: 15 × 104, 20 × 104, 25 × 104, 30 × 104, 35 × 104 Lx/m2. Transfer all groups (including the dark group) simultaneously into a light incubator and process them at the same temperature (22 ℃) and same light exposure time (1 week). Ensure that all conditions are completely consistent except for light intensity.

Topic：Detecting the expression levels of HY5 protein, CER, and wax content in tobacco plants treated with different light intensities

Recorder:SONGYIMING

Process：

Tobacco plants treated with light were taken from leaves of the same size, and the total protein content was extracted. Western Blot and gel electrophoresis were used to obtain band plots, and anti-HY5 antibodies were used to detect the induced HY5 protein content. The grayscale values of each sample's HY5 band and internal reference band were measured separately, and the HY5/internal reference ratio of each sample was calculated. This ratio represents the relative expression level of HY5 protein, i.e. [HY5] in the equation.

Detection of HY5 protein levels (Western Blot) and CER1 mRNA levels (quantitative real-time fluorescence PCR, qPCR).

The detection method for HY5 protein level is the same as above. The detection of CER1 mRNA level uses a reagent kit (such as TRIzol method) to extract high-quality total RNA. After determining the concentration, an equal amount of RNA is reverse transcribed into cDNA using reverse transcriptase. CER1 specific fluorescent quantitative primers are designed, and qPCR reaction is performed on each cDNA sample to amplify CER1 and internal reference genes, respectively. Calculate the relative expression level of CER1 gene using the Δ Δ Ct method, i.e. [CER1\_mRNA]=2 ^ (- Δ Ct), which will obtain the relative expression level of CER1 mRNA in each sample relative to the control group (such as Mock treated samples), i.e. the dependent variable [CER1\_mRNA].

The RNA extraction, reverse transcription, and qPCR processes are exactly the same as the previous protocol. Using GC-MS to determine the wax content of tobacco leaves, quantitative analysis was conducted by comparing the peak areas of wax components (alkanes, aldehydes, alcohols, acids, etc.) in the sample with internal standards.

1. 2025.08.06-09.06

Topic：Identification of positive plants

Recorder：LIU LUJIA

Process：

Number transgenic seedlings and wild-type plants, extract genomic DNA, and use it as a template for PCR identification. Among them, the positive control template used is a vector plasmid with correct sequencing, and the negative control template is wild-type tobacco genomic DNA and sterile water. After gel imaging, determine whether the transgenic plants are positive seedlings according to the size of the bands. Send the PCR products of correctly identified positive plants to a biotech company for sequencing, and determine the editing type based on the sequencing results.

Topic：Analysis of wax content and composition in CER1 knockout plants

Recorder：LIU LUJIA

Process：

Select healthy and mature tobacco leaves. Place the leaves in a test tube containing 20 ml of chloroform at room temperature. Soak for 1 minute, then transfer to a new test tube containing 30 ml chloroform at 60 ℃ and soak for 2 minutes. Then remove the leaves and mix the two chloroform extracts. And add 30 μ g of heptane standard sample, dry and concentrate in a nitrogen dryer until the dissolved substance is slightly wet. Add 100 to the test tube μl N,O-bis（trimethylsilyl）trifluoroacetamide）:trimethylchlorosilane（99:1） Derive the reagent and seal it, then incubate in an oven at 115 ℃ for 30 minutes. Dry the remaining residue in a nitrogen blow dryer, dissolve the extract in 500 μ l of hexane, and transfer it to an injection bottle for storage. The wax content and composition of leaves were determined using gas chromatography-mass spectrometry (GC-MS) analysis method. The initial setting of the instrument is to maintain the temperature of the mass spectrometer detector at 280 ℃, the injection port temperature at 260 ℃, and the power supply voltage at 70eV. Siphon injection was performed through a 30 m × 0.25 mm HP-5 column, with high-purity helium gas as the carrier gas at a constant flow rate of 1.0 mL/min, without splitting the injection. The measurement procedure is to set the initial temperature of the capillary column at 60 ℃, then raise it to 200 ℃ at a rate of 20 ℃/min and maintain it for 2 minutes, and then raise it to 280 ℃ at a rate of 5 ℃/min and maintain it for 8 minutes, using ion selective mode for measurement.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

### Content

[Background](#a1)
[Design](#a2)
[Build](#a3)
[Test](#a4)
[CER1 gene is capable of regulating wax synthesis](#b1)
[Analysis of the wax synthesis pathway mediated by HY5-CER1](#b2)
[Increasing light intensity can promote the synthesis of waxy substances in plants](#b3)
[Learn](#a5)
[References](#a6)

Background

Plant epidermal wax is a layer of hydrophobic lipid compounds covering the surface of all aerial organs of plants (such as leaves, fruits, stems), primarily composed of ultra-long chain fatty acids and their derivatives (such as aldehydes, alkanes, ketones, primary and secondary alcohols, esters, etc.) [1].It is akin to a "smart coat" for plants. This "coat" is a crucial substance formed by plants during their long-term evolution to cope with terrestrial environments. It plays a significant role in preventing non-stomatal water loss (anti-transpiration), resisting pathogen invasion, and preventing mechanical damage. Nowadays, it is commonly used in the agricultural sector. By selecting waxy-layered crop varieties, the resistance of crops to stress can be enhanced, and the freshness of plants can be prolonged through artificial waxing [2].In the industrial field, epidermal wax can also serve as a raw material for the production of paints, polishing machines, cosmetics, and other products [3].In addition to its widespread application in agriculture and industry, epidermal wax has shown potential research prospects in the medical and pharmaceutical fields. This is a highly cutting-edge and actively researched area with immense potential. For instance, plant waxes, such as carnauba wax and beeswax, are excellent hydrophobic pharmaceutical excipients that can be used for drug coating and slow/controlled release formulations. Furthermore, the hydrophobic and film-forming properties of waxes make them suitable for developing novel wound dressings [4].

The ultimate goal of this project is to explore the most effective method for maximizing wax synthesis by modifying the wax synthesis pathway in Nicotiana benthamiana plants. It aims to provide insights for enhancing plant stress resistance and create low-cost, short-cycle, high-wax plant materials, offering abundant raw materials for agricultural and industrial production. Initially, we constructed the pKSE402-CER1 recombinant plasmid vector and utilized CRISPR-Cas9 technology to verify that the key regulatory gene for wax synthesis in plants is CER1. Subsequently, by constructing the pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1 recombinant plasmid vectors, we discovered that the wax synthesis regulatory network in plants is mediated by HY5-CER1, providing a breakthrough point for our modification. Afterwards, we modified the HY-CER1-wax synthesis pathway by altering light intensity to promote wax synthesis, and further investigated the optimal light intensity for maximizing wax synthesis.

Design

Based on the powerful function of tobacco genome and the mature genetic transformation technology, we chose the model plant Nicotiana benthamiana for research in order to identify the key regulatory genes involved in wax synthesis in plants. Through extensive reading and consulting of literature, we first identified the CER1 gene as our candidate gene. However, to verify its function, we needed to utilize some molecular biology experimental methods. We edited the CER1 gene using the gene knockout vector pKSE402, confirming that CER1 can control the synthesis of waxy substances and plays a positive regulatory role in this process.After clarifying the function of CER1, we need to identify suitable sites for modification. We further verified through interaction experiments that HY5 can bind to the promoter region of CER1, affecting the expression of CER1 and subsequently influencing the synthesis of waxy substances.

![](https://static.igem.wiki/teams/6002/engineering-success/img1-new.webp)

Schematic diagram of the HY5-CER1-mediated wax synthesis pathway regulated by light in plants

Most studies have shown that HY5 is directly linked to light exposure, and the intensity of light directly affects the expression of HY5, which in turn influences plant traits. As illustrated in the figure, we have designed a wax high-yield production system driven by light intensity. By increasing light intensity, we promote the expression of HY5, enhance the binding ability of HY5 and downstream target gene CER1, and artificially modify the HY5-CER1 module through physical means to increase the wax content in tobacco plants. To evaluate the effectiveness of our project, we also used gas chromatography-mass spectrometry (GC-MS) to detect the specific wax content in tobacco plants under different light treatment conditions, finding the optimal light intensity of 40×104 Lx/m2, with wax production reaching 400 ug/dm2, and the synthesis amount increased by more than three times compared to natural conditions.

Build

To explore the function of CER1, we designed target sites on the website (http://crispr.hzau.edu.cn/CRISPR2/). To enhance editing efficiency, we selected two target site, we select the two terminal sequences serve as targeting sites the GTAGCAGCATTGCTGCACGC and the TTGTTGGTGACAGTGCTAAC, amplify the target sequence through the method of four-primer amplification（Picture 1A）. Establishing an enzyme digestion and ligation system, perform double enzyme digestion using BsaI endonuclease, remove the SpR structure from the pKSE402 vector, and insert the target sequence we cloned, Construct the recombinant plasmid vector pKSE402-CER1（Picture 1B），transforming into Escherichia coli DH5α, and after a single colony grows, identify the positive clone through colony PCR（Picture 1C）. The striped sample was sent to QinKe Biotechnology Co., Ltd. for sequencing. The sequencing results aligned correctly with our target sequence, indicating that we successfully constructed the pKSE402-CER1 recombinant plasmid vector.

