In this section, we present all the results of the experiments that served as important checkpoints. These include the validation of successful vector construction, the successful validation of PAL-TBH interactions by yeast one-hybrid assay color development, the successful phenotypic characterization of PAL-TBH overexpression plants, and the significant increase of flavonoid substances in PAL-TBH overexpression plants.
The following are the results of our successful cloning of CsTBH gene with the help of TOPO vector. We verified the consistency of our cloned CsTBH gene with that in the cucumber genome database (http://cucurbitgenomics.org/) by both PCR and sequencing of a single colony of E. coli, indicating that the cucumber material we selected in the natural state did not undergo mutation.
In order to further study the gene function and explore the relationship between genes and genes, we need to construct different vectors to carry out our research, the following are the gel electrophoresis graphs obtained from the PCR of E. coli colonies and single colonies of overexpression of PCY, yeast monoheterozygous PJG and PLacZi vectors constructed by us, respectively, in which the red box, yellow box and blue box represent the length of the target fragments of PCY, PJG and PLacZi, respectively, and the length of the bands are consistent with our expectation. PLacZi, and the lengths of the target fragments shown by the bands were all consistent with our expectations.
We directly sequenced the single colonies picked from the plates coated after ligation transformation to verify whether they were consistent with the sequence of our target gene. Below are the graphs of the sequence results comparison.
By observing our color development results, we found that the yeast round spots in the positive control group turned blue, the yeast round spots in the experimental group turned blue, while the yeast round spots in the negative control group did not undergo any color change, which proved that our selected CsTBH gene would bind with the downstream cis-acting regulatory element on the promoter of the CsPAL gene, and the two of them would interact.
Observation of the overexpression plants showed that their cucumber fruits grew many small black spines compared with the wild type, which might be the key to the production of flavonoids, and further testing is needed to verify this.
In order to clarify whether the fruits of CsTBH overexpression plants that we cultivated using gene editing could produce more flavonoids, by referring to the methods of some scientists [1], we quantified the flavonoid content within the wild-type and mutant fruits by using liquid chromatography mass spectrometry (LC-MS), and the following are the results of the assay, which are presented in the form of bar graphs.
[1] Feng Z, Sun L, Dong M, et al. Novel players in organogenesis and flavonoid biosynthesis in cucumber glandular trichomes. Plant Physiol. 2023;192(4):2723-2736. doi:10.1093/plphys/kiad236