Results

Experimental Outcomes

Plasmid Construction and Preliminary Verification Process

In 2025, we attempted to construct two plasmids: one was a dsRNA plasmid targeting superfold green fluorescence protein (sGFP), and the other was an expression plasmid for chitinase driven by the T7 promoter (BBa_25YSLUTM).

For safety reasons, we cannot culture the pathogenic fungi that cause peach gummosis, and thus cannot verify whether the designed dsRNAs can interfere with their growth.

Instead, if we can develop dsRNA that interferes with the synthesis of sGFP, we will be able to determine the success of the experiment with the naked eye.

So a dsRNA plasmid was constructed to silence sGFP, providing us with a visible indicator of RNA interference efficiency to confirm the experimental workflow used for dsRNA design and plasmid construction. Meanwhile, the T7-driven peach chitinase plasmid aimed to express an enzyme predicted to be capable of degrading fungal chitin. Both constructs were transformed into E. coli DH5α for amplification.

Subsequently, plasmid DNA was extracted and verified by restriction enzyme digestion and Sanger sequencing. The results showed that only part of the dsRNA was inserted into the vector, but Sanger sequencing confirmed the correct insertion of the full-length peach chitinase. Therefore, pET-CH1 was transformed into E. coli BL21 for amplification and induced for expression.

Protein Expression Analysis and Optimization Directions

Total proteins from different IPTG treatments were harvested and analyzed by SDS-PAGE. Faint bands indicated partial expression, and further optimization of the protein extraction protocol, induction time, and IPTG concentration is needed to improve yield.

Although we did not obtain the expected clear bands for robust expression, the established experimental workflow—from vector construction to transformation and expression—provides a foundational framework for future attempts. The partial success in constructing the pET-CH1 plasmid demonstrates the feasibility of our cloning strategy, and we successfully performed SDS-PAGE analysis under limited conditions, confirming that our system can support protein expression with further refinement.

Currently, the laboratory is in continuous development, with ongoing efforts to enhance technical capabilities and infrastructure. We are actively optimizing expression conditions, including temperature, induction duration, and lysis buffers, to improve the solubility and detection of recombinant proteins. We may collaborate with other institutions or universities to conduct nickel-affinity chromatography purification trials and further validation via Western blotting, which are essential steps to verify the identity and functionality of the expressed protein.

Discussion

With the cost reduction of gene sequencing, thousands of species' genomes have been decoded, but gene function annotation remains a critical gap. Our project addresses this by:

  • Exploring unannotated gene value (e.g., peach chitinase)
  • Developing a simple, high school-friendly protocol to popularize gene function verification.

Plans

  • AI-based Functional Prediction and Experimental Validation of the PpCH1

    Use AI tools (e.g., AlphaFold for structure prediction, BLAST for homology analysis) to hypothesize PpCH1’s role in fungal cell wall degradation. Validate via enzymatic activity assays (e.g., chitin hydrolysis experiments).

  • Chitinase Extraction Workflow

    Standardize steps for microbial/plant chitinase: sample pretreatment-centrifugation-column purification-activity detection. Compare processes by purity (SDS-PAGE) and yield (BCA assay) to enable industrial application.

  • Optimization of the dsRNA Fragment Design and Validation Workflow

    dsRNA plays a core role in RNA interference technology. We will combine the latest bioinformatics algorithms and experimental data to optimize dsRNA design parameters, such as sequence length, GC content, and target region selection, in order to improve the silencing efficiency of dsRNA on target genes and reduce off-target effects. At the same time, we will establish a fast and accurate dsRNA validation system.