![](https://static.igem.wiki/teams/6002/engineering-success/img2-new.webp)

Picture 1 pKSE402-CER1Diagram of the construction process of recombinant plasmid vector

* Picture 1A Four-primer amplification of target sequence
* Picture 1B Construction of enzyme digestion and ligation system
* Picture 1C Colony PCR gel electrophoresis results
* Picture 1D pKSE402-CER1Comparison of sequencing results of recombinant plasmid vectors

To further explore the regulatory network of wax synthesis in plants, we have artificially applied external control measures to identify suitable entry points. We will utilize interaction experimental methods to verify the potential HY5-CER1 regulatory pathway within plants. Firstly, we cloned the coding sequence of HY5 and the promoter sequence of CER1. Specific primers for cloning the HY5 coding region were designed at both ends, and PstI and HindIII restriction enzyme sites were added. HindIII and PstI restriction enzyme sites were added at both ends of the primers for cloning the CER1 promoter sequence. The PCR product was obtained through amplification (Picture 2A),pGreenII 62-SK and pGreenII 0800-LUC were digested with HindIII and PstI endonucleases, respectively, and then ligated using T4 DNA ligase for recombination. Afterwards, they were transformed into E. coli DH5α（Picture 2B），after PCR, the bacterial colonies were sent for sequencing, and the sequencing results were aligned and analyzed. It indicates that we have successfully constructed pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1 recombinant plasmid vector（Picture 2C and Picture 2D）.

![](https://static.igem.wiki/teams/6002/engineering-success/img3-new.webp)

Picture 2 pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1Process diagram of recombinant plasmid vector

* Picture 2A PCR amplification with added enzyme cleavage sites, showing the gel electrophoresis results of the HY5 coding region and CER1 promoter region. The blue box represents the amplification result of HY5, and the green box represents the amplification result of CER1.
* Picture 2B The constructed recombinant plasmid vectors pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1 were transformed into Escherichia coli.
* Picture 2C Sequencing result alignment
* Picture 2D pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1Comparison of sequencing results of recombinant plasmid vectors

After confirming the presence of the HY5-CER1-mediated wax synthesis pathway in plants, we decided to approach from the perspective of light exposure, given that HY5 is a well-known star gene responsive to light. By controlling the light intensity, we aimed to alter the binding of HY5 and CER1 in plants, thereby regulating wax synthesis.After transplanting the Nicotiana benthamiana, we primarily utilize a plant light-controlled incubator to treat the tobacco plants with varying light intensities, exploring their impact on wax synthesis and seeking the optimal light intensity for maximum wax production.

![](https://static.igem.wiki/teams/6002/engineering-success/img4-new.webp)

Picture 3 tobacco cultivation process

* Picture 3A plant light-controlled incubator
* Picture 3B tobacco plants grow under different light intensities

Test

CER1 gene is capable of regulating wax synthesis

We transformed the constructed pKSE402-CER1 recombinant plasmid vector into agrobacterium cells, prepared an agrobacterium suspension, and after planting the tobacco for two weeks, took tobacco leaves as explants for genetic transformation on a clean bench. Using the agrobacterium suspension of pKSE402-CER1 that we have prepared for infection and vacuumize. Subsequently, the infected explants were placed in a co-culture medium, after reacting with agrobacterium and tobacco leaf explants for three days, the explants were transferred to a differentiation medium, and after one month, they were transferred to a rooting medium. Positive plants were identified through fluorescence observation（ Picture 4A），Taking tobacco plant leaves and taking them into the company for sequencing. Analyzing the sequencing results to determine whether the CER1 gene has been edited. The results show that there is a 4-base pair (bp) deletion at the first target site, there is a 2bp base deletion at the second target site（Picture 4B）, it causes the premature termination of the transcription process，it indicates the loss of CER1 function. Furthermore, we utilized gas chromatography-mass spectrometry (GC-MS) for analysis. The wax content of wild-type and CER1 functionally deficient mutants was detected. It was found that the wax content significantly decreased after the loss of CER1 function compared to the wild-type（Picture 4C）. It indicates that CER1 can control the synthesis of waxy substances.

![](https://static.igem.wiki/teams/6002/engineering-success/img5-new.webp)

Picture 4: Functional verification of CER1 gene

* Picture 4A Positive knockout mutant with loss of CER1 function
* Picture 4B Editing type of CER1 loss-of-function mutant
* Picture4C Comparison of wax content between wild-type and CER1 loss-of-function mutants

Analysis of the wax synthesis pathway mediated by HY5-CER1

To identify entry points for regulating wax synthesis, we utilized the constructed recombinant plasmid vectors pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1, employing a dual-luciferase reporter gene system to verify the relationship between the light-responsive factors HY5 and CER1 (Picture 5A). Preparing agrobacterium GV3101 carrying pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1 plasmids into suspensions, respectively, for infecting tobacco leaves. The experiment set up two treatment groups: one group received a mixed bacterial solution of pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1, while the other group received a mixed bacterial solution of pGreenII 62sk-HY5 and an empty vector pGreenII 0800-luc as a control. Inject two sets of bacterial solutions onto the two sides of the same tobacco leaf, and after dark cultivation, observe and detect the luciferase activity（Picture 5B and 5C）. The luciferase complementation imaging results showed that the treatment group combining pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1 exhibited a strong fluorescence signal; further quantitative analysis using a microplate reader indicated that the luciferase activity in this group was significantly higher than that in the control group. The above results indicate that the light-responsive factor HY5 can bind to the CER1 promoter region and promote the expression of CER1, suggesting the existence of a wax synthesis pathway mediated by HY5-CER1 in plants.

![](https://static.igem.wiki/teams/6002/engineering-success/img6-new.webp)

Picture 5 HY5 and CER1 Interactive verification

* Picture 5A Schematic diagram of vector construction
* Picture 5B Complementary imaging map of luciferase.
* Picture 5C analysis of dual luciferase activity results

Increasing light intensity can promote the synthesis of waxy substances in plants

We took light intensity as the starting point. Since light has a positive regulatory effect on the HY5-CER1-mediated wax synthesis pathway, we continuously increased the light intensity and detected changes in wax content. By controlling the light intensity, we achieved high-yield wax synthesis. We set up different light intensity gradients and used GC-MS to detect the wax content and the content of each component in tobacco leaves. The results showed (Picture 6). As the light intensity increases, the total wax content and the total content of each component in plants continue to rise, gradually leveling off, with C29 paraffins being the most abundant.Further analysis also revealed that when the light intensity reached 40×104 Lx/m2 and was further increased, the wax content in plants no longer increased, indicating that the wax synthesis in plants had reached a certain threshold. However, excessive light may also have adverse effects on plant growth. Therefore, if only increasing the light intensity to increase the wax content in plants, a light intensity of 40×104 Lx/m2 is the most appropriate.

![](https://static.igem.wiki/teams/6002/engineering-success/img7-new.webp)

Picture 6 Content changes of alkanes with different carbon chain lengths under varying light intensities （Picture A and Picture B）and the changes in total wax content under different light intensities (Picture A and Picture C)

Learn

It is not difficult to observe from our results that when we increase the light intensity to 40×104 Lx/m2, the wax content in the plant no longer increases with the increase in light intensity, indicating that wax synthesis reaches a certain threshold. However, in our production applications, the wax content increased through this method is far from meeting our needs. For some crops that are highly susceptible to adverse environments and pathogens, the improvement in stress resistance is still not significant. As raw materials for industrial production, the wax content remains insufficient. Therefore, we plan to further modify and optimize the HY5-CER1-mediated wax synthesis pathway in the future.

We plan to start with the HY5 protein and modify its protein structure, allowing it to partially or completely escape the constraints of upstream signals and obtain stronger transcriptional activation ability. Therefore, in the future, by point mutation or domain fusion, we aim to modify the HY5 protein to make it more sensitive to light or have stronger binding ability with the CER1 promoter, which is expected to further increase the content of wax synthesis in plants.

Of course, we can also utilize strong promoters (such as 35S or plant epidermal-specific promoters) to simultaneously overexpress HY5 and CER1, providing both ample "activation signals" (HY5) and sufficient "factories" (CER1), allowing the two to synergize at high levels and potentially produce a synergistic effect.

References

[1] Zhu, L., Guo, J., Zhu, J., & Zhou, C. (2014). Enhanced expression of EsWAX1 improves drought tolerance with increased accumulation of cuticular wax and ascorbic acid in transgenic Arabidopsis. Plant physiology and biochemistry : PPB, 75, 24–35.

[2] Zhu, J., Huang, K., Cheng, D., Zhang, C., Li, R., Liu, F., Wen, H., Tao, L., Zhang, Y., Li, C., Liu, S., & Wei, C. (2022). Characterization of Cuticular Wax in Tea Plant and Its Modification in Response to Low Temperature. Journal of agricultural and food chemistry, 70(43), 13849–13861.

[3] Zhang, Z., Ye, J., Fei, T., Ma, X., Xie, X., Huang, H., & Wang, Y. (2019). Interesterification of rice bran wax and palm olein catalyzed by lipase: Crystallization behaviours and characterization. Food chemistry, 286, 29–37.

