Parts

This year, we contributed five basic plasmid vectors and four recombinant plasmid vectors, aiming to explore gene interactions and help plants synthesize more flavonoids with the help of different vectors.

We successfully relied on Escherichia coli (DH5α) to construct four plasmid vectors for gene overexpression and in vitro realization of gene interactions.

Plasmid construction: We integrated the CDS sequence of the cucumber PAL gene into our PCY-35S overexpression vector with His tag by homologous recombination, and the recombinant plasmid is shown below（Fig.1）.

RESULTS: After we transformed PCY-35g integrating the CDS sequence of the PAL gene into E. coli DH5α, we picked single colonies for colony PCR. and chose the ones that were successfully transformed for amplification and plasmid extraction, and then transformed them into Agrobacterium to infest plants. The following figure shows the colony PCR results（Fig.2）.

To further confirm that the constructed plasmids were correct, we sent them for sequencing. Below are the sequencing results（Fig.3）.

Plasmid construction: we integrated the CDS sequence of cucumber TBH gene into our PCY-35g overexpression vector with His tag by homologous recombination, and the following figure shows the recombinant plasmid（Fig.4）.

RESULTS: After we transformed PCY-35g integrating the CDS sequence of TBH gene into E. coli DH5α, we picked single colonies for colony PCR. and chose the successful ones for amplification and plasmid extraction, and then transformed them into Agrobacterium to infest plants. The following figure shows the colony PCR results (yellow box part)（Fig.5）.

To further confirm that the constructed plasmids were correct, we sent them for sequencing. Below are the sequencing results（Fig.6）.

Plasmid construction: we integrated the CDS sequence of cucumber TBH gene into our PJG vector by homologous recombination, and the following figure shows the recombinant plasmid（Fig.7）.

RESULTS: After we transformed PJG integrating the CDS sequence of TBH gene into E. coli DH5α, we picked single colonies for colony PCR. and chose the ones that were successfully transformed for amplification and plasmid extraction, and then transformed them into yeast cells, which in turn were subjected to color development experiments. The following figure shows the colony PCR results (red box part)（Fig.8）.

To further confirm that the constructed plasmids were correct, we sent them for sequencing. Here are the sequencing results（Fig.9）.

Plasmid construction: we integrated the cis-acting regulatory elements on the promoter of the cucumber PAL gene into our PlacZi vector by homologous recombination, and the recombinant plasmid is shown in the figure below (Fig.10).

RESULTS: After we transformed PlacZi, which integrates a homeopathic-acting regulatory element on the promoter of the PAL gene, into E. coli DH5α, we picked a single colony for colony PCR. and chose the transformed ones that were successfully amplified and plasmid extracted, and then transformed them into yeast cells, which in turn were subjected to a color development experiment. The following figure shows the colony PCR results (Fig.11).

To further confirm that the constructed plasmids were correct, we sent them for sequencing. Below are the sequencing results (Fig.12).

In order to construct plasmids that can be transferred into plants for interactions and functional validation, we utilized five basic plasmid vectors.

The vector contains kanamycin sulfate resistance gene (KanaR), which is used for selection of E. coli; the vector also provides multiple restriction enzyme sites for insertion of exogenous genes; it also contains replicon for E. coli, which ensures stable replication of the vector in E. coli; the core of the vector is the CaMV 35S constitutive promoter, which has various cis-acting elements, including -343 to -46bp upstream of the transcription start site, the transcriptional enhancement region, -343 to -208 and -208 to -90bp, the transcriptional activation region, and -208 to -90bp. The core part of this vector is the CaMV 35S constitutive promoter, which has various cis-acting elements, and the upstream of its transcriptional start site, -343～-46bp, is the transcriptional enhancement region, -343～-208 and -208～-90bp are the transcriptional activation region, -90～-46bp is the transcriptional activation region, and -90～-46bp is the transcriptional activation region. -90 to -46bp is a transcriptional enhancement region, -343 to -208 and -208 to -90bp are transcriptional activation regions, and -90 to -46bp is a region that further enhances the transcriptional activity. The action of this vector can help the gene to realize overexpression (Fig.13).

This vector contains an ampicillin resistance gene (AmpR) for selection against E. coli; it also provides multiple restriction sites for insertion of exogenous genes; and it contains a replicon for E. coli, which ensures stable replication of the vector in E. coli; in addition to that, this vector contains tryptophan- and In addition, the vector also contains tryptophan and uracil on PlacZi for screening of second-deficiency plates. The characteristics of this vector ensure the smooth progress of the experiment (Fig.14).

This vector contains the ampicillin resistance gene (AmpR) for selection against E. coli; it also provides multiple restriction enzyme sites for insertion of exogenous genes; and it contains a replicon for E. coli, which ensures stable replication of the vector in E. coli; in addition to that, this vector contains uracil - and PJG In addition, the vector also contains uracil- and PJG-tryptophan for the screening of two-deficient plates. The characteristics of this vector ensure the smooth progress of the experiment (Fig.15).

CsPAL-CDS is the abbreviation of the coding sequence of the cucumber PAL gene. Similar to CsTBH-CDS, it encodes a series of PAL protein products, participates in the transcriptional regulation of PAL, and serves as a template throughout the entire process of gene transcription, translation, and processing. CsPAL participates in regulating the synthesis of phenylalanine ammonia lyase in the initial reaction of cucumber phenylpropanoid metabolism, thus becoming the master switch of the cucumber flavonoid synthesis pathway.

CsTBH-CDS is the abbreviation of the coding sequence of the cucumber TBH gene. CDS is a sequence that encodes a protein product and is a term in structural genomics. DNA is transcribed into mRNA, and mRNA

is translated into protein after splicing and other processing. The so-called CDS is a DNA sequence that corresponds one-to-one with the protein sequence, and the sequence does not contain other sequences that do not correspond to the protein, regardless of sequence changes during mRNA processing. In short, it completely corresponds to the codons of the protein.