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.

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.

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.

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.

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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.