[4] Zou, M., Wang, Y., Xu, C., Cheng, G., Ren, J., & Wu, G. (2009). Wax-matrix tablet for time-dependent colon-specific delivery system of sophora flavescens Aiton: preparation and in vivo evaluation. Drug development and industrial pharmacy, 35(2), 224–233.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

### Content

[Acquisition of target gene NbCER1 and NbHY5 sequences](#a1)
[Extraction of DNA from tobacco leaves](#a2)
[Extraction and quality assessment of RNA from tobacco leaves](#a3)
[RNA extracting](#b1)
[RNA concentration determination](#b2)
[Agarose gel electrophoresis for detecting RNA integrity](#b3)
[cDNA synthesis](#a4)
[Clone primer design](#a5)
[PCR amplification of CDS sequences of NbCER1 and NbHY5 genes](#a6)
[Agarose gel electrophoresis](#a7)
[PCR Product purification and recovery](#a8)
[Construction of pTopo-CER1 vector](#a9)
[Preparation before Connection Reaction](#b4)
[Connection reaction and transformation of Escherichia coli competent state](#b5)
[Coating](#b6)
[Selecting monoclonal bacterial colonies](#b7)
[Sequencing result comparison](#b8)
[Construction of NbCER1 gene knockout vector](#a10)
[NbCER1 gene target design](#b9)
[Four primer amplification](#b10)
[Placing it in a PCR machine and react according to the following reaction procedure](#b11)
[Protecting bacteria (Escherichia coli/Agrobacterium)](#b12)
[Extracting plasmids from Escherichia coli](#b13)
[Transforming Agrobacterium GV3101](#b14)
[Construction of pGreenII 62-SK-HY5 Vector](#a11)
[Cloning of CDS fragment of HY5 gene](#b15)
[HY5 gene linked to pGreenII 62 SK homologous B](#b16)
[Plasmid digestion (pGreenII 62 SK and pGreenII 0800-LUC)](#b17)
[Separation and Purification of Enzyme Cut Products](#b18)
[homologous recombination](#b19)
[Construction of pGreenII 0800-LUC-CER1 Vector](#a12)
[Cloning of NbCER1 gene promoter fragment](#b20)
[NbCER1 promoter sequence connected to pGreenII 0800-LUC homologous B](#b21)
[Tobacco undergoes genetic transformation](#a13)
[Identification of positive plants](#a14)
[Experimental steps for dual luciferase reporter gene detection system](#a15)
[Agrobacterium infection in tobacco](#b22)
[Sampling and luciferase activity detection](#b23)
[Instrumentation](#b24)
[Data processing](#b25)
[Analysis of wax content and composition](#a16)

Acquisition of target gene NbCER1 and NbHY5 sequences

Looking through the official website of Arabidopsis (https://www.arabidopsis.org/), select the gene option, search for CER1, click on the gene ID corresponding to CER1, AT1G02205 (CER1), select Protein in Sequences, and copy the protein sequence. Logging in to the tobacco database (https://solgenomics.net/organism/Nicotiana\_benthamiana/genome), click on the Blast option, select blastp (protein to protein), and choose N.benthamiana Genome V1.0.1 predicted proteins as the Database. Enter the protein sequence of Arabidopsis CER1 for alignment.

Based on the alignment results, selecting the gene with the highest Score value, an evalue of 0, and a Wax2 domain-containing feature. The gene ID is Niben101Scf17024g00002.1. Click on the ID, select "View in genome context", right-click and select "View details" to obtain the gene DNA sequence and CDS sequence. Use the same method to obtain the HY5 gene. The Arabidopsis thaliana HY5 gene ID is AT5G11260 (HY5). Through Blast alignment, the gene ID of HY5 in Nicotiana benthamiana is Niben101Scf15744g00003.1.

Extraction of DNA from tobacco leaves

1. 1.Take approximately 50 to 100 mg of fresh tobacco leaf tissue and grind it thoroughly in liquid nitrogen.
2. 2.Collect the ground powder into a centrifuge tube (self-provided), add 700μl of preheated solution A at 65℃ (add β-mercaptoethanol to the preheated solution A before use to make its final concentration 0.1%), quickly invert and mix, then place the centrifuge tube in a 65℃ water bath for 20 minutes, during which invert the centrifuge tube several times to mix the sample.
3. 3.Note: If RNA removal is required, after completing the above steps, add 4μl of RNase A solution with a concentration of 100 mg/ml, shake and mix, and leave it at room temperature for 5-10 minutes.
4. 4.Add 700μl of chloroform, mix thoroughly, and centrifuge at 12,000 rpm for 5 minutes.
5. 5.Carefully transfer the upper aqueous phase into a new centrifuge tube (provided by yourself), add 700μl of solution B, and mix thoroughly.
6. 6.Add all the solution obtained in Step 4 into the spin column (Spin Column DM) that has been placed in the collection tube. If the solution cannot be added all at once, transfer it in multiple portions. Centrifuge at 10,000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the spin column back into the collection tube.
7. 7.Add 500μl of solution C to the adsorption column (check if anhydrous ethanol has been added before use), centrifuge at 10,000 rpm for 1 minute, discard the waste liquid in the collection tube, and replace the adsorption column back into the collection tube.

Extraction and quality assessment of RNA from tobacco leaves

RNA extracting

1. 1.Using scissors treated with RNase inhibitor to cut 50-100 mg of young tobacco leaves (approximately 2.0 cm \* 2.0 cm in size), place them into a 2.0 ml RNase-free centrifuge tube, add steel balls that have been sterilized with alcohol, and quickly place the tube into liquid nitrogen. Note: Place the materials in the dark for 24 hours before the experiment to reduce the starch content in the materials.
2. 2.Placing the centrifuge tube containing the sample into the plant tissue grinding instrument, grind for 30 seconds, and repeat the grinding process twice.
3. 3.Taking out the sample, add 1ml of Trizol reagent, vortex on a vortex mixer for 30 seconds to mix thoroughly, let it stand at room temperature for 5 minutes to allow for complete lysis, and centrifuge at 12000 rpm for 5 minutes.
4. 4.Pipette the supernatant (no more than 700ul) into another clean 1.5ml RNase-free centrifuge tube, add 200ul of chloroform, and mix quickly by shaking. At this point, the solution should be in a uniform emulsion state. Let it stand at room temperature for 15 minutes.
5. 5.Centrifuge at 12000 rpm for 10 minutes using a low-temperature centrifuge (4°C), aspirate the supernatant into another centrifuge tube, add 500 μl of isopropanol, mix well (to precipitate RNA), and let it stand at room temperature for 5-10 minutes.
6. 6.Centrifuge at 12000RPM for 10 minutes at 4℃, discard the supernatant, and the RNA will precipitate at the bottom of the tube. Add 1ml of 75% ethanol, gently shake the centrifuge tube, and suspend the precipitate.
7. 7.Centrifuge at 8000RPM for 5 minutes at 4℃, and discard as much of the supernatant as possible.
8. 8.Let it dry at room temperature or dry in vacuum for 5-10 minutes. Note: The RNA sample should not be too dry, otherwise it will be difficult to dissolve.
9. 9.Dissolve the RNA sample with 50ul of RNase-free ultrapure water at 55-65℃ for 5-10 minutes to obtain total plant RNA. Store the extracted RNA at -80℃.

RNA concentration determination

Using a pipette to aspirate 2μl of the extracted solution, open the Nanodrop software, use elution buffer as a blank control, and measure the RNA concentration and the relative absorbance of A260/A280 on the RNA Nanodrop 2000 Spectrophotometer. The A260/A280 ratio for pure RNA should be between 1.8 and 2.2.

Agarose gel electrophoresis for detecting RNA integrity

1. 1.Making gel：Weigh 0.9g of agarose and pour it into a 200ml conical flask. Use a graduated cylinder to measure 60ml of 1×TAE solution and heat it in the microwave for 2 minutes. Wrap the opening of the flask with a towel, rinse the body of the flask with running water for 10 seconds, add 6μl of nucleic acid dye, mix well, pour it into a gel preparation plate, remove any air bubbles, insert a comb, and wait for it to cool down.
2. 2.RNA Treatment：In a clean RNase-free PCR tube

![](https://static.igem.wiki/teams/6002/experiment/img1.webp)

1. 3.Point sample：Pipette the liquid from the PCR tube and dispense it into the sample wells in order；Put the condensed gel into the electrophoresis tank, for sample loading, the voltage is approximately 50V~70V. After 10 minutes of electrophoresis, the samples were placed in a gel imaging system for observation and analysis of the results.

The main components of the total RNA sample are 28s rRNA, 18s rRNA, and 5s rRNA. After electrophoresis, three distinct bands appear on the gel. The brightness of the 28s rRNA band is twice that of the 18s rRNA band, indicating that the result is relatively complete.

cDNA synthesis

In this experiment, we utilized the AMV First-strand cDNA Synthesis Kit [Sangon Biotech (Shanghai) Co., Ltd.] with product number B532445.

