Our project not only proposes innovative ideas for improving plant wax synthesis, but also makes solid contributions to scientific research. We have successfully revealed and validated a novel pathway in Nicotiana benthamiana through molecular biology and synthetic biology methods, in which the key wax synthesis gene NbCER1 is directly regulated by the light controlled transcription factor NbHY5, providing crucial genetic and molecular biology evidence for this. Our specific contributions mainly include basic research contributions, synthetic biology contributions, and application and conceptual contributions.
Although previous studies have shown that NbHY5 is involved in light signal transduction and NbCER1 is a key enzyme in wax synthesis, it is still unclear whether there is a direct regulatory relationship between the two in Nicotiana benthamiana.
- We constructed a knockout vector (pKSE402-CER1) for the NbCER1 gene using CRISPR-Cas9 technology and successfully obtained mutant plants. This mutant exhibits a significantly reduced wax synthesis phenotype, which directly and strongly proves that NbCER1 is an essential key gene for wax synthesis, especially alkane synthesis, in Nicotiana benthamiana. This work has laid a solid foundation for subsequent regulatory research.
- To investigate whether NbHY5 regulates NbCER1, we conducted the following key experiments:
Construction of effector and reporter vectors: We constructed effector vectors for NbHY5 (pGreenII 62-SK-HY5) and reporter vectors for luciferase (LUC) driven by CER1 natural promoter (pGreenII 0800-LUC-CER1).
Dual Luciferase Assay: By co injecting the above two vectors into tobacco leaves, we found that the expression of NbHY5 can significantly activate the activity of the CER1 promoter. This provides strong evidence that NbHY5 protein can directly or indirectly act on the promoter region of CER1, thereby upregulating its transcriptional expression.
- Our work has demonstrated for the first time this direct connection within plants, providing new insights into the molecular mechanisms by which plants respond to environmental light to regulate their epidermal barrier.
- This is a modular CRISPR-Cas9 vector. We first used the pCBC-DT1T2 (anti chl) vector as a template, which is an intermediate vector specifically designed for the Golden Gate cloning method and can serve as a template for creating expression cassettes containing multiple gRNA target sites. PCBC is the name of the carrier skeleton, which typically includes a replication origin and a resistance screening marker. DT1T2 refers to the vector containing two BsaI restriction enzyme sites designed for cloning. In the Golden Gate clone, after BsaI enzyme cleavage, T1 and T2 will reveal four base overhangs. This design ensures that the inserted fragments can only be connected to the vector in the correct direction, avoiding self circularization and achieving seamless and directed cloning, helping us efficiently and accurately install the designed gRNA sequence onto the final CRISPR vector.
- In addition, we introduced mutations through four primer amplification, changing the original dual primer approach. The four primer method locks the mutation in the center of the product through two rounds of PCR. The first round of PCR generates two independent small fragments that are 100% mutated. In the second round of PCR, these two fragments were spliced through overlapping regions and finally amplified into full-length products by external primers. The mutation rate of the final product is close to 100%. Because the original wild-type template is only used as the starting point in the first round of PCR, the final full-length product is assembled from two mutated fragments and is almost not contaminated by the original wild-type template. Therefore, in the subsequent cloning steps (such as transformation), even if a very small amount of the original template plasmid is introduced into the reaction, it is extremely difficult to form clones due to the lack of correct cloning sites, greatly reducing the background of false positives (non mutated clones).
The traditional dual primer mutation introduction requires the synthesis of two very long primers (usually>40nt), with one or more mismatched bases in the middle. Long primers not only have high synthesis costs, but their annealing effect is also more prone to errors. And the mutation efficiency is low, the false positive rate is high, which exacerbates the workload of subsequent screening. And the four primer amplification has high flexibility and is suitable for long fragment mutations. If the mutation site is close to the end of the DNA sequence, the two primer method is feasible. But if the mutation site is located in the center of a long sequence, the PCR efficiency of the two primer method will sharply decrease because the most critical part at the 3 'end of the primer (the part that fully matches the template) is too far from the end.
In addition, although our four primer amplification is essentially a two round PCR, due to the clever design of our four primers, we directly added different concentrations of four primers to the system for PCR reaction. The second round of primers can only use the product from the first round as a template, and the two rounds of PCR reaction can be carried out simultaneously without separating and purifying the product from the first round, greatly saving experimental costs and time.
And we found that the first round of primers DT1-F0/DT2-R0 should be diluted 20 times, but the concentration cannot be consistent with that of DT1 BsF/DT2 BsR. Otherwise, there will be a large number of non target bands close to the target band, which increases the difficulty of separation and purification.
We believe that there are three main reasons for diluting pCBC-DT1T2 and DT1-F0/DT2-R0: firstly, the design of the internal primers (DT1-F0/DT2-R0) is complementary to the pCBC-DT1T2 sequence. If they are added at high concentrations (such as 10 μ M), it is very easy for primer dimers to form between them, or for them to bind incorrectly with other similar sequences on the template, resulting in the production of a large number of non target bands, consuming dNTPs and enzymes in the reaction system, thereby seriously reducing the yield of target intermediate segments. After diluting it 20 times, the effective concentration decreased, greatly reducing the probability of this side reaction, making the first round of PCR cleaner and more efficient. Secondly, it is to reserve the dominance of external primers for the second round of PCR. The design of this reaction system is a variant of Nested PCR. After the first round of PCR is completed, purification will not be carried out, but a small amount of product will be directly taken as a template for the second round of PCR. If the concentration of internal primers is high, they will continue to compete with external primers for binding to the template (i.e. the product of the first round) in the second round of PCR. By diluting the internal primers, at the beginning of the second round of PCR cycle, the concentration of internal primers in the system has been depleted or become extremely low, while the newly added external primers (DT1 BsF/DT2 BsR) occupy an absolute advantage with their high concentration, thus efficiently and specifically amplifying the final full-length product. Thirdly, the internal primers only need to be sufficient to complete the synthesis of the first round of PCR, without excess. Diluting usage is an economical approach.
This is a plant expression vector containing the HY5 coding sequence (CDS) derived from Nicotiana benthamiana, driven by a strong promoter. This component is a powerful tool for studying plant light signal networks, which can be used to activate a range of downstream light responsive genes, not limited to wax synthesis, but also for studying growth and development processes regulated by light.
This is a high-throughput reporting system that includes the natural promoter of the tobacco CER1 gene, which drives the expression of the luciferase reporter gene. This component can be used for rapid screening of other transcription factors or signaling molecules that can regulate CER1 expression, and can also be used for real-time monitoring of the dynamic response of plant wax synthesis pathways under different environmental stresses, such as drought and strong light.
We did not simply overexpress CER1 using strong promoters (such as 35S), but instead chose to modify and enhance its upstream natural light controlled switch (HY5 system). This strategy has two major advantages.
- To avoid excessive metabolic burden, synchronize wax synthesis with plant photosynthetic capacity (light conditions), and reduce unnecessary energy waste.
- Maintaining internal homeostasis preserves the plant's own feedback regulation of pathways, making metabolic flow changes more in line with physiological rhythms.
We provide a classic example of functional gene research process for the field of synthetic biology: from CRISPR gene editing to validate functionality, to reporting system analysis of regulatory mechanisms, and then to rational design based on this mechanism. This method can be widely applied to the mining and transformation of other economic traits.