1. 1.Add 2μg of total RNA, 1μl of Oligo dT, and RNase-free ddH2O to a RNase-free PCR tube in an ice bath, and dilute to 12μl.
2. 2.After gently mixing, centrifuge for 3-5 seconds. The reaction mixture is then placed in a 65°C water bath for 5 minutes, followed by an ice bath for 30 seconds, and finally centrifuged for another 3-5 seconds.
3. 3.Add the following components to a clean RNase-free PCR tube:

![](https://static.igem.wiki/teams/6002/experiment/img2.webp)

1. 4.Gently mix and centrifuge for 3-5 seconds.
2. 5.Perform reverse transcription reaction in the PCR machine according to the following conditions.42℃, 30-60 minutes; terminate the reaction, 85℃, 5 minutes (enzyme inactivation), after treatment, cDNA is obtained and stored in a 4℃ refrigerator for future use.

Clone primer design

1. 1.Open Snapgene software, select File, enter new DNA or RNA software, and input the CDS sequences of NbCER1 and NbHY5 respectively.
2. 2.According to the primer design principles, the primer length should be 15-35 bp, avoiding the presence of more than five consecutive purine or pyrimidine nucleotide sequences. The content of G+C should be preferably between 40%-60%. Complementarity between primers should be avoided to prevent primer dimer formation. Select a sequence from the ATG site, click "Add Primer", and choose the top strand or bottom strand. At this point, you can see the annealing temperature, binding site, and GC content values. Ensure that the difference in Tm values between upstream and downstream primers is less than 5℃.
3. 3.Send the primer name and the sequence from the 5' end to the 3' end of the primer to the biotechnology company for primer synthesis.

PCR amplification of CDS sequences of NbCER1 and NbHY5 genes

1. 1.Firstly, prepare the cDNA template, primer, ddH2O, and M5-Taq polymerase.
2. 2.In a sterile and clean PCR tube, establish the reaction system according to the following requirements.

![](https://static.igem.wiki/teams/6002/experiment/img3.webp)

1. 3.Place it in the PCR machine and proceed according to the following reaction program

![](https://static.igem.wiki/teams/6002/experiment/img4.webp)

(The annealing time can be adjusted based on the transcription speed of the enzyme used and the maximum length of the fragment that needs to be copied.)

Agarose gel electrophoresis

1. 1.Preparation of TAE buffer: Dilute 50× TAE solution to 1× TAE solution according to the ratio.
2. 2.Prepare agarose gel mixture: According to the size of the DNA/RNA fragments to be separated, prepare an agarose solution of appropriate concentration using electrophoresis buffer. In this experiment, the concentration of the agarose gel is 1.5% (suitable for separating linear DNA fragments ranging from 0.2 to 3Kb). Weigh 0.9 g of agarose and mix it with 60 ml of 1×TAE solution in a conical flask.
3. 3.Dissolving agarose: Gently cover the neck of the conical flask with KimWipes wiping paper and place it in the microwave for heating and melting. If it is not completely dissolved, wear heat-resistant gloves and carefully rotate the conical flask to ensure that the unmelted agarose particles sticking to the wall enter the solution. Repeat the heating process until it is completely melted.
4. 4.Transfer the conical flask to a 55°C water bath using heat-resistant gloves. After the melted gel has cooled slightly, add 6 μl of GelRed nucleic acid dye. Gently swirl to mix the gel solution thoroughly.
5. 5.Pour into gel mold: Pour the warm liquid gel into the gel mold, remove any air bubbles, insert a comb, and wait for about 20 minutes until the gel is completely dry. Gently pull out the comb and then remove the gel from the mold (depending on the gel plate, choose a comb with large or small holes as needed).
6. 6.Prepare the electrophoresis tank: Pour the TAE solution into the electrophoresis tank and place the gel inside. Continue to add electrophoresis buffer until it just covers the gel by about 1mm.
7. 7.Sample loading: Add the PCR product and Marker to the wells of the gel. The amount added can be determined based on the size of the gel. (Extracted RNA should be added with 0.2 times the volume of 6×loading buffer).
8. 8.Gel running: Close the lid of the electrophoresis tank and connect the electrode plugs. DNA migrates towards the positive pole (red plug) side. The loading well should be positioned at the negative pole. Turn on the power supply, and when dense small bubbles emerge from the electrophoresis tank, it indicates that the program is running. Electrophoresis is performed at a constant voltage of 150V for 15 minutes. If necessary, increase the running time until the product migrates to at least the middle of the gel. To obtain clearer results, the voltage can be reduced and the running time can be extended.
9. 9.Observation result: Observe the position of the band with a gel imager.

PCR Product purification and recovery

1. 1.Column equilibration step: Add 500μl of equilibration buffer BL to the adsorption column CA2 (with the adsorption column placed in the collection tube), centrifuge at 12,000 rpm for 1 minute, discard the waste liquid in the collection tube, and replace the adsorption column back into the collection tube. (Please use the column processed on the same day)
2. 2.Cut the single target DNA band from the agarose gel (try to remove excess parts) and place it into a clean centrifuge tube, then weigh it.
3. 3.Add 3 times the volume of sol solution PN to the gel block; when the target fragment to be recovered is <150 bp or the agarose gel concentration is >2%, 6 times the volume of sol solution PN is required (if the gel weight is 0.1 g, its volume can be considered as 100 μl, and so on).
4. 4.Place the centrifuge tube in a 50℃ water bath for 10 minutes, gently flipping it up and down during the process to ensure complete dissolution of the gel block. If there are still undissolved gel blocks, add some more sol solution or continue to place it for a few more minutes until the gel blocks are completely dissolved (if the volume of the gel block is too large, it can be cut into smaller pieces beforehand). (Note: After the gel block is completely dissolved, the temperature of the gel solution needs to be lowered to room temperature before loading onto the column, as the adsorption column has a weaker ability to bind DNA at higher temperatures.)
5. 5.Add the solution obtained in the previous step to an adsorption column CA2 (with the adsorption column placed in a collection tube) and let it stand at room temperature for 2 minutes.
6. 6.Centrifuge at 12,000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA2 into the collection tube. Note: The adsorption column has a capacity of 800 μl. If the sample volume is greater than 800 μl, it can be added in batches.
7. 7.Add 600 μl of rinsing solution PW (please check if anhydrous ethanol has been added before use) to the adsorption column CA2, centrifuge at 12000 rpm for 30-60 seconds, discard the waste liquid in the collection tube, and place the adsorption column CA2 into the collection tube. Repeat the previous step.
8. 8.Place adsorption column CA2 in the recovery manifold, centrifuge at 12000 rpm for 2 minutes, and try to remove the rinse solution as much as possible. Place the adsorption column CA2 at room temperature for several minutes and thoroughly air dry it to prevent residual rinsing solution from affecting the next step of the experiment. Attention: The residual ethanol in the rinse solution can affect subsequent enzyme reactions (such as enzyme digestion, PCR, etc.) experiments.
9. 9.Place the adsorption column CA2 in a clean centrifuge tube and suspend an appropriate amount of elution buffer E in the middle of the adsorption membrane. (If the recovered target fragment is>10 kb, the elution buffer EB should be preheated in a water bath at 65-70 ℃.) Let it stand at room temperature for 2 minutes. Collect DNA solution by centrifugation at 12000 rpm for 2 minutes.

(Note: The elution volume should not be less than 30 μ l, as a small volume can affect the recovery efficiency.). The pH value of the eluent has a significant impact on the elution efficiency. If sequencing is to be performed later, deionized water should be used as the eluent, and its pH value should be kept within the range of 7.0-8.5 (NaOH can be used to adjust the pH value of the water to this range). A pH value below 7.0 will reduce the elution efficiency; And DNA products should be stored at -20 ℃ to prevent DNA degradation. DNA can also be eluted with buffer solution (10 mM Tris CI, pH 8.0). To improve the recovery of DNA, the solution obtained by centrifugation can be added back to the centrifugal adsorption column, left at room temperature for 2 minutes, centrifuged at 12000 rpm for 2 minutes, and the DNA solution collected in a centrifuge tube）

Construction of pTopo-CER1 vector

Preparation before Connection Reaction

Establish the following reaction system (10 μ l system) at room temperature (20-30 ℃)

![](https://static.igem.wiki/teams/6002/experiment/img5.webp)

Recommended usage for inserting fragments of different sizes:

![](https://static.igem.wiki/teams/6002/experiment/img6.webp)

Connection reaction and transformation of Escherichia coli competent state

1. 1.Connect at room temperature (20-30 ℃) for 5 minutes, and the connecting product can be directly transformed into competent cells or stored at -20 ℃.
2. 2.Transforming Escherichia coli competent state DH5 α:
3. 3.Take out 100 μ l of E. coli competent cells from the -80 ℃ refrigerator, quickly insert them into an ice bath, thaw and melt (about 3 minutes).
4. 4.Immediately add 5-10 μ l of the connecting product, gently mix by hand at the bottom of the centrifuge tube (avoid using a gun to suck), and ice bath for 30 minutes. A 30 minute ice bath helps to effectively bind the product to Escherichia coli cells.
5. 5.Heat shock in a 42 ℃ water bath for 90 seconds, then quickly ice bath for 2-3 minutes. Do not shake the centrifuge tube during this process.
6. 6.Add 400 μ l of LB free medium,
7. 7.Place the transformed Escherichia coli in a shaking table at 37 ℃ and 200 rpm for 45-60 minutes.

Coating

1. 1.Perform the following operations on the ultra clean workbench: pipette 100-200 μ l of bacterial solution onto the ampicillin plate.
2. 2.Apply the bacterial solution evenly onto the bacterial culture plate using a coating machine, blow dry it, and seal the plate with a sealing film. Invert the plate and culture it in a 37 ℃ biochemical incubator. (Cultivate for 12-16 hours).

Selecting monoclonal bacterial colonies

Perform the following operation in the ultra clean workbench: add 1ml of LB medium containing ampicillin resistance to a sterile centrifuge tube of 1.5/2.0 ml. Open the resistance plate with well grown colonies, insert a 10 μ l/2.5 μ l pipette into the white tip, gently pick up 5 monoclonal colonies with the tip of the pipette, place them in a centrifuge tube, cover with a lid, label them, and incubate them on a shaking table at 37 ℃ and 200 rpm for 4-5 hours.

Establish a reaction system in a sterile and clean PCR tube according to the following requirements:

![](https://static.igem.wiki/teams/6002/experiment/img7.webp)

Placing it in a PCR machine and react according to the following reaction procedure

![](https://static.igem.wiki/teams/6002/experiment/img8.webp)

The agarose gel electrophoresis detection operation is as shown in process 7 (omitted), and then the bacterial solution with correct bands is sent to the biological company, and the universal primer M13F/R is used for sequencing, waiting for the sequencing result (1 day).

Sequencing result comparison

Open DNAMAN software, select Alignment in Sequence, click Mutiple Sequence Alignment, click File to enter the corresponding file, select DNA for alignment, and determine if there are sequence differences. If there is no difference from the CDS in the database, the CDS in the database will be used as the final gene result for the subsequent construction of other vectors. If there are differences, there may be natural evolutionary mutations. Repeat the above steps multiple times to ensure the reliability of the experimental results, and the final sequencing results shall prevail.

Construction of NbCER1 gene knockout vector

![](https://static.igem.wiki/teams/6002/experiment/img9.webp)

NbCER1 gene target design

Firstly, logging into the website http://crispr.hzau.edu.cn/CRISPR2/ ， then clicking on the button of starting design; Selecting U6 for snoRNA promoter, 19 for Guide Sequence Length, Nicotiana benthamiana (v0.4.4) for Target Genome, and default for other values. After setting the parameters, input the CDS sequence or segmented exon sequence of NbCER1 in Sequence, and then select two suitable targets with a hit rate greater than 0.6 and a miss rate less than 0.1, mainly in the non coding region. Generally, the target not far behind ATG is chosen because editing often leads to premature termination of translation. At the same time, the score is relatively high, there is no off target, and the GC content is moderate. For example, we selected two sgRNAs and confirmed that both ATAGCAATTGCTGCACGCTGG (sg1) and GTTAGCACTGTCACCACAATGG (sg2) sequences were identical to the sequencing results mentioned above, and we can proceed to the next step.

DT1-BsF: ATATATGGTCTCGATTGATAGCAGCATTGCTGCACGCGTT

DT1-F0: TGATAGCAGCATTGCTGCACGCGTTTTAGAGCTAGAAATAGC

DT2-R0: AACTTGTTGGTGACAGTGCTAACCAATCTCTTAGTCGACTCTAC

DT2-BsR: ATTATTGGTCTCGAAACTTGTTGGTGACAGTGCTAACCAA

Four primer amplification

![](https://static.igem.wiki/teams/6002/experiment/img10.webp)

![](https://static.igem.wiki/teams/6002/experiment/img11.webp)

Placing it in a PCR machine and react according to the following reaction procedure

![](https://static.igem.wiki/teams/6002/experiment/img12.webp)

The connecting product can be directly transformed into E. coli competent cells or stored at -20 degrees Celsius.

Afterwards, the following experiments were conducted in sequence: transforming E. coli competence, coating with kanamycin resistance, selecting monoclonal antibodies, and using U626-IDF and U629-IDR primers for colony PCR identification. Use U626-IDF forward sequencing (around 700bp to detect 2 targets).

Protecting bacteria (Escherichia coli/Agrobacterium)

Preparation: Preparation of Bacillus subtilis glycerol: Pour 50 ml of glycerol into a blue capped glass bottle using a measuring cylinder, add an equal volume of deionized water, and place in a high-pressure sterilization pot at 121 ℃ for 15 minutes until cooled at room temperature.

Perform the following operations on the ultra clean workbench: pipette 500ml of glycerol into a clean centrifuge tube, add an equal volume of bacterial solution, shake and mix well, and store at -80 degrees Celsius.

Extracting plasmids from Escherichia coli

1. 1.Preparation work: Shake the bacteria the night before extracting plasmids in an ultra clean workbench. Add 50 μ l of kanamycin and 80-100 μ l of bacterial solution to 50ml of LB liquid medium, and shake overnight at 37 ℃ and 200 rpm.
2. 2.Take 1.5-4.5ml of overnight cultured bacterial solution, centrifuge at 12000 rpm for 30 seconds, try to dry the supernatant as much as possible, and collect the bacterial cells. Collect more than 1.5ml of bacterial solution. After centrifuging and discarding the supernatant, add more bacterial solution into the same 1.5ml tube and repeat step 1 until enough bacterial cells are collected.
3. 3.Resuspend the bacterial precipitate in 250 μ l solution P1 and vortex until completely suspended.
4. 4.Add 250 μ l of solution P2, gently flip up and down 6-8 times to fully lyse the bacterial cells, and leave at room temperature for 4 minutes. Mix gently without vigorous shaking to prevent damage to the plasmid.
5. 5.Add 350 μ l of solution P3 and gently flip it up and down 6-8 times immediately. When thoroughly mixed, white flocculent precipitates will appear. Centrifuge at 12000 rpm for 10 minutes and carefully remove the supernatant. After adding solution P3, it should be mixed immediately to avoid local precipitation of SDS.
6. 6.Column equilibrium: Take a new silica gel membrane adsorption column and install it in a collection tube. Draw 100 μ l of equilibrium solution into the adsorption column. Centrifuge at 13000rpm for 1 minute, discard the waste liquid from the collection tube, and place the adsorption column back into the recovery collection tube. At this point, the equilibrium liquid pretreatment column is complete. Follow the subsequent steps.
7. 7.Add the supernatant obtained in the previous step into the adsorption column (place the adsorption column in the collection tube), centrifuge at 12000 rpm for 30-60 seconds, and discard the waste liquid in the collection tube.
8. 8.Add 600 μ l of rinse solution WB (please check if anhydrous ethanol has been added first!)! ）Centrifuge at 12000rpm for 30 seconds and discard the waste liquid.
9. 9. Repeat step 6.
10. 10.Put the adsorption column back into the empty collection tube, centrifuge at 12000rpm for 2 minutes, and try to remove the rinsing solution as much as possible.
11. 11. Remove the adsorption column AC and place it in a clean centrifuge tube. Add 50-100 μ l of elution buffer EB to the middle of the adsorption membrane (the elution buffer is better heated in a 65-70 ℃ water bath beforehand), let it stand at room temperature for 2 minutes, and centrifuge at 12000rpm for 1 minute. The liquid obtained from the centrifuge tube is the desired plasmid. Store the extracted plasmid at -20 degrees Celsius or directly use it to transform Agrobacterium into a competent state.

Transforming Agrobacterium GV3101

Take out the Agrobacterium susceptible state (100ul) from the -80 °C refrigerator and quickly place it on ice to melt (about 5 minutes). Use a pipette to extract 3-5 ul of the extracted plasmid and add it to the Agrobacterium susceptible state. Ice bath for 5 minutes, liquid nitrogen for 5 minutes, heat shock at 37 °C for 5 minutes, and ice bath for 5 minutes. Add 400ul of non resistant LB liquid culture medium to the ultra clean workbench, place it in a shaking table at 28 ℃ and 200rpm for 3-4 hours, and then coat the plate (LB kana+Rif plate). After coating, cultivate in a 28 degree biochemical incubator for 48 hours.

Afterwards, monoclonal antibodies were also selected, and U626 IDF+U629 IDR=726bp colony PCR identification was performed; The strip can be used correctly to preserve bacteria.

Construction of pGreenII 62-SK-HY5 Vector

Cloning of CDS fragment of HY5 gene

HY5-F: ATGCAGGAGCAAGCGACGAG （Tm:60℃）

HY5-R: TCACTCACCGCTTCTCCTACT (Tm:59℃)

The cloning method is as described in process 6, where the annealing temperature is modified to 57 ℃, the CDS fragment size of HY5 is set to 477bp, the extension time is set to 20s, and the number of cycles is 32. Then the CDS fragment of HY5 was purified by agarose gel electrophoresis and gel recovery. (Using a marker of 1000)

HY5 gene linked to pGreenII 62 SK homologous B

1. 1.Using Snapgene software, open the pGreenII 62 SK vector map and select two unique endonuclease sites, PstI and HindIII
2. 2.Select the left PstI enzyme cleavage site 6bp+the left 15bp on the carrier skeleton as the left homologous B, and select the right HindIII enzyme cleavage site 6bp+the right 15bp on the carrier skeleton as the right homologous B. Add them to the 5 'end of the HY5 cloning primer F/R for primer synthesis.
3. 3. The primer name sequence is as follows:

62-SK-HY5-F:AGTGGATCCCCCGGGCTGCAGATGCAGGAGCAAGCGA (Tm:69℃)

62-SK-HY5-R:GTCGACGGTATCGATAAGCTTTCACTCACCGCTTCTCCT (Tm:68℃)

1. 4.Continue the PCR reaction, set the annealing temperature to 62 ℃, the extension time to 30s, and set other conditions as in process 6.

Plasmid digestion (pGreenII 62 SK and pGreenII 0800-LUC)

1. 1.Determine the required endonuclease based on different carrier maps, and then perform enzyme cleavage operations according to the properties of the endonuclease.
2. 2.The endonucleases required for pGreenII 62-SK are PstI and HindIII, while the endonucleases required for pGreenII 0800-LUC are HindIII and PstI
3. 3. Take clean PCR tubes and add enzyme digestion system according to the following components (added on ice)

![](https://static.igem.wiki/teams/6002/experiment/img13.webp)

1. 4. Place the PCR tube in the PCR machine and perform enzyme digestion at 37 ℃ for 2 hours.

Separation and Purification of Enzyme Cut Products

Use a pipette to aspirate 6 μ l of plasmid before enzyme digestion and 1 μ l of 6 × Loading Buffer. Use the pipette to beat and mix well as a control sample. At the same time, 8.8 μ l of 6 × Loading Buffer was added to the PCR tube after enzyme digestion, and the mixture was shaken and mixed with a pipette. Then, agarose electrophoresis was performed. The detailed steps are shown in process 7, which is omitted here.

After electrophoresis, place the gel in the gel imager to observe whether the enzyme is cut. The cut vector fragment is linear, and during electrophoresis, the migration rate is slower than that of the circular plasmid. Then, the target band is cut for gel recovery and purification. The detailed steps of the gel recovery and purification process are shown in process 8.

homologous recombination

1. 1.The principle of homologous recombination

![](https://static.igem.wiki/teams/6002/experiment/img14.webp)

1. 2.Take clean PCR tubes and add homologous recombination system according to the following components (added on ice)

![](https://static.igem.wiki/teams/6002/experiment/img15.webp)

(Optimal cloning vector dosage=[0.02 × base pairs] ng)

(Optimal insertion fragment dosage=[0.04 x base pairs] ng)

1. 3.Calculate based on the measured concentration and add the corresponding fragments with homologous B and the vector after cleavage. Recombinant at 37 ℃ for 30 minutes. (After homologous recombination, E. coli is transformed, then the plasmid is extracted and transformed into Agrobacterium competent state for preservation and future use.)

Construction of pGreenII 0800-LUC-CER1 Vector

Cloning of NbCER1 gene promoter fragment

CER1-6QD-F：TATATTACCCTCGCATACAAT Tm:51℃

CER1-6QD-R：TGTTATTGTTTTAGAAGGTTACAAT Tm:51℃

The cloning method is as follows: the annealing temperature is 50 ℃, the fragment size of the CER1 promoter sequence is 2000bp, the extension time is set to 40s, and the number of cycles is 32. Then the CDS fragment of NbCER1 was purified by agarose gel electrophoresis and gel recovery. (Using a marker of 5000)

NbCER1 promoter sequence connected to pGreenII 0800-LUC homologous B

1. 1.Using Snapgene software, open the 0800-LUC vector map and select two unique endonuclease sites, HindIII and PstI
2. 2.Select the left HindIII enzyme cleavage site 6bp+the left 15bp on the carrier skeleton as the left homologous B, and select the right PstI enzyme cleavage site 6bp+the right 15bp on the carrier skeleton as the right homologous B. Add them to the 5 'end of the CER1 cloning primer F/R for primer synthesis. The primer name sequence is as follows:

0800-LUC-CER1-F: TCGACGGTATCGATAAGCTTGGTTCTTCACAAATTATATT (Tm:63)

0800-LUC-CER1-R: AGTGGATCCCCCGGGCTGCAGTGTTATTGTTTTAGAAGG (Tm:69)

1. 3.Continue the PCR reaction, set the annealing temperature to 62 ℃, the extension time to 1 minute, and other conditions as in process 6; The homologous recombination operation is described in process 11.5. After homologous recombination, transform Escherichia coli in sequence; Coating board; Picking bacteria; Bacterial P; Sequencing; Shaking bacteria; Quality enhancing granules; Transforming Agrobacterium GV3101; Coating board; Picking bacteria; Bacteria P; Protecting bacteria.

Tobacco undergoes genetic transformation

This experiment uses Agrobacterium mediated leaf disc method for genetic transformation of tobacco

1. 1.Plant sterile tobacco seedlings, take healthy and tender leaves, rinse the leaves to remove surface dust, use a punch to make circular leaf discs with a diameter of about 5-10 millimeters, then soak them in 2% NaClO (sterile distilled Tween 20 ddH2O) and gently shake them on a horizontal shaker to sterilize.
2. 2.Remove the Agrobacterium previously stored in the -80 ℃ refrigerator, take 100 and add it to liquid LB medium. Incubate overnight at 28 ℃ and 200rpm on a shaker for 16 hours to activate the Agrobacterium, and adjust the OD600 of the bacteria to 0.2-0.3 using infection solution.
3. 3.Immerse the leaf disc in the prepared Agrobacterium solution for 15 minutes to allow the Agrobacterium to adhere to the wound site. Then remove the leaf disc, absorb excess bacterial liquid, and place it on a co culture medium (without antibiotics). Incubate in the dark at an appropriate temperature for 2-3 days. During this process, Agrobacterium transfers and integrates T-DNA into the genome of tobacco cells.
4. 4.After 48 hours of dark cultivation at 23 ℃, transfer the leaves to a selected medium (containing kanamycin, 6-BA, NAA, Tim) and continue to observe the cultivation until new shoots grow.
5. 5.Cut off the callus tissue as much as possible, insert the buds vertically into the rooting medium, with about 3-5 plants per bottle. Continue to cultivate under the conditions of 23 ℃ and light/dark cycle of 16 h/8 h until the root system of the plants develops and matures, and then proceed with seedling refinement.
6. 6.Clean the culture medium of Nicotiana benthamiana and plant it in a soil mixture of peat soil and vermiculite in a 1:1 ratio. Wrap the plants with plastic film, keep the soil moist and ventilated, at 23 ℃ with a light/dark cycle of 16 h/8 h. After 30 days of cultivation, the transgenic Nicotiana benthamiana plants can be identified.

Identification of positive plants

Number transgenic seedlings and wild-type plants, extract genomic DNA, and use it as a template for PCR identification. Among them, the positive control template used is a vector plasmid with correct sequencing, and the negative control template is wild-type tobacco genomic DNA and sterile water. After gel imaging, determine whether the transgenic plants are positive seedlings according to the size of the bands. Send the PCR products of correctly identified positive plants to a biotech company for sequencing, and determine the editing type based on the sequencing results.

Experimental steps for dual luciferase reporter gene detection system

Agrobacterium infection in tobacco

1. 1.Add the Agrobacterium that has been transferred into the recombinant plasmid to 1-5ml LB liquid medium containing Rif and Kan, and shake at 28 ℃,200rpm early activation culture;
2. 2.Add an appropriate amount of activated Agrobacterium tumefaciens to 30ml of LB liquid medium containing Rif and Kan, and shake at 28 ℃on a shaker Incubate at a constant temperature of 200rpm until OD600 reaches 1.0;
3. 3.Take an appropriate amount of bacterial solution and centrifuge at 4000rpm for 10 minutes at room temperature. Discard the supernatant and resuspend the bacteria using MMG solution or MES solution ,Wait until OD600 reaches 0.8-1.0 for later use;
4. 4.Mix the recombinant plasmid GENE-PGreenII 62-SK infection solution and PROMOTER-pHreenII 0800-LUC infection solution in a volume ratio of 9:1, and let them stand in the dark at 28 ℃ for at least 3 hours;
5. 5.Select Nicotiana benthamiana during its vigorous growth period (about a month, without flowering), use a 1ml syringe to aspirate the mixed infection solution, remove the needle, and select leaves in good condition for injection from the back. After treating the infected Nicotiana benthamiana under dark conditions for 12-24 hours, continue to culture under normal day night alternation light for 24-48 hours before sampling.

Attention: Remember to water the tobacco thoroughly the day before infection; When injecting tobacco, it is recommended to use a little force to preserve the circular imprint generated during injection for later sampling; The control and experimental groups were injected on the left and right sides of the same leaf, and attention was paid to avoiding overlapping areas of infection on both sides as much as possible.

![](https://static.igem.wiki/teams/6002/experiment/img16.webp)

Picture 1 Schematic diagram of tobacco injection

Sampling and luciferase activity detection

1. 1. Sampling: Use a punch to select 1-2 locations near the injection site of Benshi cigarettes, place them in a 2ml centrifuge tube containing steel balls, freeze them in liquid nitrogen, and shake and grind them in a grinder at 30-40Hz for 30 seconds until they are in powder form. Follow the steps of the Genbiyun Tian dual luciferase assay kit (item number RG027/RG028) for subsequent operations.
2. 2.Add 200-300 μ l of cell lysate and shake well. Centrifuge at room temperature at 15000 × g for 5 minutes, and transfer the supernatant to a new 1.5ml centrifuge tube. The supernatant can be directly used for subsequent testing. If it cannot be measured in a timely manner, it can be stored at -20 ℃ for one month until further testing. The frozen sample needs to be thawed and restored to room temperature before measurement.
3. 3.Prepare reagents

Dissolve the firefly luciferase detection reagent and sea kidney luciferase detection buffer in advance and allow them to reach room temperature; The substrate for detecting sea kidney luciferase (100x) is stored on ice for future use. Sea kidney luciferase detection working solution: Add 1 volume of 100 x sea kidney luciferase detection substrate to 100 times the volume of sea kidney luciferase detection buffer and mix well. Prepare and use immediately. After preparation, wrap it in tin foil and store it in the dark. If there is any remaining detection working solution, it can be stored at -20 ℃ for one week, but the detection effect will decrease with prolonged storage time. Therefore, it cannot be prepared into a working solution and stored for a long time.

1. 4. Add 100 μ l of the test sample to the enzyme-linked immunosorbent assay (ELISA) plate and mix it evenly with 100 μ l of firefly luciferase detection reagent. Then, measure the firefly luciferase activity in the ELISA reader to obtain the LUC value; After the measurement is completed, add 100 μ l of sea kidney luciferase detection working solution and mix it evenly. Measure the sea kidney luciferase and obtain the REN value; Record data.

(Choosing a white opaque plate for the enzyme-linked immunosorbent assay (ELISA) plate, which can be washed and dried before reuse. Set 6-10 replicates for each sample group. After measuring the control and experimental groups of one sample group, proceed to measure the next set of data.)

Instrumentation

This experiment used the DEVICES multifunctional enzyme-linked immunosorbent assay (ELISA) reader. After opening the SoftMax pro 6.5.1 software, select "Settings" in the Plate Tools menu at the top to set the software parameters. After completing the software parameter settings, prepare the test samples.

The relevant settings are as follows:

Optical Configuration: Select Monochromator

Read Modes: Select LUM, chemiluminescence

Read Type: Select Endpoint, endpoint method detection, which obtains the sample detection value at a single time point

Category dropdown menu:

Wavelength：All Wavelengths

Plate Type Settings: Select 96 Wells from the drop-down menu for Plate Format

Select Specific: 96 Well Standard opaque

Read Area Settings: Select the area where the test sample has been added

PMT and Optics Settings: 1000-5000ms (Biyun Tian Kit)

Shake Settings: Optional pre detection oscillation mixing (optional)

More Settings: Select Row from the dropdown menu for Read Order

Data processing

Based on the real-time fluorescence intensity values of LUC and REN, calculate the LUC/REN ratio to compare the activation level of target reporter genes among different samples, and conduct subsequent data analysis.

Analysis of wax content and composition

1. 1. Select healthy and mature tobacco leaves. Place the leaves in a test tube containing 20 ml of chloroform at room temperature
2. 2. Soak for 1 minute, then transfer to a new test tube containing 30 ml chloroform at 60 ℃ and soak for 2 minutes.
3. 3. Then remove the leaves and mix the two chloroform extracts. And add 30 μ g of heptane standard sample, dry and concentrate in a nitrogen dryer until the dissolved substance is slightly wet.
4. 4. Add 100 to the test tube μl N,O-bis（trimethylsilyl）trifluoroacetamide）:trimethylchlorosilane（99:1） Derive the reagent and seal it, then incubate in an oven at 115 ℃ for 30 minutes.
5. 5. Dry the remaining residue in a nitrogen blow dryer, dissolve the extract in 500 μ l of hexane, and transfer it to an injection bottle for storage.
6. 6. The wax content and composition of leaves were determined using gas chromatography-mass spectrometry (GC-MS) analysis method. The initial setting of the instrument is to maintain the temperature of the mass spectrometer detector at 280 ℃, the injection port temperature at 260 ℃, and the power supply voltage at 70eV. Siphon injection was performed through a 30 m × 0.25 mm HP-5 column, with high-purity helium gas as the carrier gas at a constant flow rate of 1.0 mL/min, without splitting the injection. The measurement procedure is to set the initial temperature of the capillary column at 60 ℃, then raise it to 200 ℃ at a rate of 20 ℃/min and maintain it for 2 minutes, and then raise it to 280 ℃ at a rate of 5 ℃/min and maintain it for 8 minutes, using ion selective mode for measurement.

Lighting treatment (core step): Randomly divide etiolated seedlings into six experimental groups. Set up a dark chamber (L = 0) and five or more different light intensity (L) treatments: 15 × 10⁴, 20 × 10⁴, 25 × 10⁴, 30 × 10⁴, and 35 × 10⁴ Lx/m². Simultaneously move all groups (including the dark chamber) into a lighted incubator and maintain the same temperature (22°C) and bulb on time (1 week). Ensure that all other conditions, except bulb intensity, remain identical.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block page\_content %}

### Content

[Overview](#a1)
[Gene cloning and plasmid construction](#a2)
[CER1 gene cloning](#b1)
[Construction of pKSE402-CER1, pGreenII 62-SK-HY5, and pGreenII 0800-LUC-CER1 plasmid vectors](#b2)
[CRISPR Cas9 technology for editing CER1 gene](#a3)
[pKSE402-CER1 plasmid transformation into Agrobacterium and tissue culture operation](#b3)
[Editing of CER1 gene in Nicotiana benthamiana plants](#b4)
[GC-MS technology verification of wax content in CER1 edited plants](#a4)
[Using Dual Luc technology to verify the joint regulation of plant wax synthesis by CER1 and light regulatory factors](#a5)
[Controlling light intensity in vitro to achieve maximum wax synthesis in plants](#a6)
[Improving the Light Intensity and Modifying the HY5-CER1 Wax Synthesis Pathway](#b5)
[Exploring the optimal light intensity for wax synthesis](#b6)
[Summary](#a7)

Overview

Our project aims to increase the wax content in plants, which not only enhances their stress resistance but also provides low-cost raw materials for the production of industrial wax. Multiple studies have shown that the CER1 gene is a star gene for wax synthesis in plants, but the wax synthesis pathway has not yet been elucidated. We cloned the CER1 gene using gene cloning technology and constructed it into the pKSE402 vector plasmid. The function of CER1 was verified through gene editing, and its relationship with wax was clarified. Afterwards, in order to explore the synthesis of wax in plants and provide a starting point for us to modify the wax content in plants, we used pGreenII 62-SK to connect the HY5 coding region, pGreenII 0800-LUC to connect the CER1 promoter region, and Dual Luc assay to verify that HY5 can bind to the promoter region of CER1. HY5 is a typical light regulated gene. Therefore, we approached from the perspective of light and adjusted the light intensity to affect the wax synthesis pathway mediated by HY5-CER1 in plants, thereby improving the content and efficiency of wax synthesis in plants. This study demonstrates how to use synthetic biology thinking to solve practical production problems, providing valuable technical routes and experience for designing plant production systems for other high-value metabolites such as medicinal ingredients, specialty oils, and biofuel precursors in the future.

Gene cloning and plasmid construction

CER1 gene cloning

In order to obtain the gene cloning template, we extracted the total RNA of Bens tobacco plants, obtained the cDNA library of tobacco plants through reverse transcription, designed the primer for cloning CER1 gene as the template, obtained the product through PCR amplification, and connected it to the pTopo plasmid after gel purification, successfully constructed the pTopo-CER1 plasmid, laying the foundation for subsequent experiments.

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Picture 1 PCR technology cloning CER1 running gel results and transformation into Escherichia coli results

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Picture 2 Comparison of CER1 gene cloning and sequencing results

The results of agarose gel electrophoresis show that we have successfully amplified the CER1 gene in tobacco, and the stripe results are consistent with the template sequence in the gene library, both of which are 1857bp (Picture 1A). After transformation with Escherichia coli, a single colony was successfully grown, (Picture 1B) Beijing Qingke Biological Science and Technology Co., Ltd. was entrusted to carry out sequencing, and the results show that we have successfully cloned the sequence of CER1 gene, which is consistent with the sequence in the gene library (Picture 2), indicating that our pickle materials have not changed under natural conditions, and successfully constructed pTopo-CER1 recombinant plasmid vector for subsequent experiments.

Construction of pKSE402-CER1, pGreenII 62-SK-HY5, and pGreenII 0800-LUC-CER1 plasmid vectors

We used CRISPR-Cas9 technology to knock out the CER1 gene in Nicotiana benthamiana plants and verify its function（ http://crispr.hzau.edu.cn/CRISPR2/ ）The website is designed with targets. In order to improve editing efficiency, we selected two targets. The company synthesized them and amplified them by PCR. After gel purification, they were constructed into pKSE402-CER1 plasmid vector. With pKSE402, we realized the function damage of CER1 gene. As shown in Picture 3, the gel band of the PCR product was consistent with the expected size. The company's sequencing results also showed that we successfully constructed the CER1 gene target onto pKSE402.

A

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B

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Picture 3 PCR amplification of CER1 dual target running gel and sequence alignment of constructed pKSE402-CER1 plasmid vector

In order to further investigate the regulatory pathway of wax synthesis controlled by CER1, we searched literature and identified that the light regulated gene HY5 may be involved in this process. To verify our hypothesis, we used a dual luciferase reporter gene detection system to construct the HY5 gene coding region and CER1 promoter region onto pGreenII 62-SK and pGreenII 0800-LUC plasmids, respectively. We used these two plasmid vectors to investigate whether CER1 and HY5 could bind. As shown in Picture 4, we designed primers added with restriction sites to amplify the HY5 coding region and CER1 promoter region by PCR (Picture 4A), recycled their gel, connected them to pGreenII 62-SK and pGreenII 0800-LUC empty plasmids, and transformed them into competent cells of E. coli (Picture 4B), sent them to the company for sequencing. The sequencing results were compared with the template sequence by SnapGene software, and found that the sequencing results were completely consistent with the template sequence (Picture 4C and Picture 4D), indicating that we successfully constructed pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1 recombinant plasmid vectors.

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Picture 4: Process diagram of pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1 recombinant plasmid vectors

* Picture 4A PCR amplification with the addition of enzyme cleavage site HY5 coding region and CER1 promoter region running gel plot, where the blue box represents the HY5 amplification result and the green box represents the CER1 amplification result.
* Picture 4B The constructed pGreenII 62-SK-HY5 and pGreenII 0800-LUC-CER1 recombinant plasmid vectors were transformed into Escherichia coli.
* Picture 4C Comparison of sequencing results

CRISPR Cas9 technology for editing CER1 gene

pKSE402-CER1 plasmid transformation into Agrobacterium and tissue culture operation

The plasmid pKSE402-CER1, which was successfully sequenced, was transferred into Agrobacterium GV3101 using Agrobacterium transformation technology. After the growth of a single bacterium, the plate was coated and selected for colony PCR identification. After the bands were correct, subsequent infection experiments were conducted. (Picture 5A and 5B) Using tobacco leaves as explants, Agrobacterium suspension carrying the target gene CER1 was brought into contact with tobacco leaves, and the Agrobacterium was allowed to infect and transfer DNA to recipient cells by vacuuming. Co culture explants and Agrobacterium in a 28 ℃ biochemical incubator for a period of time to promote DNA transfer and integration. Transfer the screened transformants to an appropriate culture medium, induce their differentiation and rooting, and form complete transgenic tobacco plants.

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Picture 5: Growth status and colony PCR results of pKSE402-CER1 plasmid transformed agrobacterium tumefaciens

The colony PCR results showed that the size of the selected single colony PCR product of agrobacterium was around 500bp, which was consistent with our expectations. My editing site was successfully connected to agrobacterium and can be used for infection experiments.

Editing of CER1 gene in Nicotiana benthamiana plants

As shown in Picture 6A, we successfully obtained tobacco regenerated plants with fluorescent signals (indicating that the target gene may have been successfully edited) and sent them to Beijing Qingke Biotechnology Co., Ltd. for high-throughput sequencing. It was found that the CER1 gene was successfully knocked out, with a deletion of 4 bases at the first target and 2 bases at the second target, resulting in premature termination of the transcription process. This indicates that we have successfully knocked out the CER1 gene in Nicotiana benthamiana plants, causing it to lose its function. We can further detect changes in wax content to determine the relationship between CER1 and wax synthesis.

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Picture 6: CER1 Editing Plant Fluorescence Signal and Editing Form

GC-MS technology verification of wax content in CER1 edited plants

In order to further investigate the relationship between CER1 gene and wax synthesis, we used gas chromatography-mass spectrometry (GC-MS) to measure the wax content in wild-type and cer1 edited plants. The specific process involved taking tobacco leaves of the same size, grinding them into powder, separating the wax components from the mixture by gas chromatography, and then measuring the mass to charge ratio (m/z) of charged particles to determine the specific molecular weight of wax. As shown in Picture 7, the wax content in the cer1 edited plants was around 90ug/dm ², while the wax content in the control plants was around 130ug/dm ². Compared with the control plants, the total wax content in the cer1 edited plants decreased significantly, indicating that the CER1 gene is indeed involved in the process of plant wax synthesis.

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Picture 7 : Determination of Total Wax Content in Plants Edited by Gas Chromatography Mass Spectrometry and Cer1

Using Dual Luc technology to verify the joint regulation of plant wax synthesis by CER1 and light regulatory factors

Although we understand the regulatory genes involved in wax synthesis, there is still a lack of a regulatory medium for how to regulate wax content in plants in vitro. Reports have shown that light may affect wax content in plants, but whether there is a correlation with CER1 is still unknown. Therefore, we used dual luciferase reporter technology to investigate whether there is a relationship between CER1 and the light regulatory factor HY5. Transform the pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1 plasmids into agrobacterium GV3101, respectively (Picture 8A), and prepare a suspension of agricultural stems to infect tobacco leaves. Mix pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1 stem suspensions to prepare one group, and prepare pGreenII 62sk-HY5 and pGreenII 0800-luc empty stem suspensions to prepare one group. Transfer the two groups into the same tobacco plant on both sides and observe the luciferase activity after dark cultivation (Picture 8B and Picture 8C). The luciferase complementary imaging instrument showed that the combination of pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1 agricultural stem suspension exhibited strong activity signals, and the specific LUC/REN values detected by the enzyme-linked immunosorbent assay also showed that the group had significantly higher values than the control group. The above results indicate that the light regulating factor HY5 can bind to the CER1 promoter region and promote the expression of CER1, suggesting the existence of a HY5-CER1 mediated wax synthesis pathway in plants.

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* Picture 8A: The pGreenII 62sk-HY5 and pGreenII 0800-luc-CER1 plasmids were transformed into Agrobacterium GV3101, respectively;
* Picture 8B: Fluorescent enzyme complementary imaging results;
* Picture 8C：LUC/REN values of experimental group and control group

Controlling light intensity in vitro to achieve maximum wax synthesis in plants

Improving the Light Intensity and Modifying the HY5-CER1 Wax Synthesis Pathway

We have identified that the wax synthesis pathway in plants may be affected by light, so we artificially changed the light intensity in a light controlled incubator to modify the wax synthesis pathway mediated by HY5-CER1, promote the expression of CER1, and increase the wax content in plants. The natural light intensity is generally 100000 Lx. We used a light controlled incubator to create environments with intensities of 150000 Lx and 200000 Lx, respectively. Tobacco was placed under different conditions during the same period, and the transcription levels of HY5 and CER1 genes were detected.

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* Picture 9A: Light controlled incubator;
* Picture 9B: Cultivation of tobacco plants under different light intensities;
* Picture 9C Expression levels of HY5 and CER1 genes under natural light intensity (CK), 150000 Lx, and 200000 Lx light intensities

The results showed that increasing the light intensity could alter the expression levels of HY5 and CER1 (Picture 9C). Therefore, increasing the light intensity could modify the binding ability of HY5 and CER1, enhance their expression, and with the increase of light intensity, the expression levels of both genes continued to increase.

Exploring the optimal light intensity for wax synthesis

Although increasing light intensity can increase the expression levels of HY5 and CER1, thereby increasing wax content, in order to maximize wax synthesis in plants, we further explored the optimal light conditions for wax synthesis through gradient experiments and GC-MS methods. We set eight gradient light intensities of 15 × 104, 20 × 104, 25 × 104, 30 × 104, 35 × 104, 40 × 104, 45 × 104, and 50 × 104 Lx/m2, respectively, while keeping other variables (such as humidity and temperature) constant. After one month of cultivation, we investigated the content of different carbon chain alkanes and total wax under different light intensities, explored the changing patterns, and found the optimal light intensity.

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Picture 10: Changes in the content of carbon chain alkanes of different lengths under different light intensities (A and B) and total wax content under different light intensities (A and C)

The results show (Picture 10) that the content of C25, C27, C29, and C31 alkanes continuously increases with the increase of light intensity, and gradually stabilizes and no longer increases after the light intensity reaches 40 × 104 Lx/m2. We also observed the trend of changes in total wax content and found that it was consistent with the trend of changes in the content of different carbon chain alkanes. This indicates that under the influence of 40 × 104 Lx/m2 light intensity, the synthesis of wax in the plant body reaches its maximum, and the plant itself achieves the transformation of high wax materials.

Summary

Our project successfully modified the wax synthesis pathway mediated by HY5-CER1 in tobacco plants by changing the light intensity. Increasing the light intensity can enhance the binding ability between HY5 and CER1, promote the expression of CER1 genes, and thereby increase the wax content in plants. But we also found that when the light intensity increases to a certain value, changing the light intensity no longer increases the wax content in the plant body, causing the wax synthesized in our plant body to reach a bottleneck.

Improving wax content can also start from the genes themselves. As a transcription factor, HY5's activity, stability, and DNA binding ability are precisely regulated by post-translational modifications such as phosphorylation and ubiquitination. By modifying its protein structure, it can partially or completely break free from the constraints of upstream signals and gain stronger transcriptional activation ability. Therefore, in the future, HY5 protein can be modified through point mutations or domain fusion to increase its sensitivity to light or enhance its binding ability to the CER1 promoter. This optimization strategy can greatly improve the efficiency and content of wax synthesis in plants.

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